WO2018099148A1 - 微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途 - Google Patents

微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途 Download PDF

Info

Publication number
WO2018099148A1
WO2018099148A1 PCT/CN2017/099249 CN2017099249W WO2018099148A1 WO 2018099148 A1 WO2018099148 A1 WO 2018099148A1 CN 2017099249 W CN2017099249 W CN 2017099249W WO 2018099148 A1 WO2018099148 A1 WO 2018099148A1
Authority
WO
WIPO (PCT)
Prior art keywords
microcystins
blm
expression
tgf
rats
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/CN2017/099249
Other languages
English (en)
French (fr)
Inventor
王亚平
徐力致
王洁
赵庆亚
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Nanjing University
Nanjing Tech University
Original Assignee
Nanjing University
Nanjing Tech University
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Nanjing University, Nanjing Tech University filed Critical Nanjing University
Priority to US16/343,750 priority Critical patent/US20190275103A1/en
Priority to EP17876506.1A priority patent/EP3549596B1/en
Publication of WO2018099148A1 publication Critical patent/WO2018099148A1/zh
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P11/00—Drugs for disorders of the respiratory system

Definitions

  • the invention belongs to the field of medical technology.
  • the present invention relates to the use of Microcystins (MCs) for the preparation of a medicament for the prevention or treatment of fibrotic diseases in human organ tissues.
  • MCs Microcystins
  • Fibrosis is a pathological reaction caused by damage to tissues and organs of the body, characterized by abnormal deposition of extracellular matrix (ECM) (mainly collagen). Fibrosis of different tissues and organs has similar molecular pathological mechanisms: it involves tissue and body damage caused by internal and external factors, increased reactive oxygen species (ROS), infiltration of inflammatory cells in damaged tissues, and transforming growth factor- ⁇ (Transforming Growth factor- ⁇ , TGF- ⁇ ) release, activation of TGF- ⁇ /Smad signaling pathway, formation of myofibroblast (1-3). Fibrosis generally progresses steadily, eventually leading to tissue and organ failure, and the clinical prognosis is poor (4).
  • ROS reactive oxygen species
  • Pulmonary fibrosis is a severe pulmonary interstitial chronic disease caused by a variety of causes. It can be divided into secondary pulmonary fibrosis (SPF) and Idiopathic pulmonary fibrosis (IPF).
  • SPF secondary pulmonary fibrosis
  • IPF Idiopathic pulmonary fibrosis
  • the cause of the former is clear, such as pneumoconiosis, silicosis, asbestosis and lungs caused by industrial and ore dust exposure, and pulmonary fibrosis based on autoimmune diseases of patients; the latter cause is unknown.
  • IPF is one of the common diseases associated with respiratory aging and one of the most difficult clinically high mortality cases (5,6). With the rapid development of society, the incidence of IPF in the population is on the rise.
  • IPF histopathology and molecular pathology are characterized by generalized interstitial pneumonia and pulmonary fibrosis, characterized by progressive labor dyspnea, restricted ventilatory dysfunction and limited oxygen diffusion, and eventually death from respiratory failure. The average survival time is only about 3 years.
  • bleomycin (BLM)-induced rat or mouse pulmonary fibrosis models are routine animal models used to study pulmonary fibrosis (7, 8).
  • Microcystins are a common class of toxins produced by cyanobacterial blooms in environmental waters. It is thought to have activity inhibiting serine/threonine protein phosphatases 1 and 2A (PP1 and PP2A) and mainly produces hepatorenal toxicity (9, 10). No related research reports have been reported so far on the biological activity against fibrosis and the function of developing clinical drugs.
  • the main structure of MCs is a kind of monocyclic heptapeptide compound. Due to the difference of constituent amino acids, there are many isomers in nature, and the main mutation occurs in the second and fourth amino acids.
  • the present invention first selects the development of microcystins (MC-LR) of the second and fourth amino acids of the monocyclic heptapeptide structure, which are leucine (Leucine, L) and arginine (Arginine, R), respectively. Preparation of therapeutic and interventional drugs for pulmonary fibrosis. On the basis of observing the therapeutic effect, the present invention further selects the development of microcystins (MC-RR) in which the second and fourth amino acids of the monocyclic heptad structure are arginine (Arginine, R). Preparation of therapeutic and interventional drugs for pulmonary fibrosis. The toxicity of MC-RR was significantly less than that of MC-LR.
  • MC-RR The toxicology test (LD 50) of mice exposed to cells and cultured cells in vitro showed that the toxicity of MC-RR was less than 1/5 (10) of the toxicity of MC-LR (Fig. 1). However, MC-RR also has a good effect in treating and intervening pulmonary fibrosis.
  • the invention is based on the construction of an animal model of pulmonary fibrosis induced by Bleomycin (BLM), and is administered by daily drinking water to MC-RR or MC-LR, and histopathological observation and tissue fibrosis are key. Molecular, signaling pathway analysis to determine the effectiveness of intervention therapy.
  • RESULTS MC-RR and MC-LR interventions could alleviate and improve the pathological state of lung fibrosis induced by BLM; significantly reduce collagen I (Collagen 1) and ⁇ -SMA ( ⁇ -smooth muscle actin) in the lung tissue of model rats.
  • TGF- ⁇ and its signaling pathway protein were significantly inhibited by MC-RR or MC-LR intervention in rat lung tissue cells, mainly TGF- ⁇ , smad2/smad3 mRNA and protein expression. Significantly down-regulated, while the mRNA and protein expression of the fibrosis-inhibiting molecule smad7 was up-regulated.
  • Activation of the TGF- ⁇ /Smad signaling pathway promotes epithelial-mesenehymal transition (EMT) and myofibroblast formation (11-14); at the same time, structural collagen formation in the lung tissue of rats
  • EMT epithelial-mesenehymal transition
  • myofibroblast formation 11-14
  • structural collagen formation in the lung tissue of rats The key enzyme P4HA3 expression is inhibited.
  • P4HA3 is a downstream pro-fibrotic factor regulated by TGF- ⁇ (15).
  • VEGF Vascular endothelial growth factor
  • ET-1 Endothelin 1
  • microcystins can be used to develop and prepare novel drugs for preventing and treating organ tissue fibrotic diseases.
  • the present invention also provides a method for inhibiting myofibroblast differentiation and collagen synthesis using microcystins, which can be used in scientific research, medical treatment and the like. It will be apparent that the above two aspects of the invention share a common technical feature that one or more of the microcystins that inhibit the TGF-[beta]/Smad signaling pathway.
  • the invention discloses the medical use of the monocyclic heptapeptide compound Microcystins (MCs), especially the microcystins-RR and LR (MC-RR and MC-LR) are developed and prepared for prevention or treatment. Use in drugs for organ tissue fibrotic diseases.
  • MCs monocyclic heptapeptide compound Microcystins
  • MC-RR and MC-LR microcystins-RR and LR
  • the organ is a lung.
