柚皮素调控巨噬细胞极化和肌卫星细胞增殖修复骨骼肌损伤

Naringenin repairs skeletal muscle injury by regulating polarization of macrophages and proliferation of muscle satellite cells

背景:研究表明,巨噬细胞的极化精确调控在组织损伤的修复进程中十分重要,肌卫星细胞增殖及分化在骨骼肌损伤的修复中也有重要作用,并显示柚皮素能改善骨骼肌损伤修复中的过度纤维化。目的:探讨柚皮素对于骨骼肌修复的作用及其机制,为用于骨骼肌损伤提供理论依据。方法:取80只10周龄SPF级SD大鼠随机分为2组(n=40),采用重物击打方法构建骨骼肌损伤模型后,实验组腹腔注射柚皮素(2μg/g)14 d,对照组注射等剂量1%二甲基亚砜。于伤后12 h及1,3,5,7,14 d分别每组采集6只大鼠腓肠肌。Masson和苏木精-伊红染色观测骨骼肌组织纤维化程度;酶联免疫吸附法(ELISA)检测炎症因子白细胞介素4、白细胞介素13、干扰素α、干扰素γ表达;实时荧光定量PCR检测促纤维化基因(Ⅰ、Ⅲ型胶原蛋白)和成肌分化抗原的m RNA表达;流式细胞术检测巨噬细胞极化程度;免疫荧光(Pax7、成肌分化抗原)染色观察肌卫星细胞增殖情况。结果与结论:(1)组织学观察:伤后5,7,14 d时实验组纤维化面积比值低于对照组(P <0.05),再生肌纤维百分比均明显大于对照组(P <0.05);(2)ELISA检测:与对照组相比,实验组伤后7,14 d白细胞介素4以及5,7,14 d时白细胞介素13和干扰素α的表达增加(P<0.05),而干扰素γ的表达出现下降;(3)实时荧光定量PCR检测:与对照组比较,实验组伤后3,5,7,14 dⅠ、Ⅲ型胶原m RNA相对表达量均下降(P<0.05);3,5,7 d成肌分化抗原m RNA相对表达量增加(P<0.05);(4)流式细胞术检测:伤后3,5,7,14 d实验组M1型巨噬细胞数量均少于对照组,而M2型巨噬细胞数量均多于对照组(P <0.05);(5)免疫荧光染色:术后3 d的实验组肌卫星细胞增殖高于对照组(P <0.05);(6)结论:柚皮素能够通过调控巨噬细胞发生M2型极化从而促进肌卫星细胞增殖的方式治疗骨骼肌损伤。

BACKGROUND:Studies have shown that the precise regulation of macrophage polarization is extremely important for tissue repair,the prolife ration and differentiation of muscle satellite cells during the repair of skeletal muscle injury.Naringenin can improve excessive fibrosis during skeletal muscle injury repair.OBJECTIVE:To explore the effect and mechanism of macrophage polarization in skeletal muscle repair,thereby providing a theo retical basis for the treatment of skeletal muscle injury.METHODS:Eighty 10-wee k-old Sprague-Dawley rats of specific pathogen-free grade were randomly divided into two groups(n=40).A skeletal muscle injury model was constructed by weight drop method.The experimental group was intraperitoneally injected with naringenin(2 μg/g) for 14 days and the control group was injected with the same amount of 1% dimethyl sulfoxide.Samples of gastrocnemius muscle were collected from 6 rats in each group at 12 hours and 1,3,5,7,and 14 days after injury.Masson and hematoxylin-eosin staining were used to observe the degree of skeletal muscle tissue fibrosis,and enzymelinked immunosorbent assay was used to detect the expression of inflammatory factors interleukin-4,interleukin-13,interferon-a,and interferon-y.The mRNA expressions of pro-fibrotic genes(type Ⅰ and Ⅲ collagens) and myogenic differentiation a ntigen(MyoD) were detected by real-time fluorescence quantitative PCR,Pola rization degree of macrophages was detected by flow cytometry.Prolife ration of muscle satellite cells was observed by immunofluorescence(Pax7,MyoD) staining.RESULTS AND CONCLUSION:Histological observations showed that the fibrosis area ratio of the experimental group was lower than that of the control group at 5,7,and 14 days after injury(P <0.05),and the size of regenerated muscle fibers was significantly larger than that of the control group(P <0.05).The ELISA res ults showed that compared with the control group,the expression of interleukin-4 at 7 and 14 days after injury and the expression of interleukin-13 and interferon-a at 5,7,and 14 days after injury increased significantly in the expe rimental group(P <0.05),while the expression of interferon-y declined.Real-time fluorescence quantitative PCR results revealed that compared with the control group,the relative expression of type Ⅰ and Ⅲ collagens decreased significantly in the expe rimental group at 3,5,7,and 14 days after injury(P<0.05),while the relative expression of MyoD increased significantly at 3,5,and 7 days after injury(P <0.05).Flow cytometry res ults showed that the number of M1-type macrophages in the expe rimental group was lower than that of the control group at 3,5,7,and 14 days after injury,while the number of M2-type macrophages was significantly higher than that of the control group(P <0.05).Immunofluorescence staining res ults showed that the prolife ration of muscle satellite cells in the experimental group was significantly higher than that in the control group at 3 days after injury(P <0.05).To conclude,naringenin can treat skeletal muscle injury by regulating the M2-type polarization of macrophages to promote the proliferation of muscle satellite cells.