白藜芦醇通过HIF-1α/BNIP3信号通路促进骨骼肌缺氧损伤修复的机制

    雷斯?陈睿?佘燕玲?周珊瑶?史华彩

    【摘要】目的 探討白藜芦醇(Res)对二氯化钴(CoCl2)诱导的骨骼肌细胞缺氧损伤的保护机制。方法 将分化的小鼠骨骼肌细胞系C2C12按不同干预方法分为4组,即对照组、 CoCl2组、 Res组、 CoCl2+Res组。观察免疫荧光染色后的细胞形态,统计肌管融合指数;采用荧光定量PCR技术检测肌球蛋白重链(MyHC) mRNA水平的变化;采用蛋白免疫印迹法检测低氧诱导因子-1α(HIF-1α)、 Bcl2/腺病毒E1B相互作用蛋白3(BNIP3)、微管相关蛋白1轻链3(LC3)以及p62和Beclin1蛋白水平。结果 CoCl2诱导的缺氧损伤使肌细胞形态异常、肌管分化减少,与对照组比较,CoCl2组肌管融合指数降低(P < 0.001),MyHC mRNA水平和蛋白表达量降低(P均 < 0.05),HIF-1α、BNIP3、Beclin1蛋白水平和LC3升高(P均 < 0.001),p62蛋白水平降低(P < 0.001)。加入Res处理后,细胞形态恢复,肌管分化增多;与CoCl2组比较,CoCl2+Res组肌管融合指数升高,MyHC亚型(Myh7、Myh2、Myh4)的mRNA和MyHC蛋白水平升高,HIF-1α、BNIP3和Beclin1蛋白水平降低,p62蛋白水平升高(P均 < 0.05)。结论 CoCl2诱导的缺氧可抑制MyHC表达,导致肌细胞分化和融合能力下降。Res可增强缺氧条件下成肌细胞的分化和融合能力,对肌细胞的损伤修复有保护作用,可能通过抑制HIF-1α/BNIP3信号通路诱导的自噬来促进肌细胞的损伤修复。

    【关键词】骨骼肌细胞;缺氧;白藜芦醇;分化;自噬

    Resveratrol promotes the repair of hypoxia-induced skeletal muscle injury through HIF-1α/BNIP3 signaling pathway Lei Si, Chen Rui, She Yanling, Zhou Shanyao, Shi Huacai. Guangdong Traditional Medical and Sports Injury Rehabilitation Research Institute, Guangdong Second Provincial General Hospital, Guangzhou 510317, China

    Corresponding author, Chen Rui, E-mail: rui.c.med@ 163. com

    【Abstract】Objective To investigate the protective mechanism of resveratrol (Res) against the CoCl2-induced hypoxia injury in the skeletal muscle cells.? Methods According to different intervention methods, the murine skeletal muscle C2C12 cells were divided into the control, CoCl2, Res and CoCl2+Res groups, respectively. The cell morphology was observed after myosin heavy chain (MyHC) immunofluorescence staining. The fusion index of the myotubes was calculated. The expression level of MyHC mRNA was detected by quantitative fluorescence PCR. The expression levels of hypoxia-inducible factor-1α (HIF-1α), Bcl2/ adenovirus E1B interacting protein 3 (BNIP3), microtubule associated protein 1 light chain 3 (MAP1LC3, LC3), p62 and Beclin1 proteins were measured by Western blot. Results After CoCl2-induced hypoxia, the morphology of myotubes was abnormal and the quantity of differentiated myotubes was reduced. Compared with the control group, the fusion index of differentiated myotubes was significantly decreased (P < 0.001), the expression levels of MyHC mRNA and protein were significantly down-regulated (both P < 0.05), those of HIF-1α, BNIP3 and Beclin1 proteins and LC3 were significantly up-regulated (all P < 0.001), and that of p62 protein was significantly down-regulated (P < 0.001) in the CoCl2 group, respectively. Following the Res intervention, the cellular morphology was recovered and the quantity of differentiated myotubes was increased. Compared with the CoCl2 group, the fusion index of differentiated myotubes was significantly elevated, the expression levels of? mRNA of MyHC subtypes (Myh7, Myh2 and Myh4) and MyHC protein were significantly up-regulated, those of HIF-1α, BNIP3 and Beclin1 proteins were significantly down-regulated, and that of p62 protein was significantly up-regulated (all P < 0.05). Conclusions CoCl2-induced hypoxia can down-regulated the expression of MyHC, resulting in decreased muscle differentiation and fusion. Rescan enhance the differentiation and fusion of myoblasts under hypoxia condition, and extert protective effect upon the repair of muscle cell injury probably by suppressing the autophagy induced by the HIF-1α/BNIP3 signaling pathway, thereby accelerating the repair of muscle cell injury.

