温补肾阳方通过ROS/p38 MAPK通路对骨髓增生异常综合征细胞株自噬及凋亡的影响

Effect of Wenyang Bushen prescription on autophagy and apoptosis of myelodysplastic syndrome cell lines through ROS/p38 MAPK pathway

目的探讨温阳补肾方对骨髓增生异常综合征(MDS)细胞株SKM-1凋亡和自噬的影响及其机制。方法SD大鼠分别以5,10,20 g/kg温阳补肾方药液和等量生理盐水灌胃,获得低、中、高剂量温阳补肾方含药血清及对照血清;将体外培养的SKM-1细胞分为空白对照组、生理盐水组和药物低、中、高剂量组(分别加入低、中、高剂量温阳补肾方含药血清),采用噻唑蓝(MTT)法和流式细胞术分别检测SKM-1细胞增殖和凋亡,比色法检测SKM-1细胞含半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)活性,2,7-二氯二氢荧光素二乙酸酯(DCFH-DA)荧光探针法检测SKM-1细胞内活性氧(ROS)水平,免疫印迹法(Western blot)检测SKM-1细胞中自噬相关蛋白Beclin1、P62、微管相关蛋白1轻链3(LC3)-Ⅰ、LC3-Ⅱ和p38丝裂原活化蛋白激酶(p38 MAPK)通路相关蛋白P38 MAPK、p-P38 MAPK表达水平。结果与空白对照组相比,生理盐水组细胞增殖活力、凋亡率、Caspase-3活性、ROS水平以及P62、Beclin1蛋白表达和LC3-Ⅱ/LC3-Ⅰ、p-P38 MAPK/P38 MAPK比值差异无统计学意义(P>0.05)。与生理盐水组比较,药物低、中、高剂量组SKM-1细胞增殖活力和P62蛋白表达水平明显降低,而细胞凋亡率、Caspase-3活性、ROS水平、Beclin1蛋白表达水平、LC3-Ⅱ/LC3-Ⅰ值和p-P38 MAPK/P38 MAPK值均明显升高(P<0.05),且呈一定的药物浓度依赖性(P<0.05)。结论温阳补肾方可能通过激活ROS/p38 MAPK信号通路,调控细胞凋亡和自噬相关蛋白的表达,进而促进MDS细胞发生凋亡自噬。

Objective To investigate the effect of Wenyang Bushen prescription on apoptosis and autophagy of myelodysplastic syndrome(MDS)cell line SKM-1 and its mechanism.Methods SD rats were treated with Wenyang Bushen decoction(5,10,20 g/kg)and equal amount of normal saline respectively by gavage to obtain serum containing low,medium and high doses of Wenyang Bushen prescription and normal saline serum.SKM-1 cells cultured in vitro were divided into blank control group,normal saline group and low,medium,high does groups,and the cells were treated with the corresponding serum,respectively.The proliferation and the apoptosis of SKM-1 cells by MTT assay and flow cytometry,the activity of cysteinyl aspartate specific proteinase 3(Caspase-3)in SKM-1 cells was detected by colorimetry,and the level of reactive oxygen species(ROS)in SKM-1 cells was detected by 2,7-dichlorodi-hydrofluorescein diacetate(DCFH-DA)fluorescence probe.Western blot was used to detect the expression levels of autophagy-related proteins Beclin1,P62,microtubule-associated protein 1 light chain 3(LC3)-Ⅰ,LC3-Ⅱand p38 mitogen activated protein kinase(p38 MAPK)pathway-related proteins P38 MAPK and phosphorylation(p)-P38 MAPK in SKM-1 cells.Results There was no statistical significance in all indexes between control group and normal saline group.Compared with normal saline group,the proliferation activity of SKM-1 cells and P62 protein expression in low,medium and high dose groups were significantly decreased,the apoptosis rate,Caspase-3 activity,ROS level,Beclin1 protein expression,LC3-Ⅱ/LC3-Ⅰvalue and p-P38 MAPK/P38 MAPK were significantly increased(P<0.05).In addition,the above indexes showed concentration-dependent changes.Conclusion Wenyang Bushen prescription can inhibit the proliferation of SKM-1 cells and promote cell apoptosis and autophagy.The mechanism may be related to the activation of ROS/p38 MAPK pathway.