To Explore The Effect And Mechanism Of Dahuang Huanglian Xiexin Decoction On Type 2 Diabetes Mellitus Based On "Microbiota-Th17 Cells"

Objective:1.To evaluate the efficacy of Dahuang Huanglian Xiexin Decoction(DHXD)on type2 diabetes mellitus(T2DM).2.To investigate the therapeutic effect of DHXD on T helper 17(Th17)cells-mediated inflammation and insulin resistance in AT.3.To clarify the regulatory effect of gut microbiota on Th17 cells and evaluate the influence of DHXD on gut microbiota and Th17 cells.To elucidate the potential mechanism of DHXD in the treatment of T2DM,we explored the“crosstalk”between intestine and adipose tissue,which was mediated by Th17 cells.Methods:Firstly,we searched numerous studies and did meta-analysis to find the relationship between Th17 cells and T2DM.In this study,high-fat diet and streptozocin were used to establish T2DM mouse model.The experimental groups were divided into control group,model group,metformin group,low-,middle-,and high dose group of DHXD.We observed the general conditions,blood lipid and blood glucose of mice to evaluate the glucose and lipid metabolism.Oral glucose tolerance test,insulin tolerance test and homeostasis model assessment for insulin resistance were used to estimate insulin resistance.HE staining was utilized to observe the pathologies of white adipose tissue(WAT),brown adipose tissue and colon.Quantitative real-time PCR(qPCR)was used to detect the levels of Interleukin 17(IL-17)and Interferonγ(IFN-γ)mRNA in WAT and intestine.Western blot(WB)was used to detect the protein expression levels of JNK/p-JNK,IRS-1/p-IRS-1 and AKT/p-AKT in insulin signal transduction pathway.The levels of ZO-1、Occludin and Claudin-1 in colon were also detected by WB.To observe the quantities of goblet cells in colon,AB-PAS staining was used.Intestinal permeability was evaluated by fluorescein isothiocyanate-dextran.Intestinal Th17,Th1/17 and Th1 cells were detected by flow cytometry.The changes of gut microbiota were analyzed by 16S rRNA sequencing.Results:1.The levels of Th17 cells and IL-17 in peripheral blood were up-regulated in patients with T2DM.A total of 10 studies from 2014 to 2021 involving 586 patients with T2DM and healthy people were included in the study,with a mean age of 55.48 years(range 43.0to 68.1 years)and a mean BMI of 25.3 kg/m~2(range 20 to 30.4 kg/m~2).51.7%of patients were male(range 30 to 67%).Meta-analysis suggested that compared with the controls,the patients with T2DM had significantly increased levels of Th17 cells(SMD,1.74;95%CI,0.47-3.01;P<0.01)and IL-17(SMD,2.17;95%CI,0.06-4.28;P<0.01)in peripheral blood.2.DHXD improved general conditions,glucose metabolism and insulin resistance in T2DM mice.The state was improved in T2DM mice,who was more active.The rough hair and hair loss were improved.Loose but normal volume stool and excessive urination were observed with the treatment of DHXD.DHXD can significantly improve the polydipsia(P<0.01).Fasting blood glucose and random blood glucose were down-regulated by DHXD in T2DM mice(P<0.01).The glucose tolerance and insulin tolerance were improved by DHXD.The homeostasis model assessment for insulin resistance level was decreased by DHXD in T2DM mice(P<0.01).3.Inflammation and insulin resistance in adipose tissue were ameliorated by DHXD.DHXD could reduce the WAT/body weight ratio of T2DM mice.It also reduced the white adipocyte volume.Reduced IL-17 and IFN-γmRNA were observed in WAT(P<0.01),which suggested that DHXD decreased inflammation in WAT.As for the insulin signal transduction pathways,DHXD inhibited the phosphorylation of JNK(P<0.05)and IRS-1(P<0.01),and promoted the phosphorylation of AKT(P<0.01).4.DHXD improved the diversity of gut microbiota and enriched the structure of it.DHXD may decrease intestinal Th17 cells by promoting relative abundance of Bacilli、Lactobacillales and Lactobacillus,and/or decreasing relative abundance of Bacteroides.Flow cytometry suggested that the levels of intestinal Th17 and Th1/17 cells were decreased by DHXD(P<0.05).The nuclear transcription factor RORγt mRNA of Th17 cell was also down-regulated(P<0.01).This kind of effect depended on gut microbiota(P<0.05).However,the reduction of Th1 cells was independent of gut microbiota(P<0.05).Correlation analysis showed that intestinal Th17,Th1/17 and Th1 cells were positively correlated,which were gradually on the rise(P<0.01).It was showed that T2DM reduced gut microbiota diversity and changed the structure of it.DHXD improvedαandβdiversity of gut microbiota(P<0.05)and decreased relative abundance of Bacteroides(P<0.05).Bacilli,Lactobacillales and Lactobacillus may be the biomarker of DHXD through LEfse analysis.Metagenome Seq analysis showed that compared with the model group,the Helicobacter was up-regulated and the[Ruminococcus]torques group was down-regulated through DHXD(P<0.01).5.DHXD could repair intestinal barrier as well as reduce intestinal permeability.The shape and number of intestinal crypts were improved by DHXD.DHXD also limited the infiltration of inflammatory cells into gut.The quantities of goblet cells were elevated,which could secrete mucus to form an intestinal barrier.DHXD could up-regulate the expression of colon tight junction proteins Occludin and Claudin-1(P<0.05).DHXD significantly reduced intestinal permeability(P<0.01),which was observed by fluorescein isothiocyanate-dextran.6.The“corsstalk”between gut and AT may be mediated by Th17 cells.Elevated intestinal Th17 cells,IL-17 and IFN-γwere found in T2DM mice(P<0.01),along with damaged intestinal barrier and increased intestinal permeability(P<0.01).Likewise,high levels of IL-17(P<0.01)and IFN-γ(P<0.05)were observed in AT,which suggested that there was inflammation in AT.Correlation analysis indicated that inflammation in AT was positively correlated with intestinal Th17 cells.However,due to small samples,it did not reach statistical significance.There was a positive correlation between intestinal permeability and AT inflammation(P<0.05),which suggested the gut may“communicate”with AT.Conclusions:1.Glucose metabolism and insulin resistance were ameliorated by DHXD in T2DM mice.2.Inflammation and insulin resistance in adipose tissue were ameliorated by DHXD.3.DHXD reduced the levels of intestinal pro-inflammatory Th17 cells through the regulation of gut microbiota.This kind of mechanism may participate into the down-regulation of inflammation and insulin resistance in AT by DHXD.There may be a“crosstalk”between gut and AT,and Th17 cells were the“bridge”of this connection.Targeting“gut microbiota-Th17 cells”may be a potential therapy for T2DM.4.DHXD may decrease intestinal Th17 cells by promoting relative abundance of Bacilli,Lactobacillales and Lactobacillus,and/or decreasing relative abundance of Bacteroides.5.In the context of T2DM,intestinal Th17 cells,Th1/17 cells and Th1 cells were positively correlated,which were gradually on the rise.