Study On The Effect And Mechanism Of Punicalagin On Improving Diabetic Kidney Disease By Regulating Intestinal Mucosal Barrier

Objective:Diabetic kidney disease（DKD）is a chronic kidney disease caused by diabetes mellitus（DM）,which is the main cause of end-stage kidney disease.Intestinal mucosal barrier plays an important role in the occurrence and development of diabetes and renal disease.Damage of intestinal mucosal barrier will lead to bacterial translocation,toxins in intestinal lumen enter the body,induce chronic systemic inflammation and affect kidney health.Polyphenolamine punicalagin（PU）from pomegranates has potential effects on microbial ecosystem,intestinal mucosal barrier and renal function.In this study,a population study was conducted to explore the relationship between intestinal barrier function and the risk of DKD in patients with diabetes,and to further explore whether PU improves renal injury in DKD mice by regulating intestinal mucosal barrier.Methods:1.The population study:A cross-sectional study was conducted by cluster sampling.According to the clinical diagnostic criteria of DM established by the American Diabetes Association,patients with DM≥65 years old in rural communities in Qingdao city were included in the study,according to the clinical criteria of DKD formulated by the Nephrology Branch of the Chinese Medical Association,it is divided into diabetic non-kidney disease（DNKD）group and DKD group.The general demographic characteristics were collected,and serum fasting blood glucose（FBG）,total cholesterol（TC）,triglyceride（TG）,low-density lipoprotein cholesterol（LDL-C）,high-density lipoprotein cholesterol（HDL-C）,blood urea nitrogen（BUN）and serum creatinine（Scr）were detected by automatic biochemical analyzer,white blood cell count,neutrophil count and lymphocyte count were measured by automatic hematology analyzer.Estimated glomerular filtration rate（e GFR）was calculated by modification of Diet in Renal Disease（MDRD）formula.The levels of lipopolysaccharide（LPS）,lipopolysaccharide binding protein（LBP）,diamine oxidase（DAO）and zonulin were detected by enzyme linked immunosorbent assay（ELISA）.The correlation between intestinal barrier function and e GFR was evaluated by Spearman correlation analysis.Conditional logistic regression model was used to calculate odds ratio（OR）and 95%confidence interval（CI）to evaluate the correlation between intestinal barrier function and the risk of DKD.2.The animal study:a high-fat diet-induced DKD mouse model was established,and C57BL/6J mice were randomly divided into 5 groups（15 mice/group）:healthy control group（NC）,DKD model group（DKD）,metformin group（MET）,low dose of punicalagin group（LPU）,high dose of punicalagin group（HPU）.NC group and DKD group were gavaged with saline solution every day,MET group was gavaged with 150 mg/kg/day of metformin solution every day,LPU group was gavaged with 50 mg/kg/day of PU solution every day,and HPU group was gavaged with 100 mg/kg/day of PU solution every day.After 8 weeks of intervention,the intraperitoneal injection glucose tolerance test was performed,and the mice were sacrificed after 5 days of resting after the experiment,and blood,colon and kidney tissues were retained for follow-up index detection.The pathological changes of intestines and kidneys were observed by hematoxylin-eosin（H&E）staining.ELISA test was used to detect the expression of insulin（INS）,TC,TG,LDL-C,HDL-C,BUN,Scr,uric acid,LPS and DAO in serum,as well as inflammatory factors and chemokines in kidney tissue.The expression of nephroblastoma 1（WT-1）in kidney was detected by immunohistochemistry,and the expression of tight junction proteins such as ZO-1 and occludin in colon was detected by immunofluorescence.Intestinal microbial composition and short-chain fatty acid（SCFAs）levels were detected by 16S r RNA sequencing and gas chromatography-mass spectrometry,and renal RNA sequencing analysis,such as differentially expressed genes and kyoto encyclopedia of genes and genomes（KEGG）enrichment analysis.Results:1.The population study:（1）A total