Therapeutic Effect And Mechanism Of Shugan Method On Non-alcoholic Fatty Liver Disease With Phlegm And Blood Stasis

ObjectiveNowadays,the incidence of NAFLD(non-alcoholic fatty liver disease)continues to increase,the age range of the disease gradually expanded.Clinical studies have found that some patients with NAFLD are significantly associated with social stress.Most casea of this disease caused by social stress are because of liver qi stagnation,soothing the liver is an effective treatment.It was found that the abnormal state of HPA axis after stress can lead to IR(insulin resistance)and abnormal lipids metabolism.Therefore,it is suggested that attention should be paid to the treatment of NAFLD related with stress,which may be reflected in the abnormal state of HPA axis,abnorma lipid metabolism and transport,IR and oxidative stress.In this study,we use high fat diet and chronic stress to establish the animal model of NAFLD with liver stagnation and phlegm dampness.Syndrome Evaluation,pathological section,etc,are used to evaluate the pathological features of nonalcoholic fatty liver disease caused by liver depression.The levels of cortisol,Insulin resistance index,lipid metabolism enzymes and liver function were detected to evaluate the changes of HPA axis,lipid metabolism and transport,IR and oxidative stress,and to explore the therapeutic effect of Four inverse scattered.Thereby,we can explore the role and mechanism of liver depression on NAFLD,and the importance of the treatment of fatty liver with soothing the liver,to provide reference for clinical treatment of NAFLD caused by social stress.Method(1)70 healthy male Wistar rats,weight 250±10 g,adaptive feeding 1 week before experiment,free to eat and drink.After OFT(Open-field Test),divided the rats into 7 groups by score of OFT.Blank group(n=8),fed with normal conditions,4/cage,,do not receive any stimulation;HFD group(n=8),fed with high fat diet,4/cage,do not receive any stimulation;CS group(n=8),fed with normal conditions,4 cages,are restrained for 6h(7:00am-1:00pm)per day;Control group(n=8),fed with high fat diet,4/cage,are restrained for 6h(7:00am-1:00pm)per day;Sini san treatment group(n=8),fed with high fat diet,4/cage,are restrained for 6h(7:00am-1:00pm)per day,given daily dose of 4.0g/(kg*d)from the fourth week to eighth week;Hongqu treatment group(n=8),fed with high fat diet,4/cage,are restrained for 6h(7:00am-1:00pm)per day,given daily dose of 0.12g/(kg*d)from the fourth week to eighth week;Lianhe treatment group(n=8),fed with high fat diet,4/cage,are restrained for 6h(7:00am-1:00pm)per day,given daily dose of 4.12g/(kg*d)from the fourth week to eighth week.(2)Observe and record the general situation of animals and food intake everyday;the test records of body weight,,open field test scores and degree of sugar preference were measured every 2 weeks;weigh the visceral fat to calculate visceral adiposity index at last.All datas are used to evaluate TCM syndrome model.(3)ALT and AST were detected by the method of UV-lactate dehydrogenase method and UV-malic dehydrogenase method.Pathological section and liver index are used to evaluat NAFLD.(3)rt-PCR is applied to detect Hippocampal cortisol receptor mRNA,the contents of serum ACTH and cortisol are detected by radioimmunoassay.All datas are used to evaluate the state of HPA axis.(4)Serum total cholesterol,triglyceride,HDL,LDL,liver HMG-CoA reductase mRNA are detected by glycerol phosphate oxidase method,glycerol phosphate oxidase method,direct method and rt-PCR,to evaluate the level of lipid metabolism and transport.(5)Serum insulin,fasting blood glucose,liver tissue ROS are detected by radio immunoassay,glucose oxidase method,fluorescence assay of frozen section,to evaluate the level of IR and oxidative stress.Results(1)Compared the weight of rats in each group shows that,there is no significant change between not only composite model group and the blank group(P>0.05),but also the group of Sini san and control(P>0.05).The results of OFT shows that composite model group is significantly less than the blank group(P<0.01),and the OFT score significangtly increases after the treatment of Sini san(P<0.05).Sucrose preference test shows that the mean value of composite model group is less than the blank group,and the result increases after Sini san treatment.The record of appetite of each group shows that composite model group is less than the blank group significantly(P<0.01),and the appetite of rats in Sini san group increases significantly(P<0.05).Visceral adiposity index shows that composite model group is higher than the blank group significantly(P<0.05),and no significant change after Sini san treatment(P>0.05).(2)Pathological sections shows that the area dyed by rathonum red of composite model group increases,and decreases after the treatment of Sini san.Compared the AST of rats in each group shows that,the AST content of composite model group increases significantly(P<0.01)and decreases after the treatment of Sini san(P<0.05).The recorded content of ALT shows composite model group is higer than the blank group(P<0.01),and there is no change after Sini san treatment(P>0.05).Liver index of rats in composite model group is higher than the rats in the blank group significantly(P<0.01),and liver index significantly decreases after the treatment of Sini san(P<0.05).(3)Relative expression of Cortisol receptor mRNA in hippocampus shows that composite model group decreases significantly(P<0.01)and increases after the treatment of Sini san(P<0.05).Serum cortisol shows that composite model group is higher than the blank group(P<0.05),and decrease significantly after Sini san treatment(P<0.05).(4)Results of serum total cholesterol content shows that there is no significant change between composite model group and the blank group(P>0.05),but a significant decreasing appears after the treatment of Sini san(P<0.01).Results of serum triglyceride content shows no significant difference between composite model group and the blank group(P>0.05),even using Sini san to treat(P>0.05).Results of serum FFA content shows that composite model group increases after model established(P<0.01),and serum FFA content decrease after Sini san treatment(P<0.05).Results of serum HDL content shows that composite model group decreases after model established(P<0.05),and there is no significant change after Sini san treatment(P>0.05).Results of serum LDL content shows that composite model group increases after model established(P<0.05),and there is a significant decrease after the treatment of Sini san(P<0.01).Relative expression of HMG-CoA reductase mRNA in liver shows that composite model group is significantly higher than the blank group(P<0.01),and Sini san is effective to decrease the relative expression(P<0.01).(5)Frozen section of superoxide anion red fluorescence shows that fluorescence intensity of composite model group increases significantly,and decreases after the treatment of Sini san.The result of insulin resistance index shows that,compared with the blank group,composite model group increases significantly after the model established(P<0.01),and insulin resistance index decreases after the treatment of Sini san(P<0.05).Serum TNF-a results shows compared with the blank group,composite model group increases significantly after the model established(P<0.05),and the content decreases after the treatment of Sini san(P<0.05).Conclusions(1)Stress factors can directly induce the occurrence of NAFLD,leading to liver damage.In addition,stress factors can Influence lipid metabolism and transport,increase IR to aggravate NAFLD.Therefore,depression of liver-QI,stagnation of liver-QI can induce NAFLD.(2)Sini san can adjust the balance of HPA axis,oxidative stress state,lipid metabolism and transport to treat NAFLD caused by social stress,which can be said as NAFLD caused by liver depression and phlegm.