CN120419528B - Construction method and application of kidney protection model for tail remote ischemia pretreatment of mice - Google Patents

Abstract

The invention discloses a construction method and application of a kidney protection model for remote ischemia pretreatment of a tail part of a mouse, belongs to the technical field of ischemia pretreatment, and aims to solve the technical problems of poor stability and reliability of kidney protection experiment results in the prior art. The method comprises the steps of respectively constructing a tail distal ischemia pretreatment group, a sepsis acute kidney injury model group and a tail control group, binding a tourniquet at the tail of a mouse when constructing the tail distal ischemia pretreatment group, blocking blood flow, recovering blood flow and repeating circulation, collecting blood and kidney tissues of the mouse in each group, detecting serum creatinine and serum urea nitrogen indexes, evaluating pathological injury conditions of renal tubules and evaluating the expression level of kidney active oxygen. The method proves that the tail ischemia reperfusion pretreatment can improve the kidney pathological damage of the acute sepsis kidney injury and relieve the tubular injury, and can inhibit the kidney oxidative stress of the acute sepsis kidney injury and relieve the inflammatory response of the acute sepsis kidney injury.

Description

Construction method and application of kidney protection model for tail remote ischemia pretreatment of mice

Technical Field

The invention belongs to the technical field of ischemia pretreatment, relates to construction of a kidney protection model based on mouse ischemia pretreatment, and particularly relates to a construction method and application of a remote ischemia pretreatment kidney protection model of a tail part of a mouse.

Background

Ischemic Preconditioning (IPC) is a protective mechanism that enhances tolerance of organs or tissues to subsequent more severe ischemic injury by transient, non-lethal ischemic stimuli. The core principle of the method is to excite endogenous adaptive response, and through activating a cell signal channel and reducing oxidative stress and inflammatory response, the organ can resist subsequent ischemia-reperfusion injury, thereby reducing injury caused by long-term ischemia, and being widely applied to the protection of important organs such as heart, brain, liver and the like. However, ischemia Pretreatment (IPC) acts directly on the target organ, which causes stress reaction, damages vascular structure, causes poor prognosis, and limits its clinical application.

In the prior art, when ischemia reperfusion is performed on a target organ (i.e., kidney), a corresponding experimental apparatus is generally required. The utility model patent with the application number 202021983041.4 discloses an experimental bench for animal ischemia reperfusion experiments, which comprises a flat plate, wherein two parallel sliding rails along the front and rear directions are fixedly arranged on the top surface of the flat plate, two first sliding blocks are respectively matched and arranged on the left sliding rail and the right sliding rail, a cylinder body with an opening at the upper end is fixedly arranged above the first sliding blocks, a rotating shaft is rotationally connected between the front inner side wall and the rear inner side wall of the cylinder body, a round wheel is sleeved on the rotating shaft, a section of arc-shaped tooth is arranged below the round wheel, worms with mutually perpendicular axes are meshed below the arc-shaped tooth, first through holes are respectively arranged on the outer sides of the side walls of the cylinder body corresponding to the worms, and the outer ends of the worms penetrate through the first through holes and are fixedly provided with coaxial turntables. The patent of the utility model with the application number 202120170725.0 also discloses an animal ischemia reperfusion experimental device, which comprises a spring clamp and an air pump, wherein the spring clamp is formed by connecting a first clamping plate and a second clamping plate through a torsion spring, the right sides of the first clamping plate and the second clamping plate are respectively and fixedly connected with a first handle and a second handle, the bottom of the inner wall of the first clamping plate and the top of the inner wall of the second clamping plate are respectively and fixedly connected with a mounting frame, and the interiors of the two mounting frames are respectively and fixedly connected with a first air bag and a second air bag.

For ischemia reperfusion, it is also of vital importance to construct animal models, in addition to the corresponding experimental setup. The aim of constructing an ischemia animal model is to generate kidney ischemia reperfusion injury in an animal body, so that the growth condition of cells capable of repairing kidney injury in the animal body and the effect of repairing kidney injury are observed.

In the prior art, the construction of an animal model for ischemia reperfusion of the kidney of an animal body is lacking, but the construction of other animal models exists. For example, patent application number 202110816267.8 discloses a rat renal lower abdominal aortic aneurysm model constructed by a retroperitoneal access and a construction method, which comprises the steps of fully freeing and ligating abdominal aortic branches of a perfusion section by using a mu wire, fully exposing an operation visual field by using a mastoid spreader, blocking the proximal end of the abdominal aortic branch by using a microscopic vascular clamp, extruding blood in a blood vessel of the perfusion section to the distal end by using a microscopic hemostatic clamp, blocking the distal end of the abdominal aortic branch by using the microscopic vascular clamp, completely collapsing blood vessels to indicate good vascular tightness, extracting 0.2mL of a blood vessel containing 10U elastase by using a disposable insulin syringe, slightly bending a syringe needle by using the vascular clamp, puncturing the abdominal aortic branch slightly in an L shape, slowly injecting 0.1mL of elastase containing 5U after puncture success, fully filling the abdominal aortic branch of the perfusion section by using a puncture needle, retaining for 20min, keeping the abdominal aortic branch of the perfusion section in a fully filled state if a little elastase is infiltrated and supplemented in time, removing Bi Huichou intravascular drugs, pulling the needle, covering a puncture point by using a sponge, pressurizing cotton, taking out a small vessel clamp, checking the blood vessel, and taking out a small vessel clamp for 10 mm, draining a small vessel clip, and cleaning a small vessel clip, and draining a small vessel, and checking the surface by a small vessel clip, if a small vessel clip is closed after the surface is closed, and a small-end of a small vessel clip is not in a small-shaped, and a small-diameter capillary tube is left, and a small-sized. The method can shorten the infusion time to 20min, and can stop bleeding by local compression after the infusion. The invention patent application with the application number 202411332193.0 also discloses a construction method for the myocardial ischemia animal model, which comprises the steps of calculating morphological characteristics of contrast mesenchymal stem cells in a pretreatment image, carrying out principal component analysis on the morphological characteristics, outputting and analyzing cell activity corresponding to the principal components through an activity analysis model, constructing an animal three-dimensional model of a myocardial injury animal, collecting a culture environment of experimental mesenchymal stem cells, selecting a growth environment in the myocardial injury animal by utilizing the culture environment, collecting an environment image of the growth environment, selecting a transplanting position of the myocardial injury animal from the environment image, determining a three-dimensional position of the transplanting position from the animal three-dimensional model, constructing a transplanting path from the surface position of the animal three-dimensional model to the three-dimensional position, and transplanting the experimental mesenchymal stem cells to the transplanting position to obtain an in-situ transplanting animal model.

With reference to the above animal model construction mode, when an animal model based on ischemia reperfusion is constructed, the model is realized by adopting the model based on abdominal aortic ischemia, lower limb femoral ischemia and the like, and although the kidney protection mechanism and screening related therapeutic drugs can be observed and researched through the two models of tourniquet induced abdominal/lower limb ischemia and direct abdominal aortic ischemia/lower limb femoral ischemia, the middle mode still has certain defects, mainly in the aspects of 1, trauma and injury, the two methods can cause larger trauma to the animal abdominal/lower limb, which is unfavorable for the subsequent survival and recovery of animals. 2. In the aspect of nerve injury, the tourniquet induces the ischemia of the abdomen/lower limb to press the nerve, the long-time ischemia can cause the ischemia and hypoxia of the nerve, cause the injury and necrosis of nerve fibers, cause the weakness and paralysis of the muscle of the abdomen/lower limb, and the operation of the ischemia of the femoral artery of the abdomen/lower limb can damage the peripheral nerve, thereby causing the symptoms of numbness, tingling, hypoesthesia and the like in the pressed nerve innervation area. 3. In terms of operation and stability, the method has higher requirements on operation skills and postoperative nursing, increases experiment difficulty and workload, and is easy to cause adverse consequences and increase experiment complexity. 4. The method has the advantages of great trauma to animals, unstable animal state, great result difference among different experiments and poor stability and repeatability of the experiments. Therefore, it is necessary to construct an animal model which is less traumatic to animals and more convenient to operate, and the reliability and scientificity of the experiment are improved.

