CN102772425A - Anti-pulmonary fibrosis application of small interfering RNA of Fstl 1 - Google Patents

Abstract

The invention discloses the use of small interfering RNA of Fstl1 in anti-fibrosis. The present invention is verified by pharmacological experiments that the small interfering RNA of Fstl1 significantly reduces the death rate of mice infused with bleomycin; reducing the expression of Fstl1 can significantly reduce lung injury and pulmonary fibrosis induced by bleomycin. It shows that the small interfering RNA of Fstl1 can be used to prepare drugs for preventing and/or treating pulmonary fibrosis.

Description

Anti-pulmonary fibrosis application of small interfering RNA of Fstl1

technical field

The invention relates to a new application of small interfering RNA of Fstl1 in preventing and treating pulmonary fibrosis, and belongs to the technical field of medicine.

Background technique

Idiopathic pulmonary fibrosis (IPF) is a chronic inflammatory interstitial lung disease characterized by pathological changes of usual interstitial pneumonia (UIP). The course of the disease is generally progressive and the prognosis is poor. , The average survival time after diagnosis is 2-4 years. Its etiology is complicated, its pathogenesis is not yet clear, and there is no effective treatment in clinical practice. Although the research on the pathogenesis of idiopathic pulmonary fibrosis has been carried out in depth from the aspects of cells, cytokines, extracellular matrix (ECM), cell signal transduction pathways, etc., the exact pathogenesis has not yet been clarified, so its treatment is mainly It starts from inhibiting pulmonary fibrosis and improving pulmonary fibrosis, so it also lacks specificity and effectiveness in treatment. In-depth research on the pathogenesis and molecular mechanism of IPF can provide assistance for the treatment and drug development of IPF.

It is of great significance to establish a pulmonary fibrosis disease model by using specific methods to induce pulmonary fibrosis in animals. The animal model of the disease can help us reveal the molecules involved in the pathogenesis and the mechanism of action. At present, there are many methods for inducing idiopathic pulmonary fibrosis to make animal models, and bleomycin, as the most commonly used induction reagent, has been widely used because of its relatively simple and low-cost modeling method. The use of bleomycin-induced mouse pulmonary fibrosis model can help researchers better describe the pathological characteristics of IPF and provide a strong guarantee for the identification of new treatment methods and new drugs for IPF.

TGF-β is an extremely critical factor in the development of pulmonary fibrosis. A large number of drugs that can affect TGF-β have been used in the study of bleomycin mouse models, and some of them have shown promising application prospects. Fstl1 (Follistatinlike 1) is a secreted extracellular glycoprotein. It was initially cloned from the mouse osteoblast cell line MC3T3-E1, and its expression can be induced and upregulated by TGF-β1 (transforming growth factor β1). Existing studies have shown that Fstl1 is related to embryonic development, tumor migration and inflammatory response. Recent microarray data analysis showed that the expression of FSTL1 in the lungs of IPF patients was significantly higher than that in normal tissues (GEO_accession: GDS1252). This suggests that Fstl1 may have an effect on pulmonary fibrosis.

Contents of the invention

Aiming at the technical problems existing in the prior art, especially the side effects of pulmonary fibrosis that often occur in tumor patients during drug treatment, the present invention provides a new technical solution, that is, the use of Fstl1 interfering small RNA for the prevention and treatment of pulmonary fibrosis fibrosis.

The present invention finds through systematic research that for the animal model of pulmonary fibrosis caused by tracheal injection of bleomycin (5U/Kg) in C57BL/6 mice, 3, 7, 11, 15 and 19 days after the modeling, respectively, the Lower tracheal spray of Fstl1 siRNA and nonsense siRNA. The changes of lung injury and fibrosis in bleomycin-infused mice were studied by histopathological analysis, and it was found that reducing the expression of Fstl1 could significantly reduce the mortality of bleomycin-treated mice; reducing the expression of Fstl1 significantly reduced the bleomycin Toxin-induced lung injury and pulmonary fibrosis.

