人脐带血间充质干细胞通过抑制MEK/ERK通路改善LPS诱导的AECⅡ凋亡机制的研究

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摘要目的探讨人脐带血间充质干细胞（hUC-MSC）对脂多糖（LPS）诱导的肺泡Ⅱ型上皮细胞（AECⅡ）凋亡的影响，及MEK/ERK信号通路在其中的作用。方法采用LPS与A549细胞共培养建立AECⅡ凋亡模型；将凋亡模型按随机数字表法分成6组，分别与MSC条件培养基（MSC-CM）、MEK/ERK通路抑制剂（PD98059）及PBS体外共培养，分别为A549+LPS+MSC、A549+LPS+PBS、A549+LPS+PD98059，阴性对照组为A549+PBS、A549+MSC、A549+PD98059，48 h后采用流式细胞术检测A549细胞凋亡情况；对MEK/ERK通路及凋亡相关信号（BAX/BCL-2、FAS、caspase 3及caspase 8）进行qPCR检测，对A549+LPS+MSC、A549+LPS+PBS、A549+PBS、A549+MSC四组组进行Western blot检测。结果建立A549+LPS凋亡模型。A549+LPS+MSC、A549+LPS+PD98059组与A549+LPS+PBS比较，凋亡率显著下降。A549+LPS+MSC及A549+LPS+PD98059的MEK、ERK、BAX/BCL-2、caspase 3及caspase 8 mRNA扩增倍数均显著低于A549+LPS+PBS（P < 0.05），而蛋白水平上FAS、BAX、cleaved Caspase 3、p-MEK、p-ERK、A549+LPS+MSC较A549+LPS+PBS明显降低（P < 0.05），而TNF-R1差异无统计学意义（P ＞ 0.05）。结论 hUC-MSCs可能通过旁分泌作用抑制MEK/ERK信号通路磷酸化，影响内源性线粒体及外源性FAS/FASL途径进而减轻LPS诱导的AECⅡ凋亡。

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	刘坚1 龙婷婷2 李晨彦1 易军1 潘子阳1 杨根辉1 贺喜强1

关键词 ：间充质干细胞, 细胞外信号调节激酶, 凋亡, 肺泡Ⅱ型上皮细胞, 脂多糖

Abstract：Objective To discuss the effect of human umbilical cord blood mesenchymal stem cells (hUC-MSC) on alveolar type Ⅱ epithelial cells (AEC Ⅱ) apoptosis induce by lipopolysaccharide (LPS), and the role of MEK/ERK signaling pathway. Methods The AECⅡ apoptosis model was established by LPS co-cultured with A549 cells. The apoptosis model was randomly divided into 6 groups according to random number method, they were co-cultured with MSC conditioned medium (MSC-CM), MEK/ERK pathway inhibitor (PD98059) and PBS, which were named A549+LPS+MSC, A549+LPS+PBS, A549+LPS+PD98059, and the negative control groups were A549+PBS, A549+MSC, A549+PD98059. Apoptosis of A549 cells was detected by Annexin V/PI staining flow cytometry 48 h later. MEK/ERK pathway and apoptosis-related signals (BAX/BCL-2, FAS, caspase 3 and caspase 8) were examined by qPCR. The groups of A549+LPS+MSC, A549+LPS+PBS, A549+PBS, and A549+MSC were selected for Western blot detecting. Results The apoptosis model was established by A549+LPS. Compared with A549+LPS+PBS, the apoptosis rate of A549+LPS+MSC and A549+LPS+PD98059 were decreased significantly (P < 0.05). The mRNA expression levels of MEK, ERK, BAX/BCL-2, caspase 3 and caspase 8 in A549+LPS+MSC and A549+LPS+PD98059 were significantly lower than those in A549+LPS+PBS (P < 0.05). The expression of FAS, BAX, cleaved Caspase 3, p-MEK, and p-ERK protein in the A549+LPS+MSC and A549+LPS+PD98059 were significantly lower than A549+LPS+PBS(P < 0.05). There was no significant different in TNF-R1 among each group (P > 0.05). Conclusion hUC-MSCs may inhibit the phosphorylation of MEK/ERK signaling pathway affecting mitochondria pathway and FAS/FASL pathway by paracrine action, thereby attenuating apoptosis of AECⅡ induced by LPS.

Key words： Mesenchymal stem cells Extracellular signal-regulated kinases Apoptosis Alveolar type Ⅱ epithelial cells Lipopolysaccharide

基金资助:湖南省自然科学基金面上项目（2019JJ40287）；
湖南省卫生计生委科研基金项目（湘卫科教发[2016]521号B2017168）。

通讯作者: 贺喜强（1963-），男，主任医师，教授；研究方向：急危重症抢救治疗、重症急性胰腺炎、多器官功能障碍。

作者简介: 刘坚（1990-），男，硕士；研究方向：脓毒症、急性呼吸窘迫综合征、多器官功能障碍及干细胞转化医学。

引用本文:

刘坚1 龙婷婷2 李晨彦1 易军1 潘子阳1 杨根辉1 贺喜强1. 人脐带血间充质干细胞通过抑制MEK/ERK通路改善LPS诱导的AECⅡ凋亡机制的研究[J]. 中国医药导报, 2020, 17(6): 13-18.
LIU Jian1 LONG Tingting2 LI Chenyan1 YI Jun1 PAN Ziyang1 YANG Genhui1 HE Xiqiang1. Human umbilical cord blood mesenchymal stem cell protects apoptosis of AECⅡ induced by LPS via inhibition of MEK/ERK-mediated pathway. 中国医药导报, 2020, 17(6): 13-18.

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