This study established a pyroptosis injury model by stimulating insulinoma cells(INS-1) of rats with high glucose(HG) and observed the impact of additional ethanol(ET) exposure on cell pyroptosis, as well as the intervention effect of salidroside(SAL). INS-1 cells were cultured and divided into a normal control group(NG), an HG group, an HG + ET(100 mmol·L~(-1)) group, and an HG + ET + SAL(1-100 μmol·L~(-1)) group. After 72 hours of treatment, cell viability was assessed using the cell counting kit-8(CCK-8) assay. The number of pyroptotic bodies was observed under a microscope. Western blot was used to detect changes in the intracellular Nod-like receptor protein 3(NLRP3)/gasdermin D(GSDMD) signaling pathway and adenosine monophosphate-activated protein kinase(AMPK) activity. A fluorescence probe was used to detect changes in intracellular reactive oxygen species(ROS) levels. Time-resolved fluorescence resonance energy transfer(TR-FRET) technology was employed to observe the effect of SAL on recombinant AMPK protein kinase activity in vitro. The results showed that compared to the NG group, HG exposure induced an increase in the number of pyroptotic bodies, elevated ROS levels, and activation of the NLRP3/GSDMD signaling pathway in INS-1 cells. Compared to the HG group, HG + ET exposure further exacerbated these changes. Compared to the HG + ET group, SAL dose-dependently increased cell viability, reduced the formation of pyroptotic bodies in INS-1 cells, and inhibited excessive ROS production, overactivation of the NLRP3/GSDMD signaling pathway, and the decrease in AMPK activity. TR-FRET experiments indicated that SAL could directly activate AMPK. When INS-1 cells were pretreated with an AMPK inhibitor, the effects of SAL on increasing cell viability, alleviating the formation of pyroptotic bodies, and inhibiting excessive ROS production were abolished. These results suggest that SAL can alleviate HG combined with ET-induced exacerbation of INS-1 pyroptosis by activating AMPK.
Keywords: INS-1; diabetes; ethanol exposure; pyroptosis; salidroside.