Study On Preventive Mechanism Of Jiangtang Xiaozhi Prescription On Type 2 Diabetes Mellitus In ZDF Rats Based On Gut Microbiology And Metabolomics

Type 2 Diabetes mellitus（T2DM）is a chronic metabolic disease that has been widely prevalent worldwide.Therefore,it is necessary to explore effective interventions for the prevention and treatment of type 2 Diabetes mellitus.It is well known that gut microbes play a crucial role in host energy homeostasis,glucose and lipid metabolism and other processes.Current studies have observed an association between gut microbiota and type 2 diabetes.Intestinal microbiota metabolites,such as short-chain fatty acids,lipopolysaccharides,and bile acids,directly or indirectly involved in glucose metabolism,especially bile acid metabolism Farnesoid X receptor（FXR）,as an endogenous ligand of bile acid,can activate a series of signaling pathways leading to abnormal glucose metabolism.Studies have shown that type 2 diabetes mellitus is often accompanied by obesity.Under the guidance of traditional Chinese medicine theory and combined with clinical diagnosis and treatment,the syndrome of type 2 diabetes mellitus is often characterized by deficiency of Qi and Yin,and inter-combination of phlegm and blood stasis.In this study,Jiang Tang Xiao Zhi Prescription has been proved to reduce insulin resistance,improve fibrinolytic activity and inflammatory state,and inhibit the apoptosis of pancreatic cells in clinical application and previous studies.In addition,multiple pharmaceutical components of Jiang Tang Xiao Zhi Prescription can adjust the composition and diversity of intestinal microorganism through different mechanisms to achieve the purpose of improving glucose and lipid metabolism.Therefore,this study intends to evaluate the effectiveness of Jiang Tang Xiao Zhi Prescription in preventing and delaying type 2 diabetes by intervening ZDF rats with impaired glucose tolerance,reveal the relationship between intestinal microbial structure disorder,bile acid metabolism disorder and the progression of type 2 diabetes by 16S rDNA sequencing and metabolomics analysis,and explored the mechanism of Jiang Tang Xiao Zhi Prescription in helping improve glucolipid metabolism and prevent type 2 diabetes mellitus.This study includes the following parts:Literature review:This paper systematically reviewed the biological function of gut microbiota,the changes of gut microbiota in type 2 diabetes mellitus,and the possible mechanism of gut microbiota disturbance causing type 2 diabetes mellitus.Based on the "Spleen and Stomach Theory",the physiological function of gut microbiota was understood and the pathological mechanism of gut microbiota involved in type 2 diabetes was discussed from the perspective of traditional Chinese medicine.Experimental research:Part I:Efficacy evaluation of Jiang Tang Xiao Zhi Prescription on the intervention of glucose and lipid metabolism in ZDF ratsObjective:To observe the effects of Jiang Tang Xiao Zhi Prescription on related indexes of glucose and lipid metabolism in ZDF rats,to evaluate insulin sensitivity and islet β-cell function,and to evaluate the preventive effect of Jiang Tang Xiao Zhi Prescription on type 2 diabetes mellitus.Methods:ZDF rats were fed with high fat diet to establish a prediabetic rat model with impaired glucose tolerance.These pre-diabetes rat models were randomly divided into model group,metformin group（MET,300 mg/Kg）,Jiang Tang Xiao Zhi low dose group（JTXZ-LD,4 g/Kg）,Jiang Tang Xiao Zhi high dose group（JTXZ-HD,student 8 g/Kg）,and with the strains with weeks of ZL rats as normal control group（NC）.NC group and MC group were given the same volume of pure water.During the intervention period,breeding feed was adopted and the drug was administered continuously for 8 weeks.Body weight and OGTT test were measured every 2 weeks,AUC area was calculated,and the changes of body weight,blood glucose and incidence of type 2 diabetes were compared at different time points.After the intervention,blood samples were collected from the abdominal aorta to detect the levels of blood lipids,insulin,C-peptide,glycosylated hemoglobin and liver and kidney function in ZDF rats.HOMA-IR and HOMA-β were calculated to evaluate insulin sensitivity and islet β-cell function.Outcomes:1.In terms of incidence of type 2 diabetes mellitus,there was no difference in the incidence of type 2 diabetes among all groups at 2 weeks of drug intervention（P>0.05）.MET,JTXZ-LD,JTXZ-HD intervention can effectively reduce the incidence of T2DM at 4 weeks of drug intervention（P<0.05）.At 6 to 8 weeks of drug intervention,different doses of Jiang Tang Xiao Zhi Prescription intervention cannot effectively prevent T2DM（P>0.05）and metformin is still significant preventive effect（P<0.05）.2.In terms of body weight,the body weight of MC,MET,JTXZ-LD,JTXZ-HD group increased significantly from 0 to 6 weeks of drug intervention,and there was no difference among all groups（P>0.05）.3.In terms of blood glucose and glucose tolerance,compared with