    【Key words】Skeletal muscle cell;Hypoxia;Resveratrol;Differentiation;Autophagy

    骨骼肌在为机体提供运动和保持姿势等方面起着至关重要的作用。暴露在高海拔地区、肢体制动、长时间静卧、贫血和COPD等环境和病理条件下,机体会出现低氧或缺氧诱导的肌肉萎缩现象[1-4]。二氯化钴(CoCl2)可稳定细胞内低氧诱导因子-1α(HIF-1α)的表达,以达到和缺氧条件类似的结果,被广泛运用于各种研究[5]。本课题组前期采用CoCl2建立了骨骼肌细胞缺氧模型,并发现缺氧损伤可引起骨骼肌细胞萎缩、肌细胞蛋白降解增加[6-7]。

    据报道,白藜芦醇(Res)是一种具有多种功能的多酚类化合物,具有抗炎和抗氧化作用,能延长多种物种的寿命,预防早期动脉粥样硬化,治疗肥胖和糖尿病[8-14]。近期有研究表明,Res能够抑制过氧化物酶体增殖蛋白激活性受体γ(PPARγ)活性和抑制核转录因子(NF)-κB的激活,促进间充质干细胞成骨分化[15]。另外,Res可以阻断NF-κB途径激活,上调蛋白激酶 B(Akt)mRNA表达,减少骨骼肌蛋白质分解代谢,促进蛋白质合成代谢[16]。然而,Res对缺氧下骨骼肌细胞损伤修复的影响尚未明确,本研究采用小鼠骨骼肌细胞系C2Cl2构建缺氧细胞模型,观察缺氧损伤对C2Cl2细胞的影响及Res的保护作用。充分研究缺氧环境下骨骼肌损伤的发生发展机制,对促进肌再生修复具有重要的意义。

    材料与方法

    一、材 料

    1. 实验细胞系

    小鼠骨骼肌细胞系C2C12购于中国科学院干细胞库。

    2. 试剂及仪器

    主要试剂包括:DMEM高糖培养基、胎牛血清和马血清(美国Gibco公司),Res(德国Merck Millipore公司),BCA蛋白浓度测定试剂盒(上海碧云天公司), 化学发光ECL试剂盒(德国Merck Millipore公司),肌球蛋白重链(MyHC)一抗(美国R&D Systems公司),DAPI染色液(江苏凯基生物技术有限公司),荧光定量PCR试剂(日本TaKaRa公司),HIF-1α 和Bcl2/腺病毒E1B相互作用蛋白3 (BNIP3)一抗(英国abcam公司),微管相关蛋白1轻链3 (MAP1LC3, LC3) 一抗、Beclin1一抗、β-Tubulin一抗、辣根过氧化物酶标记山羊抗兔、辣根过氧化物酶标记山羊抗兔和MyHC染色荧光二抗(Alexa Fluor 594-conjugated AffiniPure Goat Anti-Mouse IgG)(美国ABclonal公司),p62一抗(美国CST公司)。主要仪器包括:二氧化碳培养箱(美国Thermo Fisher Scientific公司),荧光显微镜(德国Leica公司),电泳转印系统(北京凯元信瑞仪器有限公司),化学发光成像系统(上海天能科技有限公司),StepOnePlus PCR仪(美国Applied Biosystems 公司)。

    二、方 法

    1.细胞培养

    将C2C12置于含10%胎牛血清的高糖DMEM培养基中,于含5% 二氧化碳的37℃培养箱中进行培养,隔日换液,观察细胞生长情况[17]。当细胞达到80% ~ 90%密度时吸去培养液,使用含2%马血清的高糖DMEM培养基进行诱导分化,隔日换液。分化72 h后将细胞分为4组,分别加入等量培养基(Control,对照组)、200 μmol/L CoCl2(CoCl2组)、20 μmol/L Res(Resveratrol,Res组)、200 μmol/L CoCl2+20 μmol/L Res(CoCl2+Res組)。

