Study On The Mechanism Of Sanhuangtangshenkang On TLR4/MyD88/NF-κB Mediated By Intestinal Flora In Rats With Type 2 Diabete

Purpose:The network pharmacology method was used to screen the potential targets of Sanhuangtangshenkang on type 2 diabetes,and the molecular mechanism of treatment was predicted.The effect of Sanhuangtangshenkang on intestinal flora and TLR4/MyD88/NF-κB in type 2 diabetic rats was analyzed by 16S rDNA high-throughput sequencing and animal experiments.To provide a new theoretical basis for Sanhuangtangshenkang in the treatment of type 2 diabetes.Materials and Methods:1.Using TCMSP,SymMap,TCMID and TCM Databases@Taiwan databases to collect the active components of Sanhuangtangshenkang,and search the literature to supplement the active components of hirudo.Obtain the corresponding Target in the above database and Swiss Target Prediction data platform.GeneCards,OMIM and TTD databases were used to obtain targets related to type 2 diabetes.Cytoscape 3.9.0 and String 11.5 were used to construct the protein interaction network(PPI)map of drug-disease common targets.The core targets were selected and analyzed using DAVID database for gene ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG).Autodock tools 1.5.7 was used to perform molecular docking between the selected targets and the corresponding active components.2.Fifty SPF SD rats were fed adaptive diet for one week,then 10 rats were selected as blank control group by random number method and fed standard diet,and the other 40 rats were fed high sugar and high fat diet as type 2 diabetes group.After feeding for 4 weeks,streptomycin with 2%sodium citrate buffer(pH4.5)was injected intrabacely at a rate of 30mg/kg.After successful modeling,they were randomly divided into model group,high-dose group,low-dose group and metformin group.The model group was not fed the drug,and the treatment group was fed the drug for 12 weeks.At the 12th week of feeding(from day 81),rat stool samples were collected using sterilized EP tubes,then frozen in liquid nitrogen for testing,and 16S rDNA high-throughput sequencing of intestinal flora was performed.At the end of the experiment,fasting for 12 hours and free drinking water,abdominal aorta blood and colon tissue were taken under anesthesia.Serum fasting blood glucose(FBG),cholesterol(TG),triglyceride(TC),high density lipoprotein(HDL-C),low density lipoprotein(LDL-C)levels were measured by automatic biochemical analyzer.Serum fasting insulin(FINS),lipopolysaccharide(LPS)and interleukin-1β(IL-1β)levels were detected by enzyme-linked immunosorbent assay(Elisa),and insulin resistance index(HOMA-IR)was calculated.The colonic morphology was observed by HE staining.The levels of toll-like receptor 4(TLR4),myeloid differentiation factor(MyD88)and nuclear transcription factor-κB(NF-κB)protein in colon tissues were detected by Western Blot.16S rDNA high-throughput sequencing was used to detect the microbial diversity of intestinal flora in feces.Results:1.There are 63 effective components and 159 potential targets of Sanhuangtangshenkang in the treatment of type 2 diabetes.54 core targets such as STAT3,INS,AKT1,TLR4,IL-1β,and 58 key components such as quercetin,berberine,luteolin and rhein were obtained through screening.The core targets mainly involved HIF-1 signaling pathway,PI3K-Akt signaling pathway,TNF signaling pathway,Toll-like receptor signaling pathway and other 115 pathways.It is associated with positive regulation of RNA polymerase Ⅱ promoter transcription,negative regulation of apoptotic process,lipopolysaccharide-mediated signaling pathway,protein binding,identical protein binding,nucleus,cytoplasm,and other GO enrichment functions.The selected TLR4 and IL-1β targets docking well with berberine,respectively.2.1 Serum FBG,FINS and HOMA-IR levels in rats:Compared with the normal group,the serum FBG,FINS and HOMA-IR in the model group were significantly increased(P<0.01).Compared with the model group,the levels of serum FBG and HOMA-IR in the high-dose group,low-dose group and metformin group were decreased(P<0.05 or P<0.01),the levels of serum FINS in the low-dose group and metformin group were decreased(P<0.05 or P<0.01),and there was no significant difference between the high-dose group and the model group.Compared with the metformin group,the levels of FINS in the high-dose and low-dose group were increased(P<0.05 or P<0.01).2.2 Serum lipid levels in rats:Compared with normal group,the serum levels of TG,TC and LDL-C in model group were significantly increased(P<0.01),while the level of HDL-C was significantly decreased(P<0.01).Compared with model group,serum TG in low-dose group and metformin group