葛根素调控SLC7A11/GPX4轴抑制高糖诱导的成骨细胞铁死亡

Puerarin inhibits high glucose-induced ferroptosis in osteoblasts by modulation of the SLC7A11/GPX4 axis

目的探讨葛根素对高糖环境下成骨细胞铁死亡的保护作用及其分子机制。方法用高糖培养基诱导MC3T3-E1细胞铁死亡,使用不同浓度葛根素进行干预。通过CCK-8检测葛根素对MC3T3-E1细胞活性的影响;通过ALP染色、ARS染色、qRT-PCR检测葛根素对MC3T3-E1细胞成骨分化能力的影响;通过流式细胞技术、DHE染色和试剂盒测定MC3T3-E1骨细胞中ROS、GSH、SOD和MDA水平;通过Western blot检测MC3T3-E1细胞中SLC7A11和GPX4的表达情况。结果CCK-8结果表明,100μmol/L以下浓度的葛根素对MC3T3-E1细胞活性无明显抑制作用;与高糖组相比,100μmol/L的葛根素能够有效减轻高糖对MC3T3-E1细胞增殖的抑制作用(P<0.05);与高糖组相比,葛根素干预后能明显提高ALP、RUNX2、COL1A1、OPN的基因表达,增加MC3T3-E1细胞ALP活性和矿化水平(P<0.05);与高糖组相比,葛根素干预后明显降低了MC3T3-E1细胞中ROS、MDA水平,增加了GSH、SOD活性(P<0.05);Western blot结果表明,高糖干预明显降低了MC3T3-E1细胞中SLC7A11和GPX4的表达,葛根素干预后上调了SLC7A11和GPX4的表达(P<0.05)。结论葛根素能够减轻高糖诱导的成骨细胞铁死亡,其作用机制可能是通过调控SLC7A11/GPX4信号通路实现的。

Objective To investigate the protective effect of puerarin against ferroptosis in high glucose-induced ferroptosis in osteoblasts and its molecular mechanism.Methods Ferroptosis in MC3T3-E1 osteoblasts was induced by high glucose medium,using different concentrations of puerarin for the intervention.The effect of puerarin on the activity of MC3T3-E1 cells were detected with CCK-8.The effect of puerarin on the osteogenic differentiation ability of MC3T3-E1 cells were detected using ALP staining,ARS staining,and qRT-PCR.The levels of ROS,GSH,SOD,and MDA in MC3T3-E1 were measured with flow cytometry,DHE staining,and kits.The expressions of SLC7A11 and GPX4 were detected with Western blotting.Results CCK-8 result showed that puerarin at concentrations below 100μM had no significant inhibitory effect on MC3T3-E1 cell activity.Compared with that in the high glucose group,100μM of puerarin effectively attenuated the inhibitory effect of high glucose on proliferation of MC3T3-E1 cells(P<0.05).In addition,puerarin significantly increased the expressions of ALP,RUNX2 COL1A1,and OPN,and increased ALP activity and mineralization level in MC3T3-E1 cells compared to those in the high glucose group(P<0.05).Compared with the high glucose group,puerarin markedly decreased ROS and MDA levels and increased GSH and SOD activities in MC3T3-E1 cells(P<0.05).Western blotting result showed that high glucose intervention obviously decreased the expressions of SLC7A11 and GPX4 in MC3T3-E1 cells,whereas puerarin upregulated the expressions of SLC7A11 and GPX4(P<0.05).Conclusion Puerarin is able to attenuate high glucose-induced ferroptosis in osteoblasts.Its mechanism may be achieved by regulating the SLC7A11/GPX4 signaling pathway.