Study On The Therapeutic Mechanism And Compatibility Effect Of Scutellaria-Coptis Herbal Couple On T2DM Based On The Gut Microbiota And The Intestinal Mucosal Barrier

Purpose:This dissertation explored the related problems of herbal medicine Scutellaria-Coptis（SC）herbal couple in treating type 2 diabetes mellitus（T2DM）from the perspective of he gut microbiota and the intestinal mucosal barrier.After confirming that SC could inhibit LPS mediated metabolic inflammation,we attempted to explore the molecular mechanism of SC in repairing the intestinal mucosa damage.Using16Sr RNA high-throughput sequencing and SD rats as well as ICR mice animal models,we attempted to explore whether the pharmacological effect of SC was achieved by targeting a certain intestinal bacteria.In addition,from the perspective of the gut microbiota and the intestinal mucosal barrier,we also wanted to explore the relevant evidence of the compatibility of SC（mutual reinforcement）,dose dependent（heavy dosage for severe diseases）and side effects（hurt the spleen and stomach）,so as to provide a reference of clinical rational use of herbal medicine.Methods:In this study,T2DM rats and mice induced by high-fat diet+STZ were used as experimental subjects.The rats were divided into nomall control group（NC group）,T2DM model group（DC group）,T2DM+Scutellaria-Coptis high,medium and low dose group(8.4,4.2 and 2.1g·kg^-1·day^-1,DHSC,DMSC,DLSC group),normal rats+Scutellaria-Coptis medium dose group(4.2g·kg^-1·day^-1,NSC group)and metformi-n group(194.25mg·kg^-1·day^-1,DME group).Evaluated the overall state of the rats by observing/recording/detecting the general condition,amount of food and drinking water,weight,blood sugar,etc;By measuring the serum insulin level,calculating the homeostasis model assessment-Insulin resistance（HOMA-IR）,oral glucose tolerance test（OGTT）,insulin tolerance test（IPITT）,blood lipid and free fatty acid（FFA）levels to assess glucose and lipid metabolism in rats;HE staining and light microscopy were used to observe the pathological changes of pancreas,ileum and colon;ELISA method was used to detect the levels of the markers of intestinal mucosal damage in serum such as LPS,diamine oxidase（DAO）,D-lactate（D-LA）,and inflammatory factors in serum and intestinal tissues such as interleukin-1β（IL-1β）,interleukin-6（IL-6）and tumor necrosis factor-α（TNF-α）;biochemical method was used to detect the level of superoxide dismutase（SOD）,catalase（CAT）,glutathione peroxidase（GSH-PX）,and Malondialdehyde（MDA）;immunohistochem-ical method was used to detect the levels of the tight junction protein such as Claudi-n-1,ZO-1 and Occludin protein in ileum.The levels of toll like receptor-4（TLR-4）,nuclear factor kappa Bp65（NF-κBp65）in pancreas and TLR-4,TRIF-related adaptor molecule（TRAM）,β-interferon TIR domain adaptor protein（TRIF）,tumor necrosis factor receptor 1（TNFR-1）,TNFR-associated factor-2（TRAF-2）,receptor-interacting serine/threonine protein kinases（RIPK-1）,phosphorylated inhibitor of nuclear factor kappa-B kinase（p-IKKβ）and NF-κBp65 protein in ileum were detected by Western blotting;16S r RNA amplicon sequencing was used to detect the change of gut micro-biota,and the contents of acetic acid,propionic acid and butyric acid in rats feces were detected by high performance liquid chromatography（HPLC）.The mice were divided into normal control group（NC group）,T2DM model group（MC group）,Scutellaria group(6.7g·kg^-1·day^-1,DS group),Coptis group(6.7g·kg^-1·day^-1,DC group),Scutellaria-Coptis Group(13.4g·kg^-1·day^-1,DSC group)and metformin group(308.3mg·kg^-1·day^-1,DME group).Evaluated the overall state of the mice by observing/recording/detecting the general condition,amount of food and drinking water,weight,blood sugar,etc;by measuring the serum insulin level,calculating HOMA-IR index,OGTT and ITT to evaluate Insulin sensitivity;HE staining and light microscopy were used to observe the pathological changes of ileum and colon;ELISA method was used to detect the levels of the markers of intestinal mucosal damage in serum such as LPS,DAO,D-LA,and inflammatory factors in serum and intestinal tissues such as IL-1β,IL-6,IL-18and TNF-α;RT-PCR was used to detect the gene and protein expression of the tight junction proteins such as Claudin-1,Claudin-2,ZO-1 and Occludin in ileum;16S r RNA amplicon sequencing was used to detect the change of gut microbiota,and the contents of acetic acid,propi-onic