肉仔鸡卫星细胞氧化应激时MAPK信号通路

 【目的】在地塞米松(DEX)诱导骨骼肌卫星细胞氧化应激的体外条件下,筛选丝裂原活化蛋白激酶(MAPKs)信号系统中起关键作用的信号通路。【方法】 将体外培养的肉仔鸡胸肌骨骼肌卫星细胞(SCs)分别进行处理：Ⅰ、对照组;Ⅱ、DEX;Ⅲ、p38 MAPK抑制剂;Ⅳ、DEX+p38 MAPK抑制剂;Ⅴ、JNK抑制剂;Ⅵ、DEX+JNK抑制剂;Ⅶ、ERK5抑制剂;Ⅷ、DEX+ERK5抑制剂;Ⅸ、ERK1/2抑制剂;Ⅹ、DEX+ERK 1/2抑制剂。除处理Ⅰ和处理Ⅱ外,其它处理首先用抑制剂预处理30 min,弃去培养基后,再按处理设置分别加入含DEX的培养基或者正常培养基继续处理24 h。处理结束后,分别测定细胞培养液中丙二醛(MDA)、活性氧自由基(ROS)、超氧化物歧化酶(SOD)和谷胱甘肽硫转酶(GST)含量或活性;同时采用RT-PCR方法检测通路蛋白及SOD与GST基因表达量。【结果】 抑制剂单独处理组MDA和ROS的产生量显著低于DEX处理(P＜0.05);DEX+ERK5抑制剂处理与DEX+ERK 1/2抑制剂处理的MDA和ROS产生量显著高于ERK 5抑制剂处理和ERK1/2抑制剂处理(P＜0.05); DEX+p38 MAPK抑制剂处理与 DEX+JNK抑制剂处理组的MDA和ROS产生量与p38 MAPK处理和JNK处理的差异不显著(P＞0.05)。抑制剂单独处理组的SOD和GST活性比DEX处理高(P＜0.01);DEX+ERK5抑制剂处理与 DEX+ERK 1/2抑制剂处理的SOD和GST活性极显著低于ERK5抑制剂处理组和ERK1/2抑制剂处理组(P＜0.01),DEX+p38 MAPK抑制剂处理与DEX+JNK抑制剂处理的SOD和GST活性与p38 MAPK抑制剂处理和 JNK抑制剂处理的差异不显著(P＞0.05)。基因表达结果表明,DEX能够抑制GST和SOD基因的表达,抑制率达37%以上。与抑制ERK5和ERK1/2通路不同,抑制p38 MAPK和JNK通路后,DEX对GST和SOD基因的表达无明显抑制作用。【结论】 在DEX诱导的肉仔鸡胸肌骨骼肌SCs产生的氧化应激过程中,p38 MAPK和JNK通路起着关键作用。

Study on Oxidative Stress-Activated MAPK Signaling Pathway in Broilers Breast Muscle Satellite Cells

【Objective】 Mitogen-activated protein kinases (MAPKs) signaling pathway was investigated to screen which one plays a key role in broiler skeletal muscle satellite cells (SCs) under oxidative stress induced by dexamethasone (DEX) in vitro. 【Method】 The broilers skeletal muscle SCs were assigned into one of the ten treatments: control (I), DEX (Ⅱ), inhibitor p38 MAPK (Ⅲ), DEX plus inhibitor p38 MAPK (Ⅳ), inhibitor JNK (Ⅴ), DEX plus inhibitor JNK (Ⅵ), inhibitor ERK5 (Ⅶ), DEX plus inhibitor ERK5 (Ⅷ), inhibitor ERK1/2 (Ⅸ), DEX plus inhibitor ERK1/2 (Ⅹ). The treatments exclude treatments I and Ⅱ were treated with inhibitors for 30 min before treating with DEX and normal medium for 24 h. The relative production of malonaldehyde (MDA), reactive oxidative species (ROS), the activities of superoxide dismutase (SOD) and glutathione S-transferase (GST) were measured, respectively. Polymerase chain reaction (PCR) was employed to assess SOD and GST gene expression.【Result】 Compared with DEX, the MDA and ROS concentrations were decreased (P＜0.05) in all the inhibitors-treated groups. The concentration of MDA and ROS in treatment with inhibitors were higher than that in treatment with DEX (P＜0.05) The MDA and ROS concentrations of treatment with DEX plus inhibitor ERK5 and of treatment with DEX plus inhibitor ERK 1/2 were significantly higher than that of treatment with inhibitor ERK5 and treatment with inhibitor ERK1/2 (P＜0.05). However, there were no significant differences in MDA and ROS concentrations among treatment with DEX plus inhibitor p38 MAPK, treatment with DEX plus inhibitor JNK and treatment with inhibitor p38 MAPK, treatment with inhibitor JNK (P＞0.05). The activities of SOD and GST of all the inhibitors-treated groups were significantly higher than those of DEX group (P＜0.01). The activities of SOD and GST of treatment with DEX plus inhibitor ERK5 and treatment with DEX plus inhibitor ERK 1/2 were significantly lower than those of treatment with inhibitor ERK5 and treatment with inhibitor ERK1/2 (P＜0.01), while there were no significant differences in treatment with DEX plus inhibitor p38 MAPK, treatment with DEX plus inhibitor JNK and treatment with inhibitor p38 MAPK, and treatment with inhibitor JNK (P＞0.05). DEX could inhibit the expression of SOD and GST gene, the inhibition rate was more than 37%. On the contrary to ERK5 and ERK1/2 pathway, DEX treatment had no significant effects on the expression of SOD and GST gene while the p38 MAPK and JNK pathway were inhibited. 【Conclusion】 The p38 MAPK and JNK pathway play a key role in broiler breast muscle SCs under oxidative stress induced by DEX.