南方医科大学学报 ›› 2024, Vol. 44 ›› Issue (11): 2192-2200.doi: 10.12122/j.issn.1673-4254.2024.11.16
蔡祺晖1(), 蓝海强2, 冼柏俊1, 刘连3, 王楠1, 黄晓蕾1, 牛晓璐1, 胡欣雨1, 李辰1, 谢俊毅1, 廖钊宏1,2(
)
收稿日期:
2024-05-16
出版日期:
2024-11-20
发布日期:
2024-11-29
通讯作者:
廖钊宏
E-mail:m19802076244@163.com;liao1219315353@163.com
作者简介:
蔡祺晖,在读本科生,E-mail: m19802076244@163.com
基金资助:
Qihui CAI1(), Haiqiang LAN2, Bojun XIAN1, Lian LIU3, Nan WANG1, Xiaolei HUANG1, Xiaolu NIU1, Xinyu HU1, Chen LI1, Junyi XIE1, Zhaohong LIAO1,2(
)
Received:
2024-05-16
Online:
2024-11-20
Published:
2024-11-29
Contact:
Zhaohong LIAO
E-mail:m19802076244@163.com;liao1219315353@163.com
摘要:
目的 探究骨骼肌E2信号对Cardiotoxin (CTX)诱导的小鼠急性损伤骨骼肌内巨噬细胞胞葬作用的影响。 方法 选择野生 C57BL/6雌鼠150只、C57BL/6雄鼠33只,对一部分雌鼠进行卵巢去势(OVX)操作。CTX胫骨前肌注射诱导小鼠急性肌损伤,后给予β-雌二醇(β-Estradiol)、4-他莫昔芬(4-OHT)等试剂肌注,分为Control(Female)组、Male组、Control+β-Estradiol组、Control+4-OHT组、OVX组、OVX+β-Estradiol组。采用ELISA法比较各组小鼠损伤肌内血清中E2表达。采用免疫荧光、流式细胞术等方法比较损伤肌内炎性渗出巨噬细胞数量、表型、胞葬作用与肌再生修复的差异。紫外照射法体外诱导细胞凋亡。体外分化培养C2C12细胞,分化为成熟肌管后,将之于炎性环境中与巨噬细胞、凋亡细胞共培养,且给予β-Estradiol、4-OHT等处理,主要分为Control(C2C12+HS+Mac+ACs)组、C2C12+HS+IFN-γ+Mac+ACs组、C2C12+HS+IFN-γ+β-Estradiol+Mac+ACs组、C2C12+HS+IFN-γ+4-OHT+Mac+ACs组、C2C12+HS+IFN-γ+β-Estradiol+4-OHT+Mac+ACs组。采用免疫荧光、流式细胞术等方法比较各组巨噬细胞胞葬作用的差异。 结果 较之于Control组,OVX鼠损伤肌内炎性单核巨噬细胞渗出增加,M1细胞比例增加(P<0.05),但M2细胞比例、胞葬作用下调(P<0.05),肌纤维再生修复延迟。体外炎性环境中,β-Estradiol共培养体系中的M2巨噬细胞数目、巨噬细胞胞葬较之于Control组均上调(P<0.05),而4-OHT组的趋势则相反(P<0.05)。 结论 骨骼肌纤维E2信号通过促使损伤肌内M1向M2的转变,以促进损伤肌内巨噬细胞胞葬作用,继而促进炎症撤退与肌再生修复。
蔡祺晖, 蓝海强, 冼柏俊, 刘连, 王楠, 黄晓蕾, 牛晓璐, 胡欣雨, 李辰, 谢俊毅, 廖钊宏. 肌纤维E2信号促进小鼠急性损伤骨骼肌内的巨噬细胞胞葬[J]. 南方医科大学学报, 2024, 44(11): 2192-2200.
Qihui CAI, Haiqiang LAN, Bojun XIAN, Lian LIU, Nan WANG, Xiaolei HUANG, Xiaolu NIU, Xinyu HU, Chen LI, Junyi XIE, Zhaohong LIAO. E2 signaling in myofibers promots macrophage efferocytosis in mouse skeletal muscles with cardiotoxin-induced acute injury[J]. Journal of Southern Medical University, 2024, 44(11): 2192-2200.
