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,�
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 pr
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)。