Compared with the prior art, the advantages of the present invention are: TGF-β is the most important cytokine in the process of fibrosis, which can increase the expression of Fstl1, and the expression of Fstl1 can be significantly inhibited by using Fstl1 interfering small RNA to reduce the expression of Fstl1 The resulting pulmonary fibrosis process greatly reduces its mortality rate, reduces the side effects of bleomycin, and wins valuable treatment time for patients.

Description of drawings

Sham: Sham group

ScRNA: nonsense interfering small RNA group

Si-Fstl1: Fstl1 interfering small RNA panel

n=10-15

^** P<0.01 vs. sham group

^## P<0.01 vs model group

Figure 1 Survival rate of mice in three groups

Figure 2 Three groups of mouse lung HE staining

Figure 3 Masson staining of lungs of three groups of mice treated with bleomycin for 21 days

specific implementation plan

The invention will be further described below in conjunction with the examples, but the scope of the invention is not limited.

Example 1

【Materials and methods】

Main reagents and experimental animals

The Fstl1 interfering small RNA used in the experiment was purchased from (Dharmacon RNAi Technologies, Thermo); the nonsense small RNA was purchased from (Dharmacon RNAi Technologies, Thermo); the bleomycin (Bleomycin) was purchased from Nippon Kayaku Co., Ltd. Y91450).

The animal size used in the experiment of Fstl1 interfering small RNA on bleomycin-induced pulmonary fibrosis is SPF grade C57BL/6J mice (male, 8-10 weeks, 20-25g), which were purchased from the Institute of Model Animals, Nanjing University , animal certificate number SCXK (Su) 2010-0001. Animals were kept in the Institute of Model Animals, Nanjing University, with constant temperature and humidity, and free diet.

Pulmonary fibrosis animal model preparation

Male C57BL/6J (age 8-10 weeks) mice were anesthetized by intraperitoneal (I.P.) injection of 7.5% chloral hydrate at 0.6ml/100g, and intratracheal injection of bleomycin (5U/kg). The specific scheme is as follows: after weighing and recording the body weight, fix the mouse on the operating table, disinfect the neck with 70% alcohol, use a scalpel to cut vertically about 1 cm in the neck of the mouse, use micro-tweezers to separate the tissue to expose the trachea, Insert the syringe into the trachea through the gap between the tracheal cartilage rings towards the heart, then slowly inject a volume of bleomycin saline solution (5u/ml) corresponding to its body weight at a rate of 5U/kg, and immediately turn the animal upright and rotate left and right, Make the liquid evenly distributed in the lungs. The same volume of normal saline for injection was injected into the trachea of the sham operation group. During the experiment, the small interfering RNA was given according to the standard of 10ug/animal, and the body weight and the death rate of the animals were recorded at the same time, and the experiment was ended on the 21st day.

The experimental group design is as follows:

The experiment was divided into control group (Sham), Fstl1 interfering small RNA group and nonsense small RNA group. On the 3rd, 7th, 11th, 15th and 19th days of bleomycin modeling, 10ug/20ul Fstl1 interfering small RNA, nonsense interfering small RNA and corresponding volume of normal saline were sprayed through the sublingual trachea as the control group. The experiment was ended on the 21st day. See Table 1 for detailed group design.

Table 1. Experimental design of Fstl1 interfering small RNA on bleomycin-induced pulmonary fibrosis

Mouse Survival Statistics

There were 15 mice in each group, and they were weighed at the same time (10:00) every day from the 1st day to the 21st day after the administration of bleomycin. The number of dead mice in each. The 21-day survival rates of the three groups of mice were compared using GraphPad Prism software.

Pathomorphological analysis and Masson staining pathological imaging analysis of pulmonary fibrosis induced by bleomycin

The left lung tissue of the animal was taken, fixed in neutral formalin, embedded in paraffin, and sliced from the hilum of the lung tissue. Hematoxylin/eosin (HE) staining was used to observe pathological changes, and Masson staining was used to observe fibrosis, collagen deposition.

