低氧调控EIMD后肌细胞膜损伤MAPK机制的探索与验证

发布时间：2018-07-28 14:51

【摘要】：目的:探索运动性肌损伤(EIMD)后低氧调控肌细胞膜损伤的MAPK机制,并进行验证。方法:健康雄性SD大鼠随机分为对照组、常氧24 h、48 h、72 h组和低氧24 h、48 h、72 h组。大鼠以EIMD运动模型进行间歇性下坡跑离心运动,腹腔注射伊文氏蓝荧光染色剂(EBD),取腓肠肌样本,用激光共聚焦显微镜观察EBD切片和阳性细胞率(PRC)评价膜损伤。用RT-qPCR和Westernblot测定MAPK的ERK1/2、JUN、p38和BMK1的mRNA表达、蛋白含量和磷酸化水平。再开展抑制剂实验以确认低氧调控膜损伤MAPK机制中的关键信号通路。结果:EIMD后,低氧与常氧各组均出现明显膜损伤,低氧各组阳性细胞率显著高于常氧各组和对照组(P0.01)。低氧组ERK1、p38和BMK1通路的mRNA表达、蛋白含量和磷酸化水平显著高于常氧对应组(P0.05),JNK信号通路蛋白含量和磷酸化水平无显著变化(P0.05)。抑制剂实验结果表明,ERK1/2和p38抑制剂组的蛋白含量和磷酸化水平无显著变化(P0.05)。BMK1抑制剂组24 h、48 h的磷酸化水平、阳性细胞率显著低于安慰剂对应组(P0.001),且与对照组无显著差异(P0.05)。结论:1)EIMD后低氧明显加剧膜损伤并造成损伤峰值前移。2)低氧调控膜损伤主要通过BMK1通路实现,抑制BMK1磷酸化能阻止低氧对膜损伤的加剧作用。BMK1/ERK5通路是低氧调控EIMD后膜损伤MAPK机制的关键途径。
[Abstract]:Aim: to explore the MAPK mechanism of hypoxic regulation of muscle cell membrane injury after (EIMD). Methods: healthy male Sprague-Dawley rats were randomly divided into control group, normoxic 24 h + 48 h + 72 h group and hypoxia 24 h + 48 h + 72 h group. Rats were subjected to intermittent downhill centrifugal exercise with EIMD model. Samples of gastrocnemius muscle were collected by intraperitoneal injection of Yi Wen blue fluorescent staining (EBD),. EBD sections and positive cell rate (PRC) were observed by confocal laser microscopy to evaluate membrane damage. The mRNA expression, protein content and phosphorylation level of ERK1 / 2 JUNN p38 and BMK1 in MAPK were measured by RT-qPCR and Westernblot. Inhibitor experiments were carried out to identify the key signaling pathways in the mechanism of hypoxic membrane damage to MAPK. Results the membrane damage was observed in both hypoxic and normoxic groups, and the positive cell rate in hypoxic group was significantly higher than that in normoxic group and control group (P0.01). The mRNA expression, protein content and phosphorylation level of ERK1 p38 and BMK1 pathway in hypoxia group were significantly higher than those in normoxic group (P0.05). The results of inhibitor experiment showed that the protein content and phosphorylation level of ERK1 / 2 and p38 inhibitor groups had no significant change (P0.05). The phosphorylation level of BMK1 inhibitor group was significantly lower than that of placebo corresponding group (P0.001), and there was no significant difference between BMK1 inhibitor group and control group (P0.05). Conclusion hypoxia after EIMD significantly increases membrane damage and causes the peak value of injury to shift forward. 2) hypoxic regulatory membrane damage is mainly achieved through the BMK1 pathway. Inhibition of BMK1 phosphorylation can prevent hypoxia from exacerbating membrane damage. BMK1 / ERK5 pathway is the key pathway to regulate the mechanism of EIMD membrane damage after hypoxia.
【作者单位】：杭州师范大学体育与健康学院;北京体育大学;
【基金】：浙江省自然科学基金面上项目(LY18C110002) 国家自然科学基金资助项目(31271276) 杭州市社科优秀青年人才培育计划资助项目(2017RCZX40)
【分类号】：R873

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