Andrographolide Prevents Renal Tubulointerstitial Injury And Fibrosis By Attenuating Mitochondrial Dysfunction And NLRP3 Inflammasome Activation

Purpose: Diabetic nephropathy(DN)is associated with renal interstitial injury and fibrosis.Among them,renal tubulointerstitial fibrosis leads to epithelial-mesenchymal transformation(EMT),excessive accumulation of extracellular matrix(ECM),tubule atrophy,and nephron loss.Therein EMT of renal tubular epithelial cells is one of the major pathogenesis of renal tubulointerstitial fibrosis,the feature is decreased expression of E-cadherin and increased expression of α-smooth muscle actin(α-SMA).Moreover,excessive deposition of ECM is an important feature of diabetic renal tubulointerstitial fibrosis,where there is a mass of collagen fibers.Renal tubular injury and tubulointerstitial inflammation contribute to the progression of DN.Our previous study found that andrographolide had a protective effect on DN progression and mesangial dysfunction induced by high glucose(HG).However,the protective roles of andrographolide in renal tubular epithelial cells has not been fully elucidated.In order to investigate the protective effect of andrographolide on renal tubular injury and to observe whether andrographolide alleviates renal tubulointerstitial fibrosis by targeting the activation of NLRP3 inflammasome,we conducted the following studies to explore the mechanism of andrographolide in renal tubular injury in diabetic mice.Methods: In vitro assay,human tubular epithelial cells(HK-2 cells)were treated with andrographolide under HG conditions.Western blot,q-PCR,immunofluorescence and flow cytometry were used to detect the effects of andrographolide on the renal tubular injury and fibrosis mechanisms in HK-2cells.Mitochondrial dysfunction and NLRP3 inflammasome are the potential mechanisms of andrographolide’s effective inhibition of HG-induced renal tubulointerstitial fibrosis.In vivo assay,the blood glucose of mice was measured after 5 days of continuous intritoneal injection of streptozotocin(STZ).Mice with a fasting blood glucose level above 11.1 mM were considered to be diabetes.Diabetic mice were treated with andrographolide(i.p.2 mg/kg,twice a week).The protective effects of andrographolide against the renal tubulointerstitial injury and fibrosis was investigated in diabetic mice fed by high fat diet(HFD).Renal interstitial tissues were isolated for immunohistochemistry,immunofluorescence,q-PCR and Western blotting at sacrifice to analyze the effects of andrographolide on renal tubular injury and fibrosis.The results of in vivo experiments were verified to confirm the protective effect of androholide on the mitochondrial dysfunction and the activation of NLRP3 inflammasome in diabetic mice.Results: In vitro assay results,after treatment with andrographolide,the protein expression of Bcl2 decreased,and the protein expression of Bax and cleaved caspase-3 increased in HK-2 cells induced by HG,indicating that andrographolide effectively inhibited HG-induced apoptosis.Andrographolide treatment significantly reduced the expression levels of KIM-1,α-SMA,FN and collagen induced by HG.In addition,HG-induced decreased E-cadherin expression,while andrographolide significantly increased the expression of Ecadherin,suggesting that andrographolide can effectively improve HG-induced EMT and collagen deposition in HK-2 cells.Mechanistically,q-PCR and Western blot results showed that andrographolide also improve mitochondrial dysfunction and inhibit the production of mitochondrial reactive oxygen species activated by high glucose,and further inhibit the activation of NLRP3 inflammasome and renal tubule cell inflammation.In vivo results were consistent with in vitro results,q-PCR and Western blot assays showed that andrographolide treatment inhibited renal tubular cell apoptosis,EMT and tubulointerstitial fibrosis in diabetic mice.Andrographolide also improved the mitochondrial dysfunction and NLRP3 inflammasome in diabetic mice.Conclusion: Andrographolide has a protective effect on the progression of experimental DN by suppressing renal tubular injury,mitochondrial damage,inflammation and fibrosis.Our study provides a new mechanism for the prevention and treatment of diabetic renal fibrosis with andrographolide.