| Objective In recent years,with the change of lifestyle and aging of the society,the incidence of diabetes is increasing.How to effectively prevent and delay the incidence of diabetes have become the top problem of medical and health work.Increased level of plasma lipopolysaccharide(endotoxinemia),one of the cell wall component of gram negative bacteria,has been confirmed to be closely related to chronic inflammation of diabetes.The role of gut hormones,intestinal flora and intestinal mucosal barrier in the pathogenesis of T2 DM has been paid more and more attention.In order to observe the effect of Huang-lian-Jie-Du Decoction on preventing and/or delaying the onset of type 2 diabetes mellitus in rats with impaired glucose tolerance(IGT),ZDF rats(Zucker Diabetic Fatty rat,fa/fa),suffering from type2 diabetes mellitus spontaneously due to mutation of leptin receptor,were selected in this research for drug intervention,and the possible mechanism was studied.MethodsA high fat diet,Purina#5008,was used,and 20 male Specific pathogen Free ZDF rats were randomly divided into model group(n=10)and Huang-lian-Jie-du-Decoction intervention group(Huanglian Group,n=10)once they entered into stage of impaired glucose tolerance.The normal ZL(Zucker Lean rats,fa/+)rats were defined as Normal Group to contrast(n=5).The Huanglian Group was treated with Huang-lian-Jie-du-Decoction at dose of 0.075g/d,the Model Group and Normal Group were received only same volume of distilled water as vehicle.Weekly oral glucose tolerance test(OGTT)was used to determine the incidence of rats,the intervention were terminated if all rats in Model Group developed diabetes.Incidence of diabetes was calculated.Changes of blood glucose,body weight and food intake were recorded every week,and blood for each period was taken.Plasma insulin(INS),glucagon(GC),lipopolysaccharide(LPS),glucagon-like peptide-1(GLP-1)and glucagon-like peptide-2(GLP-2)were measured by enzyme linked immunosorbent assay(ELISA),and HOMA-IR index was calculated to evaluate insulin resistance.Effect of Huang-lian-Jie-Du-Decoction on blood lipid was also been observed.Liver and kidney functions were measured to to evaluate the side effects of drugs.The fresh fecal samples of rats in each group were taken to observe microbiota structure changes by 16 Sr DNA high throughput sequencing method.The animals were sacrificed at the end of the experiment to take fresh intestinal tissues,and the ability of intestinal L cells to secrete GLP-1 and GLP-2 was evaluated.Immunohistochemistry method was used to observe the expression of occludin,ZO-1,TLR-4,NF-κB and tumor necrosis factor-α(TNF-α)in different groups.Results1.Incidence of diabetes.All rats were impaired in glucose tolerance at the age of 9weeks when high fat diet Purina#5008 was started at the age of 6 weeks.After intervention for 3 weeks,all rats in Model Group developed diabetes.But in Huanglian Group,50 percent developed diabetes and 50 percent returned to normal.2.General situation.Body weight of each period in Huanglian Group were lower than the Model Group when drug intervention started(P <0.05).After 2 weeks of intervention,food intake of Huanglian Group significantly decreased(P <0.05);After 3 weeks of Huang-lian-Jie-Du-Decoction treatment,the fasting blood glucose and blood glucose at different time points were significantly decreased compared with the model group(P <0.01).3.Plasma levels measured by ELISA.During the natural course of diabetes,the fasting plasma glucagon level(P<0.01),insulin resistance(P<0.01),LPS level(P<0.05),GLP-1 level(P<0.01)of ZDF rats were gradually increased,while insulin level(P<0.01)and GLP-2 level(P<0.01)were decreased.After 3 weeks of intervention,compared with Model Group,the fasting plasma insulin level(P<0.05),HOMA-IR(P<0.01),glucagon level(P<0.05)and LPS level(P<0.01)declined in various degree,The level of GLP-1(P<0.05)was further increased,but there was no significant change in fasting