| Objective: To explore the relationship between the Chinese visceral adipose index(CVAI),visceral fat area(VFA),and diabetic nephropathy(DN),as well as their predictive value for disease progression through clinical data collection.Meanwhile,through in vitro cell culture,the study investigates the effect of the inflammatory response mediated by Tumor Necrosis Factor Receptor-Associated Factor 6(TRAF6)on the high glucose and visceral fat-induced cell differentiation of human renal tubular epithelial cells(HKC),providing new idea for the prevention of diabetic nephropathy.Methods:Part Ⅰ: A total of 442 patients with type 2 diabetes who were hospitalized in the Department of Endocrinology,Gansu Provincial People’s Hospital from October 2020 to October 2022 were selected,and their relevant clinical data(general data,physical examination,biochemical indicators,etc.)were collected.The patients were divided into DN group(n=209)and non-DN group(n=233)according to the presence or absence of diabetic nephropathy.CVAI and VFA were analyzed for Spearman correlation with urinary albumin excretion rate,urinary albumin/creatinine ratio and glomerular filtration rate.Multi-factorial logistic stepwise regression analysis was used to screen independent risk factors for the occurrence of DN in T2 DM patients,and a multiple logistic regression model was established.The predictive value of the model and each risk factor was evaluated by the subject operating characteristic curve.Part Ⅱ: HKC cell lines were cultured in vitro and treated with different concentrations of visceral adipose tissue cytokines(0ng/m L,25 ng/m L,100ng/m L,200ng/m L,400ng/m L,800ng/m L,1600ng/m L)for 48 hours.The relative activity of the cells and the levels of inflammatory cytokines TNF-α and IL-6 were detected by CCK8 and ELISA,respectively,to determine the optimal concentration of Visfatin for this experiment.HKC was treated with different concentrations(0μM,5μM,10μM,20μM,40μM,80μM)of MG-132(TRAF6inhibitor)for 48 hours,and the relative cell activity and expression of TRAF6 gene in the cells were detected respectively by CCK8 and q RT-PCR to screen out the optimal concentration of MG-132 for inhibiting TRAF6.The cells were randomly divided into 4 groups: Control group,H-Glu group,H-Glu+Visfatin group,and H-Glu+Visfatin+MG-132 group.After HKC was treated with high glucose and visceral adiposity cytokines for 48 hours,the protein expression of TRAF6 in the Control group,H-Glu group,and H-Glu+Visfatin group was detected by immunofluorescence,and q RT-PCR was used to detect the m RNA expression of TLR4,TRAF6,NF-κB,α-SMA,and E-cadherin in the cells.After treating the cells with MG-132 for 48 hours,the m RNA expressions of NF-κB,α-SMA and E-cadherin in each group(Control group,H-Glu group,H-Glu+Visfatin group,H-Glu+Visfatin+MG-132 group)were detected by q RT-PCR,and TNF-α and IL-6 were detected by ELISA.Results:Part Ⅰ: 1.Of the 442 subjects in this study,with a total of 209 in the DN group and 233 in the non-DN group.Compared with the non-DN group,the DN group had a longer duration of diabetes,greater weight,body mass index,waist circumference,higher systolic blood pressure,diastolic blood pressure,triglycerides,lipid accumulation index,CVAI,VFA and subcutaneous fat area,all with statistically significant differences(P < 0.05).2.Spearman correlation analysis of the two groups showed that UAER,UACR,and Scr were positively correlated with CVAI and VFA(rs=0.239,0.194,0.160,0.102,0.146,and 0.094,respectively;all P<0.05).There was a negative correlation between e GFR and CVAI(rs=-0.197,P<0.05).3.Binary logistic regression analysis showed that CVAI,VFA,systolic blood pressure,and diabetes duration were independent risk factors for DN in patients with T2DM(P<0.05).4.ROC curve analysis showed that the AUC of CVAI was 0.645,the sensitivity was 0.507,and the specificity was 0.734.The AUC of VFA was 0.632,with the sensitivity 0.636,and the specificity 0.631.The AUC of the multiple logistic regression model was 0.700,with the sensitivity 0.670,and the specificity0.652.These results were superior to the individual factor parameters.Part Ⅱ: 1.CCK8 tests showed that different concentrations of visfatin had no significant effect on HKC cell activity(P>0.05).As the concentration of Visfatin increased,the concentration of IL-6 and TNF-α in the cell culture supernatant gradually increased.When the concentration of visfatin was 200 ng/m L,the content of IL-6 and TNF-α reached the maximum.Therefore,200 ng/m L was selected as the concentration of visfatin for subsequent experiments.2.As the concentration of TRAF6 inhibitor(MG-132)increased,the inhibitory effect on HKC cell proliferation became stronger,and the expression of TRAF6 m RNA in cells was significantly reduced.Based on the ability to inhibit cell proliferation and TRAF6 gene expression,a concentration of 20μM of MG-132 was selected as the concentration for subsequent experiments.3.The immunofluorescence of each group was detected.TRAF6 was expressed in all groups.Compared with the control group,TRAF6 in HKC cells treated with high glucose for 48 hours was enhanced in immunofluorescence.Compared with the high glucose group,H-Glu+Visfatin group could further enhance the green fluorescence of TRAF6 in the cells.4.The q RT-PCR test results showed that compared with the Control group,the relative m RNA expression levels of TLR4,TRAF6,NF-κB,and α-SMA in HKC cells in the HGal group and H-Gal+Visfatin group increased significantly(P<0.05),while the relative m RNA expression level of E-cadherin in HKC cells in the H-Glu group and H-Glu+Visfatin group decreased(P<0.05).Compared with the H-Glu group,the relative m RNA expression levels of TLR4,TRAF6,NF-κB,and α-SMA in HKC cells in the H-Glu+Visfatin group increased significantly(P<0.05).Compared with the H-Glu group,the relative m RNA expression levels of E-cadherin in HKC cells in the H-Glu+Visfatin group decreased significantly(P<0.05).5.After inhibiting the TRAF6 expression,compared with the H-Glu group and H-Glu+Visfatin group,the H-Glu+Visfatin+MG-132 group showed a decrease in m RNA expression of NF-κB and α-SMA in cells(P<0.05),an increase in m RNA expression of E-cadherin(P<0.05),and a decrease in release of inflammatory factors IL-6 and TNF-alpha in cells(P<0.05).Conclusion: 1.CVAI,VFA,and T2 DM are independent risk factors for DN in T2 DM patients.providing an important theoretical basis for the management of visceral obesity patients.2.Under high glucose and visfatin intervention upregulate α-SMA gene expression,down-regulate E-cadherin gene expression,and cause renal tubular epithelial cell transdifferentiation.When TRAF6 expression is inhibited,the content of IL-6 and INF-α in the cells can be reduced,α-SMA expression can be inhibited,and E-cadherin expression can be promoted,thereby preventing the transdifferentiation of renal tubular epithelial cells.In theory,appropriate doses of MG-132 can inhibit the expression of TRAF6,effectively inhibiting the transdifferentiation of HKC cells,reducing the production of fibrotic cells and extracellular matrix,and inhibiting TRAF6 can effectively inhibit the production of inflammatory responses,providing an important theoretical basis for delaying and treating diabetic complications,as well as providing new action targets for drugs in DN. |
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