Disclosure of Invention

The invention aims to solve the technical problems that a model constructed in the prior art is large in animal trauma, large in operation difficulty and poor in experimental result stability and reliability caused by difficulty in fixing animals, and provides a construction method and application of a tail remote ischemia pretreatment kidney protection model of a mouse.

The invention adopts the following technical scheme for realizing the purposes:

The construction method of the kidney protection model for the remote ischemia pretreatment of the tail part of the mouse comprises the following steps:

step 1, selecting materials and grouping;

And selecting a plurality of healthy mice with male C56BL/6J, body weight of 23-25 g and week age of 6-8 weeks, and randomly dividing the healthy mice into a tail control group, a sepsis acute kidney injury model group and a tail remote ischemia pretreatment group.

Step 2, inducing anesthesia;

The mice of the tail distal ischemia pretreatment group are slowly injected by using the uratam with the concentration of 15-25% (the dosage standard is 1.5-2.0 mg/kg) through the abdominal cavity, so that the mice are anesthetized, and the mice of the sepsis acute kidney injury model group and the tail control group are not anesthetized.

Step 3, constructing an animal model;

The tail remote ischemia pretreatment group is constructed, namely after the mice of the tail remote ischemia pretreatment group are anesthetized, the tail of the mice is bundled by using a tourniquet, blood flow is blocked for 4-8 min, then the tourniquet is loosened to restore the blood flow for 4-8 min (blood flow blocking time=blood flow restoring time), the ischemia-reperfusion process is repeated for 3-6 cycles, after the ischemia reperfusion cycle is finished for 12-20 min, lipopolysaccharide LPS with the concentration of 8-12 mg/kg is injected into the abdominal cavity (the dosage standard is 80-120 ul/kg). The time interval from the time of anesthesia to the time of lipopolysaccharide LPS injection was T.

A sepsis acute kidney injury model group is constructed by injecting lipopolysaccharide LPS with equal concentration and equal dosage standard into mice through abdominal cavity after the same time period as the tail distal ischemia pretreatment group (namely, injecting lipopolysaccharide LPS after the time T is elapsed after the mice of the tail distal ischemia pretreatment group are anesthetized).

The tail control group is constructed by sleeving the tourniquet on the tail of the mouse but not binding the tourniquet, keeping the blood flow of the tail of the mouse normal, injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail far-end ischemia pretreatment group (namely, injecting the physiological saline after the time T passes after the anesthesia of the mouse of the tail far-end ischemia pretreatment group, and the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

The specific injection method for the lipopolysaccharide LPS comprises the following steps:

The method comprises the steps of grabbing and fixing a mouse by the left hand, enabling the abdomen of the mouse to face upwards and enabling the head to be lower than the tail so as to avoid damage to internal organs, disinfecting the abdomen of the mouse by using an alcohol cotton ball, enabling an injector to penetrate into the abdomen of the mouse at a position of 0.3-0.7 cm on any side of the white line of the abdomen, enabling a needle head to push downwards for 3-5 mm, enabling the needle head to penetrate into the abdominal cavity of the mouse at an angle of 40-48 degrees with the skin, enabling the needle head to have a falling feel when penetrating, enabling liquid to be slowly injected when the needle head is pulled back, and enabling the needle head to be rotated and pulled out slowly after injection is completed so as to prevent liquid leakage.

Step 4, collecting and detecting for evaluating the model effect;

after lipopolysaccharide LPS and physiological saline are injected into the abdominal cavity for 12-20 hours, blood and kidney tissues of the mice are synchronously collected.

After the collected blood is subjected to centrifugal treatment, supernatant is taken and serum creatinine and serum urea nitrogen indexes are measured (the specific method for measuring the serum creatinine and serum urea nitrogen indexes according to the supernatant can be directly applied to the prior art without the need of creative labor) and are used for evaluating the kidney functions of mice in each group.

The harvested kidney tissue is divided into three parts:

The first part is used for extracting protein and RNA, detecting a tubular injury marker such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and the like, and detecting the expression level of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like (wherein, the specific method for detecting the expression level of the tubular injury marker and the inflammatory factors by using kidney tissues can be directly applied to the prior art without the need of creative labor):

When protein is extracted from kidney tissue, the required tissue homogenate preparation conditions are that 20-30 mg of kidney tissue is placed in a 2-5 ml grinding tube, 450-600 ul of protein lysate is added, 1-2 stainless steel grinding steel balls with the diameter of 4mm and 2-4 stainless steel grinding steel balls with the diameter of 3mm are placed in a high-speed tissue grinding instrument with the temperature of 3-6 ℃ for grinding, and the grinding conditions are 60-70 Hz, 50-75 s and grinding for 3-4 times.

When the kidney tissue is used for extracting RNA, the required tissue homogenate preparation conditions are that 15-20 mg of kidney tissue is placed in a 2-5 ml grinding tube, 300-380 ul of lysate is added, 1-2 stainless steel enzyme-free grinding steel balls with the diameter of 4mm and 2-4 stainless steel enzyme-free grinding steel balls with the diameter of 3mm are placed in a high-speed tissue grinding instrument at normal temperature for grinding, and the grinding conditions are 60-70 Hz, 50-75 s and grinding for 3-4 times.

The second part is used for preparing paraffin sections, carrying out renal tubular injury scoring after hematoxylin-eosin (H & E) staining, and evaluating the pathological injury condition of the renal tubular (wherein, the specific method for carrying out the renal tubular injury scoring and the renal tubular pathological injury evaluating by using the renal paraffin sections can be directly applied to the prior art without the need of creative labor);

The third section is used to prepare kidney frozen sections by OCT embedding, and the level of expression of kidney Reactive Oxygen Species (ROS) is assessed by ethidium (DHE) staining (wherein the specific method of assessing the level of expression of kidney reactive oxygen species using kidney frozen sections can be directly applied to the prior art without the need for creative effort).

Further, the method also comprises a step 5;

step 5, analyzing and comparing;

Comparing the sepsis acute kidney injury model group with a tail control group, and analyzing serum creatinine and serum urea nitrogen levels, wherein if the serum creatinine of the sepsis acute kidney injury model group rises more than 2 times, and the difference between the two groups has statistical significance through statistical test, the sepsis acute kidney injury model group is judged to be successfully constructed, otherwise, the construction is failed;

Comparing the sepsis acute kidney injury model group with a tail remote ischemia pretreatment group, analyzing serum creatinine and serum urea nitrogen levels of the two groups, verifying the improvement effect on the sepsis acute kidney injury kidney function, detecting whether RNA and protein expression levels of tubular injury markers and inflammatory factors are inhibited by the remote ischemia pretreatment, and judging whether the remote ischemia pretreatment can alleviate the kidney pathological injury and inhibit the expression level of kidney ROS by combining the kidney pathological injury condition, the tubular injury score and the kidney active oxygen expression level.

Furthermore, in the step 3, a double-limb remote ischemia pretreatment group and a lower limb control group are also constructed;

Constructing a double-lower limb remote ischemia pretreatment group, namely binding the double lower limbs of a mouse by using a tourniquet after the mouse of the double-lower limb remote ischemia pretreatment group is anesthetized and blood flow is blocked for 4-8 min, loosening the tourniquet and recovering blood flow for the same time, repeating the ischemia-reperfusion process for 3-6 cycles, and injecting lipopolysaccharide LPS (the dosage standard is 80-120 ul/kg) with the concentration of 8-12 mg/kg by abdominal cavity after the ischemia-reperfusion cycle is finished for 12-20 min.

