| Diabetes mellitus(DM)is a metabolic disease characterized by chronic hyperglycemia caused by multiple causes,the increasing blood sugar level caused by the disorder of glucose metabolism can cause damage to multiple systems,leading to chronic progressive disease in tissues and organs such as the kidneys,blood vessels and eyes,among them,diabetic nephropathy(DN)is one of the common diabetic microvascular complications.Intestinal barrier dysfunction is a hot spot in the field of clinical medicine.It is closely related to many diseases,such as systemic inflammatory response syndrome,inflammatory bowel disease,intestinal flora disorders,etc.More pharmacological papers have showed that Gegen Qinlian Decoction(GQD)has anti-inflammatory and antiviral effects,and has a certain therapeutic effect on enteritis and other inflammatory diseases.At the same time,GQD can also adjust the structure of intestinal flora;In addition to inhibiting the growth of pathogenic bacteria,GQD can also protect the integrity of the intestinal mucosal barrier;and in clinical practice,GQD also has a good treatment for diabetes effect.However,the mechanism of GQD on diabetic nephropathy is still unclear.Therefore,we intend to establish a model of diabetic rats,and further study the protective mechanism of GQD on the kidneys of diabetic rats based on the intestinal barrier,so as to provide new therapeutic strategies and methods for the treatment of DN.Purposes: To study the protective effect of GQD on the kidneys of diabetic rats,and further study the related mechanism.Methods: First,establish a diabetic rat model,group them,and then sacrifice the rats after gavage treatment;the materials were taken.The changes in serum urea nitrogen levels was detected by urease method;the serum creatinine level changes was detected by colorimetric method;the pathological structure changes of rat kidney tissues was observed and compare by Hematoxylin-eosin staining(HE)method in the control group,model group and treatment group;the serum malondialdehyde(MDA)level changes was detected by TBA method;the changes in serum catalase(CAT)activity was detected by ammonium molybdate method;the changes in serum superoxide dismutase(SOD)activity levels of rats in each group was detected by WST-1 method;the changes in serum glutathione peroxidase(GSH-PX)activity was detected by UV colorimetric method;the changes in the levels of TNF-a and IL-6 m RNA in the kidney and intestine tissue was detected by RT-q PCR method;the TLR4,NF-κB,My D88,Occludin,Claudin-1,β-actin protein expression changes was detected by Western blot and Immunohistochemistry method;transmission electron microscope was used to observe the changes in the morphology of the tight junctions between the intestinal epithelial cells of rats in each group;intestinal microbial sequencing was used to detect the differences in the composition and proportion of the intestinal flora of each group of rats;fecal microbiota transplantation test was used to detect the effects of intestinal microbes on the kidney and intestinal barrier function of diabetic rats.Results:1.The protective effect of GQD on the kidneys of diabetic rats:(1)The results showed that the levels of BUN and Cr of diabetic rats were significantly higher than those of normal rats(P <0.01),and after GQD and metformin intervention,these indicators of rats were lower than those of the model group(P<0.01);(2)HE staining observed severe kidney damage in diabetic rats: renal tubule lumen enlargement,renal tissue visible cell disorder,granular degeneration,nuclear psychosis,glomerular contraction,and inflammatory cell infiltration;after the intervention of GQD and metformin,the pathological damage of kidney in rats was improved in different degrees;2.Study on the protective mechanism of GQD on the kidneys of diabetic rats based on the intestinal barrier(1)The results showed that the level of MDA in the serum of diabetic rats was significantly increased,while the levels of SOD,CAT,and GSH-PX all decreased.After treatment with Gegen Qinlian Decoction and metformin,the level of MDA decreased(P<0.01),the activities of these three enzymes have been enhanced(P<0.01);(2)Compared with the control group,the m RNA expression of TNF-a and IL-6 in the kidney and intestine tissues of the model group increased(P <0.01),after treatment,these indexes were lower than the model group(P<0.01);(3)Western blot results showed that compared with the model group,the treatment group can improve the expression of TLR4,NF-κB,My D88 protein in the kidney of diabetic rats and can enhance the expression of Occludin and Claudin tight junction related protein in the intestine(P<0.01);(4)Transmission electron microscope observation results showed that compared with the normal control group,the tightly connected structure of the intestines in the model group was loose and tends to be normal after treatment;(5)The results of immunohistochemistry showed that the expression of tight junction protein in the intestine of diabetic rats decreased,and after treatment,the expression of related proteins increased significantly;(6)16S r DNA sequencing found that GQD can increase the abundance of intestinal flora in rats.Firmicutes and Bacteroidetes were the dominant bacteria phyla,and the main dominant bacterial genera include Unclassified＿Lachnospiraceae,Unclassified＿Porphyromonadaceae;3.The effect of fecal microbiota transplantation on the kidney and intestinal tissues of diabetic rats(1)The results showed the intestinal flora can improve the enlargement of renal tubule lumen caused by diabetes,cell disorder,granular degeneration,nuclear psychosis,obvious glomerular contraction,inflammatory cell infiltration and other symptoms in kidney tissue;(2)The results showed that the effect of intestinal flora can repair the tight junctions of the intestines damaged by diabetes in rats,and increase the levels of intestinal tight junction proteins Occludin,Claudin-1 and ZO-1;(3)After the treatment of fecal bacteria transplantation,the first obvious change was the increase in the diversity of the flora,followed by the change in the composition of the flora,specifically: the sterile model group is mostly Proteobacteria,and the fecal bacteria transplantation is mainly two dominant bacteria,Firmicutes and Bacteroidetes.Conclusion: GQD can improve the oxidative stress state,renal function and pathological abnormalities in diabetic rats.Its effect may be related to the improvement of GQD on intestinal inflammation and abnormal intestinal barrier. |
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