中国中药杂志

2021, v.46(09) 2260-2266

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竹节参总皂苷通过调控自噬改善非酒精性脂肪性肝炎的实验研究
Effect of total saponins from Panax japonicus on non-alcoholic steatohepatitis by regulating autophagy

罗悦;刘朝奇;贺海波;王婷;何毓敏;张长城;袁丁;袁成福;
LUO Yue;LIU Chao-qi;HE Hai-bo;WANG Ting;HE Yu-min;ZHANG Chang-cheng;YUAN Ding;YUAN Cheng-fu;Hubei Key Laboratory of Tumor Microenvironment and Immunotheraphy, China Three Gorges University;College of Biology and Pharmaceuticals, China Three Gorges University;Medical College, China Three Gorges University;

摘要(Abstract):

应用高糖高脂饮食诱导小鼠非酒精性脂肪性肝炎(non-alcoholic steatohepatitis, NASH),研究竹节参总皂苷(total sa-ponins of Panax japonicus)的干预作用,并探寻其可能作用机制。使用高糖高脂饲料喂养小鼠建立NASH模型,给予不同剂量竹节参总皂苷(15,45 mg·kg~(-1))进行干预,共饲养26周。HE染色法观察肝脏组织形态及病理变化。qRT-PCR法测定小鼠肝脏中miR-199a-5p、自噬相关基因5(autophagy related gene 5,ATG5)及炎症因子白细胞介素6 (interleukin-6,IL-6)、白细胞介素1β(interleukin-1β,IL-1β)、肿瘤坏死因子α(tumor necrosis factor α,TNF-α)的转录表达水平。Western blot法检测小鼠肝脏中自噬相关蛋白ATG5,P62/SQSTM1(P62),微管相关蛋白1轻链3b(microtubule-associated protein light chain 3,LC3)-I和Ⅱ蛋白的表达情况。免疫荧光染色法检测P62蛋白的表达情况。为验证miR-199a-5p与ATG5的靶向调节关系,采用miR mimic/inhibitor NC,miR-199a-5p mimic/inhibitor转染Hepa 1-6细胞,检测ATG5 mRNA及蛋白的表达;构建pMIR-reportor ATG5-3′UTR荧光素酶报告基因质粒,将该质粒与miR mimic/inhibitor NC,miR-199a-5p mimic/inhibitor共同转染Hepa 1-6细胞,检测荧光素酶活性。体内实验中模型组HE染色表现典型脂肪样变及炎性浸润,miR-199a-5p表达上升而ATG5 mRNA及蛋白表达均下降。自噬相关蛋白P62表达水平明显上升、LC3Ⅱ/Ⅰ比值下降、炎症因子转录表达水平显著上升。竹节参总皂苷干预后,肝组织病理学表现显著改善,miR-199a-5p表达下降、ATG5 mRNA及蛋白表达升高。自噬相关蛋白P62表达水平显著下降、LC3Ⅱ/Ⅰ比值上升、炎症因子(IL-6,IL-1β,TNF-α)转录表达水平显著下降。体外实验发现,miR-199a-5p过表达细胞中ATG5 mRNA及蛋白表达水平显著下降,荧光素酶活性显著降低;而抑制miR-199a-5p表达后ATG5 mRNA及蛋白表达水平上升,荧光素酶活性增强。研究结果表明,竹节参总皂苷可改善高糖高脂饮食小鼠NASH,其作用机制可能与调节miR-199a-5p/ATG5信号通路,调节自噬水平,改善NASH的炎症反应有关。
Non-alcoholic steatohepatitis(NASH) was induced by high-sugar and high-fat diet in mice to investigate the intervention effect of total saponins from Panax japonicus(TSPJ) and explore its possible mechanism. Mice were fed with high-sugar and high-fat diet to establish NASH model, and intervened with different doses of TSPJ(15, 45 mg·kg~(-1)). The animals were fed for 26 weeks. The histomorphology and pathological changes of liver tissues were observed by HE staining. The transcriptional expression levels of miR-199 a-5 p, autophagy related gene 5(ATG5) and inflammatory cytokines interleukin-6(IL-6), interleukin-1β(IL-1β) and tumor necrosis factor α(TNF-α) in mouse liver were measured by quantitative Real-time polymerase chain reaction(qRT-PCR). Western blot was used to detect the expression of autophagy-related proteins ATG5, P62/SQSTM1(P62), and microtubule-associated protein light chain 3(LC3)-I/Ⅱ proteins in mouse liver. The expression of P62 protein was detected by immunofluorescence staining. In order to verify the targeting regulation relationship between miR-199 a-5 p and ATG5, miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor were transfected into Hepa 1-6 cells, and the expression of ATG5 mRNA and protein was detected. pMIR-reportor ATG5-3′UTR luciferase reporter gene plasmid was constructed and co-transfected with miR mimic/inhibitor NC and miR-199 a-5 p mimic/inhibitor into Hepa 1-6 cells to detect luciferase activity. In vivo, HE staining in the model group showed typical fatty degeneration and inflammatory infiltration, with increased expression of miR-199 a-5 p and decreased expression of ATG5 mRNA and protein. The expression of autophagy-associated protein P62 increased significantly, the ratio of LC3Ⅱ/Ⅰ decreased, and the transcriptional expression of inflammatory factors increased significantly. After the intervention by TSPJ, the pathological performance of liver tissue was significantly improved, the expression of miR-199 a-5 p decreased and the expression of ATG5 mRNA and protein increased, the expression of autophagy-associated protein P62 decreased significantly, the ratio of LC3Ⅱ/Ⅰ increased, and the transcriptional expression of inflammatory cytokines IL-6, IL-1β and TNF-α decreased significantly. In vitro, it was found that the expression of ATG5 mRNA and protein and luciferase activity decreased significantly in miR-199 a-5 p overexpression cells, while after inhibition of miR-199 a-5 p expression, the expression level of ATG5 mRNA and protein and luciferase activity increased. The results showed that TSPJ can improve NASH in mice fed with high-sugar and high-fat diet, and its mechanism may be related to the regulation of miR-199 a-5 p/ATG5 signal pathway, the regulation of autophagy activity and the improvement of inflammatory response of NASH.

关键词(KeyWords): 非酒精性脂肪性肝炎;竹节参总皂苷;miR-199a-5p/ATG5;自噬
non-alcoholic steatohepatitis;total saponins of Panax japonicus;miR-199a-5p/ATG5;autophagy

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金项目(81673675,81773959,81974528)

作者(Author): 罗悦;刘朝奇;贺海波;王婷;何毓敏;张长城;袁丁;袁成福;
LUO Yue;LIU Chao-qi;HE Hai-bo;WANG Ting;HE Yu-min;ZHANG Chang-cheng;YUAN Ding;YUAN Cheng-fu;Hubei Key Laboratory of Tumor Microenvironment and Immunotheraphy, China Three Gorges University;College of Biology and Pharmaceuticals, China Three Gorges University;Medical College, China Three Gorges University;

Email:

DOI: 10.19540/j.cnki.cjcmm.20210201.401

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