Complanatoside A targeting NOX4 blocks renal fibrosis in diabetic mice by suppressing NLRP3 inflammasome activation and autophagy

Abstract

BackgroundDiabetic nephropathy (DN) is an important cause of end-stage renal disease. Complanatoside A (CA), an active component from Semen Astragali Complanati, has been reported to be a potential candidate for the treatment of kidney diseases. However, the underlying mechanisms and protective effects of CA in DN remain unclear. PurposeIn this paper, the effects and the mechanism of CA against ameliorating DN were investigated in vivo and in vitro. Study designHere, a high-fat diet/streptozotocin-induced diabetic model and TGF-β1-induced HK-2 cells were used to explore the protective effects and mechanisms of CA on DN in vivo and in vitro. MethodsMajor biochemical indexes, Histopathological morphology, and Immunohistochemistry have explored the therapeutic effect of CA on DN. Subsequently, TGF-β1-induced HK-2 cells were utilized to investigate the anti-renal fibrosis effect of CA. Finally, the mechanism of CA against renal fibrosis was studied via western blotting, immunofluorescence, transfection, and molecular docking. ResultsThe results showed that CA attenuated glomerular hypertrophy, collagen matrix deposition, and tubular interstitial fibrosis in diabetic mice. Moreover, the activation of TGF-β1-inducible epithelial-mesenchymal transition (EMT) was hindered by CA treatment in HK-2 cells. Mechanistically, the data suggested that upregulated NOX4 during diabetes and TGF-β1 in HK-2 cells was prominently diminished after CA treatment. Furthermore, CA exposure inhibited NLRP3 inflammasome activation and downstream inflammation gene expression such as IL-18 and IL-1β in vivo and vitro. These findings indicated that CA obstructed the EMT to protect renal tubular epithelial cells against fibrosis via blocking NLRP3 activation, which was associated with inhibiting NOX4. Besides, the markedly raised autophagy levels in the diabetic model characterized by increasing LC3II/LC3I and Beclin1 were reversed after CA treatment, which is also a pivotal mechanism against renal fibrosis. More importantly, specific NOX4 overexpressed in HK-2 cells abolished that CA exposure blocked TGF-β1-induced-EMT, ROS generation, NLRP3, and autophagy activation. Meanwhile, the inhibition of cell migration, ROS generation, autophagy, and renal inflammation after CA treatment was more pronounced in NOX4-deficient HK-2 cells. ConclusionOur findings provided evidence that CA might be a potential therapeutic agent for DN by ameliorating NLRP3 inflammasome and autophagy activation via targeting NOX4 inhibition.