  • the medicament of the present invention inhibits myofibroblast formation (reducing ⁇ -SMA levels) and inhibiting collagen I (collagen) in pulmonary tissue of pulmonary fibrosis rats induced by bleomycin (BLM). 1) Expression.
  • the medicament of the present invention inhibits mRNA and protein expression of a key molecule TGF- ⁇ in tissue fibrosis in lung tissue cells of BLM-induced pulmonary fibrosis rats.
  • the medicament of the invention inhibits the expression of Smad2 and Smad3.
  • Smad2 and Smad3 are key signaling pathways (TGF- ⁇ /Smad signaling pathway) in the lung tissue of BLM-induced pulmonary fibrosis rats, and are molecules that promote fibrosis.
  • the medicament of the invention upregulates the expression of Smad7.
  • Smad7 is a negative regulator of a key signaling pathway (TGF- ⁇ /Smad signaling pathway) for fibrosis in lung tissue cells of rats with pulmonary fibrosis induced by BLM, and is a molecule that inhibits fibrosis formation.
  • the medicament of the present invention inhibits the expression of a key enzymatic molecule P4HA3 in pulmonary fibrosis in lung tissue cells of BLM-induced pulmonary fibrosis rats.
  • the present invention also discloses a method of inhibiting myofibroblast differentiation and collagen synthesis by using one or more microcystins to suppress the TGF- ⁇ /Smad signaling pathway.
  • the microcystin is a microcystin-LR containing a monocyclic heptad structure, the second and fourth amino acids of which are leucine and arginine, respectively, or a single ring The heptad structure, the second and fourth amino acids are all microcystin-RR of arginine.
  • the invention is based on the classic animal model of bleomycin (BLM)-induced pulmonary fibrosis in rats, and is treated by MC-RR and MC-LR respectively. Based on histopathology and analysis of key signaling pathway molecules for fibrosis, MC-RR and MC-LR were proposed to alleviate and treat the pathological state of pulmonary fibrosis induced by BLM and effectively prevent pulmonary fibrosis. Based on this, the present invention provides molecular targets and mechanisms of action for microcystin intervention, treatment of fibrosis.
  • BLM bleomycin
  • Natural microcystins have multiple variants based on the basic structure of monocyclic heptapeptide due to differences in amino acid composition, and the toxicity between different variants varies greatly.
  • the present invention is based on the use of less toxic MC-RR and more toxic MC-LR intervention to treat pulmonary fibrosis in model rats, both of which show good intervention and therapeutic effects. It is proved that the anti-fibrotic effect of MCs is related to the molecular structure of monocyclic heptapeptide and the resulting biological effects, but not related to the toxicity of MCs.
  • the present invention discloses that the microcystin monocyclic heptapeptide structure has the biological effect of relieving and retarding the pathogenesis of pulmonary fibrosis, and can be used for the development and preparation of a treatment and intervention drug for organ tissue fibrotic diseases.
  • Figure 1 shows the chemical structure of Microcystins, where A is the general structure of microcystins (MCs); B is the structure of MC-LR; C is the structure of MC-RR; D shows real-time dynamic cell analysis Real time cellular analysis (RTCA) showed the toxic effects of MC-LR and MC-RR on cultured liver cell line (L02), indicating that the cytotoxicity of MC-LR was significantly higher than MC-RR (NC was the control group). .
  • RTCA Real time cellular analysis
  • Figure 2 shows the changes in body weight of rats in the BLM-induced pulmonary fibrosis model and MCs intervention, in which NS is the saline control group; BLM is 5 ⁇ g/kg bleomycin (BLM) airway instillation to construct pulmonary fibrosis Rat model group; LR7, LR14 and LR28 started MC-LR intervention group on the 7th, 14th and 28th day after BLM airway instillation; RR14 started MC on the 14th day after BLM airway infusion - RR intervention group.
  • BLM is 5 ⁇ g/kg bleomycin (BLM) airway instillation to construct pulmonary fibrosis Rat model group
  • LR7, LR14 and LR28 started MC-LR intervention group on the 7th, 14th and 28th day after BLM airway instillation
  • RR14 started MC on the 14th day after BLM airway infusion - RR intervention group.
  • Figure 3 shows the liver, kidney and lung indices of each group of rats, where A is the lung index.
  • A is the lung index.
  • "*” P ⁇ 0.05 compared with the NS group;
  • "#” P ⁇ 0.05 compared with the BLM group.
  • B is the liver index;
  • C is the kidney index.
  • Figure 4 shows the results of liver and kidney function analysis of each group of rats, wherein A and B reflect serum levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT); C and D reflect renal function of serum creatinine (CRE) And urea nitrogen content (BUN).
  • a and B reflect serum levels of serum aspartate aminotransferase (AST) and alanine aminotransferase (ALT);
  • C and D reflect renal function of serum creatinine (CRE) And urea nitrogen content (BUN).
  • Figure 5 shows the establishment of a model of pulmonary fibrosis induced by BLM in rats, in which A shows Masson staining of lung tissue sections of normal saline (NS) control rats; B shows lung tissue sections of lung fibrosis rats constructed by BLM airway instillation Masson Staining; C showed the evaluation of lung fibrosis in the NS group and the BLM model group.
  • the lung tissue of the model group showed fibrotic lesions; D showed the expression of collagen I in the lung tissue of the NS group and the BLM model group.
  • Figure 6 shows the effect of MC-RR or MC-LR intervention on BLM-induced pulmonary fibrosis in rats.
  • AD is Masson staining of rat lung tissue section: A shows that MC-LR intervention (LR7) starts to relieve pulmonary fibrosis in rats on the 7th day of BLM modeling, and B shows that MC-LR intervention begins on the 14th day of BLM modeling ( LR14) alleviated pulmonary fibrosis in rats, C showed the effect of intervention on the 28th day of BLM modeling (LR28) on pulmonary fibrosis in rats, and D showed that MC-RR intervention (RR14) was relieved on the 14th day of BLM modeling.
  • LR7 shows that MC-LR intervention starts to relieve pulmonary fibrosis in rats on the 7th day of BLM modeling
  • B shows that MC-LR intervention begins on the 14th day of BLM modeling ( LR14) alleviated pulmonary fibrosis in rats
  • C showed the effect of intervention on the 28th day of BLM modeling
  • Pulmonary fibrosis in rats E showed that MC-RR and MC-LR intervention groups were compared and evaluated in the treatment of pulmonary fibrosis induced by BLM. The results showed that LR7 and RR14 had the most obvious relief effect; F showed MC-RR and MC.
  • the expression of collagen I in the lung tissue of LR-intervention group showed that the expression of collagen I was down-regulated in LR7 and RR14 groups.
  • G showed that the expression of ⁇ -SMA protein in lung tissue of MC-RR and MC-LR groups was inhibited. RR14 and RR28 have the largest reductions.
  • Figure 7 shows that MC-RR or MC-LR intervention inhibits BLM-induced TGF- ⁇ expression in rat lung tissue cells, where "*": P ⁇ 0.05 compared to the BLM group.
  • Figure 8 shows the effect of MC-RR and MC-LR intervention on BGF-induced TGF- ⁇ /Smad signaling pathway in rat lung tissue cells.
  • A shows the results of Western Blot analysis of TGF- ⁇ /Smad pathway protein in lung tissue of each group.