acid and butyric acid in mice feces were detected by HPLC.Results:1. The effect of Scutellaria-Coptis on glucose and lipid metabolism of T2DMSC herbal couple could significantly reduce the blood glucose and body weight,improve the level of blood lipid（TG,TC,LDL-C,HDL-C）and FFA,promote insulin secretion and relieve insulin resistance in T2DM rats,and the effect of DHSC group was the best;both single and combined use of Scutellaria and Coptis could significan-tly improve the disorders of glucose metabolism in T2DM mice,and the effects of DC and DSC group was better.2. The effect of Scutellaria-Coptis on metabolic inflammation of T2DMSC could significantly reduce the level of LPS,IL-6 and TNF-αin serum of T2DM rats（P<0.05,and the effect of DHSC group is the best）,improve islet cells necrosis and inflammatory cell infiltration in T2DM rats,and significantly reduce the expression of TLR-4 and NF-κBp65 protein in the pancreas of T2DM rats,and the effect of DHSC group was the best,which presented a significant difference in dose and efficacy.The correlation analysis indicated that there was a positive correlation between serum LPS and fasting blood glucose,HOMA-IR,TC,LDL-C,TG,FFA,IL-6,TNF-α,and a negative correlation with body weight and insulin.3. The effect of Scutellaria-Coptis on intestinal mucosal barrier of T2DM3.1. The levels of DAO and D-LA in serum markersIn the rat experiment,compared with NC group,the levels of DAO and D-LA in DC group increased significantly（P<0.05）.After taking SC,the levels of DAO and D-LA decreased,but only DHSC group had statistical significance（P<0.05）.The results in the mouse experiment were similar to those in the rat experiment.3.2. Intestinal histopathologyThe experiment of rats and mice suggested that in the model of T2DM induced by high-fat diet and streptozotocin（STZ）,the pathological changes of ileum and colon showed necrosis of epithelial cells,pyknosis and fragmentation of nuclei,infiltration of inflammatory cells,atrophy of intestinal glands,etc,in the SC intervention group,the above pathological changes were alleviated in varying degrees.3.3. The intestinal tight junction proteinsThe results of immunohistochemistry in rats suggested that compared with NC group,the expression of ZO-1,Occludin and Claudin-1 protein in DC group decrea-sed significantly（P<0.05）,and after the treatment of SC,the expression of protein showed an increasing trend（P<0.05）,meanwhile,the effect of high dose of SC was more obvious（P<0.001）;the results of RT-PCR and immunofluorescence in mice showed that the level of ZO-1,Occludin and Cludin-1 in MC group decreased significantly,but the level of Claudin-2 increased significantly,and after SC treatment,the corresponding gene and protein expression tended to recover to the nomall control group.4. The molecular mechanism of Scutellaria-Coptis in improving intestinal mucosa injury of T2DM4.1. AntiinflammatoryThe experimental results of rats suggested that the expression of IL-1β,IL-6,TNF-αin colon and ileum tissue of DC group was significantly higher than that of NC group（P<0.05）,after using SC,the level of inflammatory factors was signifi-cantly lower（DHSC group P<0.01）.The experiment of mice showed that the expres-sion of IL-1β,IL-6,TNF-α,IL-18 in ileum tissue of MC group was significantly higher than that of NC group（P<0.05）,and after SC intervention,the level of inflammatory factors decreased significantly.4.2. AntioxidantThe experimental results of rats suggested that compared with NC group,the levels of SOD,CAT,GSH-Px in DC group were lower,while the level of MDA was higher（P<0.05）.SC could significantly improve the levels of CAT and MDA.4.3. TLR-4/TRIF and TNFR-1/TNFR-1/NF-κB signaling pathwayIn the rat experiment,compared with NC group,the expression of TLR-4,TRAM,TRIF,TNFR1,TRAF-2,RIPK-1 protein in DC group was significantly up-re-gulated（P<0.05）.SC could significantly inhibit the expression of these molecules,and DHSC group had the best effect,showing significant dose-response difference.5. The effect of Scutellaria-Coptis on the gut microbiota5.1. Normal rat modelSC could reduce the number and diversity of the gut microbiota in normal rats:at the level of phylum,it could increase Firmicutes/Bacteroides（F/B）,reduce the abun-dance of Proteobacteria;On the level of family and genus,it could increase the abund-ance of Lachnospiraceae,Erysipelototrichaeae,Lachnospiraceae NK4A136 