三七总皂苷对H2O2诱导BMSCs 凋亡的保护作用及机制研究
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R285.6

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湖州市科技局公益性资助项目(2019GY06)


Study on Protective Effect and Mechanism of Total Panax Notoginseng Saponins on Apoptosis of BMSCs Induced by H2O2
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    摘要:

    目的:体外观察三七总皂苷(tPNS) 对过氧化氢(H2O2) 诱导的骨髓间充质干细胞(BMSCs) 凋亡的影响,并通过ERK1/2 通路初步探讨tPNS 的作用分子机制。方法:全骨髓贴壁法体外分离、培养SD 大鼠BMSCs。不同浓度tPNS (25、50、100 和200 μg/mL) 处理BMSCs 24 h 后,用500 μmol/L H2O2 孵育细胞24 h 诱导凋亡,后行MTT 实验检测细胞活性,流式细胞术检测细胞周期。100 μg/mL tPNS 预处理24 h 后,加ERK1/2 抑制剂PD98059(25 μmol/L) 1 h,后用500 μmol/L H2O2 孵育细胞24 h 诱导凋亡,用Annexin V-FITC/PI 双染法检测细胞凋亡率;Western Blot 检测细胞ERK1/2 及其蛋白磷酸化的表达。结果:与对照组比较,其他各组存活率均显著降低(P<0.01);与H2O2 组比较,100 μg/mL 和200 μg/mL tPNS 组均能显著提高BMSCs 的存活率(P<0.01,P<0.05),其中100 μg/mL 效果更佳,所以采用最佳浓度组100 μg/mL tPNS 组进行后期研究。BMSCs 经tPNS干预24 h 后,100 μg/mL 和200 μg/mL tPNS 组细胞周期中G2+S 期细胞数的比例较H2O2组明显增加(P<0.05),其中100 μg/mLtPNS 组较200 μg/mL tPNS 组更多;其他浓度tPNS 组与H2O2 组比较,差异无统计学意义(P>0.05)。与对照组比较,其他各组凋亡率均升高(P<0.01);与H2O2 组比较,tPNS 组能显著降低BMSCs 的凋亡率(P<0.01);与tPNS 组比较,tPNS+PD98059 组能显著升高BMSCs 的凋亡率(P<0.05)。与对照组比较,H2O2 组、tPNS+PD98059 组能显著下调ERK1/2 蛋白的磷酸化表达(P<0.01);与H2O2 组比较,tPNS 组、tPNS+PD98059 组能显著上调ERK1/2 蛋白的磷酸化表达(P<0.01);tPNS 组与tPNS+PD98059 组之间并没有显著差异。结论:tPNS 对H2O2 诱导的BMSCs 凋亡有保护作用,其机制可能与促进ERK1/2 蛋白的磷酸化表达有关。

    Abstract:

    Abstract: Objective: To observe the effect of total Panax notoginseng saponins(tPNS) on apoptosis of bone marrow mesenchymal stem cells(BMSCs) induced by hydrogen peroxide(H2O2) in vitro,and to explore the molecular mechanism of tPNS through ERK1/2 pathway. Methods: BMSCs of SD rats were isolated and cultured in vitro by whole bone marrow adherent method. After BMSCs were treated with different concentrations of tPNS(25 , 50 , 100 and 200 μg/mL) for 24 hours,the cells were incubated with 500 μmol /L H2O2 for 24 hours to induce apoptosis. MTT assay was used to detect cell viability,and flow cytometry was used to detect cell cycle. After pretreatment with 100 μg/mL tPNS for 24 hours and treatment with ERK1/2 inhibitor PD98059(25 μmol/L) for 1 h,and the cells were incubated with 500 μmol/L H2O2 for 24 hours to induce apoptosis. The apoptosis rate was detected by Annexin V-FITC/PI double staining assay,and the Western Blot was used to detect the expression of ERK1/2 and its protein phosphorylation. Results:The survival rates in the other groups were significantly decreased when compared with those in the control group(P<0.01);the 100 μg/mL group and the 200 μg/mL tPNS group could significantly increase the survival rate of BMSCs when compared with that in the control group(P<0.01), and the 100 μg/mL has better effect,and therefore,the 100 μg/mL tPNS group with the best optimal concentration was used for further study. After BMSCs were treated with tPNS for 24 hours,the proportions of cell number in the cell cycle of G2 + S in the 100 μg/mL group and the 200 μg/mL tPNS group were significantly increased when compared with that in the H2O2 group(P<0.05),and the number in the 100 μg/mL tPNS group was larger than that in the 200 μg/mL tPNS group;there was no significant difference being found between the tPNS groups with the other concentrations and the H2O2 group(P> 0.05). The apoptosis rates in the other groups were increased when compared with those in the control group(P<0.01);the tPNS group could significantly decrease the apoptosis rate of BMSCs when compared with that in the H2O2 group(P<0.01); the tPNS + PD98059 group could significantly increase the apoptosis rate of BMSCs when compared with that in the tPNS group(P<0.05). The H2O2 group and the tPNS + PD98059 group can significantly down- regulate the expression of ERK1/2 protein phosphorylation when compared with that in the control group(P<0.01); the tPNS group and the tPNS + PD98059 group can significantly up- regulate the expression of ERK1/2 protein phosphorylation when compared with that in the H2O2 group(P<0.01);there was no significant difference being found in the comparison between the tPNS group and the tPNS+ PD98059 group. Conclusion:tPNS has protective effect on the apoptosis of BMSCs induced by H2O2,and its mechanism may be related to the promotion of ERK1/2 protein phosphorylation.

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王俊,陈思思,吴雪莉,陆恵琴.三七总皂苷对H2O2诱导BMSCs 凋亡的保护作用及机制研究[J].新中医,2021,53(19):1-6

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  • 在线发布日期: 2021-10-09
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