甘姜苓术汤防治蛋氨酸胆碱缺乏饮食引起的脂肪性肝病和肌少症的药效及其作用机制研究

Study on the mechanism of Ganjiang Lingzhu Decoction in improving metabolic dysfunction-associated steatohepatitis by restoring skeletal muscle function

关键词:  甘姜苓术汤  代谢功能障碍相关脂肪性肝病  肌少症  卫星细胞  肌肉因子

Key words:  Ganjiang Lingzhu Decoction  metabolic dysfunction-associated steatohepatitis  sarcopenia  satellite cells  myogenic regulatory factors

摘要:

[目的] 在中医“脾主肌肉”理论指导下，拟探究甘姜苓术汤（GJLZ）对肌肉功能的影响以及对代谢功能障碍相关脂肪性肝病（MASLD）的改善机制。[方法] 雄性6周龄C57BL/6小鼠随机分为正常组、模型组、GJLZ低剂量、GJLZ中剂量和GJLZ高剂量组，每组6只。除正常组给予普通饲粮外，其余各组小鼠给予蛋氨酸胆碱缺乏（MCD）饮食4周诱导MASLD模型，给药组同步给予对应剂量药物干预。处理前1 d测各组小鼠前肢抓力，待小鼠恢复1 d后过夜禁食，安乐各组小鼠，采集全血并分离血清。苏木精-伊红（HE）染色观察肝组织病理变化；骨骼肌采集时分取适量提取肌肉卫星细胞（SCs），HE染色观察骨骼肌组织病理变化；油红O染色观察肝脏、肌肉脂质沉积；试剂盒检测肝组织总胆固醇（TC）、三酰甘油（TG）水平。全自动生化分析仪检测各组小鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）水平。实时荧光定量聚合酶链反应（RT-qPCR）法、蛋白免疫印迹（Western blot）法和免疫荧光法检测肌肉生长相关因子水平。[结果] 与正常组比较，模型组血清ALT、AST水平升高（P<0.01），肝脏病理检测有明显气球样变和脂肪变，NAS评分升高（P<0.01），油红O染色出现较多脂滴，提示MASLD诱导成功；前肢握力降低（P<0.01），肌管萎缩（P<0.01），油红O染色结果显示肌间脂肪浸润；RT-qPCR和Western blot结果显示模型小鼠肌肉生长抑制素（MSTN）表达水平升高（P<0.05），胰岛素样生长因子-1（IGF-1）表达水平下降（P<0.05或P<0.01），成肌分化抗原（MyoD）表达水平下降（P<0.05或P<0.01），肌细胞生成素（Myog）的基因水平下降（P<0.05）。免疫荧光检测结果显示，与正常组相比，模型组小鼠配对盒7（PAX7）和MYOG的阳性表达面积均减少。SCs免疫荧光双染显示，与正常组相比，模型组SCs的MSTN表达上升。与模型组相比较，GJLZ低剂量组小鼠肝脏TG水平（P<0.01）和NAS评分（P<0.01）降低，GJLZ中剂量和高剂量组小鼠血清ALT水平（P<0.01）、肝脏TG水平（P<0.01）和NAS评分（P<0.01）降低；GJLZ低剂量、中剂量和高剂量组小鼠前肢握力（P<0.05或P<0.01）恢复，肌管萎缩（P<0.05或P<0.01）和肌间脂肪浸润改善；GJLZ低剂量组对MASLD小鼠肌肉生长相关因子水平影响不显著；GJLZ中剂量、高剂量组小鼠MSTN水平（P<0.05或P<0.01）降低，IGF-1、MyoD水平（P<0.05或P<0.01）升高；GJLZ低剂量、中剂量和高剂量组PAX7和MYOG的阳性表达面积降低，SCs的MSTN表达降低。[结论] GJLZ可能通过抑制肌肉萎缩相关因子（如MSTN）的表达，上调肌肉合成相关因子（如IGF-1、MyoD和MYOG）的表达，下调SCs中MSTN的表达水平以增加SCs的数量，从而促进小鼠肌肉量的增加、减轻肌纤维萎缩、增强前肢握力并改善MASLD。

