Study On The Mechanism Of Gegenqinlian Decoction In Protecting Pancreatic Islet Function In Rats With Type 2 Diabetes Based On Intestinal Microecolog

1 Background and PurposeType 2 diabetes mellitus(T2DM)is a progressive disease.The destruction of islet cell function is the vital pathological foundation.Islet cell function is irreversibly exhausted in all types of DM.Currently there is no effective medication that can reverse the progression of DM.Hence early protection of islet function is the key to delay this progression.Recently,researches have shown that gut microbiota imbalance in early stage of DM can destroy the gut barrier function,cause metabolic endotoxemia and nonspecific inflammatory reaction,which correlates to the pathogenesis of T2DM.Previous research has found that Traditional Chinese medicine(TCM)can lower blood glucose in DM patient effectively,and delay the progression.In latter research,we found that Gegenqinlian decoction(GQD)can lower the blood glucose by adjusting gut microbiota structure.The mechanism is yet to be illustrated.In this research,on the basis of literature screening,the effect of GQD lowering blood glucose on T2DM rat model and the islet β cell function protection via gut microbiota were explored,hoping to provide new perspectives and evidence for TCM preventing and treating DM.2 Methods2.1 Retrospective analysis of outpatientsIn this research retrospective analysis was performed.The whole process of diagnosis and treatment of T2DM outpatients were documented.Those T2DM patients accord with the inclusion criteria were enrolled.Clinical information,efficacy and safety index were collected.Correlation analysis was performed to evaluate the efficacy characteristics.Explored the efficacy and safety in T2DM patients taking TCM for long period.2.2 Experimental research40 male Wistar rats(weighed between 190g to 210g)were randomly divided into 4 groups,including normal group(normal feed,n=10)and T2DM model group(high-fat diet for 4 weeks,then intraperitoneal injection with STZ,n=30).Random blood glucose over≥11.1 mmol/L was considered T2DM model established.Then model rats were fed with high-fat diet and randomly divided into 3 groups,including model group(n=10),GQD group(n=10)and MET group(n=10).In 12 weeks’ intervention,the weight,intakes,fasting blood glucose and faeces were collected at regular intervals.OGTT and ITT test were performed before the last intervention.After last intervention,blood sample was collected from aorta abdominalis in narcosis status.Pancreas,liver,abdominal adipose tissue,skeletal muscle and small intestine were also collected.HbA1c,GSP,fasting insulin and C peptide were detected.HOMA-IR and HOMA-β were calculated.Biochemical markers and serum inflammatory factors were also detected.Inflammatory factors and connexin in adipose,liver and ileum tissue were detected via RT-PCR.IR,IRS,Akt,IKKβ and JNK and their phosphorylation levels in adipose and liver tissue were detected via Western blot.Variations of gut microbiota were detected via 16s rRNA.3 Results3.1 TCM significantly lower blood glucose in T2DM patients,protects islet β cell functionTCM can lower blood glucose in T2DM patients with uncontrolled HbAlc(over 7.0%).The control rates of HbAlc in 6th,12th,18th,24th,36th,48th and 60th month were 45.07%,52.78%,47.22%,45.83%,50.00%,57.14%and 40.00%.Multiple linear regression analysis showed HbA1c variation correlated with HbA1c baseline,course of disease and BMI(P<0.05).TCM intervention can lower FPG and 2hPG in T2DM patients significantly,lower HOMA-IR and increase HOMA-β(P<0.05).During intervention,19 patients disabled oral hypoglycemic drugs,4 patients disabled insulin,19 took TCM decoction alone and controlled blood glucose well.1 patient developed diabetic nephropathy and 1 patient developed diabetic retinopathy.1 patient had decoction-irrelevant abnormal liver function,after hepatic protectant treatment the liver function returned normal.3.2 GQD decoction improves blood glucose and insulin resistance in T2DM rat modelAfter 12 weeks’ GQD intervention,the HbAlc and C peptide were decreased,fasting insulin and HOMA-IR were increased significantly in T2DM rats.GQD intervention can lower AUC in OGTT,increase IR,Akt phosphorylation in adipose and liver tissue,which were greater in MET group.3.3 GQD decoction improves intestinal barrier function and inflammatory responseAfter 12 weeks’ GQD intervention,serum inflammatory factors including CRP,MCP-1,IL-1β,TNF-α and resistin,liver Emr1(F4/80)and adipose TNF-α,IL-1β,MCP-1 were decreased.Leptin and adiponectin were increased.The blood circulation was promoted via lowering PAI-1 and VEGF.TLR-4/MyD88/IKKβ signaling pathway was inhibited.GQD intervention can significantly decrease endotoxin and increase Occludin、Claudin-1.3.4 GQD decoction adjusts gut microbiota distribution structure in T2DM ratsAfter 12 weeks’ GQD intervention,the gut microbiota distribution structure in T2DM rats was altered.Firmicutes/Bacteroidetes were decreased.Compared to model group,the gut microbiota diversity was decreased.Beneficial Butyrate bacteria including Phascolarctobacterium,Blautia and Parasutterella were increased after GQD intervention.Pernicious Sulfate bacteria including Desulfovibrio were decreased.Functional enrichment prediction showed that carbohydrate digestion and absorption,insulin signal transduction pathway,bacterial motility protein,bacterial chemotaxis and endocytosis may be improved by GQD.Further analysis revealed that multiple bacteria were correlated with glucose metabolism,inflammatory factors related signaling pathway and intestinal barrier function.All the findings above indicated that the variation of gut microbiota structure after GQD intervention is correlated with blood glucose,inflammatory status and intestinal barrier function.4 Conclusions4.1 Based on retrospective analysis of outpatients,long-term use of TCM can control blood glucose,protect the islet β cell function and delay the progression of DM.4.2 High-fat diet combined with STZ induced T2DM rat model has dysregulated gut microbiota structure and destructed barrier transparency.Abnormal inflammatory factors and related signaling pathway,and increased blood glucose were observed.These findings provided the biological foundation of fat and sugar excessive intake leads to disease.4.3 GQD can effectively lower the blood glucose in T2DM rat model,improve the insulin resistance and adjust the insulin signaling pathway.It proved that Pidan theory based intervention can protect islet β cell function.4.4 GQD might reverse the abnormal expression of inflammatory factors and related signaling pathway by adjusting the gut microbiota structure and improving intestinal barrier function.In that can GQD lowering blood glucose and insulin resistance,exert the islet β cell protection.