NC group,fasting 2 h postprandial blood glucose and glucose AUC in MC group were significantly increased（P<0.05）.From week 0 to week 2,there was no significant difference in fasting blood glucose of ZDF rats in each intervention group（P<0.05）,but from week 4,fasting blood glucose of JTXZ-LD group was significantly decreased（P<0.05）.Fasting 2 h postprandial blood glucose in MET,JTXZ-LD and JTXZ-HD groups were significantly decreased at week 8（P<0.05,P<0.05,P<0.01）,and there was no statistical difference at week 0-6（P>0.05）.JTXZ-LD and JTXZ-HD groups significantly decreased glucose AUC at week 8（P<0.05,P<0.01）.4.In terms of HbAlc,only the MET group was significantly lower than the MC group（P<0.05）.There was no significant difference between JTXZ-LD,JTXZ-HD groups and MC groups（P>0.05）.5.In terms of fasting insulin and C-peptide levels,compared with NC group,fasting insulin and C-peptide levels in MC group were significantly increased（P<0.05）,and there was no statistical difference among intervention groups（P>0.05）.6.In the assessment of insulin sensitivity and β-cell function,HOMA-IR was significantly increased in MC group and significantly decreased only in JTXZ-LD group after intervention（P<0.05）.HOMA-β level was higher only in NC group,and no difference was found among MC,MET,JTXZ-LD and JTXZ-HD groups（P>0.05）.7.In terms of blood lipid,serum TC,TG,LDL-C and HDL-C in MC group were significantly higher than those in NC group（P<0.05）.Metformin intervention significantly decreased serum TG and LDL-C levels（P<0.05）.The levels of TC and HDL-C were not significantly improved by MET、JTXZ-LD、JTXZ-HD intervention（P>0.05）.8.There was no significant difference in liver and kidney function among the groups（P>0.05）.Conclusions:In the early stage of disease,the effect of JTXZ-LD group and JTXZ-HD group on the prevention of T2DM was the same as that of MET.In the late stage of disease,the prevention effect of traditional Chinese medicine is inferior to that of metformin.MET,JTXZ-LD,and JTXZ-HD did not have significant intervention effects on the body weight of ZDF rats,but showed good effects in reducing fasting and 2h postprandial blood glucose and improving glucose tolerance.In general,JTXZ-LD has more significant effects,and can reduce HOMA-IR,improve insulin sensitivity,and improve insulin resistance.In addition,Jiang Tang Xiao Zhi Prescription and metformin have good safety.Part Ⅱ:Discussion on the mechanism of Jiang Tang Xiao Zhi Prescription regulating intestinal microbiota-bile acid metabolism to prevent type 2 diabetes mellitus in ZDF ratObjective:To observe the changes of intestinal microflora and bile acid of Jiang Tang Xiao Zhi Prescription in ZDF rats,and to further explore the mechanism of Jiang Tang Xiao Zhi Prescription in preventing type 2 diabetes in ZDF rats.Methods:After the experiment,intestinal contents of ZDF rats were collected for 16S rDNA sequencing and metabolomics analysis,and microbial species and bile acid composition in intestinal contents were identified.Correlation analysis was conducted based on phylum and genus levels for different species and different metabolites,and key strains and bile acid types affecting glucose and lipid metabolism were found.The levels of ileum tissue and serum ceramides were detected by ELISA,and the expression of endogenous bile acid receptor FXR,ER stress and apoptotic genes GRP78,CHOP,Bcl-2 and Bax were detected by qPCR.The structure of endoplasmic reticulum of β cells was observed by transmission electron microscopy.The apoptosis of islet β cells was further observed by immunofluorescence.Outcomes:1.Gut microbiota analysisIn terms of a diversity,the Observed species index,Chaol index,Shannon index and Simpson index of the other four groups were significantly decreased except for the higher species abundance in NC group,and there was no statistical difference between the intervention groups and MC group（P>0.05）.In terms of β-diversity,the species composition of NC group and MC group was significantly different,the sample scattered points of MET group,JTXZ-LD group and JTXZ-HD group were relatively aggregated,and the overall similarity of intestinal microbial community was relatively high.After drug intervention,MET group and JTXZ-LD group showed a trend of deviation toward NC group.In terms of microbial composition,at the phylum level,compared with the NC group,the abundance of Firmicutes decreased and Bacteroidetes increased in the MC group.After 8 weeks of intervention,both JTXZ-LD and JTXZ-HD could effectively increase Firmicutes and reduce Bacteroidetes（P<0.05）,and the effect of JTXZ-LD was more significant（P<0.001）.MET reduced the relative abundance of Bacteroidetes（P<0.01）,but had no significant regulatory effect on Firmicutes（P>0.05）.The abundance of Actinobacteria,Proteobacteria,Epsilonbacteraeota,Spirochaetes,Acidobacteria,Gemmatimonadetes,Deferribacteres and Fusobacteria were similar among the groups,but no difference was shown between the groups（P>0.05）.At the genus