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基于JAK2/STAT3信号通路探讨三棱-莪术含药血清对人异位子宫内膜间质细胞增殖、凋亡和迁移的影响 被引量:2

Effect of Sparganii Rhizoma-Curcumae Rhizoma-medicated serum on proliferation,apoptosis,and migration of human ectopic endometrial stromal cells:based on JAK2/STAT3 signaling pathway
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摘要 基于酪氨酸蛋白激酶(Janus kinase, JAK)2/信号传导及转录激活因子(signal transducer and activator of transcription, STAT)3信号通路,研究三棱-莪术含药血清对异位的子宫内膜间质细胞(ectopic endometrial stromal cells, ESCs)增殖、凋亡、迁移和炎症因子分泌的影响。原代培养ESCs,噻唑蓝法(methyl thiazolyl tetrazolium, MTT)检测不同质量分数(5%、10%、20%)三棱、莪术、两药配伍的含药血清和AG490溶液(50μmol·L^(-1))对ESCs增殖的影响,并由此选择最优剂量用于后续实验;将细胞分为空白血清组、三棱组(10%)、莪术组(10%)、配伍组(10%)和AG490组,以流式细胞术检测ESCs凋亡水平,细胞划痕实验检测细胞迁移能力,酶联免疫吸附测定法(enzyme linked immunosorbent assay, ELISA)检测细胞白细胞介素(interleukin, IL)-1β、IL-6和肿瘤坏死因子(tumor necrosis factor, TNF)-α分泌水平,蛋白质印迹法(Western blot)检测含半胱氨酸的天冬氨酸蛋白水解酶(cysteinyl aspartate specific proteinase, caspase)-3、B淋巴细胞瘤(B-cell lymphoma, Bcl)-2、Bcl-2相关X蛋白(Bcl-2 associated X protein, Bax)的蛋白表达以及JAK2、STAT3蛋白的磷酸化水平。结果显示,与空白血清组相比,各给药组均能使ESCs的活力显著降低(P<0.01),尤其10%含药血清细胞活力低于其他组,因此后续实验采用10%的含药血清进行观察。10%的三棱、莪术和两药配伍的含药血清均能使细胞凋亡率显著增加(P<0.01),细胞中caspase-3和Bax蛋白表达均显著升高(P<0.05或P<0.01),Bcl-2蛋白表达显著降低(P<0.01),细胞迁移率显著降低(P<0.05或P<0.01),细胞分泌的IL-1β、IL-6和TNF-α显著减少(P<0.05或P<0.01),JAK2和STAT3的磷酸化水平显著降低(P<0.05或P<0.01)。与三棱、莪术组相比,10%配伍组含药血清孵育后细胞活力更低(P<0.01),caspase-3和Bax蛋白表达较高(P<0.05或P<0.01),Bcl-2蛋白表达更低(P<0.05),JAK2蛋白磷酸化程度较低(P<0.05)。10%配伍组含药血清孵育后,细胞凋亡率明显高于莪术组(P<0.05),细胞的迁移率明显低于莪术组(P<0.01),对STAT3蛋白磷酸化的抑制作用明显强于三棱组(P<0.05)。三棱、莪术及其配伍应用改善子宫内膜异位症的作用机制可能与阻断JAK2/STAT3信号通路,抑制ESCs增殖,促进细胞凋亡,减弱其迁移能力,减少炎症因子的分泌有关,并且三棱-莪术等比配伍效果优于两药单用。 Based on the Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway,this study investigated the effect of medicated serum of Spargani Rhizoma(SR)and Curcumae Rhizoma(CR)on the proliferation,apoptosis,migration,and secretion of inflammatory factors of ectopic endometrial stromal cells(ESCs).Specifically,human ESCs were primary-cultured.The effect of different concentration(5%,10%,20%)of SR-,CR-,and SR-CR combination-medicated serum,and AG490 solution(50μmol·L^(-1))on the proliferation of ESCs was detected by methyl thiazolyl tetrazolium(MTT)assay,and the optimal dose was selected accordingly for further experiment.The cells were classified into normal serum(NS)group,SR group(10%),CR group(10%),combination(CM)group(10%),and AG490 group.The apoptosis level of ESCs was detected byflow cytometry and the migration ability was examined by wound healing assay.The secretion of interleukin(IL)-1β,IL-6,and tumor necrosis factor(TNF)-αwas determined by enzyme-linked immunosorbent assay(ELISA).The protein levels of cysteinyl aspartate specific proteinase-3(caspase-3),B-cell lymphoma(Bcl-2),and Bcl-2-associated X protein(Bax)and the levels of phosphorylated(p)-JAK2 and p-STAT3 were detected by Western blot.The results showed that the viability of ESCs cells was lowered in the administration groups compared with the blank serum group(P<0.01),especially the 10%drug-medicated serum,which was selected for further experiment.The 10%SR-medicated serum,10%CR-medicated serum,and 10%CM-medicated serum could increase the apoptosis rate(P<0.01),up-regulate the protein expression of caspase-3 and Bax in cells(P<0.05 or P<0.01),down-regulate the expression of Bcl-2(P<0.01),decrease the cell migration rate(P<0.05 or P<0.01),and reduce the secretion levels of IL-1β,IL-6,and TNF-α(P<0.05 or P<0.01),and levels of p-JAK2 and p-STAT3(P<0.05 or P<0.01).Compared with the SR and CR groups,CM group showed low cell viability(P<0.01),high protein expression of caspase-3 and Bax(P<0.05 or P<0.01),and low protein expression of Bcl-2 and p-JAK2(P<0.05).After incubation with CM,the apoptosis rate was higher(P<0.05)and the migration rate was lower(P<0.01)than that of the CR group.The p-STAT3 protein level of CM group was lower than that of the RS group(P<0.05).The mechanism of SR,CR,and the combination underlying the improvement of endometriosis may be that they blocked JAK2/STAT3 signaling pathway,inhibited ESC proliferation,promoted apoptosis,weakened cell migration,and reduced the secretion of inflammatory factors.The effect of the combination was better than that of RS alone and CR alone.
作者 王焱 聂晓博 金霞 王建平 刘丽华 刘姣 WANG Yan;NIE Xiao-bo;JIN Xia;WANG Jian-ping;LIU Li-hua;LIU Jiao(the Fourth Hospital of Shijiazhuang,Shijiazhuang 050035,China;Hebei Provincial Key Laboratory of Maternal Fetal Medicine,Shijiazhuang 050035,China;Shijiazhuang Medical College,Shijiazhuang 050599,China;Hebei University of Chinese Medicine,Shijiazhuang 050200,China)
出处 《中国中药杂志》 CAS CSCD 北大核心 2023年第12期3199-3206,共8页 China Journal of Chinese Materia Medica
基金 河北省卫健委医学科研项目(20201383) 国家自然科学基金项目(81803774) 河北省自然科学基金面上项目(H2020423222)。
关键词 酪氨酸蛋白激酶2/信号传导及转录激活因子3(JAK2/STAT3)信号通路 三棱 莪术 配伍 异位子宫内膜间质细胞(ESCs) Janus kinase 2/signal transducer and activator of transcription 3(JAK2/STAT3)signaling pathway Sparganii Rhizoma Curcumae Rhizoma compatibility of medicines ectopic endometrial stromal cells(ESCs)
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