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基于蛋白质组学研究七味清肝散的抗肝纤维化作用及机制
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篇名: 基于蛋白质组学研究七味清肝散的抗肝纤维化作用及机制
TITLE: Effects and Mechanism of Qiwei Qinggan Powder on Hepatic Fibrosis Based on Proteomics
摘要: 目的:探讨七味清肝散的抗肝纤维化(HF)作用,并挖掘其潜在机制。方法:将雄性Wistar大鼠随机分为空白组、HF模型组和七味清肝散低、中、高剂量组[135、270、405mg/(kg·d),以生药总量计],每组12只。除空白组外,其余各组均灌胃50%四氯化碳花生油溶液(2mL/kg,每周2次,连续8周)以复制HF模型;与此同时,空白组和HF模型组大鼠均灌胃等容0.5%羧甲基纤维素钠溶液,各给药组大鼠均灌胃相应药物,每日1次,连续8周。观察各组大鼠的一般情况,于末次给药后观察其肝脏形态,并测定肝脏指数;检测大鼠血清肝功能指标[丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)、碱性磷酸酶(ALP)、羟脯氨酸(HYP)]含量以及肝组织中α-平滑肌肌动蛋白(α-SMA)的表达情况,并采用苏木精-伊红染色和Masson染色进行组织病理学观察。采用串联质谱标签(TMT)技术,以蛋白表达量的差异倍数为指标,筛选药物组(合并七味清肝散各剂量组肝组织样品)和HF模型组的差异表达蛋白,并借助Uniprot-GOA数据库以及KAAS、KEGGmapper在线工具等对其进行基因本体(GO)和KEGG通路富集分析。结果:空白组大鼠健康状况好;肝脏呈鲜红色且表面光滑,肝小叶完整,未见变性坏死、炎症细胞浸润或纤维组织增生。与空白组比较,HF模型组大鼠饮食减少、精神萎靡、皮毛杂乱且无光泽;肝脏呈暗红色或黄色,表面粗糙,质地较硬,可见炎症细胞浸润、纤维组织破坏、桥间连接等现象;其肝脏系数、肝功能指标含量和α-SMA的表达水平均显著升高(P<0.05)。与HF模型组比较,七味清肝散各剂量组大鼠上述症状均有不同程度的改善,且低剂量组肝脏系数、高剂量组ALP含量以及各剂量组ALT、AST、HYP含量和α-SMA的表达水平均显著降低(P<0.05)。共筛选出与HF有关的差异表达蛋白42个,其中表达上调15个、表达下调27个,包括脂肪酸结合蛋白4(FABP4)、胆固醇7α-羟化酶(CYP7A1)等。富集分析结果显示,差异表达蛋白主要富集于细胞外空间、血液微粒等细胞部位,涉及氧化还原酶活性、脂肪酸结合等分子功能,异型细胞间黏附的调控、蛋白质活化级联等生物过程以及视黄醇代谢、花生四烯酸代谢、过氧化物酶体增殖物激活受体(PPAR)等信号通路。结论:七味清肝散可降低HF模型大鼠的肝脏系数以及血清中ALT、AST、ALP、HYP含量,下调α-SMA的表达,改善肝组织的炎症及纤维化程度,具有一定的肝保护作用。该方潜在抗HF作用机制可能涉及FABP4、CYP7A1、PPAR等多个靶点和信号通路。
ABSTRACT: OBJECTIVE:To investigate the anti- hepatic fibrosis (HF)effects of Qiwei qinggan powder and explore its possible mechanism. METHODS :Male Wistar rats were randomly divided into blank group ,HF model group ,Qiwei qinggan powder low-dose,medium-dose and high-dose groups [ 135,270,405 mg/(kg·d),by total amount of crude drugs] ,with 12 rats in each group. Except for blank group ,other groups were given 50% CCl4-peanut oil solution intragastrically (2 mL/kg,twice a week ,for consecutive 8 weeks) to induce HF model. At same time , blank group and model group were given constant volume of 0.5% CMC-Na solution intragastrically ;administration groups were given relevant medicine intragastrically ,once a day ,for consecutive 8 weeks. General situation of rats were observedand liver morphology was observed after last administration and hepatic indexes were detected. The contents of liverfunction indexes (ALT,AST,ALP,HYP)in serum and the expression of α-SMA in hepatic tissue were determined , and HE and Masson staining were performed to observe the histopathology. Using the difference multiple of expression quantity as the index ,TMT technology was used to screen the differentially expressed protein in medicine group (combining the liver tissue samples of Qiwei qinggan powder groups )and HF model group. Uniprot-GOA database and KAAS ,KEGG mapper online tools were used to analyze GO and KEGG pathway enrichment. RESULTS :The rats in the blank group were in good health ;the liver was bright red and smooth ,the liver lobules were intact ,no degeneration and necrosis ,inflammatory cell infiltration or fibrous tissue proliferation was found. Compared with blank group ,the rats in HF model group had poor diet ,depressed spirit ,disordered and lusterless fur ;the liver was dark red or yellow with rough surface ,hard texture ,inflammatory cell infiltration ,fiber tissue destruction ,bridge connection and so on ;the hepatic index ,the contents of liver function indexes and the expression of α-SMA were increased significantly (P<0.05). Compared with HF model group ,above symptoms of rats were improved to different extent in different dose groups of Qiwei qinggan powder ;hepatic index in Qiwei qinggan powder low-dose group ,the content of ALP in high-dose group ,the contents of ALT,AST and HYP and the expression of α-SMA in different dose groups were decreased significantly (P<0.05). A total of 42 differentially expressed proteins related to HF were screened ,of which 15 were up-regulated and 27 were down-regulated in expression,including fatty acid binding protein 4(FABP4),cholesterol 7α-hydroxylase(CYP7A1). The results of enrichment analysis showed that the differentially expressed proteins were mainly enriched in extracellular space ,blood particles and other cell parts,involving the molecular functions of oxidoreductase activity and fatty acid binding ,the biological processes of the regulation of heterotypic cell adhesion ,protein activation cascade ,as well as retinol metabolism ,arachidonic acid metabolism ,PPAR and other signal pathway. CONCLUSIONS :Qiwei qinggan powder can reduce the hepatic index ,ALT,AST,ALP and HYP contents in serum ,down-regulate the expression of α-SMA,improve the degree of inflammation and fibrosis of liver tissue ,and have a certain protective effect on rats. The anti-HF mechanism of it involves multiple targets and signal pathways ,such as FABP 4, CYP7A1 and PPAR.
期刊: 2020年第31卷第11期
编辑: 梁洁,孟根斯立木,颜羽昕,金蓉,包小妹,马丽杰,那日苏,苏晓丽,谢敏琦,马月宏
AUTHORS: LIANG Jie, Menggensilimu,YAN Yuxin,JIN Rong,BAO Xiaomei,MA Lijie,Narisu,SU Xiaoli,XIE Minqi,MA Yuehong
关键字: 肝纤维化;七味清肝散;蛋白质组学;串联质谱标签技术;差异表达蛋白;大鼠
KEYWORDS: Hepatic fibrosis ;Qiwei qinggan powder ;Proteomics;TMT technology ;Differentially expressed proteins ;Rats
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