淫羊藿苷通过Toll样受体4/核因子-κB信号通路抑制高糖诱导H9c2细胞凋亡与细胞焦亡的作用及其机制研究OA

Objective To investigate the inhibitory effect of icariin(ICA)on high glucose-induced apoptosis and pyroptosis in H9c2 cells,and to investigate its mechanism from the perspective of Toll-like receptor 4/nuclear factor-κB(TLR4/NF-κB)signaling pathway.Methods H9c2 cells in logarithmic growth phase were divided into the control group(group A),high-glucose group(group B),and ICA low-and high-dose groups(group C and group D).The cells in group A were incubated with a 1∶1 mixture of Dulbecco's modified Eagle medium and Ham's F-12 nutrient mixture[DMEM/F12(1∶1,m/m)]medium(containing 11 mmol/L glucose)for 48 h,while the cells in groups B,C,and D were incubated with DMEM/F12(1∶1,m/m)medium(33 mmol/L glucose)for 48 h to construct diabetic cardiomyopathy(DCM)cell models.During modeling,the cells in groups C and D were incubated with 10 and 20 µmol/L ICA for 48 h.Cell apoptosis level was detected by the Hoechst 33258 staining solution,mitochondrial membrane potential level was detected by the JC-1,the levels of interleukin-6(IL)-6,IL-1β,IL-18,tumor necrosis factor-α(TNF-α)were detected by the enzyme-linked immunosorbent assay(ELISA)method,and expression levels of cleaved caspase-3,cleaved caspase-1,nucleotide binding domain leucine rich repeat and heat containing protein domain receptor 3(NLRP3),leptin D(GSDMD),IL-1β,TLR4,phosphorylated NF-κB p65(p-NF-κB p65)protein were detected by the Western blot.The three-dimensional(3D)structures of ICA and TLR4 proteins were imported into the AutoDock 1.5.7 software for molecular docking.In addition,a high-dose ICA+lipopolysaccharide(LPS)group(group E,20 µmol/L ICA+1 µg/mL LPS)was added,and the expression levels of cleaved caspase-3,cleaved caspase-1,NLRP3,GSDMD,and IL-1β protein were detected by the Western blot to investigate the reversal effect of LPS.Results Compared with those in group B,the apoptosis rate,ratio of JC-1 green fluorescence to red fluorescence,and levels of IL-6,IL-1β,IL-18,and TNF-α,and the expression levels of cleaved caspase-3,NLRP3,cleaved caspase-1,GSDMD,IL-1β,TLR4,p-NF-κB p65 protein in H9c2 cells significantly decreased in groups C and D(P<0.05).Compared with those in group D,the expression levels of cleaved caspase-3,NLRP3,cleaved caspase-1,GSDMD,and IL-1β protein in H9c2 cells significantly increased in group E(P<0.01).Conclusion ICA has a clear protective effect on high glucose-induced apoptosis and pyroptosis in H9c2 cells,and its mechanism may be related to the inhibition of the TLR4/NF-κB signaling pathway.