  • B showed the analysis results of Smad3 gene mRNA transcription and expression in each group of lung tissue cells;
  • C showed the analysis results of Smad7 gene mRNA transcription expression in each group of lung tissue cells.
  • Figure 9 shows that MC-RR and MC-LR inhibited the transcriptional expression of the profibrotic molecule P4HA3, wherein "*": P ⁇ 0.05 compared to the BLM group.
  • Figure 10 shows that MC-RR and MC-LR inhibited the expression of fibrosis-associated cytokines VEGF and ET1, with "*" compared with the BLM group, P ⁇ 0.05.
  • Bleomycin induces pulmonary fibrosis in rats (or mice), which is a common animal model used in international and domestic laboratories to study the mechanism of pulmonary fibrosis, intervention targets and therapeutic drugs.
  • the model was constructed stably and histopathological changes were similar to human pulmonary fibrosis.
  • the rat BLM exposure method used was a commonly used orotracheal intubation, instilled in the airway; the control was intratracheally instilled with an equal volume of saline.
  • mice SPF Sprague Dawley (SD) rats, male, weighing 200g-250g (provided by Changzhou Cavans Laboratory Animal Co., Ltd.), animal certificate number: SCXK (Su) 2011-0003. Breeding conditions: a well-ventilated 20 ° C constant temperature environment, alternating light and dark every 12 hours, drinking water and rat food freely. Bleomycin (BLM, Nippon Kayaku Co., Ltd.) induced the construction of a rat pulmonary fibrosis model: SD male rats, approximately 8 weeks old. General anesthesia was performed by intraperitoneal injection of 10% chloral hydrate (0.4 ml/kg).
  • the rats were fixed, and tracheal intubation was performed orally, and BLM was instilled in the airway (dose was 5 mg/kg). After the rats were observed to have stable breathing, the dry and clean squirrel cages were returned. After the rats awakened, they were raised normally. At the same time, a control group of equal volume saline infusion was established.
  • a total of 36 SD rats were randomly divided into 6 groups (6 in each group): 1 saline control group (NS); 2BLM induced pulmonary fibrosis model group (BLM); 3BLM modeling + MC-LR began on the 7th day Intervention group (LR7); 4BLM Modeling + MC-LR Day 14 Intervention Group (LR14); 5BLM Modeling + MC-LR Day 28 Intervention Group (LR28); 6BLM Modeling + MC-RR Day 14 Intervention Group (RR14).
  • NS group and BLM group were treated with natural drinking sterilized ultrapure water; LR7 group was added to MC-LR (final concentration: 20 ⁇ g/L) on the 7th day after BLM infusion, and continued to drink; LR14 group On the 14th day after the BLM instillation, the drinking water contained MC-LR (final concentration: 20 ⁇ g/L) and continued to drink; the LR28 group began to drink water containing MC-LR on the 28th day after the BLM instillation (final concentration: 20 ⁇ g/L) , continued drinking; RR14 group began to drink water on the 14th day after BLM infusion containing MC-RR (final concentration: 20 ⁇ g / L), continued drinking.
  • MC-RR or MC-LR did not produce significant toxic effects on the model rats by dose and administration: 1Compared with the body weight change of the model (BLM group) during the experiment, BLM+MC-RR , or BLM+MC-LR rats gained good weight (Fig. 2); the liver and kidney index (rat liver or kidney weight/rat weight) of rats were not significantly different among groups; BLM induced pulmonary fibrosis The lung index of the rats was significantly higher than that of the NS control group, while the lung index of the MC-RR and MC-LR intervention groups was lower than that of the BLM group (Fig. 3); 2MC-RR or MC-LR intervention group rats and controls There were no significant abnormalities in liver and kidney function between the group and the BLM model group (Fig. 4).
  • TGF- ⁇ is the most known protein factor that promotes tissue fibrosis and is also fibrotic. Mechanisms and important molecular targets for anti-fibrosis research. TGF- ⁇ promotes epithelial-mesenehymal transition (EMT) and myofibroblast formation (11,12). Intratracheal instillation of BLM caused a significant increase in the release of TGF- ⁇ from rat lung tissue cells. After intratracheal instillation of BLM, MC-RR or MC-LR intervention significantly inhibited the transcription and translation of TGF- ⁇ . The LR7 intervention group and RR14 intervention group inhibited the expression of TGF- ⁇ (Fig. 7, Fig. 7 8A).
  • Intervention of MC-RR or MC-LR can significantly inhibit the activity of TGF- ⁇ /Smad signaling pathway in rat lung tissue induced by BLM: TGF- ⁇ released from tissue cells passes through TGF- ⁇ receptor on the surface of cell membrane Binding activates the TGF- ⁇ /Smad signaling pathway. It causes an increase in Smad2/3 expression (increased protein phosphorylation) and forms a complex with Smad4, which is transferred into the nucleus, alters nuclear gene expression, initiates EMT and myofibroblast differentiation, leading to tissue fibrosis (13,14) .
  • Smad2/3 expression (including protein phosphorylation level) was significantly increased, showing activation of TGF- ⁇ /Smad signaling pathway.
  • Administration of MC-RR or MC-LR drinking water to BLM modeled rats significantly reduced Smad2/3 expression (including decreased protein phosphorylation) in tissue cells.
  • Inhibition of Smad7 is also regulated by TGF- ⁇ /Smad signaling pathway in normal cells. Rats were intervened in MC-RR or MC-LR, and a significant increase in Smad7 expression was observed. The inhibitory effect of MC-RR or MC-LR on the formation of induced pulmonary fibrosis was fully demonstrated (Fig. 8).
  • P4HA3 is a pro-fibrotic factor that induces downstream up-regulation of TGF- ⁇ expression in recent years, in lung tissue of patients with pulmonary fibrosis, and lung of rat model of pulmonary fibrosis induced by BLM The expression of P4HA3 was significantly increased in tissues (15). In the BLM model rats, MC-RR or MC-LR intervention inhibited the expression of P4HA3, and the inhibitory effect was significant in the RR14 and LR7 groups (Fig. 9).
  • VEGF vascular endothelial growth factor
  • ET1 vascular endothelial growth factor
  • ET-1 promotes fibroblast proliferation, induces fibroblast differentiation into myofibroblasts, and inhibits collagen degradation (17).
  • the transcriptional expression of VEGF and ET-1 was significantly decreased in the lung tissue of rats with pulmonary fibrosis treated with MC-RR or MC-LR drinking water (Fig. 10).
  • microcystins of the present invention have new and important uses in the development and preparation of drugs for preventing and treating organ tissue fibrotic diseases.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • Veterinary Medicine (AREA)
  • Chemical & Material Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Public Health (AREA)
  • Medicinal Chemistry (AREA)
  • Pharmacology & Pharmacy (AREA)
  • General Health & Medical Sciences (AREA)
  • Epidemiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Immunology (AREA)
  • Gastroenterology & Hepatology (AREA)
  • Pulmonology (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • General Chemical & Material Sciences (AREA)
  • Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
  • Organic Chemistry (AREA)
  • Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