level,Prevotella₉,[Eubacterium]_coprostanoligenes_group,Lachnospiraceae_NK4 A13 6_group,Anaerostipes,Collinsella,Ruminococcacea E_UCG-014 was significantly different between NC group and MC group（P<0.05）.JTXZ-LD and JTXZ-HD treatments significantly decreased the relative abundance of Prevotella₉（P<0.01,P<0.05）.Anaerostipes was only significantly increased after JTXZ-LD intervention（P<0.05）,and neither MET group nor JTXZ-HD group showed intervention effect.2.Bile acid analysisChenodeoxycholic acid（CDCA）,deoxycholic acid（DCA）and hyodeoxycholic acid（HDCA）were the bile acids that had significant difference between NC group and MC group（P<0.05）.MET,JTXZ-LD and JTXZ-HD could significantly reduce CDCA level（P<0.05）,but had no significant effect on DCA and HDCA（P>0.05）.3.Correlation analysis between gut microbiota and bile acidsAt the level of phylum,Firmicutes and Bacteroidetes showed a high correlation with CDCA,and the difference was statistically significant（P<0.05）.Firmicutes and Bacteroidetes showed a negative correlation with CDCA,while Bacteroidetes showed a positive correlation with CDCA.At the level of genus,prevotella（Prevotella₉）was significantly correlated with ALCA and CDCA（P<0.05,P<0.01）.The genus Blautia was significantly related to UCA（P<0.05）.Lachnospiraceae NK4A136 group was significantly related to HDCA（P<0.01）.Allobaculum was significantly correlated with hyocholic acid（HCA）（P<0.05）.There were significant correlations of subdoligranulum with HCA and glycodesoxycholic acid（GDCA）（P<0.01,P<0.05）,and they were all positively correlated.4.Ileum and serum ceramideCompared with NC group,ileal ceramide level in MC group was significantly increased（P<0.05）,Jiang Tang Xiao Zhi Prescription could significantly reduce ileal ceramide level（P<0.05）,and metformin could also reduce the average ileal ceramide level,although the difference was not statistically significant（P>0.05）.There was no significant difference in serum ceramide among all groups（P>0.05）.5.Expression of ileum FXR and pancreas GRP78,CHOP,Bcl-2 and Bax mRNAThe expression level of FXR mRNA in ileum in MC group was significantly higher than that in NC group（P<0.05）.Meanwhile,the expression of FXR mRNA was decreased in MET group,JTXZ-LD group and JTXZ-HD group（P<0.05）.The expression level of GRP78 mRNA in pancreas of MC group was significantly increased（P<0.05）),while the expression of GRP78 in MET and JTXZ-LD groups was significantly inhibited after treatment（P<0.05）.CHOP showed no statistical difference between groups（P>0.05）.In the expression of apoptotic related genes,JTXZ-LD can promote the up-regulation of Bcl-2 mRNAexpression anddown-regulation of Bax（P<0.05）,while the JTXZ-HD group did not show similar effects（P>0.05）.In the rats with MET intervention,Bax was significantly down-regulated（P<0.05）,but the up-regulation trend of Bcl-2 was not significant（P>0.05）.6.Electron microscopic observation of endoplasmic reticulum structure of β cellsThe NC group showed normal endoplasmic reticulum structure,abundant mitochondria,Golgi apparatus,endoplasmic reticulum and other organelles could be seen in the cells,and the endoplasmic reticulum showed normal slender linear shape without expansion,with a few apoptotic bodies.The MC group had typical characteristics of ER stress,including nucleolus shrinkage,ER expansion and bubble shape and the formation of a large number of apoptotic bodies.MET group and JTXZ-LD group had full nucleolus,basically normal endoplasmic reticulum structure,abundant mitochondria,a few apoptotic bodies.In the JTXZ-HD group,the nucleolus was slightly atrophied,the endoplasmic reticulum was slightly swelled,and apoptotic bodies were also observed.7.Pancreas immunofluorescence observationIn the NC group,the islet morphology was regular,the cells were arranged inorder,and the fluorescence staining of insulin antibody was full,while in the MC group,the islet structure was disordered,the α and β cells were significantly reduced,β cells degranulation was significant.Compared with the MC group,the MET group and the JTXZ-LD group showed improvement,while the JTXZ-HD group showed no significant improvement.Conclusions:Jiang Tang Xiao Zhi Prescription can improve the intestinal microorganism disorder under the condition of chronic hyperglycemia,improve the metabolism of bile acid and restore the healthy relationship between intestinal microorganism and host to a certain extent.In particular,the effect of JTXZ-LD was better,which was consistent with the results of efficacy indicators.Jiangtang Xiaozhi prescription can effectively prevent the progression of T2DM in ZDF rats.The mechanism of action may be that Jiang Tang Xiao Zhi Prescription significantly inhibits the opportunistic pathogen Prevotella₉ in Bachteroidstes and increases the relative abundance of beneficial bacteria Anaerostipes in Firmicutes.Furthermore,CDCA synthesis was reduced,which inhibited the activation of FXR in intestinal epithelium,and reduced the ER stress induced by ceramides,thus better protecting pancreatic β cells.