葛根素对高糖诱导成骨细胞增殖、凋亡及CHOP通路的影响研究

Puerarin Has Effect on Proliferation and Apoptosis of Osteoblast Induced by High Glucose and CHOP Pathway

目的:探讨葛根素对高糖诱导成骨细胞增殖、凋亡及CHOP通路的影响。方法:设置MC3T3-E1组,葛根素低、中、高剂量组。MC3T3-E1组常规培养,葛根素低、中、高剂量组分别用浓度为20.0μg/mL、40.0μg/mL、80.0μg/mL的葛根素溶液干预培养。3-(4,5-二甲基噻唑-2)-2,5-二苯基四氮唑溴盐法(MTT)法测定各组MC3T3-E1细胞增殖水平,流式细胞法测定各组MC3T3-E1细胞凋亡水平,逆转录-定量聚合酶链反应(RT-qPCR)法测定各组MC3T3-E1细胞转录激活因子4(ATF4)、C/EBP同源蛋白(CHOP) RNA表达水平,Elisa法测定各组MC3T3-E1细胞ATF4、CHOP、超氧化物歧化酶(SOD)、丙二醛(MDA)、谷胱甘肽(GSH)蛋白表达水平。结果:与MC3T3-E1组比较,葛根素低、中、高剂量组细胞凋亡率、MDA蛋白表达水平与ATF4、CHOP RNA及蛋白表达水平均降低(P<0.05);OD值、增殖率与SOD、GSH蛋白表达水平均升高(P<0.05)。与葛根素低剂量组比较,葛根素中、高剂量组凋亡率、MDA蛋白表达水平与ATF4、CHOP RNA及蛋白表达水平均降低(P<0.05);OD值、增殖率与SOD、GSH蛋白表达均升高(P<0.05)。与葛根素中剂量组比较,葛根素高剂量组凋亡率、MDA蛋白表达水平与ATF4、CHOP RNA及蛋白表达水平均降低(P<0.05);OD值、增殖率与SOD、GSH蛋白表达水平均升高(P<0.05)。结论:葛根素能拮抗高糖对MC3T3-E1成骨细胞的增殖抑制作用和凋亡促进作用;其机制与葛根素抑制ATF4、CHOP RNA及蛋白的表达拮抗了高糖环境引起的成骨细胞内质网应激,进而减轻成骨细胞氧化应激反应有关。

Objective: To discuss the effect of puerarin on proliferation and apoptosis of osteoblast induced by high glucose and CHOP pathway. Methods:Set the MC3 T3-E1 group,the groups of puerarin in low,middle and high doses. The MC3 T3-E1 group received routine culture, while the groups of puerarin in low, middle and high doses received the interfering culture with puerarin solution in 20.0 μg/mL, 40.0 μg/mL and 80.0 μg/mL respectively. Determined the proliferation level of MC3 T3-E1 cell in each group via 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide(MTT) method. Detected the apoptosis level of MC3 T3-E1 cell in each group via flow cytometry method. Determined the expression level of MC3 T3-E1 cell activated transcription factors 4(ATF4) and C/EBP homologous protein(CHOP) RNA via reverse transcription quantitative polymerase chain reaction(RT-qPCR) method. Assessed the expression level of MC3 T3-E1 cell ATF4, CHOP, superoxide dismutase(SOD), malondialdehyde(MDA) and glutathione(GSH) via Elisa method. Results:Comparing with the MC3 T3-E1 group,the apoptosis rate and the expression level of MDA,the expression levels of ATF4,CHOP RNA and protein in the groups of puerarin in low,middle and high doses were deceased(P<0.05);the value of OD,the proliferation rate and the protein expression of SOD and GSH were increased(P<0.05). Comparing with the low-dose puerarin group,the apoptosis rate and the expression level of MDA,the expression levels of ATF4,CHOP RNA and protein in the groups of puerarin in middle and high doses were decreased(P<0.05);the value of OD,the proliferation rate and the protein expression of SOD and GSH were increased(P<0.05). Comparing with the middle-dose puerarin group, the apoptosis rate,MDA expression level and the expression levels of ATF4,CHOP RNA and protein in the group of puerarin of high dose were decreased(P<0.05);the value of OD,the proliferation rate and the protein expression of SOD and GSH were increased(P<0.05). Conclusion:Puerarin can antagonize the effect of high glucose in inhabiting the proliferation of MC3 T3-E1 osteoblasts and promoting their apoptosis. Its mechanism is relevant with the inhibiting the expression of ATF4,CHOP RNA and protein via puerarin antagonize osteogenic cytoplasm network stress,and thus reduce osteoblast oxidative stress reactions.