单环七肽结构微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途。优选地，所述微囊藻毒素是其单环七肽结构的第二和第四位氨基酸分别为亮氨酸和精氨酸的微囊藻毒素-LR，或者是其第二和第四位氨基酸均为精氨酸的微囊藻毒素-RR。还涉及一种抑制肌成纤维细胞分化和胶原蛋白合成的方法，该方法通过使用一种或多种微囊藻毒素阻抑TGF-β/Smad信号通路而实现。

Description

微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途 技术领域
本发明属于医药技术领域。具体地说,本发明涉及微囊藻毒素(Microcystins,MCs)在制备用于预防或治疗人类器官组织纤维化疾病的药物中的用途。
背景技术
纤维化(Fibrosis)是机体组织器官对损伤所产生的一种病理性反应,以细胞外基质(Extracellular matrix,ECM)(主要是胶原蛋白)异常沉积为特征。不同组织器官的纤维化发生具有相似的分子病理机制：涉及机体内、外因素引起组织细胞损伤,活性氧(Reactive oxygen species,ROS)增加，受损组织炎症细胞浸润,转化生长因子-β(Transforming growth factor-β,TGF-β)释放，TGF-β/Smad信号通路激活,肌成纤维细胞(Myofibroblast)形成(1-3)。纤维化一般渐进发展,最终导致组织器官功能衰竭,临床预后较差(4)。
肺纤维化(Pulmonary fibrosis,PF)是由多种原因引起的严重肺间质慢性疾病。可分为继发性肺纤维化(Secondary pulmonary fibrosis,SPF)和特发性肺纤维化(Idiopathic pulmonary fibrosis,IPF)。前者病因明确,如工业、矿石粉尘接触引发的尘肺、矽肺、石棉肺,及患者自身免疫性疾病基础上的肺纤维化等；后者病因不明。IPF是呼吸系统衰老相关常见疾病之一，也是临床高死亡率的疑难病症之一(5,6)。随着社会老龄化的快速发展，人群IPF的发病率呈上升趋势。IPF组织病理学和分子病理学具有一般间质性肺炎、肺纤维化的特征，表现为进行性劳力性呼吸困难，限制性通气障碍和氧弥散功能受限，最终发生呼吸衰竭而死亡，初诊后平均生存时间仅约3年。在已报导的肺纤维化模型动物研究中，博来霉素(Bleomycin,BLM)诱导的大鼠或小鼠肺纤维化模型是研究肺纤维化常规采用的动物模型(7,8)。
微囊藻毒素(Microcystins,MCs)是环境水体蓝藻水华污染产生的一类常见毒素。一般认为其具有抑制丝氨酸/苏氨酸蛋白磷酸酶1和2A(Serine/threonine protein phosphatases 1and 2A，PP1和PP2A)的活性而主要产生肝肾毒性(9, 10)。对于其抗纤维化的生物活性及开发临床药物的功能，迄今未见相关研究报道。MCs主结构是一类单环七肽化合物，由于组成氨基酸的差异，自然界存在多种异构体，主要变异发生于其第二和第四位的氨基酸。本发明首先选择这种单环七肽结构的第二和第四位氨基酸分别为亮氨酸(Leucine,L)和精氨酸(Arginine,R)的微囊藻毒素(MC-LR)开发、制备肺纤维化的治疗和干预药物。在观察到疗效的基础上，本发明又选择了这种单环七肽结构的第二和第四位氨基酸均为精氨酸(Arginine,R)的微囊藻毒素(MC-RR)开发、制备肺纤维化的治疗和干预药物。MC-RR的毒性明显小于MC-LR,小鼠染毒和体外培养细胞的毒理学实验(LD 50)显示，MC-RR的毒性不足MC-LR毒性的1/5(10)(图1)，但MC-RR同样具有治疗和干预肺纤维化的良好效果。
本发明在构建博来霉素(Bleomycin,BLM)诱导肺纤维化动物模型的基础上，通过日常饮水给予MC-RR或MC-LR干预治疗，并采用组织病理学观察及组织细胞纤维化发生关键分子、信号通路分析来确定干预治疗的效果。结果:MC-RR和MC-LR干预可以缓解和改善由BLM引发的肺组织纤维化病理状态；明显降低模型大鼠肺组织中胶原蛋白I(Collagen 1)和α-SMA(α-smooth muscle actin)的含量；在MC-RR或MC-LR干预大鼠肺组织细胞，纤维化发生关键分子TGF-β及其信号通路蛋白表达受到明显抑制，主要为TGF-β、smad2/smad3 mRNA和蛋白质表达明显下调，而纤维化抑制分子smad7的mRNA及蛋白质表达上调。TGF-β/Smad信号通路的激活可以促进上皮间质转化(Epithelial-mesenehymal transition,EMT)及肌成纤维细胞形成(11-14)；同时，在干预大鼠肺组织中，结构性胶原蛋白形成的关键酶P4HA3表达受到抑制。P4HA3是TGF-β调控的下游促纤维化因子(15)。另一方面，组织纤维化病理发生过程中常伴有其他细胞因子的释放，并对纤维化的发展具有促进作用。VEGF(Vascular endothelial growth factor)是已知功能效应最强的血管生长因子之一。组织细胞VEGF含量的增高可促进血管通透性，导致巨噬细胞迁移、聚集和活化，引起TGF-β释放增多。纤维化组织亦常见ET-1(Endothelin 1)的表达上调(16)。ET-1具有促进成纤维细胞增殖，诱导成纤维细胞向肌成纤维细胞分化,抑制胶原蛋白降解的作用(17)。在给予MC-RR或MC-LR干预的模型大鼠肺组织中,VEGF和ET-1的转录表达均显著降低。
作为提出本发明基础的上述实验结果明确显示，针对博来霉素(Bleomycin,BLM)诱导肺纤维化的病理学改变，MC-RR及MC-LR具有有效的干预和治疗作用。
其重要机制是通过下调TGF-β的表达，阻抑TGF-β/Smad信号通路，抑制肌成纤维细胞分化和胶原蛋白的合成，最终缓解和阻滞细胞外基质(ECM)的异常沉积。
本发明发现了微囊藻毒素可用于开发、制备预防和治疗器官组织纤维化疾病的新型药物。另外，本发明也提供了一种用微囊藻毒素抑制肌成纤维细胞分化和胶原蛋白合成的方法,可用于科研、医疗等领域。很显然，本发明的上述两个方面具有共同的技术特征，即一种或多种能阻抑TGF-β/Smad信号通路微囊藻毒素。
发明内容
本发明公开了单环七肽化合物微囊藻毒素(Microcystins,MCs)的医药用途，尤其是微囊藻毒素-RR和LR(MC-RR和MC-LR)在开发、制备用于预防或治疗器官组织纤维化疾病的药物中的用途。
优选地，所述器官是肺。
优选地，本发明的药物在博来霉素(Bleomycin,BLM)诱导产生的肺纤维化大鼠肺组织中，阻抑肌成纤维细胞形成(降低α-SMA水平)、抑制胶原蛋白I(Collagen 1)表达。
优选地，本发明的药物在BLM诱导肺纤维化大鼠肺组织细胞中，抑制组织纤维化发生关键分子TGF-β的mRNA和蛋白质表达。
优选地，本发明的药物抑制Smad2和Smad3的表达。所述Smad2和Smad3是在BLM诱导肺纤维化大鼠肺组织细胞中构成纤维化发生关键信号通路(TGF-β/Smad信号通路)，是促进纤维化形成的分子。
优选地，本发明的药物上调Smad7的表达。所述Smad7是在BLM诱导肺纤维化大鼠肺组织细胞中针对纤维化发生关键信号通路(TGF-β/Smad信号通路)的负调控因子，是阻抑纤维化形成的分子。
优选地，本发明的药物在BLM诱导肺纤维化大鼠肺组织细胞中抑制肺纤维化发生关键酶促分子P4HA3表达。
本发明还公开了一种抑制肌成纤维细胞分化和胶原蛋白合成的方法，该方法通过使用一种或多种微囊藻毒素阻抑TGF-β/Smad信号通路而实现。优选地，在该方法中，微囊藻毒素是含单环七肽结构，其第二和第四位氨基酸分别为亮氨酸和精氨酸的微囊藻毒素-LR，或者是含单环七肽结构，其第二和第四位氨基酸均为精氨酸的微囊藻毒素-RR。
本发明在构建博来霉素(Bleomycin,BLM)诱导大鼠肺纤维化这一经典动物模型的基础上，分别采用MC-RR、MC-LR干预治疗。依据组织病理学及针对纤维化发生关键信号通路分子的分析结果，创新提出MC-RR和MC-LR可以缓解和治疗由BLM引发的肺纤维化病理状态，有效阻止肺纤维化进程。基于此，本发明提供了微囊藻毒素干预、治疗纤维化的分子靶标和作用机制。
自然界微囊藻毒素(MCs)在单环七肽基本结构的基础上由于氨基酸组成差异存在多种变异体，不同变异体之间的毒性差异很大。本发明基于的实验采用了毒性较小的MC-RR和毒性较大的MC-LR干预、治疗模型鼠的肺纤维化，两者均表现出良好的干预和治疗效果。证明MCs的抗纤维化作用与其单环七肽分子结构及由此产生的生物学效应有关，而与MCs毒性强弱无关联。
