Study On The Efficacy And Mechanism Of "Di Zhuo Xie Du" In Type 2 Diabetes Mellitus Complicated With Non-alcoholic Fatty Liver Disease Based On AGEs/RAGE/JNK/NF-κB Signaling Pathway

Objective:To explore the possible mechanism of Dachaihu Decoction in the therapy of Type2 diabetes mellitus(T2DM)complicated with non-alcoholic Fatty Liver Disease(NAFLD)based on the theory of relativity of essence and turbidity.The potential target and signal pathway of Dachaihu Decoction for T2DM with NAFLD were predicted through Network Pharmacology.Taking the rat models with T2DM complicated with NAFLD as the research object,to explore the further mechanism of Dachaihu Decoction in the intervention of T2DM with NAFLD.Through clinical trials,to verify the clinical effect and safety of Dachaihu Decoction in the treatment of T2DM with NAFLD.Methods:1.Network Pharmacology: The molecular targets of Dachaihu decoction were obtained by using TCMSP(Traditional Chinese medicine System Pharmacology Database and Analysis Platform)database.OMIM database and Genecards database were used to collect the target genes of T2DM and NAFLD,and the intersection was obtained by Venn Diagram function.The protein-protein interaction network(PPI)was constructed by STRING database and leaded into Cytoscape.Finally,GO enrichment analysis and KEGG pathway enrichment analysis of the intersection genes were conducted by R language bioinformation software,and the results were visualized by Path View.The final results were output in the form of bar graph and bubble graph.2.Basic Research: The animal model of T2DM with NAFLD was established by the intraperitoneal injection of streptozotocin(STZ)combined with modified high-fat and high-sugar diet.Sixty SPF-grade SD(Sprague Dawley)male rats were divided into blank Control group(F,Control)(n=10)and model group(n=50)randomly.Model group was fed with modified high-fat and high-sugar foods for 4 weeks and then injected with STZ for two times totally.After model were completed,the rats were divided into Dachaihu Decoction low-dose group(A,LD-DCHD),Dachaihu Decoction medium-dose group(B,MD-DCHD),Dachaihu Decoction high-dose group(C,HDDCHD),Model control group(D,Model)and Metformin group(E,ME)randomly.Dachaihu granules or normal saline were administered daily for 4 consecutive weeks.Fasting body weight of rats were observed once a week,and the changes of diet and water intake for 24 hours,as well as the overall status of urine and feces were recorded.FPG value of rats was determined on a fixed day every week.At the end of the experiment,oral glucose tolerance test(OGTT)was used to evaluate the hyperglycemic level and insulin resistance status of rats.Blood was drawn from abdominal aorta when anesthesia was finished,and ALT,AST,TC,TG,HDL-C,LDL-C levels in serum were detected by biochemical method.The levels of FINS,TGF-β1,APN,FFA,IL-6,TNF-α,hs-CRP,AGEs in serum were detected by enzyme-linked immunoassay(Elisa).SOD,GSH and MDA levels in liver tissue homogenate were detected.The pathological changes of pancreas were examined by HE staining after collected.Besides,observation from the changes of liver tissues were operated through oil red O and HE staining,and the liver wet weight was used to calculate the liver index.The expression of RAGE,JNK,NF-κB m RNA in AGEs/RAGE/JNK/NF-κB pathway was detected by real-time PCR.The expression of RAGE,JNK,p-JNK,p65 p-p65 protein was detected through Western Blot.3.Clinical Study: Using the method of randomized controlled study,cases of T2DM with NAFLD(syndrome of stagnation of heat in liver and stomach)were collected from the inpatient and outpatient department in Endocrinology of Affiliated Hospital of Chengdu University of TCM from December 2020 to December 2021,and divide them into experimental group and control group according to the randomization principle grouping(random number table).The control group was cured with routine hypoglycemic western medicine,and the study group was cured with Dachaihu Decoction on the basis of conventional hypoglycemic western medicine for 12 weeks,so as to make sure that therapeutic effect and safety of Dachaihu Decoction on T2DM patients with NAFLD.Result:1.Results of Network pharmacology:1015 predicted target proteins were screened from the database,451 core targets of T2DM combined with NAFLD were selected,and a total of 138 targets for the interaction between Dachaihu decoction and target diseases were obtained.Through KEGG pathway enrichment analysis of common targets,a