The lower limb control group is constructed by sleeving the tourniquet on the two lower limbs of the mouse but not binding, keeping the blood flow of the tail of the mouse normal, injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail far-end ischemia pretreatment group (namely, injecting the physiological saline after the anesthesia of the mouse in the tail far-end ischemia pretreatment group or the two lower limb far-end ischemia pretreatment group for a time T, wherein the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

In the step 5, the sepsis acute kidney injury model group is compared with the tail control group and the lower limb control group, and then the sepsis acute kidney injury model group is respectively compared with the double lower limb remote ischemia pretreatment group and the tail remote ischemia pretreatment group.

The model constructed by the construction method is applied to the research of kidney protection mechanism or the screening of kidney disease treatment/protection drugs by the remote ischemia pretreatment of the tail part of the mouse.

The beneficial effects of the invention are as follows:

1. Through experiments, the method for constructing the model has high success rate, and can effectively verify that the tail ischemia reperfusion pretreatment can improve the kidney pathological damage of the acute sepsis kidney injury and lighten the tubular injury, inhibit the kidney oxidative stress of the acute sepsis kidney injury and lighten the inflammatory response of the acute sepsis kidney injury.

2. Compared with the existing lower limb ischemia model, the method selects the tail blood vessel of the mice, the tail blood vessel of the mice is distributed in a rich way, the blood supply sources are clear, tail remote ischemia pretreatment groups constructed by the tail blood vessel are relatively simple to model, have small wounds to animals, low operation and nursing difficulty, are easier to adapt to and recover, are not easy to cause serious complications, can effectively realize remote ischemia pretreatment, simultaneously reduce damage to experimental animals, reduce interference of factors such as animal stress to experimental results by adopting the tail blood vessel model with smaller influence to the animals, can more stably and accurately simulate the remote ischemia pretreatment process, has small result difference among different experiments, improves model stability and repeatability, is more beneficial to accurately evaluating the protection effect of the remote ischemia pretreatment on kidneys, and better meets the animal ethics requirements, and reflects the attention to the welfare of experimental animals.

3. In the invention, a control group, a sepsis acute kidney injury model group, a double-lower-limb remote ischemia pretreatment group and a tail remote ischemia pretreatment group are reasonably arranged, and in the pretreatment and model construction process, standardized and cross-control operation flows (such as definite anesthetic dosage, ischemia-reperfusion cycle times, time intervals, time and dosage of intraperitoneal injection of LPS and the like) are arranged for different groups, so that the kidney protection equivalence of tail remote ischemia pretreatment and double-lower-limb remote ischemia pretreatment on sepsis acute kidney injury can be more scientifically and accurately verified through strict grouping and standard treatment flows.

4. In the invention, in the model evaluation stage, not only traditional kidney function indexes such as serum creatinine, serum urea nitrogen and the like are detected, but also the small tube injury markers such as neutrophil gelatinase related lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and the like are detected, and the expression level of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like are detected, and simultaneously, the kidney pathological section (paraffin section H & E staining is combined for carrying out the small tube injury scoring, freezing section DHE staining is combined for evaluating the kidney active oxygen expression level) is observed, and the comprehensive evaluation system of multiple dimensions can be used for comprehensively and deeply evaluating the protective effect of tail distal tissue ischemia pretreatment on the kidney, so that the method has more scientificity and reliability.

Drawings

FIG. 1 is a schematic illustration of the effect of ischemia and control on blood flow in the present invention;

Wherein A is the blood flow condition of the tail of the mice in the tail ischemia group and the tail control group, B is the blood flow rate of the tail of the mice in the tail ischemia group and the tail control group, C is the blood flow condition of the lower limbs of the mice in the lower limb ischemia group and the lower limb control group, and D is the blood flow rate of the lower limbs of the mice in the lower limb ischemia group and the lower limb control group;

FIG. 2 is a graph showing the effect of lower limb control, model and lower limb ischemia on renal function and injury in mice in accordance with the present invention;

Wherein A is a construction schematic diagram of a lower limb ischemia group, B is a schematic diagram of the influence of creatinine and urea nitrogen, C is a comparative schematic diagram of staining analysis, and D is a schematic diagram of the influence of tubular injury scores and tubular injury marker levels;

FIG. 3 is a schematic representation of the effect of tail control, model and tail ischemia on kidney function and kidney injury in mice in accordance with the present invention;

Wherein E is a construction schematic diagram of a tail ischemia group, F is a schematic diagram of the influence of creatinine and urea nitrogen, G is a comparative schematic diagram of staining analysis, and H is a schematic diagram of the influence of tubular injury scores and tubular injury marker levels;

FIG. 4 is a graph showing the effect of lower limb control, model and lower limb ischemia on inflammatory factor expression levels in accordance with the present invention;

Wherein A is a schematic diagram of the influence of oxidative stress (ROS), B is a schematic diagram of the influence of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) mRNA, C is a schematic diagram of the influence of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) protein, and D is a schematic diagram of the protein abundance of the interleukins and tumor necrosis factors;

FIG. 5 is a graph showing the effect of tail control, model and tail ischemia on inflammatory factor expression levels in accordance with the present invention;

wherein E is a schematic of the effect of oxidative stress (ROS), F is a schematic of the effect of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) mRNA, G is a schematic of the effect of interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-alpha) proteins, and H is a schematic of the protein abundance of the interleukins and tumor necrosis factors.

Detailed Description

For the purpose of making the objects, technical solutions and advantages of the embodiments of the present invention more apparent, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings in the embodiments of the present invention, and it is apparent that the described embodiments are some embodiments of the present invention, but not all embodiments of the present invention.

Thus, all other embodiments, which can be made by one of ordinary skill in the art without undue burden from the invention, are intended to be within the scope of the invention.

Example 1

The present embodiment provides a method for constructing a tail remote ischemia pretreatment kidney protection model of a mouse, and aims to explore the influence of tail ischemia reperfusion on kidney injury. The method specifically comprises the following steps:

step 1, selecting materials and grouping;

Male C56BL/6J healthy mice with weight of 23g and week age of 6 weeks are selected and randomly divided into a tail control group, a sepsis acute kidney injury model group and a tail remote ischemia pretreatment group.

Step 2, inducing anesthesia;

Mice from the tail distal ischemic preconditioned group were anesthetized with a slow intraperitoneal injection of uratam at a concentration of 18% (1.6 mg/kg, standard). In the case of mice in the sepsis acute kidney injury model group and the tail control group, no anesthesia treatment was performed.

In this step, considering that the tail distal ischemia pretreatment group needs to perform ischemia and reperfusion treatment on the mice, in order to reduce pain caused by the process on the mice and reduce the experimental result and ethical problems of the mice due to the overdriving reaction caused by the pain, only the mice of the ischemia pretreatment group (including the tail distal ischemia pretreatment group in this embodiment and the two lower limb distal ischemia pretreatment groups in the following embodiments) are anesthetized, while the mice of the model group and the control group are not anesthetized. And through earlier study, whether the mice are anesthetized or not, the effect on the final test is not influenced or is very small, the effect is very little, and the effect is completely negligible.

Step 3, constructing an animal model;

Constructing a tail distal ischemia pretreatment group, namely binding a tail of a mouse by using a tourniquet after the mouse of the tail distal ischemia pretreatment group is anesthetized, blocking blood flow for 5min, then loosening the tourniquet to restore blood flow for 5min (blood flow blocking time=blood flow restoring time), repeating the ischemia-reperfusion process for 4 cycles, and injecting lipopolysaccharide LPS with the concentration of 9mg/kg (the dosage standard is 90 ul/min) through an intraperitoneal injection after the ischemia reperfusion cycle is ended for 15 min.

