Protective Mechanism Of Jiangtang Sanhuang Tablet On Islet β Cell Function Damage Induced By High Glucose And High Fat Diet In SD Rats

BackgroundDiabetes mellitus(DM)is a group of metabolic diseases characterized by chronic hyperglycemia,which is caused by the deficiency of insulin secretion and/or action.Diabetic patients are often accompanied by lipid and protein metabolic disorders,long-term metabolic disorders can eventually lead to multi-system damage,such as retina,kidney,nervous system,cardiovascular and other tissues and organs chronic lesions,dysfunction and failure.The purpose of the treatment of diabetes is to control blood glucose,control and delay the occurrence of diabetic complications and improve the quality of life.At present,the commonly used hypoglycemic drugs are sulfonylureas,biguanidine and insulin(analogues).There are also dipeptidyl peptidase Ⅳ(DPP-4)inhibitors,glucagon-like peptide-1(GLP-1)receptor agonists,sodium-glucose cotransporter 2(SGLT2)inhibitors and so on.Compared with the single target treatment strategy,the multi-component-multi-target treatment strategy of traditional Chinese medicine shows certain advantages in the treatment of diabetes.Traditional Chinese medicine has a long history of treating diabetes,and the curative effect is remarkable.Under the guidance of the concept of"syndrome differentiation and treatment",the team of the first affiliated Hospital of Guangzhou University of traditional Chinese Medicine,lead by Professor Xiong Manqi,put forward that "the combination of blood stasis and heat and deficiency of both qi and yin" is the main pathogenesis of diabetes,and established "promoting blood circulation and removing blood stasis."The basic treatment of "replenishing qi and nourishing yin".Combined with cl inical practice,Jiawei Taohe Chengqi decoction was drawn up,and the prescription was continuously optimized.Finally,the hospital preparation "Jiangtang Sanhuang tablet" was made.In the previous basic research,we confirmed that Jiangtang Sanhuang tablet can improve blood glucose,blood lipid,insulin resistance,islet function and so on.It has therapeutic effect on diabetes,diabetic nephropathy,diabetic cardiomyopathy,diabetic gastrointestinal disease and so on.However,the mechanism of Jiangtang Sanhuang tablet protecting islet function has not been clarified,so it is of great significance to study the protective mechanism of Jiangtang Sanhuang tablet on islet β cell function injury.High-sugar and high-fat diet is an important cause of type 2 diabetes and obesity,and obesity itself is a high risk factor for type 2 diabetes.Most of the patients with type 2 diabetes mellitus are accompanied by lipid metabolism disorder.Chronic hyperglycemia and lipid metabolism disorder can lead to the damage of islet function and further aggravate the progress of diabetes and its complications.However,in the previous studies on Jiangtang Sanhuang tablets,most of the methods to build the model were fed rats with high glucose and high fat diet,combined with streptozotocin(STZ)to induce the destruction of islet tissue to establish diabetes model or its related complications model.In our experiment,STZ was no longer used to induce the destruction of islet tissue,and SD rats were fed with long-term high-glucose and high-fat diet.The models of hyperglycemia,hyperlipidemia and islet function damage were more consistent with the actual occurrence and development of diabetes.On the basis of this model,it is of great significance to explore the curative effect and mechanism of Jiangtang Sanhuang tablets.The increase of islet β cell apoptosis and dedifferentiation are the mechanisms of islet β cell failure.However,recent studies have confirmed that islet β-cell dedifferentiation may be more important than apoptosis in T2DM.Studies have shown that islet cell dedifferentiation has occurred in the early stage of islet functional injury.Islet β cell dedifferentiation means that mature β cells lose the characteristics of mature cells and lose some or all of their insulin secretion ability.The expression of Fox01,MafA,Pdx-1 and Insulin in pancreatic tissue of experimental animals was detected by immunohistochemical method,which can be used to speculate whether Jiangtang Sanhuang tablet protects the function of islet β