    48 h后收集细胞,进行后续实验[18]。

    2. MyHC免疫荧光染色

    在分化的细胞中加入CoCl2、Res处理48 h后,在光学显微镜下观察不同处理组细胞形态。细胞用4%多聚甲醛溶液室温固定10 min,用山羊血清室温封闭1 h,然后加入一抗MyHC于4℃孵育过夜。第2日于室温孵育Alexa Fluor-594标记的荧光二抗1 h,然后用DAPI染液室温孵育适量时间。洗涤后用Leica荧光显微镜拍摄不同处理下的细胞形态图。用ImageJ软件统计随机选取的不同视野中的肌管融合指数。

    3. 蛋白免疫印迹法

    在细胞中加入含蛋白酶抑制剂的细胞组织裂解液,于冰上裂解30 min,然后于4℃、12 000 转/分离心15 min。用二喹啉甲酸法检测蛋白浓度。取30 μg蛋白,室温进行电泳,再将蛋白电转移至聚偏二氟乙烯膜上,室温封闭1 h。加入一抗于4℃孵育过夜。洗膜后于室温孵育二抗。洗膜后使用ECL液显色,用全自动化学发光成像分析系统扫描成像。以β-Tubulin作为内参,目的蛋白条带与内参比较作为条带的相对表达值。

    4. 实时荧光定量逆转录PCR(qRT-PCR)检测

    RNA提取按照说明书进行。使用TaKaRa PrimeScript RT Master Mix将总RNA逆转录为互补DNA。利用TaKaRa SYBR-Green Mix和PCR系统检测mRNA的丰度。以18S RNA作为内参,2-ΔΔCt 法计算mRNA的相对表达量。

    三、统计学处理

    实验重复3次,所得数据用SPSS 21.0分析,计量资料用表示,2组间比较采用独立样本t检验,多组间比较采用析因设计方差分析,交互效应有统计学意义时行单独效应分析:多组间比较采用单因素方差分析,两两比较采用LSD-t法。融合指数多组间比较采用χ2检验,各组间采用Bonferroni法进行两两比较;α = 0.05;用GraphPad Prism 6.0绘图。

    结果

    一、Res对C2Cl2肌管形成的影响

    观察MyHC免疫荧光染色后的肌管发现,对照组和Res组细胞分化情况良好,可见大量长条状肌管;经CoCl2处理缺氧损伤后,可见长条状肌管减少、部分肌管萎缩,肌管融合指数减少(P < 0.001);而CoCl2+Res组中加入Res处理后,可逆转CoCl2的抑制作用,细胞分化情况较CoCl2组改善,肌管形成相对增多,肌管融合指数增加, 差异有统计学意义(P < 0.008),见图1、表1。

    二、Res对MyHC的保护作用

    CoCl2缺氧损伤可导致MyHC不同亚型Myh7、Myh2、Myh4 mRNA表达均下降(P分别为0.0001、0.0004、0.0183),同时MyHC蛋白也下降(P = 0.0001),见图2;析因设计方差分析结果表明,Myh7、Myh2、Myh4 mRNA和MyHC蛋白表达在CoCl2缺氧损伤与Res处理间的交互作用有统计学意义(Myh7:F = 7.677,P = 0.024;Myh2:F = 15.098,P = 0.005;Myh4: F = 5.512,P = 0.047;MyHC蛋白:F = 4.458,P = 0.042),见图3。分析单独效应结果显示Myh7、Myh2、Myh4 mRNA和MyHC蛋白在各组的表达差异有统计学意义(Myh7:F = 152.010,P < 0.001;Myh2:F = 98.763,P < 0.001;Myh4: F = 10.754,P < 0.001;MyHC蛋白:F = 20.779,P < 0.001),进而对各组两两比较结果显示,CoCl2+Res组的Myh7、Myh2、Myh4 mRNA和MyHC蛋白表达均高于CoCl2组(Myh7:P = 0.016;Myh2:P = 0.020;Myh4:P = 0.034;MyHC蛋白:P = 0.015),说明加入Res处理后,可逆转CoCl2的抑制作用,上调Myh7、Myh2、Myh4 mRNA和MyHC蛋白。