group,Pr-evotellaceae UCG-003,decrease the abundance of Lactobacillaceae,Enterobacteria-leae,Lactobacillas,Escherichia-Shigella,and inhibit the production of intestinal SCFAs.5.2. T2DM rat modelSC could reduce the number and diversity of the gut microbiota in T2DM rats:at the level of phylum,it could reduce the abundance of Proteobacteria;at the family level,it could increase the abundance of Lachnospiraceae,Prevotellacae,Erysipelot-otrichaeae,and with the increase of the dosage,the effect of SC on prevotellacae proliferation was gradually enhanced,while the effect of promoting Lachnospiraceae proliferation was gradually weakened;it could inhibit the abundance of Enterobac-taceae,Lactobacillaceae,Biofidobacteria.At the genus level,the abundance of Lachnospiraceae NK4A136 group,Prevotella 9,Prevotella UCG-003,Alloprevotella,Prevotella NK3B31 group,Prevotella 1,Coprocccus 2 could be increased,and the abundance of Lachtobacillus,Ruminocaceae UCG-005,Eschericia-Shigella,Rumin-occus 2,Enterobacter,Enterococcus could be reduced;In addition,SC could increa-se the content of intestinal SCFAs,but the data was not statistically significant.5.3. T2DM mouse modelSC could increase the number and diversity of the gut microbiota in T2DM mouse:at the level of phylum,it could increase F/B and decrease the abundance of Proteobacteria;at the family level,it could increase the abundance of Lachnospi-raceae,Rikenellacae and Ruminocaceae,and decrease the abundance of Enterobac-terialeae.At the genus level,Scutellaria-Coptis could promote the proliferation of Alistipes,Ruminiclostridium 9,Latobacillus,Lachnospiraceae NK4A-136 group and reduce the abundance of Eschericia-Shigella,Enterobacter and Desulfovibrio.Furthe-rmore,SC could increase the content of SCFAs in intestine,and the results of butyric acid were statistically significant（P<0.05）.6. The compatibility effect of Scutellaria-Coptis6.1. Based on the intestinal mucosal barrierThe mouse experiment confirmed that compared with the single use of Scutella-ria-Coptis,the combined use of them could reduce the levels of serum LPS,DAO and D-LA,improve the damage of intestinal mucosa,and inhibit the expression of intest-inal inflammatory factors such as IL-6,TNF-α,IL-1βand IL-18.6.2. Based on the gut microbiotaThe experimental results of mice suggested that compared with the single use of Scutellaria-Coptis,the combined use of them could promote the proliferation of Lach-nospiraceae,Rikenellaceae,Ruminococcaceae,Alistipes,Ruminiclostridium 9,Lact-obacillus,and the accumulation of butyrate in intestine;at the same time,the combi-nation of SC could enhance the inhibition of the potential pathogenic bacteria such as Proteobacteria,Enterobacteriaeae,Eschericia-Shigella,Enterobacter,Desulfovibrio,etc.;the correlation analysis suggested that the above harmful bacteria had a positive correlation with the level of intestinal inflammatory factors.Conclusions:1.The gut microbiota disorder is related to the T2DM“hot and dirty poison”.SC as a heat-clearing herbal couple can regulate the gut microbiota,improve the mucosal barrier,inhibit the intestinal LPS“leak”,inhibit pancreas inflammatory response and glycolipid metabolism disorders of T2DM.2.SC can repair T2DM intestinal mucosa damage by inhibiting intestinal inflamm-atory response and oxidative stress.,its molecular mechanism involves the regulation of TLR-4/TRIF/NF-κB and TNFR-1/NF-κB pathway,and the high dose has the best effect.3.SC can repair intestinal mucosa and improve the glycolipid metabolism by inhibit-ing the proliferation of harmful bacteria and increasing the abundance of bacteria with promoting SCFAs production and anti-inflammatory effects;compared with single use,compatibility of SC can enhance the above pharmacological effect,which may be the important reason for mutual reinforcement of SC.4.In both rat and mouse models,SC can increase the abundance of Lachnospiraceae and Lachnospiraceae NK4A136 group,inhibit the proliferation of Proteobacteria,Ent-erobacteriaee,Eschericia-Shigella and Enterobacter,which may be the“targeted inte-stinal bacteria"of SC.5.The rat experiment suggestes that the mechanism of“hurt the spleen and stomach”of SC may be related to the inhibition of the proliferation and the diversity of bacteria and reduce the abundance of beneficial bacteria such as Bifidobacterium and Lactoba-cillus,but the results of mouse experiment don’t support this conclusion.