Hippo信号通路调控间充质干细胞抗氧化应激损伤能力的实验研究
Effects of Hippo signaling on anti-oxidative stress of mesenchymal stem cells in vitro
目的 探讨Hippo信号通路对小鼠骨髓来源间充质干细胞(mMSCs)抗氧化应激损伤的调控作用.方法 直接贴壁法分离C57BL/6小鼠原代mMSCs,通过流式细胞仪检测和诱导其成骨、成软骨、成脂分化并进行鉴定.通过2-脱氧-D-葡萄糖(2-DG)或Hippo信号通路高效特异选择性抑制剂XMU-MP-1调节Hippo信号通路.通过不同浓度双氧水(H2O2)共培养来模拟氧化应激损伤,用四甲基偶氮唑盐(MTT)法评估mMSCs存活情况;蛋白质免疫印迹试验(Western Blot)检测mMSCs中Hippo信号通路关键组分表达;Western Blot法检测凋亡相关蛋白Bcl-2及Bax的表达.结果 2-DG呈浓度依赖性地激活Hippo信号通路,其浓度为5 mmol/L时效应最明显〔与空白对照组比较,大肿瘤抑制基因1(LATS1)蛋白(灰度值):2.33±0.25比0.98±0.03,磷酸化Yes相关蛋白(p-YAP)/YAP蛋白比值(灰度值):2.30±0.35比1.01±0.05,14-3-3蛋白(灰度值):2.19±0.40比0.99±0.04,均P<0.05〕;100 nmol/L的XMU-MP-1可抑制Hippo信号通路活化〔与空白对照组比较,LATS1蛋白(灰度值):0.69±0.10比0.98±0.03,p-YAP/YAP蛋白比值(灰度值):0.65±0.06比1.01±0.05,14-3-3蛋白(灰度值):0.75±0.11比0.99±0.04,均P<0.05〕.H2O2呈浓度依赖性抑制mMSCs存活,0.1 mmol/L是其抑制mMSCs存活的最低浓度〔与空白对照组比较,mMSCs存活率:(81.25±11.85)%比(100.44±12.39)%,P<0.05〕.H2O2呈浓度依赖性抑制Hippo信号通路,0.1 mmol/L是其抑制Hippo信号通路的最低浓度〔与空白对照组比较,LATS1蛋白(灰度值):0.75±0.06比1.01±0.09,p-YAP/YAP蛋白比值(灰度值):0.69±0.05比0.98±0.05,均P<0.05〕,并可导致Bcl-2/Bax比值下降(灰度值:0.48±0.18比1.06±0.09, P<0.05).与0.1 mmol/L H2O2组比较,5 mmol/L的2-DG预处理可以通过活化Hippo信号通路〔LATS1蛋白(灰度值):0.95±0.05比0.64±0.06,p-YAP/YAP蛋白比值(灰度值):0.87±0.03比0.45±0.16,均P<0.05〕进而提高Bcl-2/Bax比值(灰度值:1.14±0.16比0.77±0.12,P<0.05),改善mMSCs存活〔(92.80±9.43)%比(75.47±9.43)%,P<0.05〕;反之,100 nmol/L的XMU-MP-1预处理可通过抑制Hippo信号通路〔LATS1蛋白(灰度值):0.39±0.03比0.64±0.06,p-YAP/YAP蛋白比值(灰度值):0.28±0.04比0.45±0.16,均P<0.05〕进而降低Bcl-2/Bax比值(灰度值:0.63±0.20比0.77±0.12,P<0.05),进一步抑制mMSCs存活〔(57.54±4.59)%比(75.47±9.43)%,P<0.05〕.此外,急性呼吸窘迫综合征(ARDS)小鼠肺组织较正常肺组织能明显促进mMSCs中Hippo信号通路活化〔LATS1蛋白(灰度值):1.71±0.08比1.00±0.10,p-YAP/YAP蛋白比值(灰度值):2.46±0.39比1.01±0.04,14-3-3蛋白(灰度值):2.27±0.52比1.01±0.08,均P<0.05〕.结论 Hippo信号通路可通过调控凋亡相关蛋白Bcl-2/Bax的平衡,进而影响mMSCs的存活和抗氧化应激损伤的能力.