A sepsis acute kidney injury model group is constructed by injecting lipopolysaccharide LPS with equal concentration and equal dosage standard into mice through abdominal cavity after the same time period as the tail distal ischemia pretreatment group (namely, injecting lipopolysaccharide LPS after the time T is elapsed after the mice of the tail distal ischemia pretreatment group are anesthetized).

The tail control group is constructed by sleeving the tourniquet on the tail of the mouse but not binding the tourniquet, keeping the blood flow of the tail of the mouse normal, injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail far-end ischemia pretreatment group (namely, injecting the physiological saline after the time T passes after the anesthesia of the mouse of the tail far-end ischemia pretreatment group, and the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

The specific injection method for the lipopolysaccharide LPS comprises the following steps:

The method comprises the steps of grasping and fixing a mouse by a left hand, enabling the abdomen of the mouse to face upwards and enabling the head to be lower than the tail so as to avoid damage to internal organs, disinfecting the abdomen of the mouse by using an alcohol cotton ball, pushing a syringe into the abdomen line of the mouse at about 0.4cm on either side of the abdomen line, pushing a needle head downwards for 4mm, penetrating the abdomen of the mouse at an angle of 42 degrees with the skin, enabling the needle head to have a falling feel when penetrating, slowly injecting liquid medicine if no liquid flows back when a needle is pulled back, rotating the needle head and slowly pulling out after injection is completed, and preventing liquid from leaking outwards.

Step 4, collecting and detecting for evaluating the model effect;

After lipopolysaccharide LPS and physiological saline are injected into the abdominal cavity for 14 hours, blood and kidney tissues of the mice are synchronously collected.

After the collected blood is subjected to centrifugal treatment, supernatant is taken and serum creatinine and serum urea nitrogen indexes are measured (the specific method for measuring the serum creatinine and serum urea nitrogen indexes according to the supernatant can be directly applied to the prior art without the need of creative labor) and are used for evaluating the kidney functions of mice in each group.

The harvested kidney tissue is divided into three parts:

The first part is used for extracting protein and RNA, detecting a tubular injury marker such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and the like, and detecting the expression level of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like (wherein, the specific method for detecting the expression level of the tubular injury marker and the inflammatory factors by using kidney tissues can be directly applied to the prior art without the need of creative labor):

When protein is extracted from kidney tissue, the required tissue homogenate preparation conditions are that 24mg of kidney tissue is placed in a 3ml grinding tube, 480ul of protein lysate is added, 1 stainless steel grinding steel ball of 4mm and 2 stainless steel grinding steel balls of 3mm are placed in a high-speed tissue grinding instrument of 4 ℃ for grinding, and the grinding conditions are 62Hz and 55s for 3 times.

When the kidney tissue is utilized to extract RNA, the required tissue homogenate preparation condition is that 16mg of kidney tissue is placed in a 3ml grinding tube, 320ul of lysate is added, 1 stainless steel enzyme-free grinding steel ball of 4mm and 2 stainless steel enzyme-free grinding steel balls of 3mm are placed, and the kidney tissue is placed in a high-speed tissue grinder at normal temperature for grinding, wherein the grinding condition is 63Hz and 56s, and the grinding is carried out for 3 times.

The second part is used for preparing paraffin sections, carrying out renal tubular injury scoring after hematoxylin-eosin (H & E) staining, and evaluating the pathological injury condition of the renal tubular (wherein, the specific method for carrying out the renal tubular injury scoring and the renal tubular pathological injury evaluating by using the renal paraffin sections can be directly applied to the prior art without the need of creative labor);

The third section is used to prepare kidney frozen sections by OCT embedding, and the level of expression of kidney Reactive Oxygen Species (ROS) is assessed by ethidium (DHE) staining (wherein the specific method of assessing the level of expression of kidney reactive oxygen species using kidney frozen sections can be directly applied to the prior art without the need for creative effort).

Step 5, analyzing and comparing;

Comparing the sepsis acute kidney injury model group with a tail control group, and analyzing serum creatinine and serum urea nitrogen levels, wherein if the serum creatinine of the sepsis acute kidney injury model group rises more than 2 times, and the difference between the two groups has statistical significance through statistical test, the sepsis acute kidney injury model group is judged to be successfully constructed, otherwise, the construction is failed;

Comparing the sepsis acute kidney injury model group with a tail remote ischemia pretreatment group, analyzing serum creatinine and serum urea nitrogen levels of the two groups, verifying the improvement effect on the sepsis acute kidney injury kidney function, detecting whether RNA and protein expression levels of tubular injury markers and inflammatory factors are inhibited by the remote ischemia pretreatment, and judging whether the remote ischemia pretreatment can alleviate the kidney pathological injury and inhibit the expression level of kidney ROS by combining the kidney pathological injury condition, the tubular injury score and the kidney active oxygen expression level.

The method of the embodiment is adopted for construction, and experiments and analysis show that the model construction is successful, and the conclusion is that the tail ischemia reperfusion pretreatment can improve the kidney pathological damage of the acute sepsis kidney injury and relieve the tubular injury, can inhibit the kidney oxidative stress of the acute sepsis kidney injury and relieve the inflammatory reaction of the acute sepsis kidney injury.

Example 2

The embodiment provides a construction method of a kidney protection model for remote ischemia pretreatment of a tail part of a mouse, and aims to explore the influence of ischemia reperfusion of lower limbs on kidney injury. The method specifically comprises the following steps:

step 1, selecting materials and grouping;

Healthy mice with male C56BL/6J, body weight of 24g and week age of 7 weeks were selected and randomly divided into a lower limb control group, a sepsis acute kidney injury model group and a double lower limb remote ischemia pretreatment group.

Step 2, inducing anesthesia;

Mice from the remote ischemic preconditioned group of the two lower limbs were anesthetized with 20% concentration of uratam (1.8 mg/kg standard) by intraperitoneal slow injection. In the case of mice in the sepsis acute kidney injury model group and the tail control group, no anesthesia treatment was performed.

Step 3, constructing an animal model;

constructing a double-lower limb remote ischemia pretreatment group, namely binding the double lower limbs of the mice with tourniquets after the mice of the double-lower limb remote ischemia pretreatment group are anesthetized and blood flow is blocked for 6min, loosening the tourniquets again to restore blood flow for the same time, repeating the ischemia-reperfusion process for 5 cycles, and injecting lipopolysaccharide LPS with the concentration of 10mg/kg (the dosage standard is 100 ul/kg) into the abdominal cavity after the ischemia-reperfusion cycle is ended for 16 min.

A sepsis acute kidney injury model group is constructed by injecting lipopolysaccharide LPS with equal concentration and equal dosage standard into mice through abdominal cavity after the same time period as the two lower limb remote ischemia pretreatment group (namely, injecting lipopolysaccharide LPS after the time T after the mice of the tail remote ischemia pretreatment group are anesthetized).

The lower limb control group is constructed by sleeving the tourniquet on the two lower limbs of the mouse but not binding, keeping the blood flow of the tail of the mouse normal, injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the two lower limb remote ischemia pretreatment group (namely, injecting the physiological saline after the period of time T passes after the anesthesia of the mouse of the tail remote ischemia pretreatment group or the two lower limb remote ischemia pretreatment group, and the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

The specific injection method for the lipopolysaccharide LPS comprises the following steps:

The method comprises the steps of grasping and fixing a mouse by a left hand, enabling the abdomen of the mouse to face upwards and enabling the head to be lower than the tail so as to avoid damage to internal organs, disinfecting the abdomen of the mouse by using an alcohol cotton ball, pushing a syringe into the abdomen line of the mouse at about 0.5cm on either side of the abdomen line of the mouse, pushing a needle head downwards for 4mm, penetrating the abdomen of the mouse at an angle of 44 degrees with the skin, enabling the needle head to have a falling feel when penetrating, slowly injecting liquid medicine if no liquid flows back when the needle is pulled back, rotating the needle head and slowly pulling out after injection is completed, and preventing liquid from leaking.

