This study aims to investigate the protective effects and mechanisms of the ethyl acetate extract of Cinnamomi Ramulus (CREAE) on oxygen-glucose deprivation/reperfusion (OGD/R)-induced injury in H9c2 cells. The effect of different concentrations of CREAE on cell viability was assessed by the MTT method, and the optimal OGD/R modeling conditions were screened also by the MTT assay. Cells were divided into the control group, OGD/R group, aspirin group (20 μmol/L), and CREAE groups (12, 6 and 3 µg/mL). Cell viability was detected by the MTT method, while the lactate dehydrogenase (LDH) release rate was measured by assay kits. The levels of interleukin-1β (IL-1β), IL-18 and creatine kinase isoenzyme MB (CK-MB) were detected by ELISA. Immunofluorescence was employed to examine the expression and nuclear translocation of high mobility group box 1 (HMGB1). Western blot analysis was used to measure the expression levels of HMGB1, NOD-like receptor thermal protein domain associated protein 3 (NLRP3), gasdermin D (GSDMD), apoptosis-associated speck-like protein containing a CARD (ASC), pro-cysteinyl aspartate specific proteinase 1 (pro-Caspase-1), IL-1β, and other related proteins. When HMGB1 was overexpression in H9c2 cells, OGD/R modeling was performed and the cells were treated with 12 μg/mL CREAE, and the expression of proteins associated with the HMGB1-NLRP3/GSDMD signaling pathway were detected by Western blot. The results showed that 12, 6, and 3 µg/mL concentration of CREAE were not significantly toxic to H9c2 cells (P>0.05). The optimal OGD/R modeling conditions were determined to be 16 hours of oxygen-glucose deprivation and followed by 3 hours of reperfusion. Treatment with 12 µg/mL CREAE significantly increased the viability rate of H9c2 cells after modeling (P<0.001), reduced the levels of LDH, CK-MB, IL-1β and IL-18 (P<0.05, P<0.01) and inhibited the expression levels of proteins associated with the HMGB1-NLRP3/GSDMD signaling pathway (P<0.05, P<0.01, P<0.001), while decreased HMGB1 nuclear translocation in H9c2 cells. Overexpression of HMGB1 could reverse the protective effect of CREAE against OGD/R-induced H9c2 cell injury and its inhibitory effect on the OGD/R-induced activation of the HMGB1-NLRP3/GSDMD signaling pathway. This study found that CREAE may alleviate OGD/R-induced H9c2 cell injury and ameliorate inflammatory responses by inhibiting the activation of the HMGB1-NLRP3/GSDMD signaling pathway, with HMGB1 potentially being one of its target proteins. The research results provided experimental evidence for the clinical treatment of cardiovascular diseases with Cinnamomi Ramulus.

ethyl acetate extract of Cinnamomi Ramulus, oxygen-glucose deprivation/reperfusion, H9c2 cells, HMGB1-NLRP3/GSDMD signaling pathway