基于上述研究，本发明公开了微囊藻毒素单环七肽结构具有缓解和阻滞肺纤维化病理发生的生物学作用，可用于开发、制备器官组织纤维化疾病的治疗和干预药物。
附图说明
图1显示微囊藻毒素(Microcystins)的化学结构，其中A是微囊藻毒素(MCs)的一般结构；B是MC-LR的结构；C是MC-RR的结构；D显示实时动态细胞分析技术(Real time cellular analysis,RTCA)检测MC-LR和MC-RR对培养肝细胞株(L02)的毒性作用的结果，表明MC-LR的细胞毒性明显高于MC-RR(NC为对照组)。
图2显示BLM诱导肺纤维化模型大鼠及MCs干预各组大鼠体重变化情况，其中NS是生理盐水对照组；BLM是5μg/kg博来霉素(BLM)气道滴注构建肺纤维化大鼠模型组；LR7、LR14和LR28分别是于BLM气道滴注后第7天、第14天和第28天开始MC-LR干预组；RR14是BLM气道滴注后第14天开始MC-RR干预组。
图3显示各组大鼠的肝、肾和肺指数,其中A是肺指数。“*”：与NS组比较，P<0.05；“#”：与BLM组比较，P<0.05。B是肝指数；C是肾指数。
图4显示各组大鼠的肝、肾功能分析结果，其中A、B反映肝脏功能的血清谷草转氨酶(AST)和谷丙转氨酶(ALT)水平；C、D反映肾脏功能的血清肌酐(CRE)和尿素氮的含量(BUN)。
图5显示BLM诱导大鼠肺纤维化模型的建立，其中A显示生理盐水(NS)对照组大鼠肺组织切片Masson染色；B显示BLM气道内滴注构建的肺纤维化大鼠肺组织切片Masson染色；C显示NS组与BLM建模组大鼠肺组织纤维化评估比较，建模组大鼠肺组织呈现纤维化病变；D显示NS组与BLM建模组大鼠肺组织中胶原蛋白I表达的比较，结果建模组胶原蛋白I的mRNA转录明显增高；E显示NS组与BLM建模组大鼠肺组织α-SMA蛋白比较，建模组肺组织α-SMA蛋白表达明显增高。*P<0.05。
图6显示MC-RR或MC-LR的干预对BLM诱导大鼠肺纤维化的影响。其中，A-D为大鼠肺组织切片Masson染色：A显示BLM建模第7天开始MC-LR干预(LR7)缓解大鼠肺纤维化作用，B显示BLM建模第14天开始MC-LR干预(LR14)缓解大鼠肺纤维化作用，C显示BLM建模第28天开始干预(LR28)对大鼠肺纤维化的影响,D显示BLM建模第14天开始MC-RR干预(RR14)缓解大鼠肺纤维化作用；E显示MC-RR和MC-LR各干预组缓解和治疗BLM诱导大鼠肺纤维化的评估比较,结果显示:LR7和RR14缓解效果最为明显；F显示MC-RR和MC-LR各干预组肺组织胶原蛋白I表达,提示LR7和RR14组胶原蛋白I转录表达下调幅度最大；G显示MC-RR和MC-LR各干预组肺组织α-SMA蛋白表达均受到抑制，其中RR14和RR28下调幅度最大。“*”：与BLM组比较，P<0.05。
图7显示MC-RR或MC-LR干预可抑制BLM诱导的大鼠肺组织细胞TGF-β表达，其中“*”：与BLM组比较，P<0.05。
图8显示MC-RR和MC-LR干预对BLM诱导的大鼠肺组织细胞TGF-β/Smad信号通路的影响，其中A显示各组肺组织细胞TGF-β/Smad通路蛋白Western Blot分析结果；B显示各组肺组织细胞Smad3基因mRNA转录表达的分析结果；C显示各组肺组织细胞Smad7基因mRNA转录表达的分析结果。
图9显示MC-RR和MC-LR抑制促纤维化分子P4HA3的转录表达，其中“*”：与BLM组比较，P<0.05。
图10显示MC-RR和MC-LR抑制纤维化相关细胞因子VEGF和ET1的表达，其中“*”与BLM组比较，P<0.05。
具体实施方式
博莱霉素(Bleomycin,BLM)诱导大鼠(或小鼠)的肺纤维化是国际、国内各实验室在研究肺纤维化发生机制、干预靶点及治疗药物时常用动物模型。该模型构建稳定，病理组织学改变与人类肺纤维化相近。我们采用的大鼠BLM暴露方式为普遍采用的经口气管插管，气道内滴注给药；对照采用等体积生理盐水大鼠气道内滴注。
实验动物：SPF级Sprague Dawley(SD)大鼠，雄性，体重200g-250g(常州卡文斯实验动物有限公司提供),动物合格证号:SCXK(苏)2011-0003。饲养条件：通气良好的20℃恒温环境，每12小时光照与黑暗交替，饮水和鼠粮自由摄取。博来霉素(BLM,日本化药株式会社)诱导大鼠肺纤维化模型的构建：SD雄性大鼠，约8周龄。腹腔注射10％水合氯醛(0.4ml/kg)实施全身麻醉，大鼠固定，经口行气管插管，气道内滴注BLM(剂量为5mg/kg)。观察大鼠呼吸稳定后，放回干燥洁净的鼠笼。大鼠苏醒后，正常饲养。同时设立等体积生理盐水滴注的对照组。
SD大鼠共36只，随机分为6组(每组6只)：①生理盐水对照组(NS)；②BLM诱导肺纤维化模型组(BLM)；③BLM建模+MC-LR第7天开始干预组 (LR7)；④BLM建模+MC-LR第14天开始干预组(LR14)；⑤BLM建模+MC-LR第28天开始干预组(LR28)；⑥BLM建模+MC-RR第14天开始干预组(RR14)。NS组、BLM组采用自然饮用灭菌超纯水；LR7组于BLM滴注后第7天开始饮用的灭菌超纯水加入MC-LR(终浓度:20μg/L)，持续饮用；LR14组于BLM滴注后第14天开始饮水含MC-LR(终浓度:20μg/L)，持续饮用；LR28组于BLM滴注后第28天开始饮水含MC-LR(终浓度:20μg/L)，持续饮用；RR14组于BLM滴注后第14天开始饮水含MC-RR(终浓度:20μg/L)，持续饮用。全部实验大鼠于实施BLM气道滴注后的第56天(8周)实施腹腔注射10％水合氯醛(0.4ml/kg)全身麻醉，腹主动脉取血(留取全血及分离血清样本)后处死；留取肺、肝、肾等组织样本。肺组织样本按肺叶分部留取。各器官样本每个留取部位的组织均分为4％中性甲醛固定样本和液氮冻存样本。中性甲醛固定样本用于组织病理学分析，液氮冻存样本主要用于蛋白质和核酸分析。检测数据的统计学分析应用SPSS11.5统计软件处理,采用单因素方差分析(one-way ANOVA)，P<0.05表示差异具有统计学意义。
结果
针对BLM诱导产生的肺纤维化模型大鼠,及MC-RR或MC-LR干预和治疗肺纤维化大鼠的观察、分析，结果显示：
(1)MC-RR或MC-LR使用剂量和给药方式对模型大鼠未产生明显的毒性效应：①与模型(BLM组)大鼠在实验过程中的体重变化比较，BLM+MC-RR，或BLM+MC-LR大鼠体重增长良好(图2)；大鼠的肝、肾指数(鼠的肝或肾重量/大鼠体重)在各组间无显著差异；BLM诱导肺纤维化大鼠的肺指数与NS对照组比较显著增高，而MC-RR及MC-LR的干预处理组肺指数低于BLM组大鼠(图3)；②MC-RR或MC-LR干预组大鼠与对照组和BLM建模组大鼠比较，肝肾功能无明显异常(图4)。
(2)大鼠气管内(单次)滴注BLM(5mg/kg)可稳定诱导大鼠肺组织纤维化病变：①Masson染色是观察病理切片组织纤维化状态的常规技术。结果显示，BLM建模组大鼠肺组织呈现明显的纤维化状态；②BLM组大鼠肺组织胶原蛋白I的表达(mRNA转录)明显高于生理盐水对照组大鼠；③BLM组大鼠肺组织α- SMA(α-smooth muscle actin)蛋白的表达明显增高，反映了BLM组大鼠肺组织中肌成纤维细胞增殖(图5)。
(3)MC-RR或MC-LR的干预可明显减轻BLM诱导大鼠产生的肺纤维化程度：①Masson染色显示，BLM建模第14天开始的MC-RR干预(RR14)，BLM建模第7天(LR7)、第14天开始的MC-LR(LR14)干预的大鼠肺纤维化程度明显降低，BLM建模第28天(LR28)开始MC-LR干预大鼠的肺纤维化也有降低趋势；②与BLM建模组大鼠比较，MC-RR和MC-LR各干预组肺组织细胞Collagen 1表达均有下降，其中LR7和RR14组的下降最为明显；③与BLM建模组大鼠比较，MC-RR和MC-LR各干预组肺组织细胞α-SMA表达均有下调，其中RR14和LR28下调幅度最大(图6)。
(4)MC-RR或MC-LR的干预可明显抑制BLM诱导的大鼠肺组织细胞TGF-β表达:TGF-β是目前已知最强的促进组织纤维化的蛋白因子，也是纤维化形成机制及抗纤维化研究的重要分子靶标。TGF-β可以促进上皮间质转化(Epithelial-mesenehymal transition,EMT)及肌成纤维细胞形成(11,12)。气管内滴注BLM引起大鼠肺组织细胞TGF-β的释放显著增高。而BLM气道内滴注后，MC-RR或MC-LR的干预均明显抑制TGF-β的转录和翻译表达，其中LR7干预组和RR14干预组抑制TGF-β表达的幅度最大(图7,图8A)。
