背景:骨骼肌处理约80%的胰岛素刺激的葡萄糖摄取,并成为发生胰岛素抵抗(IR)的主要器官。许多研究已经证实巨噬细胞和骨骼肌之间的相互作用调节了骨骼肌的炎症和再生。然而,尽管经过了几十年的研究,高糖(HG)环境下骨骼肌中巨噬细胞的浸润和极化是否会导致IR的发展尚待阐明。C2C12成肌细胞是研究生肌调节及其对刺激的反应的完善和优良的模型。有必要进一步探索巨噬细胞在成肌细胞IR中的作用及其浸润和极化的动力学。
目的:为了评估HG下成肌细胞和巨噬细胞之间的相互作用,及其对骨骼肌炎症和IR的影响。
方法:我们通过苏木精和伊红染色和免疫组织化学染色检测了IR小鼠骨骼肌浸润的巨噬细胞的极化状态。然后,我们开发了一种体外共培养系统来研究HG环境下成肌细胞和巨噬细胞之间的相互作用。通过形态学观察探讨成肌细胞对巨噬细胞的影响,CCK-8测定,流式细胞仪,和酶联免疫吸附测定。通过形态学观察检测巨噬细胞对肌生成和胰岛素敏感性的介导,CCK-8测定,免疫荧光,和2-NBDG测定。
结果:F4/80以及F4/80和CD86的共同定位增加,IR组肌纤维大小减小(P<0.01,g=6.26)。与Mc组相比,F4/80+CD86+CD206-细胞,肿瘤坏死因子-α(TNFα),白细胞介素-1β(IL-1β)和IL-6降低,McM组IL-10升高(P<0.01,g>0.8)。在McM+HG组中,F4/80+CD86+CD206-细胞,单核细胞趋化蛋白1,TNFα,IL-1β和IL-6升高,与Mc+HG组和McM组比拟,F4/80+CD206+CD86-细胞和IL-10降低(P<0.01,g>0.8)。按M组计算,肌管面积,MMc组肌管数量和E-MHC增加(P<0.01,g>0.8)。在MMc+HG组中,肌管面积,肌管编号,E-MHC,与M+HG组和MMc组比拟,GLUT4和葡萄糖摄取降低(P<0.01,g>0.8)。
结论:成肌细胞和巨噬细胞在HG环境下的相互作用导致炎症和IR,这支持巨噬细胞可以作为骨骼肌萎缩和IR的有希望的治疗靶标。
BACKGROUND: Skeletal muscle handles about 80% of insulin-stimulated glucose uptake and become the major organ occurring insulin resistance (IR). Many studies have confirmed the interactions between macrophages and skeletal muscle regulated the inflammation and regeneration of skeletal muscle. However, despite of the decades of research, whether macrophages infiltration and polarization in skeletal muscle under high glucose (HG) milieus results in the development of IR is yet to be elucidated. C2C12 myoblasts are well-established and excellent model to study myogenic regulation and its responses to stimulation. Further exploration of macrophages\' role in myoblasts IR and the dynamics of their infiltration and polarization is warranted.
OBJECTIVE: To evaluate interactions between myoblasts and macrophages under HG, and its effects on inflammation and IR in skeletal muscle.
METHODS: We detected the polarization status of macrophages infiltrated to skeletal muscles of IR mice by hematoxylin and eosin and immunohistochemical staining. Then, we developed an in vitro co-culture system to study the interactions between myoblasts and macrophages under HG milieus. The effects of myoblasts on macrophages were explored through morphological observation, CCK-8 assay, Flow Cytometry, and enzyme-linked immunosorbent assay. The mediation of macrophages to myogenesis and insulin sensitivity were detected by morphological observation, CCK-8 assay, Immunofluorescence, and 2-NBDG assay.
RESULTS: The F4/80 and co-localization of F4/80 and CD86 increased, and the myofiber size decreased in IR group (P < 0.01, g = 6.26). Compared to Mc group, F4/80+CD86+CD206- cells, tumor necrosis factor-α (TNFα), inerleukin-1β (IL-1β) and IL-6 decreased, and IL-10 increased in McM group (P < 0.01, g > 0.8). In McM + HG group, F4/80+CD86+CD206- cells, monocyte chemoattractant protein 1, TNFα, IL-1β and IL-6 were increased, and F4/80+CD206+CD86- cells and IL-10 were decreased compared with Mc + HG group and McM group (P < 0.01, g > 0.8). Compered to M group, myotube area, myotube number and E-MHC were increased in MMc group (P < 0.01, g > 0.8). In MMc + HG group, myotube area, myotube number, E-MHC, GLUT4 and glucose uptake were decreased compared with M + HG group and MMc group (P < 0.01, g > 0.8).
CONCLUSIONS: Interactions between myoblasts and macrophages under HG milieus results in inflammation and IR, which support that the macrophage may serve as a promising therapeutic target for skeletal muscle atrophy and IR.