was significantly decreased(P<0.01),but there was no significant difference between high-dose group and model group.Serum TC in high-dose,low-dose group and metformin groups was decreased(P<0.05 or P<0.01);Serum HDL-C was increased(P<0.05)and serum LDL-C was decreased(P<0.05)in high-dose group and metformin group,but there was no significant difference in serum HDL-C and LDL-C in low-dose group.Compared with the metformin group,the high-dose group significantly increased the serum TG content(P<0.01).2.3 HE staining of rat colon tissue:The results showed that in the normal group,the structure of the colon mucosa was complete,the mucosa cells were arranged neatly,the morphology was complete,and no damage was observed.Compared with the normal group,the mucosa of the model group was filled with a large number of inflammatory cells,the structure of the mucosa was destroyed,and the inflammatory cells infiltrated into the connective tissue of the submucosa.The structure and morphology of the mucosa in the high-dose,low-dose and metformin groups were improved,and the degree of inflammatory cell infiltration was significantly reduced.2.4 Expression of inflammatory cytokines and proteins in rats:Elisa was used to detect LPS and IL-1β.The results showed that compared with normal group,serum LPS and IL-1β levels in model group were significantly increased(P<0.01),while serum LPS and IL-1β levels in high-dose,low-dose and metformin groups were decreased compared with model group(P<0.05 or P<0.01).Compared with the metformin group,the serum LPS level in the high-dose group was significantly increased(P<0.01),and the serum IL-1βlevel in the high-dose group and the low-dose group was significantly increased(P<0.01).Western Blot analysis of the protein expression levels of TLR4,MyD88 and NF-κB in colon tissue showed that compared with normal group,the protein expression levels of TLR4,MyD88 and NF-κB in colon tissue of rats in model group were significantly increased(P<0.01).High-dose group,low-dose group and metformin group were significantly decreased(P<0.01).Compared with the metformin group,the high-dose of Chinese medicine group increased significantly(P<0.01).2.5 Analysis of intestinal flora in rats:Among the top ten phylum level relative abundance,there were differences among the groups of Patescibacteria,Proteobacteria and Spirochaetota(P<0.05).Among the top 20 relative abundance in genus level,there were differences among Bacteroides,Blautia,Escherichia_Shigella,Faecalibacterium,Lachnospiraceae_NK4A136_group,Lactobacillus and unclassified_Oscillospiraceae(P<0.05).Further analysis showed that compared with normal group,Proteobacteria,Escherichia_Shigella and Bacteroides were significantly increased in model group(P<0.05),while Proteobacteria,Escherichia_Shigella and Bacteroides were decreased in high-dose,low-dose and metformin groups compared with model group(P<0.05).The abundance of Spirochaetota,Lachnospiraceae NK4A136_group and unclassified_Oscillospiraceae were significantly increased in high-dose group(P<0.05),while the abundance of Lactobacillus,Blautia and Faecalibacterium in low-dose group was significantly increased(P<0.05).The abundance of Patescibacteria and in metformin group was significantly increased(P<0.05).Conclusion:1.Quercetin,berberine,luteolin and rhein in Sanhuangtangshenkang may act on HIF-1 signaling pathway,PI3K-Akt signaling pathway,TNF signaling pathway,toll-like receptor signaling pathway and other signaling pathways to play a role in type 2 diabetes through multiple targets such as STAT3,INS,AKT1,TLR4 and IL-1β Regulatory effect.2.Sanhuangtangshenkang can reduce the levels of FBG and FINS in serum of type 2 diabetic rats,improve blood lipid levels and HOMA-IR,and thus improve glucose and lipid metabolism disorders and insulin resistance.3.Sanhuangtangshenkang can reduce the content of LPS and IL-1β in serum of type 2 diabetic rats,and reduce the expression of TLR4,MyD88 and NF-κB protein in colon tissue of type 2 diabetic rats,so as to reduce the inflammatory state of type 2 diabetic rats.4.Sanhuangtangshenkang alleviates the inflammatory cell infiltration of colonic mucosa in type 2 diabetic rats and restores the morphology of colonic mucosa.5.Sanhuangtangshenkang can regulate the intestinal flora structure of rats with type 2 diabetes,reduce the abundance of harmful bacteria such as Proteobacteria,Escherichia_Shigella,and increase the abundance of beneficial bacteria.The high-dose group can increase the abundance of Spirochaetota,Lachnospiraceae_NK4A136_group and unclassified_Oscillospiraceae.Low-dose group can increase the abundance of Lactobacillus,Blautia and Faecalibacterium.