间充质干细胞抑制MEK/ERK信号通路改善盲肠结扎穿孔所致急性肺损伤中肺泡Ⅱ型上皮细胞的凋亡

Mesenchymal Stem Cell Protects Apoptosis of AECⅡ from Cecal Ligation and Puncture Induced Acute Lung Injury via Inhibition of MEK / ERK-mediated Pathway

【目的】研究间充质干细胞(MSC)可通过抑制MEK/ERK信号通路改善脓毒症所致急性肺损伤(ALI)中肺泡Ⅱ型上皮细胞(AECⅡ)凋亡水平。【方法】本研究取30只balb/c雄性小鼠,随机抽取其中20只通过盲肠结扎穿孔(CLP)建立小鼠脓毒症所致急性肺损伤的模型,CLP术后6h随机分成2组,每组10只,通过尾静脉分别注射间充质干细胞(MSC)、生理盐水,同时将剩余10只小鼠建立假手术组作为对照组。该三组分别命名为CLP/MSC、CLP/saline、SHAM。CLP术后24 h收集肺组织及肺泡灌洗液(BALF)进行模型评估及分析检测。通过与抗生素联用,分析3组7 d生存率差异。【结果】模型制备达到ALI水平。在肺损伤程度上,经MSC治疗后,髓过氧化物酶(MPO)活性、肺血管通透性及病理表现,经MSC治疗后较CLP/saline组均有明显改善。7 d生存率CLP/MSC、CLP/saline分别为32%和0(P<0.05),CLP术后24 h BALF中SP-A含量CLP/MSC组(3115±93)ng/m L较CLP/saline组(2344±192)ng/m L明显改善(P=0.002)。CLP/MSC组AECⅡ凋亡水平较CLP/saline组明显降低(P=0.001)。肺组织fas、bax/bcl-2及caspase3 m RNA扩增倍数CLP/Saline vs CLP/MSC p值分别为0.09、0.004及0.037,均<0.05,蛋白水平fas、bax/bcl-2、cleaved caspase3、p-MEK、p-ERK CLP/MSC组较CLP/saline组也明显降低。结论 MSC可能是通过抑制MEK/ERK信号通路磷酸化,影响外源性死亡受体(FAS/FASL)途径及内源性(线粒体)凋亡途径来减少ALI中肺泡上皮细胞尤其是AECⅡ凋亡,从而在一定程度上降低ALI死亡率。

[Objective] We hypothesized that MSC can reduce AEC 11 apoptosis by inhibited phosphorylation of MEK/ERK pathway. [Methods] Total 30 balb/c male mice were selected in this study. Randomly select 10 mice to receive injection of mesenchymal stem cell （MSC） 6hrs after cecal ligation and puncture （CLP）, 10 to receive injection of saline 6 hrs after CLP, the other 10 to establish Sham control group. The three groups were respectively named for CLP/MSC, CLP/saline and sham. Twenty-four hours after CLP, bronchoalveolar lavage fluid and lung tissues were collected for analyses. Longerterm studies were performed with antibiotic coadministration to assess the effect of MSC on survival. [ Results ] Models were in accordance with ALI standards. As for the level of lung injury, The myeloperoxidase （MPO）, pulmonary microvascular permeability and histology were more greately improved in CLP/MSC than those in CLP/saline. The 7-day survival rate of CLP/MSC （32%） significantly increased than that of CLP/saline （0%）. The SP-A content of CLP/saline （ （2344 ± 192） ng/mL） in BAL was significantly lower than CLP/MSCs [ （3115 ± 93 ） ng/mL, P = 0.0021. The apoptosis index of CLP/MSC（2 ± 0.6） was significantly lower than CLP/saline（7 ± 0.9, P = 0.001 ）. The expression of Bax/bcl-2, Fas, cleaved easpase 3, cleaved easpase 8 and p-MEK, p-ERK protein, which were low in the CLP/MSC, were significantly increased in the control group, as well as the mRNA expression levels of BAX/BCL-2 （P = 0.004）, FAS （P = 0.009） and CASP3 （P = 0.037）. [Conclusions] It suggests that MSC may represent a potential therapeutic strategy to alleviate ARDS via inactivation of the MEK/ERK signaling pathway, which may influence Fas/Fas ligand （FasL） pathway and mitochondrial pathway thus reducing apoptosis of AECII induced by ALI, and finally reduced mortality in some extent.