(5)MC-RR或MC-LR的干预可明显阻抑BLM诱导的大鼠肺组织细胞TGF-β/Smad信号通路活性：组织细胞释放的TGF-β通过与细胞膜表面的TGF-β受体结合，激活TGF-β/Smad信号通路。引起Smad2/3表达增高(蛋白磷酸化水平增高)，并与Smad4形成复合体，转入细胞核，改变核基因的表达，启动EMT及肌成纤维细胞分化,导致组织纤维化形成(13,14)。在气管内滴注BLM构建肺纤维化模型的大鼠肺组织细胞，Smad2/3表达(包括蛋白磷酸化水平)明显增高，显示TGF-β/Smad信号通路的激活。而针对BLM建模大鼠给予MC-RR或MC-LR饮水干预均明显降低组织细胞中Smad2/3表达(包括蛋白磷酸化水平的降低)。正常细胞内TGF-β/Smad信号通路还存在Smad7的抑制调控。在MC-RR或MC-LR干预大鼠,同时观察到Smad7的表达显著增高。充分体现了MC-RR或MC-LR对诱导性肺纤维化形成的阻抑作用(图8)。
(6)MC-RR或MC-LR的干预可明显抑制TGF-β介导的下游促进纤维化分子 P4HA3(anα-subunit of collagen prolyl hydroxylase)的表达：P4HA3是近年鉴定的TGF-β诱导下游上调表达的促纤维化因子，在临床肺纤维化患者肺组织，及BLM诱导肺纤维化模型大鼠肺组织中P4HA3表达明显增高(15)。而对BLM建模大鼠给予MC-RR或MC-LR干预可抑制P4HA3的表达，其中在RR14、LR7组抑制效果明显(图9)。
(7)MC-RR或MC-LR的干预可明显阻抑具有促进纤维化形成作用的细胞因子VEGF和ET1的释放：组织纤维化病理发生过程中常伴有其他细胞因子的释放，并对纤维化的发展具有促进作用。VEGF(Vascular endothelial growth factor)是已知功能效应最强的血管生长因子之一。组织细胞VEGF含量的增高可促进血管通透性，导致巨噬细胞迁移、聚集和活化，引起TGF-β释放增多。纤维化组织亦常见ET-1的表达上调(16)。有研究提示,ET-1具有促进成纤维细胞增殖，诱导成纤维细胞向肌成纤维细胞分化，抑制胶原蛋白降解的作用(17)。在给予MC-RR或MC-LR饮水干预肺纤维化模型大鼠肺组织中，VEGF和ET-1的转录表达均显著降低(图10)。
作为提出本发明基础的上述实验结果明确显示，针对博来霉素诱导肺纤维化的病理学改变，MC-RR及MC-LR具有有效的干预和治疗作用。其重要机制是通过下调TGF-β的表达,阻抑TGF-β/Smad信号通路，抑制肌成纤维细胞分化和胶原蛋白的合成，最终缓解和阻滞细胞外基质(ECM)的异常沉积。
因此，本发明的微囊藻毒素在开发、制备预防和治疗器官组织纤维化疾病的药物中有新的重要用途。
参考文献
1.Wallace K,Burt AD,Wright MC.Liver fibrosis.Biochem J,2008,411(1):1.
2.LeBleu VS,Taduri G,O'Connell J,Teng Y,Cooke VG,Woda C,Sugimoto H,Kalluri R.Origin and function of myofibroblasts in kidney fibrosis.Nat Med,2013,19(8):1047-53.
3.Meng XM,Tang PM,Li J,Lan HY.TGF-β/Smad signaling in renal fibrosis.Front Physiol,2015,6:82.
4.Hutchinson J,Fogarty A,Hubbard R,McKeever T.Global incidence and mortality of idiopathic pulmonary firosis:a systematic review.Eur Respir,.2015, 46(3):795.
5.Baumgartner KB,Samet JM,Coultas DB,Stidley CA,Hunt WC,Colby TV,Waldron JA.Occupational and environmental risk factors for idiopathic pulmonary fibrosis:a multicenter case-control study.Am J Epidemiol,2000,152(4):307.
6.King TJ,Pardo A,Selman M.Idiopathic pulmonary fibrosis.Lancet,2011,378(9807):1949.
7.Tager AM,LaCamera P.Shea BS,Campanella GS,Selman M,Zhao Z,Polosukhin V,Wain J,Karimi-Shah B A,Kim ND,Hart WK,Pardo A,Blackwell TS,Xu Y,Chun J,Luster AD.The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak.Nat Med,2008,14:45.
8.Zhang H,Liu XF,Chen S,Wu JH,Ye X,Xu LZ,Chen HM,Zhang DP,Tan RX,Wang YP,Tectorigenin inhibits the in vitro proliferation and enhances miR-338*expression of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis.J Ethnopharmacol,2010,131(1):165.
9.van Apeldoorn ME 1,van Egmond HP,Speijers GJ,Bakker GJ.Toxins of cyanobacteria.Mol Nutr Food Res,2007,51(1):7-60.
10.Wolf HU,Frank C.Toxicity assessment of cyanobacterial toxin mixtures,Environ.Toxicol,2002,17,395–399.
11.Allison SJ.Fibrosis:the source of myofibroblasts in kidney fibrosis.Nat.Rev.Nephrol,2013,9:494.
12.Kalluri R,Neilson EG.Epithelial-mesenchymal transition and its implications for fibrosis.J Clin Invest,2003,112:1776.
13.Lan HY.Diverse roles of TGF-β/Smads in renal fibrosis and inflammation.Int J Biol Sci,2011,7(7):1056.
14.Yan X,Liu Z,Chen Y.Regulation of TGF-beta signaling by Smad7.Acta Biochim Biophys Sin(Shanghai),2009,41(4):263.
15.Luo Y,Xu W,Chen H,Warburton D,Dong R,Qian B,Selman M,Gauldie J,Kolb M,Shi W.A novel profibrotic mechanism mediated by TGFβ-stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells.J Pathol,2015,236(3):384.
16.Wernig G,Chen SY,Cui L,Van Neste C,Tsai JM,Kambham N,Vogel H, Natkunam Y,Gilliland DG,Nolan G,Weissman IL.Unifying mechanism for different fibrotic diseases.Proc Natl Acad Sci USA,2017,114(18):4757.
17.Anguiano L,Riera M,Pascual J,Soler MJ.Endothelin Blockade in Diabetic Kidney Disease.J Clin Med,2015,4(6):1171.

