虫草菌丝调节AMPK/SirT1信号通路对四氯化碳诱导肝纤维化小鼠的作用  

Effects of cultured mycelium Cordyceps sinensis on carbon tetrachloride-induced liver fibrosis mice by regulating AMPK/SirT1 signaling pathway

作　　者：杨钊 闫阮玉 吴洪雨 黄恺 沈丽 陶艳艳 刘成海 彭渊[1] YANG Zhao;YAN Ruanyu;WU Hongyu;HUANG Kai;SHEN Li;TAO Yanyan;LIU Chenghai;PENG Yuan(Institute of Liver Diseases,Shuguang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine,Shanghai 201203,China;Shanghai Key Laboratory of Traditional Chinese Clinical Medicine,Shanghai 201203,China;Key Laboratory of Liver and Kidney Diseases,Shanghai University of Traditional Chinese Medicine,Ministry of Education,Shanghai 201203,China)

机构地区：[1]上海中医药大学附属曙光医院肝病研究所,上海201203 [2]上海市中医临床重点实验室,上海201203 [3]肝肾疾病病证教育部重点实验室,上海201203

出　　处：《中国实验动物学报》2024年第5期630-637,共8页Acta Laboratorium Animalis Scientia Sinica

基　　金：国家自然科学基金(81102701,81874363,82174057,82274305);山西中医药大学科技创新团队项目(2022TD2003);曙光四明学者计划(SGXZ-201910)。

摘　　要：目的研究虫草菌丝(cultured mycelium Cordyceps sinensis,CMCS)对四氯化碳(CCl4)诱导肝纤维化模型小鼠肝腺苷酸活化蛋白激酶/沉默信息调节因子1(AMPK/SirT1)信号通路的作用机制。方法将C57BL/6小鼠(n=40)按体重分层随机分为5组:正常对照组、CMCS对照组(3.0 g/kg)、模型对照组、CMCS 1.5 g/kg组(1.5 g/kg)和CMCS 3.0 g/kg组(3.0 g/kg),采用腹腔注射10%CCl4(2 mL/kg)诱导小鼠肝损伤模型。造模与给药2周后,检测血清ALT、AST、TBil水平;苏木素-伊红(HE)染色观察肝炎症;天狼星红染色观察肝胶原沉积;Jamall’s法检测肝羟脯氨酸(Hyp)含量;流式蛋白定量技术(cytometric bead array,CBA)检测肝组织IL-6、MCP-1、IFN-γ、TNF、IL-10、IL-12p70水平;免疫组化观察肝组织CollagenⅠ和SirT1表达情况;RT-qPCR检测肝组织Prkaa1、Prkaa2、Lkb1和p53水平。结果CCl4染毒2周后,模型对照组血清ALT、AST、TBil水平均明显升高(P<0.05);HE染色和天狼星红染色分别提示肝内大量炎症细胞浸润和胶原沉积;肝Hyp含量及IL-6、MCP-1、TNF表达明显增多(P<0.05),IL-10和IL-12p70表达明显减少(P<0.01);免疫组化染色提示肝组织Collagen I表达增多;SirT1在肝窦间隙表达减少,在胶原沉积处表达增多;RT-qPCR检测提示肝组织Prkaa1、Prkaa2、Lkb1表达下降,p53表达增多(P<0.05)。CMCS可显著降低纤维化小鼠血清ALT、AST水平;降低肝组织IL-6、MCP-1、TNF表达(P<0.05),上调IL-10、IL-12p70水平(P<0.05);减少肝炎细胞浸润、胶原形成和Hyp含量,促进肝窦内SirT1表达,上调肝Prkaa1、Prkaa2、Lkb1表达(P<0.05);减轻肝内CollagenⅠ、p53表达(P<0.05)。与CMCS 1.5 g/kg组相比,CMCS 3.0 g/kg组抑制肝炎症、胶原沉积及上调AMPK/SirT1表达更为明显(P<0.05)。结论CMCS可通过上调AMPK/SirT1信号通路而发挥抗CCl4诱导小鼠肝纤维化的作用。Objective To investigate the effects of cultured mycelium Cordyceps sinensis(CMCS)on the AMPK/SirT1 signaling pathway in carbon tetrachloride(CCl4)-induced liver fibrosis in mice.Methods Forty male SPF-grade C57BL/6 mice were divided randomly into a normal control group,CMCS control group(3.0 g/kg),model control group,CMCS 1.5 g/kg group,and CMCS 3.0 g/kg group.Mice were injected intraperitoneally with 10%CCl4(2 mL/kg)to induce liver fibrosis.Two weeks later,serum levels of alanine transaminase(ALT),aspartate transaminase(AST),and total bilirubin(TBil)were measured.Inflammation and collagen deposition in liver tissue were observed by hematoxylin and eosin(HE)and Sirius red staining,respectively.The content of hydroxyproline in liver tissue was detected by Jamall’s hydrochloric acid hydrolysis method.Levels of interleukin(IL)-6,monocyte chemoattractant protein-1(MCP-1),interferon,tumor necrosis factor(TNF),IL-10,and IL-12p70 in liver tissue were detected using a cytometric bead array analysis system.CollagenⅠand SirT1 expression in liver tissue were detected by immunohisotochemistry,and Prkaa1,Prkaa2,Lkb1,and p53 gene expression were detected by real-time fluorescent quantitative reverse transcriptase-polymerase chain reaction.Results Serum levels of ALT,AST,and TBil were significantly increased in the model control group compared with those in the normal control group(P<0.05).HE and Sirius red staining showed extensive inflammatory cell infiltration and collagen deposition in the liver,respectively.Hydroxyproline content and expression levels of IL-6,MCP-1,and TNF in the liver were significantly increased(P<0.05),while IL-10 and IL-12p70 levels were significantly decreased(P<0.01).Immunohistochemical staining revealed an increase in CollagenⅠexpression and SirT1 staining was decreased in the hepatic sinusoidal space,while collagen deposition was increased.Prkaa1,Prkaa2,and Lkb1 gene expression levels were decreased and p53 was increased in liver tissue(P<0.05).CMCS significantly reduced serum ALT and AST le

关 键 词：虫草菌丝 四氯化碳(CCl4) 肝纤维化 AMPK/SirT1信号通路 

分 类 号：Q95-33[生物学—动物学]