High-definition Masson-stained pathological pictures (200 times magnification) were obtained with an upright transmission fluorescence microscope (LeicaDFC420C), opened in Image-Pro Plus Version 6.0 (Media Cybernetics, Inc. American), and all lungs were selected using the software selection tool For the tissue area, use the automatic calculation function of the software to obtain the total pixel Pw of the selected area, and then use the same method to select the fibrosis area in the section and use the software to calculate the total pixel Pf of the fibrosis area. The ratio of the total lung pixels Pw is the proportion of fibrosis. The degree of lung tissue fibrosis was assessed according to the method provided by Szapiel et al.

Pulmonary fibrosis is divided into four grades: 1) grade 0: no fibrosis; 2) grade I: extent <20%, involving membrane and subpleural lung parenchyma, and disordered alveolar structure; 20%-50% of the whole lung, pulmonary fibrosis extends from the pleura, but is still localized; 4) Grade III: diffuse pulmonary fibrosis, range> 50%, fusion damage and extensive pulmonary parenchymal structural disorder.

Statistical Analysis

Using Excel, the data is represented by (x±s), and the grade data is converted into measurement data, with 0 points for grade 0, 1 point for grade I, 2 points for grade II, and 3 points for grade III. Chi-square test was used for the mortality rate of the three components, and t test was used for measurement data. P<0.05 was considered to be statistically significant.

【result】

Reducing Fstl1 expression in the lung reduces bleomycin-induced lung injury and fibrosis.

We used C57BL/6J mice to inject bleomycin (5U/Kg) through the trachea to induce pulmonary fibrosis animal model, and detected the regulatory effect of Fstl1 interfering small RNA on bleomycin-induced pulmonary fibrosis.

The results showed that due to severe lung damage, the survival rate of the animals in the model group was about 55%, and the administration of Fstl1 interfering small RNA to reduce the activity of Fstl1 could significantly increase the survival rate of the animals (P<0.01) (Figure 1). Compared with the sham operation group, after 7 days of bleomycin administration, the number of fibroblast hyperplasia and alveolar inflammatory cells in the lungs of the model group animals began to increase, while the number of inflammatory cells in the lungs of mice after the Fstl1 activity was reduced The infiltration was significantly reduced; when bleomycin was administered for 14 days, the degree of fibrosis in the lungs of the animals in the model group was deepened, and the area of fibrosis was enlarged, but the degree of fibrosis in the lungs of the Fstl1 interfering small RNA group was significantly lighter, and the area of fibrosis was smaller ; When bleomycin was administered for 21 days, a large area of solidified area (pointed by the arrow) appeared in the lungs of the animals in the model group, and the alveolar structure was severely damaged. To the presence of normal alveolar structure (Figure 2). In addition, compared with the sham operation group, the collagen deposition and pathological index of fibrosis in the model group were significantly increased; the collagen deposition and pathological index of fibrosis in the Fstl1 interfering small RNA treatment group were significantly decreased (P<0.01) (Fig. 3, Table 2).

Table 2 Three groups of mice pulmonary fibrosis score (x ± s) comparison

group n (only) Pulmonary fibrosis score mock surgical group 15 0±0 nonsense small RNA group 15 2.28±0.76** Fstl1 interfering small RNA group 15 1.14±0.38** ^##

【in conclusion】

The invention adopts the Fstl1 interfering small RNA to reduce the activity of Fstl1, can significantly inhibit the pulmonary fibrosis induced by bleomycin, and significantly reduce the death rate of mice with pulmonary fibrosis. Using half the dose of bleomycin combined with Fstl1 small interfering RNA can obtain the same therapeutic effect as the full dose of bleomycin, but significantly reduce the side effects of bleomycin.

Claims (3)

1. Application of small interfering RNA of Fstl1 in the preparation of drugs for preventing and/or treating pulmonary fibrosis. 2. The application according to claim 1, characterized in that the dosage form of the small interfering RNA of the Fstl1 when preparing the medicine for preventing and treating idiopathic pulmonary fibrosis is a spray solution. 3. The application according to claim 1 or 2, characterized in that the injection route of the medicine is sublingual tracheal spray.

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