of 202 patients with DM（age≥65 years old）were enrolled in this study,including 127 patients in DNKD group and 75 patients in DKD group.The proportion of males in DKD group was 23%,and the proportion of coronary heart disease and hyperlipidemia was 10%and 7%,which were significantly higher than those in the DNKD group（P<0.05）.The levels of FBG,TG,BUN and Scr,number of people positive for urine protein,white blood cell count,neutrophil count and neutrophil to lymphocyte ratio（NLR）,and intestinal barrier indexes LPS,LBP,DAO and zonulin in DKD group were significantly higher than those in DNKD group（P<0.05）.The level of e GFR in DKD group was significantly lower than that in DNKD group（P<0.001）.（2）By Spearman correlation analysis,it was found that LPS（r=-0.248,P<0.001）,LBP（r=-0.240,P<0.001）,DAO（r=-0.324,P<0.001）and zonulin（r=-0.231,P<0.001）were negatively correlated with e GFR.Through the corrected logistic regression analysis,it was found that the concentrations of LPS,LBP,DAO and zonulin were positively correlated with the risk of DKD by continuous variable analysis(P_trend<0.05).The T1 quartile（the lowest quartile）of each index was used as a reference to adjust confounding factors(sex,history of hyperlipidemia,history of coronary heart disease,FBG,TG,white blood cell count,neutrophil count and NLR,The T2 quartile（OR:2.753;95%CI:1.115,6.795）and T3 quartile（OR:7.130;95%CI:2.917,17.425）of LPS were positively correlated with the risk of DKD;the T3 quartile（OR:6.079;95%CI:2.664,13.873）of DAO was positively correlated with the risk of DKD;and the T3 quartile（OR:2.885;95%CI:1.290,6.451）of zonulin was positively correlated with the risk of DKD,and the linear trend test was statistically significant(P_trend<0.05).2.The animal study:（1）Compared with the DKD model group,supplementation of PU could reduce the body weight,FBG,INS,TC,TG and LDL-C of mice,PU supplementation could improve the structure and function of kidney,and the levels of Scr,UA and BUN in PU groups were significantly lower than those in the DKD group.（2）PU improved the intestinal morphology and intestinal permeability of DKD mice.The levels of serum LPS and DAO in PU groups were significantly lower than those in the DKD group.Compared with the DKD model group,the expression levels of ZO-1and occludin in PU groups were higher.After PU intervention,the microbial composition of intestinal tract was remodeled and the abundance of SCFAs-producing bacteria（such as Akkermansia and Eubacterium＿coprostanoligenes＿group）was increased.Further analysis showed that the level of intestinal SCFAs in PU groups was higher than that in the DKD group.（3）Through RNA sequencing to explore the differentially expressed genes,it was found that PU reversed inflammation-related signaling pathways,such as cytokine-cytokine receptor interaction signaling pathway,chemokine signaling pathway,NOD-like receptor signaling pathway and tumor necrosis factor signaling pathway,and inflammation-related genes in PU groups,such as CXC motif chemokine ligand 2（CXCL2）and CXC motif chemokine ligand 3（CXCL3）.The expression levels of inflammatory cytokines,such as Nod-like receptor pyrin domain containing 3（NLRP3）and interleukin-1α（IL-1α）,were lower than those in the DKD model group.（4）Compared with the DKD model group,the levels of renal chemokines CXCL2,CXCL3,NLRP3 and IL-1αin PU groups were lower.Spearman correlation analysis showed that intestinal barrier integrity indexes such as ZO-1 and occludin were negatively correlated with renal chemokines and inflammatory factors.Conclusion:1.The population study:a cross-sectional study found that the impairment of intestinal barrier function in patients with DM was positively correlated with the risk of DKD.2.The animal study:PU repaired the damage of intestinal mucosal barrier by regulating the composition of gut microbiota,such as increasing the abundance of SCFAs-producing bacteria,increasing the production of SCFAs,improving intestinal morphology and intestinal permeability.PU further attenuated DKD by regulating intestinal mucosal barrier.