Claims (14)

  1. 微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途。
  2. 权利要求1的用途，其中所述微囊藻毒素是含单环七肽结构，其第二和第四位氨基酸分别为亮氨酸和精氨酸的微囊藻毒素-LR。
  3. 权利要求1的用途，其中所述微囊藻毒素是含单环七肽结构，其第二和第四位氨基酸均为精氨酸的微囊藻毒素-RR。
  4. 权利要求1的用途，其中所述器官是肺。
  5. 权利要求1的用途，其中所述药物阻抑肌成纤维细胞形成。
  6. 权利要求1的用途，其中所述药物降低α-SMA水平。
  7. 权利要求1的用途，其中所述药物抑制胶原蛋白Ⅰ表达。
  8. 权利要求1的用途，其中所述药物抑制TGF-β的mRNA和蛋白质表达。
  9. 权利要求1的用途，其中所述药物抑制Smad2和Smad3表达。
  10. 权利要求1的用途，其中所述药物上调Smad7的表达。
  11. 权利要求1的用途，其中所述药物抑制P4HA3的表达。
  12. 一种抑制肌成纤维细胞分化和胶原蛋白合成的方法，该方法通过使用一种或多种微囊藻毒素阻抑TGF-β/Smad信号通路而实现。
  13. 权利要求12的方法，其中所述微囊藻毒素是含单环七肽结构，其第二和第四位氨基酸分别为亮氨酸和精氨酸的微囊藻毒素-LR。
  14. 权利要求12的方法，其中所述微囊藻毒素是含单环七肽结构，其第二和第四 位氨基酸均为精氨酸的微囊藻毒素-RR。
PCT/CN2017/099249 2016-11-30 2017-08-28 微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途 Ceased WO2018099148A1 (zh)

Priority Applications (2)

Application Number Priority Date Filing Date Title
US16/343,750 US20190275103A1 (en) 2016-11-30 2017-08-28 Use of Microcystin in Preparation of Drug for Preventing or Treating Organ and Tissue Fibrosis Diseases
EP17876506.1A EP3549596B1 (en) 2016-11-30 2017-08-28 Use of microcystins in preparation of drugs for preventing or treating organ and tissue fibrosis diseases

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
CN201611079521 2016-11-30
CN201611079521.6 2016-11-30
CN201710014483.4A CN106620647B (zh) 2016-11-30 2017-01-09 微囊藻毒素-lr在制备预防和治疗肺纤维化的药物中的应用
CN201710014483.4 2017-01-09

Publications (1)

Publication Number Publication Date
WO2018099148A1 true WO2018099148A1 (zh) 2018-06-07

Family

ID=58842853

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/CN2017/099249 Ceased WO2018099148A1 (zh) 2016-11-30 2017-08-28 微囊藻毒素在制备用于预防或治疗器官组织纤维化疾病的药物中的用途

Country Status (4)

Country Link
US (1) US20190275103A1 (zh)
EP (1) EP3549596B1 (zh)
CN (1) CN106620647B (zh)
WO (1) WO2018099148A1 (zh)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112138142A (zh) * 2020-10-09 2020-12-29 南京大学 微囊藻毒素-rr在用于制备预防或治疗肾纤维化疾病的药物中的应用
CN114470153A (zh) * 2021-05-24 2022-05-13 南京大学 微囊藻毒素-rr在制备预防或治疗肺组织纤维化疾病的药物中的用途

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106620647B (zh) * 2016-11-30 2018-12-28 南京大学 微囊藻毒素-lr在制备预防和治疗肺纤维化的药物中的应用

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102524180A (zh) * 2011-12-07 2012-07-04 安徽大学 一种微囊藻毒素mc-lr促二乙基亚硝胺den诱发大鼠肝癌模型建立的方法
US20140271923A1 (en) * 2013-03-14 2014-09-18 Christopher Brian Reid Compositions & formulations for preventing and treating chronic diseases that cluster in patients such as cardiovascular disease, diabetes, obesity, polycystic ovary syndrome, hyperlipidemia and hypertension, as well as for preventing and treating other diseases and conditions
CN106620647A (zh) * 2016-11-30 2017-05-10 南京大学 微囊藻毒素‑lr在制备预防和治疗肺纤维化的药物中的应用

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2009059425A1 (en) * 2007-11-07 2009-05-14 The University Of British Columbia Stat3 inhibitors for the treatment of fibrosis
CN104095839A (zh) * 2013-04-05 2014-10-15 滨州医学院 迷迭香酸在制备治疗或预防肺纤维化的药物中的应用
CN104490889B (zh) * 2014-12-30 2016-04-20 中国人民解放军第四军医大学 一种 ddr2 小分子抑制剂抗肺纤维化应用

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102524180A (zh) * 2011-12-07 2012-07-04 安徽大学 一种微囊藻毒素mc-lr促二乙基亚硝胺den诱发大鼠肝癌模型建立的方法
US20140271923A1 (en) * 2013-03-14 2014-09-18 Christopher Brian Reid Compositions & formulations for preventing and treating chronic diseases that cluster in patients such as cardiovascular disease, diabetes, obesity, polycystic ovary syndrome, hyperlipidemia and hypertension, as well as for preventing and treating other diseases and conditions
CN106620647A (zh) * 2016-11-30 2017-05-10 南京大学 微囊藻毒素‑lr在制备预防和治疗肺纤维化的药物中的应用

Non-Patent Citations (21)