cells by inhibiting dedifferentiation.Another important factor in the failure of islet β cells is the inflammatory reaction of the body,and the changes of inflammatory factors in the serum of experimental animals are analyzed.It can be inferred that the possible pathological mechanism of islet β cell function injury and the protective mechanism of Jiangtang Sanhuang tablet.Proteomics is a subject that elucidates the expression and functional patterns of all proteins expressed in various biological genomes of organisms.Proteome chip is a commonly used detection tool in proteomics.We used the method of protein chip to analyze the serum of SD rats,to find out the molecular differences of different groups of rats,and to analyze the protein-protein interaction,GO enrichment and KEGG enrichment of differential proteins.To explore possible mechanisms and ensure the accuracy of the results of protein chip,some of the difference factors were verified by elisa.Objectives1.Effect of Jiangtang Sanhuang tablet on glucose and Lipid Metabolism in SD Rats fed with High glucose and High Fat Diet.2.To observe the protective effect of Jiangtang Sanhuang tablet on islet function injury induced by high glucose and high fat diet in SD rats.3.Effect of Jiangtang Sanhuang tablet on dedifferentiation of islet β cells in SD rats fed with high glucose and high fat diet.4.Study on the effect of Jiangtang Sanhuang tablet on Serum inflammatory factors in SD Rats with High glucose and High Fat Diet based on ProteomicsMethods1.Effect of Jiangtang Sanhuang tablet on glucose and Lipid Metabol ism in SD Rats fed with High Sugar and High Fat Diet.30 male SD rats were randomly divided into control group,model group and Jiangtang Sanhuang tablet group.The control group was fed with common feed,the model group and Jiangtang Sanhuang tablet group were fed with high-sugar and high-fat diet.At the same time,the rats in the Jiangtang Sanhuang tablet group were given 675mg/(kg·d)Jiangtang Sanhuang tablets,and the rats in the control group and the model group were given the same volume of aseptic water once a day for 180 days.The drinking water and dietary activities of the rats were observed daily,the body weight was measured once a week,and the blood was collected on the 90th and 180th day.The levels of fasting blood glucose(FBG),high density lipoprotein cholesterol(HDL-C),low density lipoprotein cholesterol(LDL-C),total cholesterol(TC)and triglyceride(TC)were measured.After the samples were taken,the pathological examination of pancreatic tissue was carried out.2.Protective effect of Jiangtang Sanhuang tablet on islet β cell function damage induced by high glucose and high fat diet in SD rats.Fasting serum insulin(FINS)was measured on the 90th and 180th day.Then the insulin resistance index(HOMA-IR),islet cell function index(HOMA-β)and insulin sensitivity index(ISI)were calculated according to the formula.3.Effect of Jiangtang Sanhuang tablet on dedifferentiation of islet β cells in SD rats fed with high glucose and high fat diet.In order to investigate the effect of Jiangtang Sanhuang tablets on the dedifferentiation of pancreatic islet cells,the expressions of Fox01,MafA,Pdx-1 and Insulin in pancreatic tissues were detected by immunohistochemical method.4.Study on the effect of Jiangtang Sanhuang tablet on Serum inflammatory factors in SD Rats with High glucose and High Fat Diet based on Proteomics.The changes of inflammatory factors in serum of rats were analyzed by protein chip to explore the cytokines that may be involved in the differences,and some of the differential factors were verified by elisa.The differential proteins were analyzed by protein-protein interaction analysis,GO enrichment analysis and KEGG enrichment analysis,and the possible signal pathways were discussed.Results1.Effect of Jiangtang Sanhuang tablet on glucose and Lipid Metabolism in SD Rats fed with High Sugar and High Fat Diet.According to the weight change trend of rats in each group,the body weight of rats in all groups was increasing.On the 30th day,the weight gain trend of rats in all groups was basically the same.From 60 days onwards,the average body weight of the model group was significantly higher than that of the control group(P<0.05).Compared with the model group,the body weight of rats