total of 153 pathways involved in Dachaihu Decoction for the therapy of T2DM complicated with NAFLD were obtained.Among them,interleukin family,tumor necrosis factor,mitogenactivated protein kinase 8 are core target group,which is strongly related to IR,oxidative stress and inflammatory pathways,and the pathways related to this study mainly involve AGEs/RAGE pathway and its downstream pathway,namely JNK/NF-κB.2.Results of Basic Research:(1)Establishment of rat model of T2DM with NAFLD and its influence on blood glucose(1)Compared with blank group,the rats in model group gradually showed poor spirits,weight loss,loose stools,and increased intake of food and water through general observation.(2)Compared with the blank group,fasting glucose level in the model group was significantly higher(P<0.05),glucose tolerance was significantly decreased,and histopathological changes were observed by HE staining of liver.The results indicated that the modeling of T2DM rats with NAFLD was successful.(3)After intervention with Dachaihu Decoction,the general state,FBG and glucose tolerance of rats in the model group were improved.(2)The Effect of Dacaihu Decoction on curative effect index of T2DM rats with NAFLDCompared with blank group,serum ALT,AST,TC,TG,LDL-C,FINS,TGF-β1,FFA,IL-6,TNF-α,hs-CRP,AGEs and HDL-C,APN were significantly increased in model group.SOD,GSH decreased and MDA increased in liver tissue homogenate.Homa-IR,ISI,liver wet weight,liver index evidently increased(P<0.05).HE staining and oil red O staining showed obvious histopathological changes of liver and pancreas.After experimental drug intervention,ALT,AST,TC,TG,LDL-C,FINS,TGF-β1,FFA,IL-6,TNF-α,hs-CRP,AGEs were decreased and HDL-C,APN was significantly increased in rats.The level of SOD,GSH significantly increased,MDA significantly decreased;Homa-IR,ISI,liver wet weight and liver index decreased significantly,the differences were distinctly significant(P<0.05);HE staining and oil red O staining showed that the histopathology of hepatopancreas was significantly recovered.All the above indexes showed an improvement trend,and the most obvious change belong to HD-DCHD group.(3)The Effect of Dacaihu Decoction on AGEs/RAGE pathway molecular expression in rats model of T2DM with NAFLDThe protein expression of RAGE,JNK,p-JNK,p65 p-p65 and m RNA expression of RAGE,JNK,p65 in liver tissue of model group were significantly increased,and the differences were obviously significant(P<0.05),compared with blank group.After experimental drug intervention,the protein expression of RAGE,JNK,p-JNK,p65 pp65 and m RNA expression of RAGE,JNK,p65 in liver of rats were significantly lower than model rats,and the statistic differences of indexes were significant(P<0.05).3.Results of Clinical Study:A total of 60 cases were collected in this clinical test,among which 3 cases were excluded and 2 cases were dropped off.55 cases were actually completed,including 27 cases in the experimental group while 28 cases in the control group(P>0.05).After intervention of medicine,compared with the control group,body weight,BMI,FPG,2Hpg,Hb A1 c,FINS,ALT,AST,GGT,AKP,TC,TG,LDL-C,Ln(HOMAIR)level was significantly decreased,HDL-C,Ln(HOMA-β)level was significantly increased in the experimental group.B ultrasound grading of liver,TCM symptom score,total syndrome score and total effective rate were improved,and the differences of these indexes were statistically significant in the treatment group(P<0.05 or P<0.01).Conclusion:1.The AGEs/RAGE/JNK/NF-κB signaling pathway is an important signaling pathway involved in the therapy of T2DM complicated with NAFLD by Dachaihu Decoction.2.Basic Research demonstrated that Dachaihu Decoction could reduce AGEs levels and inhibit the activation of AGEs/RAGE/JNK/NF-κB signaling pathway,thereby improving the general situation and related indexes of blood glucolipid metabolism in T2DM with NAFLD,including insulin resistance,levels of inflammatory factors and oxidative stress products.Besides,Dachaihu Decoction could improved liver histomorphology,reduced liver lipid ectopic deposition,and delayed the progression of NAFLD.3.Clinical Study have preliminarily demonstrated that Dachaihu Decoction can adjust glucolipid metabolism,improve IR and liver imaging manifestations,increase insulin sensitivity and reduce TCM syndrome scores in patients with T2DM complicated with NAFLD.The results indicated that Dachaihu Decoction can effectively treat T2DM complicated with NAFLD with good efficacy and safety.