plasma GLP-2 level.4.Evaluation of secretory GLP-1 and GLP-2 capacity of intestinal L cells.After 3weeks of drug intervention,compared with the Normal Group,secretory GLP-1capacity of intestinal L cells of Model Group and Huanglian Group significantly increased,Huanglian Group is more obvious(all P <0.05).Level of GLP-2 secreted by Model Group decreased compared with the Normal Group,and Huang-lian-Jie-Du-Decoction have improved effect on intestinal L cells.5.Morphology of intestinal mucosa observed by HE staining techniques.Damage of intestinal mucosa was found in diabetic rats.Intestinal villi arranged disorderly,irregular,broadening and fusion.There were more inflammatory cells infiltrated in the lamina propria.Huang-Lian-Jie-Du-Decoction improved the intestinal mucosal morphology of rats.6.Immunohistochemistry.The expression of TLR-4 、 NF-κB and TNF-α(all P<0.01)in ileum of Model Group dramatically increased compared with the Normal Group,while the expression of occluding and ZO-1(all P<0.01)in ileum reduced.The above indexes were improved obviously in Huanglian Group.7.Changes of intestinal flora observed by 16 Sr DNA high throughput sequencing.1)There was a high degree of similarity between fecal samples in every group,while distinct structural differences were observed between these groups after 3 weeks of intervention(P<0.05)2)Compared with Normal Group,richness and diversity of Model Group declined obviously,Huang-lian-Jie-Du-Decoction has a significant improvement(all P<0.05).3)At the phylum level,dominant phyla were Bacteroidetes and Firmicutes,followed by Proteobacteria and Verrucomicrobia.Bacteroidetes significantly increased and Firmicutes distinctly decreased in diabetes rats.Huang-Lian-Jie-Du-Decoction started in IGT restored these changes markedly.At the genus level,drugs showed enriching effects on Bacteroides,Oscillospira,Akkermansia,Aggregatibacter,Clostridium,Roseburia and Eubacterium,and inhibiting effects on Prevotella,which is the main component of Model Group.At the level of species,compared with normal group,the relative abundance of Prevotella_copri significantly increased while which of Akkermansia_muciniphila,Lactobacillus_reuteri and Bacteroides_caccae declined in diabetic rats.Huang-Lian-Jie-DuDecoction improved the structure of gut microbiota.8.Biochemical index in plasma.3 weeks of Huang-Lian-Jie-Du-Decoction intervention had no significant effect on liver function(P>0.05);Increased of BUN(P<0.05)and decreased of CR(P<0.01)were found in Model Group and Huanglian Group,drugs effectively reduced UA and blood lipid levels.Conclusion1.ZDF rats were impaired in glucose tolerance at the age of 9 weeks and developed diabetes at the age of 12 weeks when high fat diet Purina#5008 was started at the age of 6 weeks.2.Intervention of Huang-Lian-Jie-Du-Decoction at IGT stage could delay the onset of T2 DM in ZDF rats.The possible mechanism:1)By promoting the GLP-1 secretion of L cells in the gut,drugs reduced food intake,improved islet function,decreased insulin resistance.It also reduced intestinal inflammation,protected the intestinal mucosal barrier,so the level of plasma LPS and chronic inflammatory response were declined,too.2)Fasting plasma GLP-1 levels in diabetic rats were ameliorated,which related to improvement of insulin resistance and impaired glucose metabolism.3)By promoting the GLP-2 secretion of L cells in the gut,drugs reduced intestinal mucosal barrier injury,the level of plasma LPS and chronic inflammatory response were also declined.4)By improving the structure of intestinal microflora,the probiotics and pathogenic bacteria ratio was adjusted,and body energy balance was promoted.Less LPS in gut protected intestinal mucosal barrier,chronic inflammatory response were declined3.The intervention of Huang-Lian-Jie-Du-Decoction showed no obvious side effects of liver function and kidney function,it could improve hyperlipidemia in diabetic rats. |
PDF Full Text Request