Effect Of Zuoguijiangtangqingganfang On Type 2 Diabetes With Nonalcoholic Fatty Liver Injury In MKR Mice

Objective1.To investigate the effect of Zuogui Jiangtang Qinggan Recipe on the expression of FOXO1 and FASL in the liver tissue of MKR rats with type 2 diabetes combined with nonalcoholic fatty liver disease.To further confirm the scientific basis of Zuogui Jiangtang Qinggan Recipe for the treatment of non-alcoholic fatty liver disease in diabetes.2.To explore the syndromes and patterns of diabetic non-alcoholic fatty liver disease,and the clinical medication rules for the treatment of this disease,and to clarify the therapeutic characteristics of Zuogui Jiangtang Qinggan prescription in the treatment of diabetic non-alcoholic fatty liver disease.Methods1.Theoretical research:Literature on traditional Chinese medicine diagnosis and treatment experience or clinical observation of diabetic non-alcoholic fatty liver published in the past 20 years was collected from various databases.After using the cloud platform of ancient and modern medical records（V1.2.7）,statistical analysis was carried out on the syndrome types,and the frequency of traditional Chinese medicine and complex network analysis were carried out.2.Experimental research:Forty eight-week-old high-fat MKR mice,half male and half female,were randomly divided into Model group（Model group）,Zuoguijiangtangqinggan prescription low-dose group(ZGJTQGF-L group:Zuoguijiangtangqinggan prescription 7.4g.kg^-1)and Zuoguijiangjiangqinggan prescription high-dose group(ZGJTQGF-H group:Zuoguijiangtangqinggan Fang14.8g.kg^-1)and metformin group(Met group:metformin 67×10^-3g.kg^-1),with 10 mice in each group.Another 10 FVB/N mice（half male and half female）were set as normal group（Control group）and fed with ordinary diet.After 28 days of drug intervention,fasting blood glucose,liver function,blood lipid,HE staining and pathological changes of liver tissue were detected by electron microscopy.The protein expression levels of FOXO1 and FASL in liver tissue were detected by Western Blot,and the m RNA expression levels of FASL and FOXO1 in liver tissue were detected by PCR.Results1.Theoretical research results:（1）There are 10 TCM syndrome types according to statistics,and the common witness types include phlegm-stasis interjunction syndrome（39.75%）,damp-heat intrinsic syndrome（39.13%）,liver depression and spleen deficiency syndrome（32.92%）,etc.（2）A total of 16 kinds of traditional Chinese medicine with a frequency greater than 28 were found,among which salvia miltiorrhiza was the first（64.60%）,followed by Hawthorn（63.98%）,diarrhea（54.04%）and Rhizoma atrictylodes（52.80%）.（3）The core prescriptions used in the treatment of non-alcoholic fatty liver were as follows:poria,Tangerine peel,bupleurum,atractylodes,alisma alisma,Hawthorn,salvia miltiorrhiza,astragalus,Yu Jin,licorice,cassia seed.2.Experimental research results:（1）Effects on FBG in each group:compared with normal group,fasting blood glucose in model group was significantly increased,the difference was statistically significant（P<0.01）;compared with model group,blood glucose in Chinese medicine group and western medicine group was significantly decreased（P<0.05）;There was no statistical significance between TCM group and western medicine group（P>0.05）.（2）ALT and AST levels in each group:compared with normal group,ALT and AST levels in model group were significantly increased,and the difference was statistically significant（P<0.01）;compared with model group,ALT and AST levels in TCM group and western medicine group were significantly decreased（P<0.05）;There was no significant difference between high dose and low dose in TCM group（P>0.05）.（3）Levels of TG and TC in each group:compared with the normal group,TG and TC in the model group were significantly increased,with statistical significance（P<0.01）,while TG and TC in the traditional Chinese medicine group and Western medicine group were significantly decreased compared with the model group（P<0.05）.（4）Protein expression levels of FASL and FOXO1 in each group:compared with the normal group,the protein expression levels of FASL and FOXO1 in the model group were significantly increased,and the difference was statistically significant（P<0.01）;compared with the model group,the protein expression levels of FASL and FOXO1 in the Chinese medicine group and the western medicine group were significantly decreased（P<0.01）.（5）FASL m RNA expression level in each group:compared with the normal group,the expression level of FASL m RNA in the model group was significantly increased,with statistical significance（P<0.01）;compared with the model group,the expression level of FASL m RNA in the Chinese medicine group and the western medicine group was significantly decreased（P<0.01）.FOXO1 m RNA expression in each group:Compared with normal group,FOXO1 m RNA expression in model group was significantly increased,the difference was statistically significant（P<0.01）,while FOXO1 m RNA expression in traditional Chinese medicine group and western medicine group was significantly decreased compared with model group（P<0.01）.There was no significant difference in FOXO1 m RNA expression between TCM group and western medicine group（P>0.05）.（6）HE staining results:the structure and arrangement of liver cells in the normal group were regular,the structure of liver lobules was complete,and the liver cells were cord-like.In the model group,the structure of hepatocytes was disordered and necrotic,the arrangement of hepatocytes was irregular,the cell swelling was significant,there were a large number of fat droplets in the hepatocyte plasma,which were mainly manifested as diffuse steatosis and fibrosis of liver tissue.There was no obvious fat vacuole phenomenon in Chinese medicine group and Western medicine group,and the degree of swelling and fibrosis was reduced.（7）Electron microscope results:lysozyme in the normal group had little or no fat granules in the outer circumference,and a small amount of fat was accumulated in hepatocytes.The liver cells of model group had a lot of fat,and the lysosomes were surrounded by a large number of fat particles.There were few or no fat granules in lysozyme circumference in western medicine group and Chinese medicine group,and fat accumulation was significantly reduced.Conclusion1.Syndrome types of NAFLD are mostly syndrome of interjunction of phlegm and blood stasis,followed by syndrome of dampness-heat connotation and syndrome of liver depression and spleen deficiency.The modern clinical efficacy of traditional Chinese medicine in the treatment of NAFLD mainly includes invigorating spleen and qi,clearing heat and dampness,eliminating phlegm and eliminating blood stasis.Its medication pattern was consistent with the etiology and pathogenesis of the disease,and was highly consistent with the composition of Zuogui Jiangtang Qinggan prescription.2.Zuogui Jiangtang Qinggan formula with nourishing Yin and qi and detoxifying and activating blood circulation can reduce the blood sugar and blood fat of MKR mice on a high fat diet,protect the liver and reduce the risk of diabetes complicated with non-alcoholic fatty liver.3.FOXO1/FASL is involved in the pathogenesis of diabetic non-alcoholic fatty liver disease.FOXO1 is combined with the death factor FASL,and FOXO1 can up-regulate the expression of FASL and induce cell apoptosis.4.By down-regulating FOXO1 protein,Zuogui Jiangtang Qinggan prescription can reduce the expression of FASL protein,reduce the apoptosis rate of liver cells,delay liver injury,and thus protect the liver and treat T2DM complicated with NAFLD.