更多Objective To explore the effects of Hippo signaling on anti-oxidative stress of mouse marrow mesenchymal stem cells (mMSCs) in vitro. Methods mMSCs derived from C57BL/6 mice were identified using fluorescence-activated cell sorting analysis and the capabilities of osteogenic, chondrogenic and adipogenic differentiation were evaluated. 2-deoxy-D-glucose (2-DG) or XMU-MP-1 was used to modulate Hippo signaling. Oxidative stress was induced by H2O2treatment and the effect of oxidative stress induced by H2O2on survival of mMSCs was evaluated using methyl thiazolyl tetrazolium (MTT) assay. The effect of oxidative stress induced by H2O2on Hippo signaling and the effect of Hippo signaling on capability of anti-oxidative stress of mMSCs were analyzed through apoptosis-regulated proteins (Bcl-2 and Bax) using Western Blot. Results Hippo signaling was activated by 2-DG in a concentration-dependent manner and the effect was most prominent by 5 mmol/L of 2-DG [compared with the blank control group, large tumor suppressor 1 (LATS1) protein (grey value): 2.33±0.25 vs. 0.98±0.03, phosphorylated Yes-associated protein (p-YAP)/YAP protein ratio (grey value): 2.30±0.35 vs. 1.01±0.05, 14-3-3 protein (grey value):2.19±0.40 vs. 0.99±0.04, all P < 0.05]; Hippo signaling was inhibited by 100 nmol/L of XMU-MP-1 [compared with the blank control group, LATS1 protein (grey value): 0.69±0.10 vs. 0.98±0.03, p-YAP/YAP protein ratio (grey value):0.65±0.06 vs. 1.01±0.05, 14-3-3 protein (grey value): 0.75±0.11 vs. 0.99±0.04, all P < 0.05]. Death of mMSCs was induced by H2O2in a concentration-dependent manner and the minimal effective concentration was 0.1 mmol/L [compared with the blank control group, survival rate of mMSCs: (81.25±11.85)% vs. (100.44±12.39)%, P < 0.05]. Inhibition of Hippo signaling was induced by H2O2in a concentration-dependent manner and the minimal effective concentration was also 0.1 mmol/L [compared with the blank control group, LATS1 protein (grey value): 0.75±0.06 vs. 1.01±0.09, p-YAP/YAP protein ratio (grey value): 0.69±0.05 vs. 0.98±0.05, both P < 0.05], those effects might associate with reduction of Bcl-2/Bax ratio (grey value: 0.48±0.18 vs. 1.06±0.09, P < 0.05). Compared with the treatment of 0.1 mmol/L of H2O2, activation of Hippo signaling by 5 mmol/L of 2-DG [ LATS1 protein (grey value):0.95±0.05 vs. 0.64±0.06, p-YAP/YAP protein ratio (grey value): 0.87±0.03 vs. 0.45±0.16, both P < 0.05] improved survival of mMSCs [(92.80±9.43)% vs. (75.47±9.43)%, P < 0.05] through an increase of Bcl-2/Bax ratio (grey value:1.14±0.16 vs. 0.77±0.12, P < 0.05); however, inhibition of Hippo signaling by 100 nmol/L of XMU-MP-1 [ LATS1 protein (grey value): 0.39±0.03 vs. 0.64±0.06, p-YAP/YAP protein ratio (grey value): 0.28±0.04 vs. 0.45±0.16, both P < 0.05] decreased survival of mMSCs [(57.54±4.59)% vs. (75.47±9.43)%, P < 0.05] through an decrease of Bcl-2/Bax ratio (grey value: 0.63±0.20 vs. 0.77±0.12, P < 0.05). Compared with normal lung tissue, acute respiratory distress syndrome (ARDS) lung tissue markedly activate Hippo signaling in mMSCs [LATS1 protein (grey value): 1.71± 0.08 vs. 1.00±0.10, p-YAP/YAP protein ratio (grey value): 2.46±0.39 vs. 1.01±0.04, 14-3-3 protein (grey value):2.27±0.52 vs. 1.01±0.08, all P < 0.05]. Conclusion Hippo signaling could affect survival and capability of anti-oxidative stress of mMSCs via modulation of Bcl-2/Bax ratio in vitro.
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