    三、Res对HIF-1α、BNIP3、LC3 、p62和 Beclin1的影响

    蛋白免疫印迹结果显示,在对照组和Res组中几乎检测不到HIF-1α,而在CoCl2组中检测到高水平表达的HIF-1α;与之相反,CoCl2组加入Res后,HIF-1α的表达降低(P < 0.001),见图4。析因设计方差分析结果表明,HIF-1α、BNIP3、 p62和Beclin1在CoCl2缺氧损伤与Res处理间的交互作用有统计学意义(HIF-1α:F = 122.539,P < 0.001; BNIP3: F = 70.937,P < 0.001;p62: F = 16.732,P = 0.001;Beclin1:F = 32.439,P < 0.001),見图5。分析单独效应结果显示,CoCl2+Res组的HIF-1α、BNIP3和 Beclin1表达均低于CoCl2组(HIF-1α:P < 0.001; BNIP3:P < 0.001;Beclin1:P < 0.001),而CoCl2+Res组的p62表达高于CoCl2组(P < 0.001),见图4。LC3在CoCl2缺氧损伤与Res处理间的交互作用无统计学意义(F = 0.469,P = 0.507),CoCl2和Res处理的主效应均有统计学意义,其中Res处理LC3表达稍低于对照组(F = 22.909,P < 0.001),见图4、5。以上结果提示Res可能通过HIF-1α/BNIP3影响缺氧损伤时骨骼肌中自噬程度。

    讨论

    MyHC是肌细胞分化标志物之一,是决定肌纤维快慢类型的主要因素[19]。在本研究中,课题组建立CoCl2缺氧模型后,采用MyHC免疫荧光染色发现C2C12形态学异常,肌管融合指数减少,同时MyHC表达量下降;课题组前期研究也显示,缺氧诱导的自噬可以抑制肌细胞分化标志物Myog生成,进而抑制肌分化[7]。据报道,Res处理小鼠成肌细胞,可以使MyHC蛋白和肌管直径增加[20]。本研究显示,CoCl2缺氧损伤与Res处理间存在交互效应,Res可使肌管融合指数增加、MyHC表达量升高,提示Res在骨骼肌缺氧损伤中发挥促融合和分化的保护作用。

    HIF-1α和BNIP3在低氧环境中发挥重要作用。1992年Semenza和Wang[21]首先发现HIF-1α,

    只有在缺氧条件下HIF-1α才可稳定表达。BNIP3是HIF-1α的靶分子,可被缺氧诱导。有报道称BNIP3可能在调控自噬体-溶酶体融合中发挥重要作用[22]。BNIP3是线粒体自噬的受体,通过直接结合LC3诱导自噬。既往研究表明,HIF-1α/BNIP3信号通路可以诱导肿瘤和肾细胞自噬[23-24]。本研究显示,在CoCl2诱导的骨骼肌细胞缺氧损伤过程中,HIF-1α、BNIP3、 LC3和Beclin1均升高,提示HIF-1α和BNIP3可能诱导自噬,导致肌分化能力降低。

    研究表明,Res可能通过不同机制发挥多种调控作用。Res可能通过降低NF-κB和肌肉特异性环指蛋白1活性来抑制肿瘤诱导的心脏萎缩[25]。Res通过改善自噬通量延缓肌肉细胞衰老[26]。骨骼肌急性钝挫伤后,Res可通过上调碱性成纤维细胞生长因子、胰岛素样生长因子1来促进骨骼肌修复[27]。目前Res对低氧或缺氧诱导肌肉损伤的影响尚未明确。在本研究中,我们通过CoCl2诱导的C2C12缺氧损伤模型推测Res可能是通过抑制HIF-1α和BNIP3介导的自噬,发挥促进肌细胞损伤修复的保护作用。

    本研究初步探讨了Res在CoCl2诱导的肌细胞缺氧损伤中的作用,实验结果显示Res可以提高肌管MyHC融合指数,促进MyHC的表达,减弱HIF-1α和BNIP3诱导的自噬,上述结果提示Res可能通过抑制自噬在骨骼肌细胞损伤修复中发挥促分化作用。本研究可为临床治疗缺氧损伤提供新的潜在靶点,为促进肌再生修复提供理论依据。

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    (收稿日期:2021-02-18)

    (本文编辑:洪悦民)