Step 4, collecting and detecting for evaluating the model effect;

after lipopolysaccharide LPS and physiological saline are injected into the abdominal cavity for 15 hours, the mice are sacrificed, and blood and kidney tissues of the mice are synchronously collected.

After the collected blood is subjected to centrifugal treatment, supernatant is taken and serum creatinine and serum urea nitrogen indexes are measured (the specific method for measuring the serum creatinine and serum urea nitrogen indexes according to the supernatant can be directly applied to the prior art without the need of creative labor) and are used for evaluating the kidney functions of mice in each group.

The harvested kidney tissue is divided into three parts:

The first part is used for extracting protein and RNA, detecting a tubular injury marker such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and the like, and detecting the expression level of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like (wherein, the specific method for detecting the expression level of the tubular injury marker and the inflammatory factors by using kidney tissues can be directly applied to the prior art without the need of creative labor):

When protein is extracted from kidney tissue, the required tissue homogenate preparation conditions are that 25mg of kidney tissue is placed in a 4ml grinding tube, 520ul of protein lysate is added, 2 stainless steel grinding steel balls with the diameter of 4mm and 3 stainless steel grinding steel balls with the diameter of 3mm are placed in a high-speed tissue grinding instrument with the temperature of 4 ℃ for grinding, and the grinding conditions are 65Hz and 62s for 4 times.

When the kidney tissue is utilized to extract RNA, the required tissue homogenate preparation condition is that 18mg of kidney tissue is placed in a 3ml grinding tube, 340ul of lysate is added, 2 stainless steel enzyme-free grinding steel balls with the diameter of 4mm and 3 stainless steel enzyme-free grinding steel balls with the diameter of 3mm are placed in a high-speed tissue grinding instrument at normal temperature for grinding, and the grinding condition is 65Hz and 62s for 4 times.

The second part is used for preparing paraffin sections, carrying out renal tubular injury scoring after hematoxylin-eosin (H & E) staining, and evaluating the pathological injury condition of the renal tubular (wherein, the specific method for carrying out the renal tubular injury scoring and the renal tubular pathological injury evaluating by using the renal paraffin sections can be directly applied to the prior art without the need of creative labor);

The third section is used to prepare kidney frozen sections by OCT embedding, and the level of expression of kidney Reactive Oxygen Species (ROS) is assessed by ethidium (DHE) staining (wherein the specific method of assessing the level of expression of kidney reactive oxygen species using kidney frozen sections can be directly applied to the prior art without the need for creative effort).

Step 5, analyzing and comparing;

Comparing the sepsis acute kidney injury model group with a lower limb control group, and analyzing serum creatinine and serum urea nitrogen levels, wherein if the serum creatinine of the sepsis acute kidney injury model group rises more than 2 times, and the difference between the two groups has statistical significance through statistical test, the sepsis acute kidney injury model group is judged to be successfully constructed, otherwise, the construction is failed;

Comparing the sepsis acute kidney injury model group with the two lower limb remote ischemia pretreatment groups, analyzing serum creatinine and serum urea nitrogen levels of the two groups, verifying the improvement effect on the sepsis acute kidney injury kidney function, detecting whether the RNA and protein expression levels of the tubular injury markers and inflammatory factors are inhibited by the remote ischemia pretreatment, and judging whether the two lower limb remote ischemia pretreatment can alleviate the kidney pathological injury and inhibit the expression level of the kidney ROS by combining the kidney pathological injury condition, the tubular injury score and the kidney active oxygen expression level.

The method of the embodiment is adopted for construction, and experiments and analysis show that the model construction is successful, and the conclusion is that the pretreatment of ischemia reperfusion of lower limbs can improve the kidney pathological damage of acute sepsis kidney injury and relieve renal tubular injury, and can inhibit the kidney oxidative stress of acute sepsis kidney injury and relieve the inflammatory reaction of acute sepsis kidney injury.

Example 3

The embodiment provides a construction method of a remote ischemia pretreatment kidney protection model of a tail part of a mouse, aiming at exploring and comparing the influence difference of tail/lower limb ischemia reperfusion on kidney injury. The method specifically comprises the following steps:

step 1, selecting materials and grouping;

a plurality of healthy mice with male C56BL/6J, weight of 25g and week age of 8 weeks are selected and randomly divided into a lower limb control group, a tail control group, a sepsis acute kidney injury model group, a double lower limb remote ischemia pretreatment group and a tail remote ischemia pretreatment group.

Step 2, inducing anesthesia;

mice from both the distal ischemic preconditioning group and the distal ischemic preconditioning group were anesthetized with 24% concentration of uratam (1.8 mg/kg standard) by intraperitoneal slow injection. In the case of mice in the sepsis acute kidney injury model group, the lower limb control group and the tail control group, no anesthesia treatment was performed.

Step 3, constructing an animal model;

The tail distal ischemia pretreatment group was constructed by binding the tail of the mice with a tourniquet after the mice of the tail distal ischemia pretreatment group were anesthetized, blocking the blood flow for 7min, then releasing the tourniquet to restore the blood flow for 7min (blood flow blocking time=blood flow restoring time), repeating the ischemia-reperfusion process for 6 cycles, and injecting lipopolysaccharide LPS (the dosage standard is 110 ul/mouse) with the concentration of 10mg/kg into the mice through the abdominal cavity after the ischemia reperfusion cycle is finished for 18 min.

Constructing a double-lower limb remote ischemia pretreatment group, namely binding the double lower limbs of a mouse by using a tourniquet after the mouse of the double-lower limb remote ischemia pretreatment group is anesthetized and blood flow is blocked for 7min, loosening the tourniquet and recovering blood flow for the same time, repeating the ischemia-reperfusion process for 6 cycles, and injecting lipopolysaccharide LPS (the dosage standard is 110 ul/min) with the concentration of 10mg/kg into the mouse through an abdominal cavity after the ischemia-reperfusion cycle is ended for 18 min.

A sepsis acute kidney injury model group is constructed by injecting lipopolysaccharide LPS with equal concentration and equal dosage standard into mice through abdominal cavity after the same time period as the tail distal ischemia pretreatment group (namely, injecting lipopolysaccharide LPS after the time T is elapsed after the mice of the tail distal ischemia pretreatment group are anesthetized).

The tail control group is constructed by sleeving the tourniquet on the tail of the mouse but not binding the tourniquet, keeping the blood flow of the tail of the mouse normal, injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail far-end ischemia pretreatment group (namely, injecting the physiological saline after the time T passes after the anesthesia of the mouse of the tail far-end ischemia pretreatment group, and the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

The lower limb control group is constructed by sleeving the tourniquet on the two lower limbs of the mouse but not binding, keeping the normal blood flow of the tail of the mouse, injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail far-end ischemia pretreatment group (namely, injecting the physiological saline after the anesthesia of the mouse in the tail far-end ischemia pretreatment group or the two lower limb far-end ischemia pretreatment group for a time T, wherein the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

The specific injection method for the lipopolysaccharide LPS comprises the following steps:

The method comprises the steps of grasping and fixing a mouse by a left hand, enabling the abdomen of the mouse to face upwards and enabling the head to be lower than the tail so as to avoid damage to internal organs, disinfecting the abdomen of the mouse by using an alcohol cotton ball, pushing a syringe into the abdomen line of the mouse at about 0.6cm on either side of the abdomen line of the mouse, pushing a needle head downwards for 5mm, penetrating the abdomen of the mouse at an angle of 46 degrees with the skin, enabling the needle head to have a falling feel when penetrating, slowly injecting liquid medicine if no liquid flows back when the needle is pulled back, rotating the needle head and slowly pulling out after injection is completed, and preventing liquid from leaking.