* Cited by examiner, † Cited by third party
Title
ALLISON SJ: "Fibrosis: the source of myofibroblasts in kidney fibrosis", NAT. REV. NEPHROL, vol. 9, 2013, pages 494
ANGUIANO LRIERA MPASCUAL JSOLER MJ: "Endothelin Blockade in Diabetic Kidney Disease", J CLIN MED, vol. 4, no. 6, 2015, pages 1171
BAUMGARTNER KBSAMET JMCOULTAS DBSTIDLEY CAHUNT WCCOLBY TVWALDRON JA: "Occupational and environmental risk factors for idiopathic pulmonary firosis: a multicenter case-control study", AM J EPIDEMIOL, vol. 152, no. 4, 2000, pages 307
DING, XIAOSHENG: "Research Progress on Microcystin and Its Kidney Toxicity", JOURNAL OF ANHUI AGRICULTURAL SCIENCES, vol. 36, no. 31, 31 December 2008 (2008-12-31), pages 13506 - 13507, XP9515681 *
GUZMAN, R.E. ET AL.: "Hepatic Oxidative Stress Following Prolonged Sublethal Microcystin LR Exposure", TOXICOLOGIC PATHOLOGY, vol. 27, no. 5, 31 December 1999 (1999-12-31), pages 582 - 588, XP055490319, Retrieved from the Internet <URL:DOI:10.1177/019262339902700512> *
HUTCHINSON JFOGARTY AHUBBARD RMCKEEVER T.: "Global incidence and mortality of idiopathic pulmonary firosis: a systematic review", EUR RESPIR, vol. 46, no. 3, 2015, pages 795
KALLURI RNEILSON EG: "Epithelial-mesenchymal transition and its implications for fibrosis", J CLIN INVEST, vol. 112, 2003, pages 1776, XP002988689, DOI: doi:10.1172/JCI200320530
KING TJPARDO ASELMAN M: "Idiopathic pulmonary fibrosis", LANCET, vol. 378, no. 9807, 2011, pages 1949, XP055227094, DOI: doi:10.1016/S0140-6736(11)60052-4
LAN HY: "Diverse roles of TGF-p/Smads in renal fibrosis and inflammation", INT J BIOL SCI, vol. 7, no. 7, 2011, pages 1056
LEBLEU VSTADURI GO'CONNELL JTENG YCOOKE VGWODA CSUGIMOTO HKALLURI R: "Origin and function of myofibroblasts in kidney fibrosis", NAT MED, vol. 19, no. 8, 2013, pages 1047 - 53
LI, XIAOYU ET AL.: "Research Progress on Relationship between Microcystin and Human Health", CHINESE JOURNAL OF PUBLIC HEALTH, vol. 24, no. 08, 31 August 2008 (2008-08-31), pages 1016 - 1017, XP009515845, ISSN: 1001-0580 *
LUO YXU WCHEN HWARBURTON DDONG RQIAN BSELMAN MGAULDIE JKOLB MSHI W: "A novel profibrotic mechanism mediated by TGF-P -stimulated collagen prolyl hydroxylase expression in fibrotic lung mesenchymal cells", J PATHOL, vol. 236, no. 3, 2015, pages 384
MENG XMTANG PMLI JLAN HY: "TGF-p/Smad signaling in renal fibrosis", FRONT PHYSIOL, vol. 6, 2015, pages 82
See also references of EP3549596A4
TAGER AMLACAMERA PSHEA BSCAMPANELLA GSSELMAN MZHAO ZPOLOSUKHIN VWAIN JKARIMI-SHAH B AKIM ND: "The lysophosphatidic acid receptor LPA1 links pulmonary fibrosis to lung injury by mediating fibroblast recruitment and vascular leak", NAT MED, vol. 14, 2008, pages 45
VAN APELDOOM MEVAN EGMOND HPSPEIJERS GJBAKKER GJ: "Toxins of cyanobacteria", MOL NUTR FOOD RES, vol. 51, no. 1, 2007, pages 7 - 60
WALLACE KBURT ADWRIGHT MC: "Liver fibrosis", BIOCHEM J, vol. 411, no. 1, 2008, pages 1
WEMIG GCHEN SYCUI LVAN NESTE CTSAI JMKAMBHAM NVOGEL HNATKUNAM YGILLILAND DGNOLAN G: "Unifying mechanism for different fibrotic diseases", PROC NATL ACAD SCI USA, vol. 114, no. 18, 2017, pages 4757, XP055459914, DOI: doi:10.1073/pnas.1621375114
WOLF HUFRANK C: "Toxicity assessment of cyanobacterial toxin mixtures", ENVIRON TOXICOL, vol. 17, 2002, pages 395 - 399
YAN XLIU ZCHEN Y: "Regulation of TGF-beta signaling by Smad7", ACTA BIOCHIM BIOPHYS SIN (SHANGHAI, vol. 41, no. 4, 2009, pages 263
ZHANG HLIU XFCHEN SWU JHYE XXU LZCHEN HMZHANG DPTAN RXWANG YP: "Tectorigenin inhibits the in vitro proliferation and enhances miR-338* expression of pulmonary fibroblasts in rats with idiopathic pulmonary fibrosis", J ETHNOPHARMACOL, vol. 131, no. 1, 2010, pages 165

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN112138142A (zh) * 2020-10-09 2020-12-29 南京大学 微囊藻毒素-rr在用于制备预防或治疗肾纤维化疾病的药物中的应用
CN112138142B (zh) * 2020-10-09 2021-08-24 南京大学 微囊藻毒素-rr在用于制备预防或治疗肾纤维化疾病的药物中的应用
WO2022073416A1 (zh) * 2020-10-09 2022-04-14 南京大学 微囊藻毒素-rr在用于制备预防或治疗肾纤维化疾病的药物中的应用
CN114470153A (zh) * 2021-05-24 2022-05-13 南京大学 微囊藻毒素-rr在制备预防或治疗肺组织纤维化疾病的药物中的用途

Also Published As

Publication number Publication date
EP3549596A1 (en) 2019-10-09
CN106620647A (zh) 2017-05-10
EP3549596A4 (en) 2020-05-20
CN106620647B (zh) 2018-12-28
US20190275103A1 (en) 2019-09-12
EP3549596B1 (en) 2022-10-05

Similar Documents

Publication Publication Date Title
Liang et al. Lycorine ameliorates bleomycin-induced pulmonary fibrosis via inhibiting NLRP3 inflammasome activation and pyroptosis
Li et al. NLRP3 inflammasome inhibition attenuates cisplatin-induced renal fibrosis by decreasing oxidative stress and inflammation
Qiu et al. Aucubin protects against lipopolysaccharide-induced acute pulmonary injury through regulating Nrf2 and AMPK pathways
Ma et al. Association of Stat3 with HSF1 plays a critical role in G-CSF-induced cardio-protection against ischemia/reperfusion injury
Liu et al. Capsaicin ameliorates renal fibrosis by inhibiting TGF-β1–Smad2/3 signaling
Lei et al. Salidroside protects pulmonary artery endothelial cells against hypoxia-induced apoptosis via the AhR/NF-κB and Nrf2/HO-1 pathways
Liu et al. Effective-components combination improves airway remodeling in COPD rats by suppressing M2 macrophage polarization via the inhibition of mTORC2 activity
Lee et al. The selective A3AR antagonist LJ-1888 ameliorates UUO-induced tubulointerstitial fibrosis
Mao et al. Ginsenoside Rg1 ameliorates psoriasis‐like skin lesions by suppressing proliferation and NLRP3 inflammasomes in keratinocytes
Ye et al. YAP1 preserves tubular mitochondrial quality control to mitigate diabetic kidney disease
Jiang et al. Coenzyme Q10 attenuates renal fibrosis by inhibiting RIP1-RIP3-MLKL-mediated necroinflammation via Wnt3α/β-catenin/GSK-3β signaling in unilateral ureteral obstruction
Abdelrahman et al. The renoprotective effect of the dipeptidyl peptidase-4 inhibitor sitagliptin on adenine-induced kidney disease in rats
Lin et al. N-acetylcysteine (NAC) attenuating apoptosis and autophagy in RAW264. 7 cells in response to incubation with mycolic acid from bovine mycobacterium tuberculosis complex
Cheng et al. LGALS3 regulates endothelial-to-mesenchymal transition via PI3K/AKT signaling pathway in silica-induced pulmonary fibrosis
Ma et al. Neutralization of interleukin-11 attenuates silica particles-induced pulmonary inflammation and fibrosis in vivo
Yan et al. USP7 promotes cardiometabolic disorders and mitochondrial homeostasis dysfunction in diabetic mice via stabilizing PGC1β
Yang et al. Activation of the α7nAChR by GTS-21 mitigates septic tubular cell injury and modulates macrophage infiltration
EP3549596B1 (en) Use of microcystins in preparation of drugs for preventing or treating organ and tissue fibrosis diseases
Cheng et al. Nobiletin alleviates macrophage M2 polarization by activating AMPK-mTOR-mediated autophagy in pulmonary fibrosis mice
Chen et al. Alpinetin ameliorates bleomycin-induced pulmonary fibrosis by repressing fibroblast differentiation and proliferation
Chen et al. 23-O-acetylshengmanol-3-O-α-L-arabinoside alleviates lipopolysaccharide-induced acute lung injury through inhibiting IκB/NF-κB and MAPK/AP-1 signaling pathways
CN111358789A (zh) Nsc228155在制备防治慢性肾纤维化药物中的用途
Huang et al. A novel caffeic acid derivative prevents renal remodeling after ischemia/reperfusion injury
Wu et al. Specneuzhenide improves bleomycin-induced pulmonary fibrosis in mice via AMPK-dependent reduction of PD-L1
Xiao et al. Mycobacterium vaccae attenuates airway inflammation by inhibiting autophagy and activating PI3K/Akt signaling pathway in OVA-induced allergic airway inflammation mouse model

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17876506

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

ENP Entry into the national phase

Ref document number: 2017876506

Country of ref document: EP

Effective date: 20190701