in the Jiangtang Sanhuang tablet group was significantly lower than that in the model group(P<0.05).At the same time,the anatomy suggested that there were more abdominal fat and inguinal fat accumulation in the model group,while the fat accumulation in the Jiangtang Sanhuang tablet group was better than that in the model group.On the 90th day,there were differences in glucose and lipid metabolism among different groups of rats.Compared with the control group,the FBG in the model group was increased(P<0.01),and the FBG in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).Compared with the control group,the TC in the model group was higher(P<0.01),and the TC in the Jiangtang Sanhuang tablet group was lower than that in the model group,but there was no significant difference(P=0.23).Compared with the control group,the TG in the model group was increased(P<0.05),and the TG in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).Compared with the control group,the HDL-C in the model group was decreased(P<0.05),and the HDL-C in the Jiangtang Sanhuang tablet group was higher than that in the model group(P<0.01).Compared with the control group,the LDL-C in the model group was increased(P<0.01),and the LDL-C in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.05).On the 180th day,FBG in the model group was higher than that in the control group(P<0.01),and FBG in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).Compared with the control group,the TC in the model group was increased(P<0.01),and the TC in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).Compared with the control group,the TG in the model group was increased(P<0.05),and the TG in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).Compared with the control group,the HDL-C in the model group was decreased(P<0.01),and the HDL-C in the Jiangtang Sanhuang tablet group was higher than that in the model group(P<0.05).Compared with the control group,the LDL-C in the model group was increased(P<0.01),and the LDL-C in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).2.Protective effect of Jiangtang Sanhuang tablet on islet β cell function damage induced by high glucose and high fat diet in SD rats.On the 90th day,compared with the control group,the FINS secretion in the model group decreased(P<0.01),and the FINS secretion in thejiangtang Sanhuang tablet group was higher than that in the model group(P<0.05).Compared with the control group,the HOMA-IR in the model group was higher(P<0.01).Compared with the model group,the HOMA-IR in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.05).Compared with the control group,the HOMA-β in the model group was decreased(P<0.01),and the HOMA-β in the Jiangtang Sanhuang tablet group was higher than that in the model group(P<0.05).Compared with the control group,the ISI in the model group was significantly lower(P<0.01),and the ISI in the Jiangtang Sanhuang tablet group was significantly higher than that in the model group(P<0.01).On the 180th day,compared with the control group,the secretion of FINS in the model group was increased(尸<0.05),and the secretion of FINS in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.05).Compared with the control group,the HOMA-IR in the model group was increased(P<0.01),and the HOMA-IR in the Jiangtang Sanhuang tablet group was lower than that in the model group(P<0.01).Compared with the control group,the HOMA-β in the model group was decreased(P<0 01),and the HOMA-βin the Jiangtang Sanhuang tablet group was improved compared with the model group(P<0.05).Compared with the control group,the ISI in the model group was significantly lower(P<0.01),and the ISI in the Jiangtang Sanhuang tablet group was significantly higher than that in the model group(P<0.01).Pathological examination of pancreatic tissue showed that in the control group,a small amount of islet volume,cell mass hyperplasia and islet interstitial fibrous tissue hyperplasia could be seen in 2 cases of control group.In the other 8 cases,the tissue structure of pancreas was intact and no abnormal changes were found.Model group:a large number of pancreatic islets were enlarged,the boundary was blurred,islet cell proliferation and interstitial fibrous tissue fibrosis were seen under the microscope in 9 cases of animal pancreas.In