Step 4, collecting and detecting for evaluating the model effect;

After the lipopolysaccharide LPS and physiological saline are injected into the abdominal cavity for 18 hours, the mice are sacrificed, and blood and kidney tissues of the mice are synchronously collected.

After the collected blood is subjected to centrifugal treatment, supernatant is taken and serum creatinine and serum urea nitrogen indexes are measured (the specific method for measuring the serum creatinine and serum urea nitrogen indexes according to the supernatant can be directly applied to the prior art without the need of creative labor) and are used for evaluating the kidney functions of mice in each group.

The harvested kidney tissue is divided into three parts:

The first part is used for extracting protein and RNA, detecting a tubular injury marker such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and the like, and detecting the expression level of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like (wherein, the specific method for detecting the expression level of the tubular injury marker and the inflammatory factors by using kidney tissues can be directly applied to the prior art without the need of creative labor):

When protein is extracted from kidney tissue, the required tissue homogenate preparation conditions are that 28mg of kidney tissue is placed in a 4ml grinding tube, 580ul of protein lysate is added, 2 stainless steel grinding steel balls with the diameter of 4mm and 4 stainless steel grinding steel balls with the diameter of 3mm are placed in a high-speed tissue grinding instrument with the temperature of 6 ℃ for grinding, and the grinding conditions are 68Hz and 70s for 4 times.

When the kidney tissue is utilized to extract RNA, the required tissue homogenate preparation condition is that 18mg of kidney tissue is placed in a 5ml grinding tube, 370ul of lysate is added, 2 stainless steel enzyme-free grinding steel balls with the diameter of 4mm and 4 stainless steel enzyme-free grinding steel balls with the diameter of 3mm are placed in a high-speed tissue grinding instrument at normal temperature for grinding, and the grinding condition is 68Hz and 70s for 4 times.

The second part is used for preparing paraffin sections, carrying out renal tubular injury scoring after hematoxylin-eosin (H & E) staining, and evaluating the pathological injury condition of the renal tubular (wherein, the specific method for carrying out the renal tubular injury scoring and the renal tubular pathological injury evaluating by using the renal paraffin sections can be directly applied to the prior art without the need of creative labor);

The third section is used to prepare kidney frozen sections by OCT embedding, and the level of expression of kidney Reactive Oxygen Species (ROS) is assessed by ethidium (DHE) staining (wherein the specific method of assessing the level of expression of kidney reactive oxygen species using kidney frozen sections can be directly applied to the prior art without the need for creative effort).

Step 5, analyzing and comparing;

Comparing the sepsis acute kidney injury model group with a tail control group and a lower limb control group, and analyzing serum creatinine and serum urea nitrogen levels, wherein if the serum creatinine of the sepsis acute kidney injury model group rises more than 2 times (namely, the serum creatinine of the sepsis acute kidney injury model group is more than 2 times of the serum creatinine of the two control groups), and statistical tests show that the difference between the two groups has statistical significance, the sepsis acute kidney injury model group is judged to be successfully constructed, otherwise, the construction is failed;

And comparing the sepsis acute kidney injury model group with a double-lower limb remote ischemia pretreatment group and a tail remote ischemia pretreatment group respectively, analyzing serum creatinine and serum urea nitrogen levels of the two groups, verifying the improvement effect on the sepsis acute kidney injury kidney function, detecting whether the RNA and protein expression levels of a tubular injury marker and inflammatory factors are inhibited by the remote ischemia pretreatment, and judging whether the remote ischemia pretreatment can alleviate the kidney pathological injury and inhibit the expression level of kidney ROS by combining the kidney pathological injury condition, the tubular injury score and the kidney active oxygen expression level.

The method of the embodiment is used for construction, experiments and analysis show that the model construction is successful, and the conclusion is that the tail remote ischemia pretreatment and the lower limb ischemia reperfusion pretreatment can improve the kidney pathological damage of the acute sepsis kidney injury and relieve the tubular injury, can inhibit the kidney oxidative stress of the acute sepsis kidney injury and relieve the inflammatory response of the acute sepsis kidney injury, and the lower limb ischemia reperfusion pretreatment and the tail ischemia reperfusion pretreatment have similar protective effects on the kidney.

Test example:

The construction method of the kidney protection model for the remote ischemia pretreatment of the tail part of the mouse comprises the following steps:

step 1, selecting materials and grouping;

The method comprises the steps of selecting 36 healthy mice purchased from Chengdu Jiuzhikang limited company, wherein the healthy mice have a weight of 25g and a week age of 8 weeks, and randomly and uniformly dividing the healthy mice into a tail control group, a lower limb control group, a sepsis acute kidney injury model group, a double lower limb remote ischemia pretreatment group and a tail remote ischemia pretreatment group.

Step 2, inducing anesthesia;

Mice from both the distal ischemic preconditioning group for both lower limbs and the distal ischemic preconditioning group were anesthetized with 25% concentration of uratam (1.75 mg/kg standard) by intraperitoneal slow injection. In the case of mice in the sepsis acute kidney injury model group, the lower limb control group and the tail control group, no anesthesia treatment was performed.

Step 3, constructing an animal model;

In this test example, two animal models were constructed, three groups in each batch, 6 groups in total, and 6 healthy mice in each group. The two animal models are respectively a lower limb control group, a sepsis acute kidney injury model group and a double lower limb remote ischemia pretreatment group in the first animal model, and a tail control group, a sepsis acute kidney injury model group and a tail remote ischemia pretreatment group in the second animal model. The construction modes of each group are as follows:

A tail distal ischemia pretreatment group (briefly referred to as a tail ischemia group in the drawing) was constructed, wherein after the tail distal ischemia pretreatment group mice were anesthetized, the tail of the mice was bundled with a tourniquet, blood flow was blocked for 5min, then the tourniquet was loosened to restore blood flow for 5min, the ischemia-reperfusion process was repeated for 4 cycles, and after the ischemia reperfusion cycle was completed for 15min, lipopolysaccharide LPS (the dosage standard was 100 ul/kg) was injected into the mice via the abdominal cavity at a concentration of 10 mg/kg.

A double-lower limb remote ischemia pretreatment group (short lower limb ischemia group in the drawing) is constructed, wherein after a mouse of the double-lower limb remote ischemia pretreatment group is anesthetized, the double lower limb of the mouse is bundled by a tourniquet and blood flow is blocked for 5min, then the tourniquet is loosened to recover the blood flow for 5min, the ischemia-reperfusion process is repeated for 4 cycles, and after the ischemia reperfusion cycle is finished for 15min, the mouse is injected with lipopolysaccharide LPS with equal concentration and equal dosage standard through an abdominal cavity.

A model group (briefly shown as a model group in the drawing) of acute sepsis kidney injury is constructed, namely, lipopolysaccharide LPS with equal concentration and equal dosage standard is injected into mice through abdominal cavities after the same duration (namely, 55min after anesthesia) as that of a tail remote ischemia pretreatment group.

A tail control group (briefly described as a tail control group in the drawing) is constructed by sleeving a tourniquet on the tail of a mouse but not binding the tourniquet, keeping the blood flow of the tail of the mouse normal, and injecting an equal amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail distal ischemia pretreatment group (namely 55min after anesthesia) (namely, the injection amount (ul) of the physiological saline is the same as the injection amount (ul) of lipopolysaccharide LPS).

