Study On The Mechanism Of DKD "internal Heat Syndrome" Based On Intestinal Flora And Microinflammatio

Diabetic kidney disease has become the primary cause of chronic kidney disease in China.It is not only an important cause of end-stage renal disease.but also a significant increase in cardiovascular mortality.However.there is no effective treatment for diabetic kidney disease，and it is urgent to study the pathogenesis and therapeutic drugs of DKD.At present，the new view is that an imbalance between proinflammatory bacteria and anti-inflammatory bacteria in the intestine of diabetes,which lead to intestinal micro-inflammation and accelerate the renal damage of DKD by activating the renal inflammatory response.Based on the viewpoint of "stagnancy of two Yang" from Yellow Emperor’s Medicine Classic and combined his clinical experience for many years with theories.Professor Wang Yaoxian put forward the point of view "internal heat causing zheng jia" of DKD.Based the view of "internal heat causing zheng jia",Qingre Xiaozheng formula was established and get best therapeutic results.We further found that "internal heat causing zheng jia" is consistent with the pathogenesis of intestinal flora disorder and micro-inflammation for DKD,and it is of great significance to explore the pathogenesis of DKD caused by "internal heat causing zheng jia" from the perspective of intestinal flora and micro-inflammation.In this paper,we will analyze the "internal heat" with the bacterial which causing the micro-inflammation from clinical observation.Based on the DKD model mice.We speculated that the mechanism of Qingre Xiaozheng formula in the treatment of DKD might be related to regulation of intestinal flora and micro-inflammation,and then reduce renal inflammatory.Objective:1.According to the observation table of internal heat syndrome,the early DKD patients was divided into two groups:internal heat syndrome and non internal heat syndrome.And the difference and relationship between two groups about proinflammatory bacteria and anti-inflammatory bacteria which were related to LPS were analyzed.2.The effect of Qingre Xiaozheng formula regulating LPS and LPS/TLR4/NF-κB inflammatory signaling pathway was observed in DKD mice model to explore the pathogenesis of intestinal flora and the micro-inflammation.Methods:1.The basic clinical information and TCM syndrome data of DKD patients about internal heat syndrome and non internal heat syndrome were collected,and the feces and serum were collected of DKD patients to analyze the difference of serum LPS between the two groups and the bacterial difference and relationship which closely related to LPS.2.The C57BL/6J DKD mice model was induced by high fat feeding and STZ intraperitoneal injection.The mice were divided into four groups:normal control group,model group,Qingre Xiaozheng formula group and irbesartan group.After the decoction of Qingre Xiaozheng formula given po for eight weeks,the weight,blood glucose,urinary albumin/creatinine（ACR）,kidney weigh/body weight,total cholesterol,triglyceride,serum creatinine,serum urea nitrogen,serum lipopolysaccharide（LPS）and serum fluorescein isothiocyanate dextran（FITC-dextran）were measured;intestinal tissue and renal sections was observed by HE;Western blot was used to detect the expression of TLR4、NF-κB p-p65 in kidney and the expression of ZO-1 in colon.Results:Part Ⅰ clinical research:1.The early DKD patients were divided into two groups:internal heat syndrome and non internal heat syndrome.And there was no significant difference in age,gender,BMI,glycosylated hemoglobin,basic history（hypertension,coronary disease,cerebrovascular disease）,smoking history,drinking history and the history of metformin and acarbose between two groups.2.Compared with the early DKD patients of non internal heat syndrome group,the serum LPS and urine ACR of the early DKD patients with internal heat syndrome increased significantly.3.Compared with the early DKD patients of non internal heat syndrome group,the abundance of LPS Bacteroidaceae,Bacteroides and Lachnoclostridium in the the early DKD patients with internal heat syndrome group increased significantly,while the abundance of Prevotellaceae,Lactobacillus,Ruminococcaceae_UCG-003 and Pseudobutyrivibrio decreased significantly.4.The relative abundance of Bacteroides and Lachnoclostridium had a positive correlation with serum LPS.while the relative abundance of Lactobacillus had a negative correlation with serum LPS.Part Ⅱ animal experiments:1.Compared with normal mice,the serum LPS and FITC-Dextran increased,the expression of ZO-1 protein in colon decreased,the expression of TLR4 and NF-κB protein in kidney increased,the level of urinary albumin concentration and ACR increased,the pathological damage of intestine and kidney serious.and the levels of total cholesterol and triglyceride increased significantly.2.Compared with the model group,Qingre Xiaozheng formula can reduce the content of serum gut derived LPS and FITC-Dextran,improve the expression of ZO-1 protein in colon,reduce the expression of TLR4 and NF-κB protein in kidney,alleviate the damage of intestine and kidney,reduce the concentration of urinary albumin and ACR,and reduce the total cholesterol and triglyceride in serum.3.Compared with the model group,Qingre Xiaozheng formula can regulate the diversity of bacterial flora in DKD mice,significantly reduce the abundance of Desulfovibrio、corynebacterium₁、Oscillibacter、Tyzzerella、Ruminicloslridium、Gemella、Anaerotruncus,improve the relative abundance of Alistipes、Parasutterella.4.Compared with the model group,Qingre Xiaozheng formula has the trend of increasing the relative abundance of Bacteroides and decreasing the relative abundance of Firmicutes.And to some extent,it has the trend of increasing Bacteroides/Firmicutes ratio.5.The serum LPS was positively correlated with FITC-Dextran:The relative abundance of Desulfovibrio was positively correlated with serum LPS and corynebacterium₁:The relative abundance of corynebacterium₁ and Osillibacter were positively correlated with FITC-Dextran.Conclusion:1.LPS may be the material basis of "internal heat" of DKD.which plays an important role in the development of urinary protein as an important inflammatory substance in the early DKD patients with internal heat syndrome.2.The imbalance of pro-inflammatory and anti-inflammatory bacteria in the intestine of the early DKD patients,which may be the pathogenesis of internal heat and micro-inflammation.3.Qingre Xiaozheng formula can alleviate DKD renal injury which may regulated intestinal flora disorder and inhibited LPS/TLR4/NF-κB inflammatory pathway mediated.4.In clinical research,the study preliminarily verified that "internal heat" is closely related to LPS and flora disorder;Qingre Xiaozheng formula can alleviate the renal inflammatory response and renal injury in DKD mice model,which may be related to the regulation of intestinal flora disorder and the reduction of micro-inflammation.And the theory of "internal heat causing zheng jia" with DKD can be explained from intestinal flora and micro-inflammation,and then it has microbiological connotation and scientificity to a certain extent.