the other 1 case,the tissue structure of pancreas was intact and no abnormal changes were found.Jiangtang Sanhuang tablet group:10 cases of pancreatic tissue showed enlarged islet volume,blurred boundary,islet cell proliferation and interstitial fibrous tissue fibrosis.In terms of degree,the islet volume and the degree of islet cell proliferation in the Jiangtang Sanhuang tablet group were better than those in the model group.3.Effect of Jiangtang Sanhuang tablet on dedifferentiation of islet β cells in SD rats fed with high glucose and high fat diet.The expression site of Insulin was located in the islet,and the cytoplasm of islet cells in each group was weak to moderately positive.Compared with the control group,the model group had a downward trend(P<0.05),and the Jiangtang Sanhuang tablet group had an upward trend compared with the model group(P<0.05).The Pdx-1 expression site was located in the islet,and the islet cells in each group were weakly to moderately positive.Compared with the control group,the model group had a downward trend(P<0.05),and the Jiangtang Sanhuang tablet group had an upward trend compared with the model group(P<0.05).The FoxO1 expression site was located in the islet,and all islet cells were moderately to strongly positive.Compared with the control group,the model group had an upward trend(P<0.05),and the Jiangtang Sanhuang tablet group had a downward trend compared with the model group(P<0.05).The expression of MafA protein was moderately to strongly positive in all islet cells,and there was a downward trend in the model group compared with the control group(P<0.05).Compared with the model group,Jiangtang Sanhuang tablet group had an upward trend(P<0.05).4.Study on the effect of Jiangtang Sanhuang tablet on Serum inflammatory factors in SD Rats with High glucose and High Fat Diet based on Proteomics.Fourteen differential factors were screened by protein chip,which were I CAM-1,TIMP-1,CNTF,Activin A,Eotaxin,Galectin-3,Gas 1,IL-13,LIX,Neuropilin-2,TWEAK R,Decorin,Erythropoietin and Galectin-1.There were four kinds of proteins,Activin A,Eotaxin,Galectin-3 and CNTF,which were different between the model group and the Jiangtang Sanhuang tablet group.There were 13 proteins in ICAM-1,TIMP-1,Activin A,Eotaxin,Galectin-3,Gas 1,IL-13,LIX,Neuropilin-2,TWEAK R,Decorin,Erythropoietin and Galectin-1 between the model group and the control group.KEGG enrichment analysis suggests that the differential factors between the model group and the control group were involved in cytokine-cytokine receptor interaction,IL-17 signaling pathway,TNF signaling pathway,asthma,rheumatoid arthritis,viral protein interaction with cytokines and cytokine receptors.TGF-β signaling pathway and HIF-1 signaling pathway.In order to ensure the accuracy of protein chip data,we selected Galectin-3,ICAM-1NRP2,TIMP-1 these four differential proteins to verify,Elisa verification results are consistent with the results of protein chip.Compared with the control group,the levels of Galectin-3(P=0.00),ICAM-1(P=0.00),NRP2(P=0.02)and TIMP-1(P=0.00)in the model group were significantly higher than those in the control group.Compared with the model group,Galectin-3(P=0.00)and TIMP-1(P=0.00)were down-regulated,the difference was statistically significant,but there was no significant difference in ICAM-1 and NRP2(P>0.05).Conclusions1.High-sugar and high-fat diet can lead to weight gain,disorder of glucose and lipid metabolism,insulin resistance and damage of islet β cell function in SD rats.2.Jiangtang Sanhuang tablet can improve the disorder of glucose and lipid metabolism and insulin resistance in SD rats with high glucose and high fat diet,slow down the weight gain and reduce the function damage of islet βcells.3.Jiangtang Sanhuang tablet can up-regulate the expression of Insulin and improve the function of islet β cells,which may be through down-regulating the expression of Fox01 and up-regulating the expression of MafA and Pdx-1,so as to inhibit the dedifferentiation of islet β cells and protect the function of islet β cells.4.Jiangtang Sanhuang tablet may also down-regulate the expression of Galectin-3 and TIMP-1 proteins in serum of SD rats fed with high-sugar and high-fat diet,improve inflammation,pancreatic fibrosis and protect islet β-cell function.