A lower limb control group (briefly referred to as a lower limb control group in the drawing) is constructed by sleeving a tourniquet on the upper limb and the lower limb of a mouse but not binding the tourniquet, keeping the blood flow of the tail of the mouse normal, and injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time period as the tail distal ischemia pretreatment group (namely 55min after anesthesia).

The specific injection method for the lipopolysaccharide LPS comprises the following steps:

The method comprises the steps of grasping and fixing a mouse by the left hand, enabling the abdomen of the mouse to face upwards and enabling the head to be lower than the tail so as to avoid damage to internal organs, disinfecting the abdomen of the mouse by using an alcohol cotton ball, enabling an injector to penetrate into the abdomen of the mouse, pushing a needle head downwards for 3-5 mm (the pushing size of the needle head is based on the falling sensation when the needle head is penetrated), penetrating into the abdominal cavity of the mouse at an angle of about 45 degrees with the skin, enabling the needle head to have the falling sensation when the needle head is penetrated, slowly injecting liquid medicine if no liquid flows back when the needle plug is pulled back, rotating the needle head and slowly pulling out after injection is completed, and preventing liquid from leaking.

When each group is constructed, the blood flow conditions of the tail control group, the lower limb control group, the double lower limb remote ischemia pretreatment group and the corresponding mouse tail and the mouse double lower limb in each group of the tail remote ischemia pretreatment group are concerned at any time, and a graph shown in figure 1 is obtained.

Step 4, collecting and detecting for evaluating the model effect;

after the lipopolysaccharide LPS and physiological saline are injected into the abdominal cavity for 16 hours, the mice are sacrificed, and blood and kidney tissues of the mice are synchronously collected.

After centrifugation of the collected blood, the supernatant was taken and serum creatinine and serum urea nitrogen indicators were determined for evaluation of the renal function of mice in each group.

The harvested kidney tissue is divided into three parts:

The first part is used for extracting protein and RNA, detecting the markers of tubular injury such as neutrophil gelatinase-associated lipocalin (NGAL), kidney injury molecule-1 (KIM-1) and the like, and detecting the expression level of inflammatory factors such as interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-alpha) and the like:

When protein is extracted from kidney tissue, the required tissue homogenate preparation condition is that about 30mg of kidney tissue is placed in a 2ml grinding tube, 500ul of protein lysate is added, 1 stainless steel grinding steel ball of 4mm and 2 stainless steel grinding steel balls of 3mm are placed in a high-speed tissue grinding instrument of 4 ℃ for grinding, and the grinding condition is 60Hz and 60s for 4 times.

When the kidney tissue is utilized to extract RNA, the required tissue homogenate preparation condition is that about 20mg of kidney tissue is placed in a 2ml grinding tube, 350ul of lysate is added, 1 stainless steel enzyme-free grinding steel ball of 4mm and 2 stainless steel enzyme-free grinding steel balls of 3mm are placed, and the kidney tissue is placed in a high-speed tissue grinding instrument at normal temperature for grinding, wherein the grinding condition is 60Hz and 60s, and the grinding is carried out for 4 times.

The second part is used for preparing paraffin sections, and carrying out renal tubular injury scoring after hematoxylin-eosin (H & E) staining to evaluate pathological injury conditions of the renal tubular;

the third section was used to prepare kidney frozen sections by OCT embedding and to evaluate kidney Reactive Oxygen Species (ROS) expression levels by ethidium Dihydrogenamide (DHE) staining.

Step 5, analyzing and comparing;

Firstly, comparing the sepsis acute kidney injury model group with a tail control group and a lower limb control group respectively, and analyzing serum creatinine and serum urea nitrogen levels, wherein if the serum creatinine of the sepsis acute kidney injury model group rises more than 2 times, and the difference between the two groups has statistical significance through statistical inspection, the sepsis acute kidney injury model group is judged to be successfully constructed, otherwise, the construction is failed;

And comparing the sepsis acute kidney injury model group with a double-lower limb remote ischemia pretreatment group and a tail remote ischemia pretreatment group respectively, analyzing and comparing serum creatinine and serum urea nitrogen levels of the three groups, verifying the improvement effect on the sepsis acute kidney injury kidney function, detecting whether the RNA and protein expression levels of a tubular injury marker and inflammatory factors are inhibited by the remote ischemia pretreatment, and judging whether the remote ischemia pretreatment can alleviate the kidney pathological injury and inhibit the expression level of the kidney ROS by combining the kidney pathological injury condition, the tubular injury score and the kidney active oxygen expression level.

Test results:

1. Analysis of blood flow rate:

After each group is constructed, blood velocity comparison is carried out between the tail control group and the tail far-end ischemia pretreatment group and between the lower limb control group and the double lower limb far-end ischemia pretreatment group respectively, as shown in fig. 1, after the tail and the lower limb of the mouse are bound by the tourniquet, the tail and the lower limb of the mouse do not have obvious blood passing through, as shown in fig. A and fig. D, the blood velocity of the tail and the lower limb of the unbound mouse is higher, the blood velocity of the tail and the lower limb of the bound mouse is obviously reduced, and the blood velocity of the bound mouse is about half of the blood velocity of the unbound mouse.

2. Renal function and renal injury analysis:

the blood and kidney tissue of each group of mice were analyzed for renal function and kidney injury, and graphs shown in fig. 2 and 3 were obtained.

For analysis of ischemia reperfusion of lower limb, as shown in FIG. 2, it can be seen from panel B that the serum creatinine of the model group is 2 times higher than that of the lower limb control group, so that the sepsis acute kidney injury model group is successfully constructed, from panel C that the renal tubular epithelial cell swelling, necrosis, cavitation degeneration, tubular lumen expansion, and the lower limb ischemia reperfusion pretreatment obviously reduce the pathological damage of the above-mentioned renal tubular, and from panel D that the mRNA expression levels of the renal tubular injury indexes KIM-1 and NGAL are obviously increased in the sepsis acute kidney injury model group, and the lower limb ischemia reperfusion pretreatment obviously reduces the mRNA expression levels of KIM-1 and NGAL. Thus, figure 2 demonstrates that the ischemia reperfusion pretreatment of lower limbs can ameliorate renal pathological damage to sepsis acute kidney injury and reduce tubular injury.

For analysis of tail ischemia reperfusion, as shown in FIG. 3, it can be seen from panel F that the serum creatinine of the model group exceeded that of the tail control group by 2 times, thus again proving that the sepsis acute kidney injury model group was successfully constructed, as can be seen from panel G that in the sepsis acute kidney injury model group, the tubular epithelial cell swelling, necrosis, cavitation denaturation, tubular lumen expansion, tail ischemia reperfusion pretreatment significantly reduced the above-mentioned kidney pathological injury, and as can be seen from panel H, the mRNA expression levels of the tubular injury indexes KIM-1 and NGAL were significantly increased, and the tail ischemia reperfusion pretreatment significantly reduced the mRNA expression levels of KIM-1 and NGAL. Thus, fig. 3 demonstrates that tail ischemia reperfusion pretreatment can ameliorate renal pathological damage to sepsis acute kidney injury and mitigate tubular injury.

3. Oxidative stress and inflammation analysis:

Oxidative stress and inflammation analysis were performed on kidney tissues of mice in each group, and graphs shown in fig. 4 and 5 were obtained.

