内质网应激与非折叠蛋白反应激活对骨骼肌纤维免疫行为的调控作用

doi:10.16098/j.issn.0529-1356.2022.06.006

解剖学报 ›› 2022, Vol. 53 ›› Issue (6): 727-736.doi: 10.16098/j.issn.0529-1356.2022.06.006

内质网应激与非折叠蛋白反应激活对骨骼肌纤维免疫行为的调控作用

黄静雯¹张任飞² 菅晓婷¹ 廖钊宏¹ 黄涛¹ 胡稷杰^3* 廖华^1*

1.南方医科大学基础医学院人体解剖学教研室，广州 510515; 2.绵阳市第三人民医院检验科，四川绵阳 612000; 3.南方医科大学南方医院创伤骨科，广州 510515

收稿日期:2021-03-17 修回日期:2021-11-18 出版日期:2022-12-06 发布日期:2022-12-06
通讯作者: 胡稷杰;廖华 E-mail:hjw0112@163.com
基金资助:
调节T细胞（Tregs）辅助的巨噬细胞胞葬对骨骼肌炎症调控及其机制研究;CAMKIV-MHC Class I-ER Stress途径对骨骼肌炎症及再生的调控及机制研究;TGF-β干预急性损伤诱发的骨骼肌内质网应激及其机制研究

Regulation effect of endoplasmic reticulum stress and unfolded protein response activation on skeletal muscle fiber immune behavior

HUANG Jing-wen¹ ZHANG Ren-fei² JIAN Xiao-ting¹ LIAO Zhao-hong¹ LAN Hai-qiang¹ HUANG Tao¹ HU Ji-jie^3* LIAO Hua^1*#br#

1. Department of Human Anatomy, School of Basic Medical Sciences, Southern Medical University, Guangzhou 510515, China; 2. Laboratory Department, Mianyang Third People’s Hospital，Sichuan Mianyang 621002, China; 3. Department of Orthopaedics, Nanfang Hospital, Southern Medical University, Guangzhou 510515, China

Received:2021-03-17 Revised:2021-11-18 Online:2022-12-06 Published:2022-12-06
Contact: HU Ji-jie;LIAO Hua E-mail:hjw0112@163.com

摘要/Abstract

摘要：

目的探讨干扰素-γ（IFN-γ）诱导的炎症环境中，骨骼肌纤维内质网应激（ERS）与非折叠蛋白反应（UPR）的激活对肌纤维免疫行为的调控作用。方法体外培养的C57BL/6小鼠原代成肌干细胞，经马血清分化成多核肌管后分成以下10组：1.对照组；2.IFN-γ组；3.衣霉素（TM）组；4.毒胡萝卜素（TG)组；5.IFN-γ+4-苯基丁酸（4-PBA）联合处理组；6.IFN-γ+TG+4-PBA联合处理组；7.IFN-γ+4μ8c联合处理组；8.IFN-γ+TG+4μ8c联合处理组；9.IFN-γ+GSK2606414联合处理组；10.IFN-γ+TG+GSK2606414联合处理组，并进行对应的处理。利用Real-time PCR检测相关肌细胞因子基因水平；免疫荧光观察UPR关键分子：真核翻译起始因子2α（eIF2α）、肌醇需要酶1α（IRE1α）、转录激活因子6（ATF6）在肌纤维内的表达；Western blotting检测肌细胞相关免疫分子和肌细胞因子及UPR关键分子；Luminex分析肌纤维内促炎症的肌细胞因子的蛋白水平。结果 IFN-γ诱导的炎症环境中，肌纤维H-2Kb、H2-Ea、Toll样受体3（TLR3）以及p-eIF2α、p-IRE1α表达上调；添加UPR抑制剂4-PBA的分组肌纤维H-2Kb、 H2-Ea、TLR3以及肌细胞因子的表达较IFN-γ组下调，添加IRE1α特异性抑制剂4μ8c的分组上述分子表达较IFN-γ组亦下调，而添加蛋白激酶R样内质网激酶（PERK）特异性抑制剂GSK2606414的分组则无明显变化。结论 IFN-γ诱导的炎症环境中，UPR-IRE1α通路激活并抑制肌纤维免疫相关分子合成，从而进一步抑制肌纤维介导的免疫反应，有利于肌再生。

关键词: 肌纤维, 内质网应激, 非折叠蛋白反应, 肌醇需要酶1α, 原代细胞培养, 免疫印迹法, 小鼠

Abstract:

Objective To investigate the regulatory effects of activated endoplasmic reticulum stress (ERS) and unfolded protein response (UPR) on the immune behavior of the stressed muscle fibers in inflammatory environments induced by interferon-γ(IFN-γ). Methods The myogenic precursor cells (MPCs) of C57 BL/6 mice cultured in vitro were differentiated into multinucleated myogenic tubes by horse serum and then to set up: 1. Control group; 2. IFN-γ group; 3. Tunicamycin group; 4. Thapsigargin group; 5. IFN-γ and 4-phenylbutyrate(4-PBA) combined treatment group; 6. IFN-γ, TG and 4-PBA combined treatment group; 7. IFN-γ and 4μ8c combined treatment group; 8. IFN-γ, TG and 4μ8c combined treatment group; 9. IFN-γ and GSK2606414 combined treatment group; 10. IFN-γ, TG and GSK2606414 combined treatment group. The level of myokines gene was detected by Real-time PCR. The expression of UPR key molecules including eukaryotic intiatio factor 2α(eIF2α), inositol requrring enzyme 1α(IRE1α) and activating transcription factor 6(ATF6) in muscle fibers was observed by immunofluorescence. Western blotting was used to detect immune molecules related to muscle cells, myokines and key molecules of UPR. Luminex analyzed the levels of pro-inflammatory myokines in muscle fibers. Results The expression of H-2Kb, H2-Ea, Toll like receptor 3(TLR3), p-eIF2α and p-IRE1α were up-regulated in IFN-γ induced inflammatory environment. The expression of H-2Kb, H2-Ea, TLR3 and myokines in the group with UPR inhibitor 4-PBA was down-regulated compared with IFN-γ group, and the expression of these molecules in the group with IRE1α specific inhibitor 4μ8c was down-regulated compared with the IFN-γ group. The addition of protein kinase R-like endoplasmic eticulum(PERK) specific inhibitor GSK2606414 showed no significant change. Conclusion In IFN-γ induced inflammatory environment, the UPR-IRE1α pathway activates and inhibits the synthesis of muscle fiber immune-related molecules, which further inhibits the muscle fiber mediated immune response and facilitates muscle regeneration.

Key words: Muscle fiber, Endoplasmic reticulum stress, Unfolded protein response, Inositol requiring enzyme 1α, Primary cell culture, Western blotting, Mouse

中图分类号:

R685.4

黄静雯张任飞菅晓婷廖钊宏蓝海强黄涛胡稷杰廖华. 内质网应激与非折叠蛋白反应激活对骨骼肌纤维免疫行为的调控作用[J]. 解剖学报, 2022, 53(6): 727-736.

HUANG Jing-wen ZHANG Ren-fei JIAN Xiao-ting LIAO Zhao-hong LAN Hai-qiang HUANG Tao HU Ji-jie LIAO Hua.

Regulation effect of endoplasmic reticulum stress and unfolded protein response activation on skeletal muscle fiber immune behavior

[J]. Acta Anatomica Sinica, 2022, 53(6): 727-736.

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内质网应激与非折叠蛋白反应激活对骨骼肌纤维免疫行为的调控作用

Regulation effect of endoplasmic reticulum stress and unfolded protein response activation on skeletal muscle fiber immune behavior

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