This study aims to investigate the role and mechanism of Shengmai Injection in oxygen-glucose deprivation/reoxygenation(OGD/R) microvascular endothelial cell injury based on mitochondrial dysfunction mediated by thioredoxin-interacting protein(TXNIP)/NOD-like receptor protein 3(NLRP3)/interleukin(IL)-1β signaling pathways. Human cardiac microvascular endothelial cells(CMEC) were cultured in vitro to establish the OGD/R model, and the experiments were performed in a normal group, a OGD/R group, OGD/R+low-dose(10 μL·mL~(-1)) and high-dose(30 μL·mL~(-1)) groups of Shengmai Injection, and OGD/R+mitochondria-targeted antioxidant agent(Mito-Tempo, 10 μmol·L~(-1)) group. The cell counting kit-8(CCK-8) method was used to screen the effective dose of Shengmai Injection on cells. The levels of IL-1β and nitric oxide(NO), along with the activity of endothelial nitric oxide synthase(eNOS) in cell supernatants, were measured by enzyme-linked immunosorbent assay(ELISA) across all groups. Additionally, the effects of low-and high-dose SMI on IL-1β, NO, and eNOS levels in cells were assessed in the absence of OGD/R treatment. In vitro blood vessel formation assay, cell scratch assay, and Transwell cell migration assay were performed to observe the effects of Shengmai Injection on cell tube formation and migration ability. The Annexin V-FITC apoptosis kit was used to detect the level of apoptosis. The fluorescent probe method and flow cytometry were used to detect the level of reactive oxygen species(ROS) of cells in each group, and the JC-1 mitochondrial membrane potential detection kit was used to detect the mitochondrial membrane potential levels in cells. The mRNA expression of related genes in the TXNIP/NLRP3/IL-1β signaling pathways was measured by using reverse transcription quantitative polymerase chain reaction(RT-qPCR). The expression of TXNIP, NLRP3, IL-1β, cleaved caspase-1, pro-caspase-3, and cleaved caspase-3 proteins was detected by Western blot, and the expression of NLRP3 and cleaved caspase-3 proteins was measured by immunofluorescence. The results showed that compared with the normal group, SMI-H(30 μL·mL~(-1)) significantly increased NO and eNOS levels without OGD/R treatment, but had minimal effect on IL-1β levels; compared with the OGD/R group, after model establishment, IL-1β level was significantly reduced, NO and eNOS levels were increased, cell tubularity, migration and cell viability were significantly increased, apoptosis rate and ROS level were significantly reduced, and mitochondrial membrane potential was increased in all groups, the expression levels of TXNIP,NLRP3,IL-1β,and cleaved caspase-1 genes and proteins were decreased, while the ratio of pro-caspase-3 to cleaved caspase-3 was increased. In conclusion, Shengmai Injection can ameliorate cell injury induced by OGD/R. Its mechanism of action may be associated with the inhibition of TXNIP/NLRP3/IL-1β signaling pathways and amelioration of mitochondrial dysfunction.
Keywords: ROS; Shengmai Injection; TXNIP/NLRP3/IL-1β pathway; cardiac microvascular I/R; mitochondrial function.