Study on themechanism of Tangshenkang regulating NLRP3-Caspase-1-IL-18/IL-1β axis to improve podocyte pyroptosis in diabetic kidney disease
Objective To explore the mechanism of Tangshenkang regulating NLRP3-Caspase-1-IL-18/IL-1β axis to improve podocyte pyroptosis in diabetic kidney disease based on the theory of kidney deficiency,dampness and stasis.Methods 18 db/db mice were randomly divided into model group,valsartan group,and Tangshenkang group,with 6 mice in each group.Six db/m mice were used as the control group.The Tangshenkang group was given 1.17 g/(kg·d)by gavage,the valsartan group was given 10.4 mg/(kg·d)of valsartan suspension by gavage,and the control group and the model group were given equal amounts of physiological saline.After 8 weeks of treatment,the general condition and glucose metabolism of db/db mice were monitored,the pathological changes of the kidney tissues were observed through the methods of hematoxylin eosin(HE),masson,periodate Schiff reaction(PAS)and transmission electron microscopy.Renal injury indicators such as urinary albumin/creatinine(UACR),blood creatinine(Scr),urea nitrogen(BUN),cystatin C(CysC),β2 microglobulin(β2-MG),N-Acetyl-β-D-glucosaminidase(NAG)were detected through biochemical analysis.The methods of ELISA,Western blot and immunofluorescence were used to detect the expression of Nephrin,as well as the pyroptotic inflammatory factors nod like receptor protein 3(NLRP3),aspartate proteolytic enzyme-1(Caspase-1),interleukin-18(IL-18)and IL-1 β changes in the kidneys of db/db mice.Cell experiment:Mouse podocyte(MPC5)cells were induced by 30 mmol/L glucose medium for 48 hours to establish the podocyte injury model.The optimal concentration of Tangshenkang drug-containing serum was selected by MTT method.The effect of Tangshenkang on the apoptosis rate of podocytes induced by high sugar was detected by flow cytometry.The cell culture supernatant in each group was collected to detect the levels of lactate dehydrogenase(LDH).NLRP3 and Caspase-1 were detected by Western blot,and the expressions of IL-18 and IL-1 β were detected by ELISA.Results Tangshenkang can improve the general condition and glucose metabolism of db/db mice.Compared with the model group,the UACR of the Tangshenkang group and the valsartan group were significantly decreased(P＜0.01),and renal injury indicators such as NAG(P＜0.01),β2-MG(P＜0.01 or P＜0.05),Scr(P＜0.01),BUN(P＜0.01),and CysC(P＜0.01)were also improved;the protein expressions of Nephrin in the Tangshenkang group were significantly increased(P＜0.01),and better than that in the valsartan group(P＜0.05).Tangshenkang could significantly reduce the expression of inflammatory factors related to pyroptosis include NLRP3(P＜0.05),Caspase-1(P＜0.01),IL-18(P＜0.01)and IL-1β(P＜0.01).Tangshenkang could improve GBM thickening,podocyte fusion,endothelial cell window structure,mesangial matrix accumulation and other pathological changes of diabetic kidney disease.The optimal concentration of Tangshenkang medicated serum screened by MTT method was 3.84 g/kg,at which the podocyte activity was the highest and the apoptosis rate was the lowest.Tangshenkang Group can significantly reduce the LDH release rate(P＜0.01).Tangshenkang could significantly increase the NLRP3(P＜0.05),Caspase1(P＜0.05),IL-18(P＜0.01)and IL-1 induced by high glucose in podocytes β(P＜0.01).Conclusion Tangshenkang Granules can inhibit pyroptosis,reduce inflammatory response,protect podocytes and alleviate kidney injury in DKD by regulating NLRP3-Caspase-1-IL-8/IL-1 β pathways.
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