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.
Zhongguo Zhong Yao Za Zhi. 2023 Jun;48(12):3199-3206. doi: 10.19540/j.cnki.cjcmm.20230118.401.
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 Sparganii 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(50 μmol·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 by flow cytometry, and the migration ability was examined by wound healing assay. The secretion of interleukin(IL)-1β, IL-6, and tumor necrosis factor(TNF)-α was determined by enzyme-linked immunosorbent assay(ELISA). The protein levels of cysteinyl aspartate specific protei-nase-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.
基于 Janus 激酶 2/信号转导与转录激活因子 3(JAK2/STAT3)信号通路,本研究探讨了三棱(SR)和莪术(CR)含药血清对异位子宫内膜间质细胞(ESCs)增殖、凋亡、迁移及炎性因子分泌的影响。具体而言,对人 ESCs 进行原代培养。采用甲基噻唑基四氮唑(MTT)法检测不同浓度(5%、10%、20%)的 SR、CR 及 SR - CR 含药血清和 AG490 溶液(50 μmol·L⁻¹)对 ESCs 增殖的影响,并据此选取最佳剂量进行进一步实验。将细胞分为正常血清(NS)组、SR 组(10%)、CR 组(10%)、联合(CM)组(10%)和 AG490 组。采用流式细胞术检测 ESCs 的凋亡水平,通过伤口愈合实验检测细胞迁移能力。采用酶联免疫吸附测定(ELISA)法测定白细胞介素(IL)-1β、IL -6 和肿瘤坏死因子(TNF)-α 的分泌水平。采用蛋白质免疫印迹法检测半胱天冬酶 -3(caspase -3)、B 细胞淋巴瘤(Bcl -2)和 Bcl -2 相关 X 蛋白(Bax)的蛋白水平以及磷酸化(p)-JAK2 和 p -STAT3 的水平。结果显示,与空白血清组相比,给药组 ESCs 细胞活力降低(P<0.01),尤其是 10%含药血清组,故选取该组进行进一步实验。10% SR 含药血清、10% CR 含药血清和 10% CM 含药血清均可提高凋亡率(P<0.01),上调细胞中 caspase -3 和 Bax 的蛋白表达(P<0.05 或 P<0.01),下调 Bcl -2 的表达(P<0.01),降低细胞迁移率(P<0.05 或 P<0.01),并降低 IL -1β、IL -6 和 TNF -α 的分泌水平(P<0.05 或 P<0.01)以及 p -JAK2 和 p -STAT3 的水平(P<0.05 或 P<0.01)。与 SR 组和 CR 组相比,CM 组细胞活力较低(P<0.01),caspase -3 和 Bax 的蛋白表达较高(P<0.05 或 P<0.01),Bcl -2 和 p -JAK2 的蛋白表达较低(P<0.05)。与 CR 组相比,CM 组孵育后凋亡率较高(P<0.05),迁移率较低(P<0.01)。CM 组的 p -STAT3 蛋白水平低于 SR 组(P<0.05)。SR、CR 及其联合用药改善子宫内膜异位症的机制可能是它们阻断了 JAK2/STAT3 信号通路,抑制了 ESCs 增殖,促进了凋亡,减弱了细胞迁移,并减少了炎性因子的分泌。联合用药的效果优于单独使用 SR 和单独使用 CR。