As shown in FIG. 4, in the acute sepsis kidney injury model group, the DHE staining of the kidney tissue shows that the oxidation level of the kidney is obviously increased, the oxidation stress level of the kidney is obviously inhibited by the pretreatment of the ischemia reperfusion of the lower limb, and the oxidation stress of the kidney is obviously inhibited by the pretreatment of the ischemia reperfusion of the lower limb, and in the acute sepsis kidney injury model group, the mRNA and protein expression levels of inflammatory factors (IL-6 and TNF-alpha) of the kidney tissue are obviously increased, and the expression level of inflammatory factors of the kidney is obviously inhibited by the pretreatment of the ischemia reperfusion of the lower limb. Thus, figure 4 demonstrates that the lower limb ischemia reperfusion pretreatment can inhibit the renal oxidative stress of sepsis acute kidney injury and alleviate the inflammatory response of sepsis acute kidney injury.

Analysis of tail ischemia reperfusion as shown in FIG. 5, it can be seen from FIG. E that the DHE staining of kidney tissue showed a significant increase in kidney oxidation level in the sepsis acute kidney injury model group, and tail ischemia reperfusion pretreatment significantly inhibited kidney oxidative stress level, demonstrating that tail ischemia reperfusion pretreatment can inhibit kidney oxidative stress of sepsis acute kidney injury, and from FIG. F-G that the mRNA and protein expression levels of kidney tissue inflammatory factors (IL-6, TNF- α) were significantly increased in the sepsis acute kidney injury model group. Thus, fig. 5 demonstrates that tail ischemia reperfusion pretreatment can suppress renal oxidative stress of sepsis acute kidney injury, alleviating inflammatory response of sepsis acute kidney injury.

In addition, the lower limb ischemia group in fig. 4 and the tail ischemia group in fig. 5 are compared and analyzed, and from the viewpoint of the index of the kidney oxidative stress, the lower limb ischemia reperfusion pretreatment and the tail ischemia reperfusion pretreatment can inhibit the kidney oxidative stress of the sepsis acute kidney injury, and from the aspect of kidney inflammation, both groups are obviously lower than the sepsis acute kidney injury model group. The data prove that the lower limb ischemia reperfusion pretreatment and the tail ischemia reperfusion pretreatment can inhibit kidney oxidative stress and inflammatory response of acute sepsis kidney injury, namely the lower limb ischemia reperfusion pretreatment and the tail ischemia reperfusion pretreatment have similar protective effects on kidneys, but compared with the lower limb ischemia reperfusion pretreatment, the tail ischemia reperfusion pretreatment has smaller wounds on animals, lower operation and nursing difficulty, easier adaptation and recovery of animals and less serious complications, reduces injury to experimental animals, and in addition, the tail ischemia reperfusion can effectively reduce animal stress response, reduce interference of factors such as animal stress response on experimental results, can more stably and accurately simulate the remote ischemia pretreatment process, has small difference of results among different experiments, improves model stability and repeatability, and is more beneficial to accurately evaluating the protective effects of the remote ischemia pretreatment on the kidneys.

Analysis of test results:

In the test example, the influence of the ischemia reperfusion pretreatment of lower limbs and the tail ischemia pretreatment on kidney functions (creatinine and urea nitrogen), oxidative stress and inflammatory factors is analyzed, and the research shows that the tail ischemia pretreatment can obviously reduce serum creatinine and serum urea nitrogen of acute sepsis kidney injury, improve kidney functions, alleviate oxidative stress and inflammatory reaction of acute sepsis kidney injury, and has the same protection effect compared with the existing lower limb ischemia pretreatment. However, the pretreatment of ischemia reperfusion of lower limbs has certain defects that firstly, large wounds are caused on the abdomen/lower limbs of animals, the subsequent survival recovery is unfavorable, secondly, nerve injury is easy to occur, such as nerve ischemia necrosis and abnormal sensation in a dominant region caused by tourniquet compression or operation, thirdly, the operation and postoperative nursing requirements are high, the experiment difficulty and the workload are increased, and fourthly, the animal wounds are large, the state is unstable, and the experiment stability and the repeatability are poor. By adopting the tail blood vessel model with smaller influence on animals, the interference of factors such as animal stress on experimental results is reduced, the remote ischemia pretreatment process can be simulated more stably and accurately, the difference of results among different experiments is small, the stability and the repeatability of the model are improved, and the protection effect of remote ischemia pretreatment on kidneys is evaluated more accurately.

Claims (5)

1. The construction method of the kidney protection model for the remote ischemia pretreatment of the tail of the mouse is characterized by comprising the following steps:

step 1, selecting materials and grouping;

Selecting a plurality of male healthy mice, and randomly dividing the mice into a tail control group, a sepsis acute kidney injury model group and a tail remote ischemia pretreatment group;

Step 2, inducing anesthesia;

The mice of the tail distal ischemia pretreatment group are slowly injected by using uratam through the abdominal cavity to anesthetize the mice, and the mice of the sepsis acute kidney injury model group and the tail control group are not anesthetized;

Step 3, constructing an animal model;

Constructing a tail distal ischemia pretreatment group, namely binding the tail of a mouse by using a tourniquet after the mouse of the tail distal ischemia pretreatment group is anesthetized, blocking blood flow for 4, 5 or 7min, loosening the tourniquet to restore blood flow for 4, 5 or 7min, repeating the ischemia-reperfusion process for 4 or 6 cycles, and injecting lipopolysaccharide LPS (LPS) into the abdominal cavity after the ischemia reperfusion cycle is finished for 15 or 18min, wherein the LPS dosage is 90, 100 or 110 ul/min;

Constructing a sepsis acute kidney injury model group, namely injecting lipopolysaccharide LPS with equal concentration and equal dosage standard into a mouse through an abdominal cavity after the same duration as that of a tail distal ischemia pretreatment group;

The tail control group is constructed by sleeving the tourniquet on the tail of the mouse but not binding the tourniquet, keeping the blood flow of the tail of the mouse normal, and injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time as the tail remote ischemia pretreatment group.

2. The method for constructing a model for protecting a kidney in remote ischemia pretreatment of a tail of a mouse according to claim 1, wherein in the step 2, the concentration of the injected urapidan is 15-25%, and the urapidan is injected according to an amount standard of 1.5-2.0 mg/kg.

3. The method for constructing a tail remote ischemia pretreatment kidney protection model according to claim 1, wherein in the step 3, when a tail remote ischemia pretreatment group is constructed, after the tail of the tail is bundled by a tourniquet to block blood flow for 4min, the tourniquet is loosened to restore blood flow for 4min, the ischemia-reperfusion process is repeated for 4 cycles, and after the ischemia reperfusion cycle is finished for 15min, the lipopolysaccharide LPS with equal concentration and equal dosage is injected into an abdominal cavity.

4. The method for constructing a rat tail remote ischemia pretreatment kidney protection model according to claim 1, wherein in step 3, a double lower limb remote ischemia pretreatment group and a lower limb control group are also constructed;

constructing a double-lower limb remote ischemia pretreatment group, namely binding the double lower limbs of a mouse by using a tourniquet after the mouse of the double-lower limb remote ischemia pretreatment group is anesthetized and blood flow is blocked for 5 or 7min, loosening the tourniquet to restore blood flow for 5 or 7min, repeating the ischemia-reperfusion process for 4 or 6 cycles, and injecting lipopolysaccharide LPS into an abdominal cavity after the ischemia-reperfusion cycle is finished for 15 or 18min, wherein the LPS dosage is 100 or 110 ul/min;

The lower limb control group is constructed by sleeving the tourniquet on the two lower limbs of the mouse but not binding, keeping the tail blood flow of the mouse normal, and injecting the same amount of physiological saline into the mouse through the abdominal cavity after the same time as the tail remote ischemia pretreatment group.

5. The method for constructing a tail remote ischemia pretreatment kidney protection model according to claim 4, wherein in step 3, the concentration of lipopolysaccharide LPS injected into tail remote ischemia pretreatment group, sepsis acute kidney injury model group and double lower limb remote ischemia pretreatment group is 8-12 mg/kg when lipopolysaccharide LPS is injected into abdominal cavity.