Abstract:

[Objective] Under the guidance of the theory of “Spleen governing muscles” in traditional Chinese medicine，this study aims to explore the effects of Ganjiang Lingzhu Decoction（GJLZ） on muscle function and the improvement mechanism of metabolic dysfunction-associated steatohepatitis（MASLD）. [Methods] Male 6-week-old C57BL/6 mice were randomly divided into normal group，model group，low-dose GJLZ group，medium-dose GJLZ group and high-dose GJLZ group，with 6 mice in each group. Except for the normal group which was fed a normal diet，the other groups were fed a methionine-choline-deficient（MCD） diet for 4 weeks to induce the MASLD model. The drug intervention groups were simultaneously given the corresponding doses of GJLZ. One day before treatment，the forelimb grip strength of each group of mice was measured. After the mice recovered for 1 day，they were fasted overnight and sacrificed. Whole blood was collected and serum was separated. Hematoxylin-eosin（HE） staining was used to observe the pathological changes in liver tissue；when collecting skeletal muscle，an appropriate amount was taken to extract muscle satellite cells（SCs），and HE staining was used to observe the pathological changes in skeletal muscle tissue；oil red O staining was used to observe lipid deposition in the liver and muscle；kits were used to detect the levels of total cholesterol（TC） and triglycerides（TG） in liver tissue. The levels of alanine aminotransferase（ALT） and aspartate aminotransferase（AST） in the serum of each group of mice were detected by an automatic biochemical analyzer. Real-time fluorescence quantitative polymerase chain reaction（RT-qPCR），Western blot and immunofluorescence were used to detect the levels of muscle growth-related factors. [Results] Compared with the normal group，the levels of ALT and AST in the serum of the model group increased（P<0.01），and the liver pathological examination showed obvious ballooning degeneration and steatosis，with an increased NAS score（P<0.01），and Oil Red O staining showed more lipid droplets，indicating successful induction of MASLD；the forelimb grip strength decreased（P<0.01），and the muscle tubes atrophied（P<0.01），and Oil Red O staining showed intermuscular fat infiltration；RT-qPCR and Western blot results showed that the expression level of myostatin（MSTN） in the muscle of model mice increased（P<0.05），the expression level of insulin-like growth factor-1（IGF-1） decreased（P<0.05 or P<0.001），the expression level of myogenic differentiation antigen（MyoD） decreased（P<0.05 or P<0.01），and the gene level of myogenin（Myog） decreased（P<0.05）. Immunofluorescence detection results showed that compared with the normal group，the positive expression areas of paired box 7（PAX7） and MYOG in the model group decreased. Double staining of SCs by immunofluorescence showed that the expression of MSTN in SCs in the model group increased compared with the normal group. Compared with the model group，the levels of TG in the liver（P<0.01） and NAS score（P<0.01） in the low-dose GJLZ group decreased，and the levels of ALT in the serum（P<0.01），TG in the liver（P<0.01） and NAS score（P<0.01） in the medium-dose and high-dose GJLZ groups decreased；the forelimb grip strength（P<0.05 or P<0.01） in the low-dose，medium-dose and high-dose GJLZ groups recovered，and the muscle tube atrophy（P<0.05 or P<0.01） and intermuscular fat infiltration improved；the low-dose GJLZ group had no significant effect on the levels of muscle growth-related factors in MASLD mice；the levels of MSTN in the medium-dose and high-dose GJLZ groups decreased（P<0.05 or P<0.01），and the levels of IGF-1 and MyoD increased（P<0.05 or P<0.01）；the positive expression areas of PAX7 and MYOG in the low-dose，medium-dose and high-dose GJLZ groups decreased，and the expression of MSTN in SCs decreased. [Conclusion] GJLZ may promote the increase of muscle mass in mice，alleviate muscle fiber atrophy，enhance forelimb grip strength and improve MASLD by inhibiting the expression of muscle atrophy-related factors（such as MSTN），up-regulating the expression of muscle synthesis-related factors（such as IGF-1，MyoD and MYOG），and down-regulating the expression level of MSTN in SCs to increase the number of SCs.