背景:骨骼肌损伤是最常见的运动损伤之一;如果没有得到适当治疗或损伤后没有得到有效的康复治疗,它可以转化为慢性累积损伤。姜黄素,一种草药成分,已发现促进骨骼肌损伤修复和再生。Wnt5a通路与生肌调节因子的表达有关,和Ca2+促进成肌细胞的分化和融合过程。本研究探讨了姜黄素对损伤骨骼肌修复再生过程中成肌细胞分化的影响及其与Wnt5a通路和Ca2+通道的关系。
方法:用2%马血清诱导C2C12细胞成肌分化,和一只老鼠(雄性,使用心脏毒素(20μL)建立了10周龄)的急性骨骼肌损伤模型。此外,我们构建了Wnt5a敲低C2C12细胞模型和Wnt5a敲除小鼠模型。此外,将姜黄素加入到细胞培养溶液(80mg/L)中并饲喂小鼠(50mg/kg)。使用荧光显微镜测定Ca2+的浓度。Westernblot和RT-qPCR检测Wnt5a蛋白和mRNA水平,CaN,NFAT2,MyoD,Myf5、Pax7和Myogenin。MyoD的表达水平,Myf5,Myogenin,MHC,Desmin,用免疫荧光技术检测NFAT2。此外,使用免疫组织化学观察MyoD的表达,HE染色观察小鼠肌肉组织的形态学变化。
结果:在成肌细胞分化和肌肉再生过程中,Wnt5a表达上调(P<0.001),Wnt5a信号通路被激活。Wnt5a过表达促进MyoD的表达,Myf5,Myogenin,MHC,和Desmin(P<0.05),反过来,敲除Wnt5a抑制其表达(P<0.001)。Wnt5a通路介导Ca2+通道的开放,调节CaN的表达水平,NFAT2,MyoD,Myf5,Myogenin,MHC,和Desmin(P<0.01),促进C2C12成肌细胞的分化和损伤骨骼肌的修复与再生。Wnt5a的表达,CaN,NFAT2,MyoD,Myogenin,姜黄素干预后C2C12成肌细胞中Myf5和MHC显著升高(P<0.05);在姜黄素干预的基础上,敲除Wnt5a后,其表达显着下降(P<0.05)。同样,在Wnt5a敲除小鼠中,姜黄素对肌肉再生的促进作用明显减弱。
结论:姜黄素可以激活Wnt5a信号通路,介导Ca2+通道的开放,加速C2C12细胞的成肌分化和损伤骨骼肌的修复再生。
BACKGROUND: Skeletal muscle injury is one of the most common sports injuries; if not properly treated or not effective rehabilitation treatment after injury, it can be transformed into chronic cumulative injury. Curcumin, an herbal ingredient, has been found to promote skeletal muscle injury repair and regeneration. The
Wnt5a pathway is related to the expression of myogenic regulatory factors, and Ca2+ promotes the differentiation and fusion process of myoblasts. This study explored the effect and mechanism of curcumin on myoblast differentiation during the repair and regeneration of injured skeletal muscle and its relationship with the Wnt5a pathway and Ca2+ channel.
METHODS: Myogenic differentiation of C2C12 cells was induced with 2% horse serum, and a mouse (male, 10 weeks old) model of acute skeletal muscle injury was established using cardiotoxin (20 μL). In addition, we constructed a
Wnt5a knockdown C2C12 cell model and a
Wnt5a knockout mouse model. Besides, curcumin was added to the cell culture solution (80 mg/L) and fed to the mice (50 mg/kg). Fluorescence microscopy was used to determine the concentration of Ca2+. Western blot and RT-qPCR were used to detect the protein and mRNA levels of
Wnt5a, CaN, NFAT2, MyoD, Myf5, Pax7, and Myogenin. The expression levels of MyoD, Myf5, Myogenin, MHC, Desmin, and NFAT2 were detected using immunofluorescence techniques. In addition, MyoD expression was observed using immunohistochemistry, and morphological changes in mouse muscle tissue were observed using HE staining.
RESULTS: During myoblast differentiation and muscle regeneration, Wnt5a expression was upregulated (P < 0.001) and the
Wnt5a signalling pathway was activated. Wnt5a overexpression promoted the expression of MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.05), and conversely, knockdown of
Wnt5a inhibited their expression (P < 0.001). The Wnt5a pathway mediated the opening of Ca2+ channels, regulated the expression levels of CaN, NFAT2, MyoD, Myf5, Myogenin, MHC, and Desmin (P < 0.01) and promoted the differentiation of C2C12 myoblasts and the repair and regeneration of injured skeletal muscle. The expression of Wnt5a, CaN, NFAT2, MyoD, Myogenin, Myf5, and MHC in C2C12 myoblast was significantly increased after curcumin intervention (P < 0.05); however, their expression decreased significantly after knocking down Wnt5a on the basis of curcumin intervention (P < 0.05). Similarly, in Wnt5a knockout mice, the promotion of muscle regeneration by curcumin was significantly attenuated.
CONCLUSIONS: Curcumin can activate the Wnt5a signalling pathway and mediate the opening of Ca2+ channels to accelerate the myogenic differentiation of C2C12 cells and the repair and regeneration of injured skeletal muscle.