[Harpagoside alleviates ethanol- and high glucose exposure-exacerbated pyroptosis in insulinoma cells].

Abstract

This study aimed to investigate the effect of harpagoside(HA) on ethanol(ET)-and high-glucose(HG)-exacerbated pyroptosis in rat insulinoma cells(INS-1) and its underlying mechanism. INS-1 were cultured and assigned to the following groups: normal glucose(NG), HG, HG+ET(100 mmol·L~(-1))(abbreviated as ET), HG+ET+HA(12.5, 25, 50 μmol·L~(-1))(abbreviated as HA), and HG+ET+HA(50 μmol·L~(-1))+AMP-activated protein kinase(AMPK) inhibitor compound C(comp.C, 2 μmol·L~(-1))(abbreviated as comp.C) to verify the mechanism of HA. CCK-8 assay was employed to evaluate cell viability. The formation of pyroptotic bodies was quantified under a microscope. Dichlorofluorescein diacetate probe was used to detect intracellular reactive oxygen species(ROS) levels. Western blot analysis was performed to determine the phosphorylation level of AMPK as well as the expression and activation of Nod-like receptor protein 3(NLRP3)/gasdermin D(GSDMD)-related proteins. Molecular docking was utilized to predict the binding affinity between HA and AMPK. Time-resolved fluorescence resonance energy transfer(TR-FRET) assay was applied to measure the effect of HA on the kinase activity of recombinant AMPK protein. The results demonstrated that compared with the NG group, the HG group showed decreased cell viability, increased number of pyroptotic bodies, reduced AMPK activity, and elevated ROS levels along with upregulated expression and activation of NLRP3/GSDMD-related proteins. Compared with the HG group, ET exposure further exacerbated these changes. Compared with the ET group, HA ameliorated the above abnormalities. Meanwhile, molecular docking analysis revealed that the binding affinity between HA and AMPK was-6.59 kcal·mol~(-1). TR-FRET assay indicated that HA enhanced the kinase activity of AMPK protein. After pretreatment of INS-1 with comp.C, the ameliorative effects of HA on the above abnormalities were abolished. In conclusion, HA ameliorates the ET-and HG-exacerbated pyroptosis in INS-1 by activating the AMPK signaling pathway.