基于“脾主肌肉”理论探讨Sirt1/Foxa1通路介导泽明红山颗粒治疗非酒精性脂肪性肝炎的机制

Mechanism of Zeming Hongshan granules in the treatment of non-alcoholic steatohepatitis through Sirt1/Foxa1 pathway:based on the theory of"spleen governs muscles"

目的探讨“脾主肌肉”理论在泽明红山颗粒治疗非酒精性脂肪性肝炎(NASH)小鼠中指导意义及作用机制。方法SPF级C57BL/6小鼠40只随机分为:对照组(control组)、NASH模型组(NASH组)、泽明红山颗粒干预组(ZMHS组)、多烯磷脂酰胆碱胶囊(易善复)西医治疗组(PPC组),采用高脂纯化饲养(MD45%添加0.5%胆固醇)法建立NASH小鼠模型。全自动生化仪检测外周血清中谷丙转氨酶(ALT)、谷草转氨酶(AST)、总胆固醇(TC)、三酰甘油(TG)、高密度脂蛋白胆固醇(HDL)、低密度脂蛋白胆固醇(LDL)表达,苏木素-伊红(HE)染色观察肝脏病理形态,酶联免疫吸附试验(ELISA)检测肌肉组织中腺嘌呤核苷三磷酸(ATP)、磷酸肌酸激酶(CPK)、α羟丁酸脱氢酶(α-HBD)、ALT、乳酸脱氢酶(LDH)表达,ATP酶染色观察肌肉组织损伤情况,免疫荧光观察肌肉组织中过氧化物酶体增殖物活化受体激活因子(PGC)-1、过氧化物酶体增殖激活受体γ(PPARα)、核呼吸因子(NRF)-1、线粒体转录因子(mtTF)A表达,Western印迹检测转录沉默信息调节因子(Sirt)1、叉头框转录因子O1(Foxa1)蛋白表达。结果与control组相比,NASH组组外周血清中ALT、AST、TC、TG、LDL表达明显升高(P<0.05),HDL明显降低(P<0.05);肌肉组织中CPK、α-HBD、ALT、LDH、Foxa1表达升高(P<0.05),ATP、PGC-1、PPARα、NRF-1、mtTFA、Sirt1表达降低(P<0.05),骨骼肌细胞、肝细胞损伤明显。与NASH组相比,ZMHS组、PPC组外周血清中ALT、AST、TC、TG、LDL表达明显降低(P<0.05),HDL明显升高,肌肉组织中CPK、α-HBD、ALT、LDH、Foxa1表达降低(P<0.05),ATP、PGC-1、PPARα、NRF-1、mtTFA、Sirt1表达升高(P<0.05),骨骼肌细胞、肝细胞损伤明显改善。结论基于“脾主肌肉”理论,泽明红山颗粒通过调节骨骼肌胰岛素抵抗,促进线粒体功能恢复,改善肝功能损伤,其作用机制可能与其调控Sirt1/Foxa1通路有关。

Objective To explore the guiding significance and mechanism of the theory of"spleen governing muscles"in the treatment of nonalcoholic steatohepatitis(NASH)mice with Zeming Hongshan granules,and to provide experimental theoretical basis for the prevention and treatment of NASH by traditional Chinese medicine.Methods Forty SPF C57BL/6 mice were randomly divided into control group(control),nonalcoholic fatty hepatitis model group(NASH),Ze MingHongShan particles intervention group(ZMHS),positive medicine group(PPC).The NASH mouse model was established by the method of high-fat purification and rearing(MD45%with 0.5%cholesterol).Alanine aminotransferase(ALT),aspartate aminotransferase(AST),total cholesterol(TC),triglycerides(TG),high-density lipoprotein cholesterol(HDL),and low-density lipoprotein cholesterol(LDL)were detected by automatic biochemical analyzer,the liver pathology was observed by HE staining,adenine nucleoside triphosphate(ATP),phosphocreatine kinase(CPK),α-hydroxybutyrate dehydrogenase(α-HBD),alanine aminotransferase(ALT)and lactate dehydrogenase(LDH)expression in muscle tissue was detected by ELISA,the muscle tissue damage was observed by ATPase staining,the peroxisome proliferation activated receptor activator(PGC-1),peroxisome proliferation activated receptorγ(PPARα),nuclear respiratory factor 1(NRF-1),mitochondria transcription factor A(mtTFA)expression in muscle tissue were detected by immunofluorescence,transcriptional silencing information regulator 1(Sirt1)and forkhead box transcription factor O1(Foxa1)protein expression was detected by Western blot.Results Compared with the control group,the expressions of ALT,AST,TC,TG and LDL in the peripheral serum was significantly increased(P<0.05),and HDL was significantly reduced(P<0.05),the expressions of CPK,α-HBD,ALT,LDH,and Foxa1 in muscle tissue increased(P<0.05),and the expressions of ATP,PGC-1,PPARα,NRF-1,mtTFA,and Sirt1 decreased(P<0.05),skeletal muscle cells damage and hepatocyte damage were obvious in the NASH group.Compared with the NASH group,the expressions of ALT,AST,TC,TG and LDL in the peripheral serum were significantly reduced(P<0.05),HDL was significantly increased,and CPK,α-HBD,ALT,LDH,Foxa1 expressions decreased(P<0.05),ATP,PGC-1,PPARα,NRF-1,mtTFA,Sirt1 expressions increased(P<0.05),and the damage of skeletal muscle cells and hepatocytes was significantly improved in the ZMHS group and PPC group.Conclusions Based on the theory of"spleen governs muscles",Zeming Hongshan granules could promote mitochondrial function recovery and improve liver function injury by regulating insulin resistance in skeletal muscle,and the mechanism might be related to its regulation of SIRT1/FOXA1 pathway.