基于凝血级联途径研究血栓通注射液对博来霉素诱导肺纤维化大鼠的作用机制  

Mechanism of Xueshuantong Injection on bleomycin-induced pulmonary fibrosis in rats based on coagulation cascade pathway

作　　者：高云航 宋玲 陈腾飞 郑志远 杨依霏[1] 严灿梅 张广平[1] 李晗 GAO Yun-hang;SONG Ling;CHEN Teng-fei;ZHENG Zhi-yuan;YANG Yi-fei;YAN Can-mei;ZHANG Guang-ping;LI Han(Institute of Chinese Materia Medica,China Academy of Chinese Medical Sciences,Beijing 100700,China;Guangxi Wuzhou Pharmaceutical(Group)Co.,Ltd.,Wuzhou 543000,China;Guangxi Key Laboratory of Notoginseng Comprehensive Uilization Technology,Wuzhou 543000,China)

机构地区：[1]中国中医科学院中药研究所,北京100700 [2]广西梧州制药(集团)股份有限公司,广西梧州543000 [3]广西三七综合利用技术重点实验室,广西梧州543000

出　　处：《中国中药杂志》2024年第16期4313-4320,共8页China Journal of Chinese Materia Medica

基　　金：国家自然科学基金青年基金项目(82204740);中国中医科学院科技创新工程重大攻关项目(CI2021A04615);中国中医科学院优秀青年科技人才培养专项(ZZ16-YQ-027);中国中医科学院科技创新工程项目(CI2023E001TS12);梧州市科技计划项目(202202009)。

摘　　要：基于凝血级联途径探讨血栓通注射液(Xueshuantong Injection,XST)对博来霉素(bleomycin,BLM)所致肺纤维化大鼠的作用机制。将60只SD大鼠随机分为假手术组、模型组、吡非尼酮组(pirfenidone,PFD,50 mg·kg^(-1))组和27、54、81 mg·kg^(-1)XST组,采用气管内注入博来霉素(5 mg·kg^(-1))建立肺纤维化大鼠模型。24 h后,给药组分别给予相应的药物,假手术组、模型组给予等体积的生理盐水。第28天取材,观察大鼠肺部影像学与胶原纤维变化,并采用免疫荧光法检测大鼠肺组织α-平滑肌肌动蛋白(alpha-smooth muscle actin,α-SMA)、Ⅰ型胶原(collagenⅠ,Col-Ⅰ)、波形蛋白(vimentin,Vim)与E-钙黏蛋白(E-cadherin,E-cad)的表达,Western blot法检测肺组织α-SMA、Col-Ⅰ、Vim和E-cad的蛋白表达水平,采用酶联免疫吸附测定(enzyme-linked immunosorbent assay,ELISA)检测大鼠肺组织中凝血酶原片段(prothrombin fragment 1+2,F1+2)、凝血酶-抗凝血酶复合物(thrombin-antithrombin complex,TAT)、可溶性纤维蛋白单体复合物(soluble fibrin monomer complex,SFMC)和大鼠血纤蛋白原降解产物(fibrinogen degradation product,FDP)含量,最后通过实时荧光定量PCR(RT-qPCR)、Western blot与免疫荧光法测定肺组织中蛋白酶激活受体-1(protease-activated receptors-1,PAR-1)mRNA水平与蛋白水平。与模型组相比,给予XST后肺部扫描也显示出斑片状、不均匀的阴影,但这些阴影的密度低于模型组;同时大鼠肺部Ⅰ型胶原纤维显著减少;并且XST抑制上皮-间充质转化并下调α-SMA、Col-Ⅰ蛋白表达。在凝血系统方面,给予81 mg·kg^(-1)XST可显著降低SFMC与FDP的含量;同时81 mg·kg^(-1)XST可显著下调PAR-1 mRNA水平与蛋白水平。XST具有抗大鼠肺纤维化的作用,其机制可能与下调PAR-1从而改善凝血途径的异常有关。This study aims to investigate the mechanism of Xueshuantong Injection(XST)on pulmonary fibrosis induced by bleomycin(BLM)in rats based on the coagulation cascade pathway.Sixty SD rats were randomly divided into sham surgery group,model group,pirfenidone(PFD,50 mg·kg^(-1))group,and 27,54,and 81 mg·kg^(-1)XST groups.The rat model of pulmonary fibrosis was established by intratracheal injection of BLM(5 mg·kg^(-1)).After 24 hours,the administration groups were given corresponding drugs,while the sham surgery group and model group were given equal volumes of saline.On the 28th day,samples were collected,and the imaging and collagen fiber changes in the lungs of rats were observed.Immunofluorescence(IF)method was used to detect the expression level of alpha-smooth muscle actin(α-SMA),collagenⅠ(Col-Ⅰ),E-cadherin(E-cad),and vimentin(Vim).Western blot was used to determine the protein expression ofα-SMA,Col-Ⅰ,Vim,and E-cad.Enzyme-linked immunosorbent assay(ELISA)was used to detect the levels of prothrombin fragment(F1+2),thrombin-antithrombin complex(TAT),soluble fibrin monomer complex(SFMC),and rat fibrinogen degradation products(FDP)in rat lung tissue.Finally,the mRNA and protein levels of protease activated receptor 1(PAR-1)were detected by RT-qPCR,western blot,and IF.Compared with the model group,the scanning of the lungs of rats receiving XST treatment also exhibited patchy and non-homogeneous shadows,but these shadows were less dense than those in the model group.At the same time,there was a significant decrease in Col-Ⅰfibers in the lungs of rats,and XST could inhibit epithelial-mesenchymal transition(EMT)and downregulateα-SMA and Col-Ⅰprotein expression.In the aspect of the coagulation system,administration of 81 mg·kg^(-1)XST significantly reduced the levels of SFMC and FDP.Meanwhile,81 mg·kg^(-1)XST significantly downregulated the mRNA and protein levels of PAR-1.XST has an anti-pulmonary fibrosis effect in rats,and its mechanism may be related to the downregulation of PAR-1 to rebalance the

关 键 词：肺纤维化 血栓通注射液 凝血级联 博来霉素 蛋白酶激活受体-1 

分 类 号：R285.5[医药卫生—中药学]