Gene | Primer sequence (5'-3') |
---|---|
ERα | For:ACTGGCCAATCTTTCTCTGC Rev:CAATTCATCCCCAAAGACATGGAC |
ERβ | For:TCACTTCTGCGCTGTCTGCAGCG Rev:CCTGGGTCGCTGTGCCAAG |
GAPDH | For:CAATGTGTCCGTCGTGGATCT Rev:GTCCTCAGTGTAGCCCAAGATG |
表1 qRT-PCR的引物序列
Tab.1 Primer sequence for qRT-PCR
Gene | Primer sequence (5'-3') |
---|---|
ERα | For:ACTGGCCAATCTTTCTCTGC Rev:CAATTCATCCCCAAAGACATGGAC |
ERβ | For:TCACTTCTGCGCTGTCTGCAGCG Rev:CCTGGGTCGCTGTGCCAAG |
GAPDH | For:CAATGTGTCCGTCGTGGATCT Rev:GTCCTCAGTGTAGCCCAAGATG |
Gene | Primer sequence(5'-3') |
---|---|
IL-1β | For:GCCCATCCTCTGTGACTC Rev:TGTGCCGTCTTTCATTAC |
IL-10 | For:TTTCAAACAAAGGACCAG Rev:GGATCATTTCCGATAAGG |
iNOS | For:CTTCCGGGCAGCCTGTGAGACG Rev:ATCCCCAGGTGTTCCCCAGGTAGG |
TNF-α | For:GCTGTCTCCCCCGAAAGATG Rev:AGGCAGGTGTAGATGTTGTGG |
Arg1 | For:CTCCAAGCCAAAGTCCTTAGAG Rev:AGGAGCTGTCATTAGGGACA |
Mrc1 | For:CTCTGTTCAGCTATTGGACGC Rev:TGGCACTCCCAAACATAATTTGA |
GAPDH | For:CAATGTGTCCGTCGTGGATCT Rev:GTCCTCAGTGTAGCCCAAGATG |
表2 qRT-PCR反应因子的引物序列
Tab.2 Primer sequence for qRT-PCR
Gene | Primer sequence(5'-3') |
---|---|
IL-1β | For:GCCCATCCTCTGTGACTC Rev:TGTGCCGTCTTTCATTAC |
IL-10 | For:TTTCAAACAAAGGACCAG Rev:GGATCATTTCCGATAAGG |
iNOS | For:CTTCCGGGCAGCCTGTGAGACG Rev:ATCCCCAGGTGTTCCCCAGGTAGG |
TNF-α | For:GCTGTCTCCCCCGAAAGATG Rev:AGGCAGGTGTAGATGTTGTGG |
Arg1 | For:CTCCAAGCCAAAGTCCTTAGAG Rev:AGGAGCTGTCATTAGGGACA |
Mrc1 | For:CTCTGTTCAGCTATTGGACGC Rev:TGGCACTCCCAAACATAATTTGA |
GAPDH | For:CAATGTGTCCGTCGTGGATCT Rev:GTCCTCAGTGTAGCCCAAGATG |
图1 肌纤维E2信号对急性骨骼肌炎症的影响
Fig.1 Effect of E2 in myofiber signaling on acute skeletal myositis in mice. A: HE and immunofluorescence staining for detecting inflammation in the tibialis anterior muscle (TA) after CTX injury (Scale bar=50 μm). B: ELISA for detection of serum E2 in male and female mice at different time points after muscle injury (*P<0.05).
图2 急性骨骼肌炎中雌激素的受体表达
Fig.2 Expression of estrogen receptors in acute skeletal myositis. A: Western blotting of ERα and ERβ expression level in mature myotubes derived from C2C12 cells. B: qRT-PCR analysis of ERα and ERβ mRNA levels in the damaged muscle of female mice. C: Immunofluorescence staining of ERβ expression level in injured mouse muscle (Scale bar=50 μm). *P<0.05, **P<0.01.
图3 肌纤维E2信号对急性骨骼肌炎症损伤肌内单核巨噬细胞渗出影响
Fig.3 Effect of E2 signaling in myofibers on exudation of intramuscular mononuclear macrophages after acute skeletal muscle injury. A: ELISA for detecting serum E2 level in female mice after different treatments. B: ELISA for detecting serum E2 levels in female mice at different time points after OVX treatment. C: HE staining for detecting muscular inflammation in female mice with different treatments after CTX injury. D: Immunofluorescence staining for detecting muscular inflammation in female mice after CTX injury with different treatments. *P<0.05, **P<0.01; Scale bar=50 μm.
图4 肌纤维E2信号对急性骨骼肌炎症损伤肌内巨噬细胞表型影响
Fig.4 Effect of E2 signaling in myofibers on phenotype of intramuscular macrophages in mice after acute skeletal muscle injury. A: Flow cytometric analysis of mononuclear macrophages in the damaged muscle of female mice after OVX treatment. B: Flow cytometric analysis of the phenotype of macrophages in the injured muscles of female mice after OVX treatment. *P<0.05, **P<0.01.
图5 肌纤维E2信号对急性骨骼肌炎症损伤肌内炎症因子的影响
Fig.5 Effect of E2 signaling in myofibers on intramuscular inflammatory cytokines after acute skeletal muscle injury. A: qRT-PCR analysis of inflammatory cytokines in female mice after OVX treatment. B: qRT-PCR analysis of inflammatory cytokines in macrophages from the injured muscles isolated by flow cytometry in female mice after OVX treatment. *P<0.05, **P<0.01.
图6 肌纤维E2信号驱动急性骨骼肌炎症损伤肌内巨噬细胞胞葬作用
Fig.6 E2 signaling in myofibers drives intramuscular macrophage efferocytosis in acute skeletal myositis. A: Immunofluorescence detection of intramuscular macrophage efferocytosis in the damaged muscle. (scale bar=50 μm). B: Flow cytometry of intramuscular macrophage efferocytosis in the damaged muscle (*P<0.05).
图7 体外共培养体系中E2信号对巨噬细胞表型与胞葬作用的影响
Fig.7 Effect of E2 signaling on macrophage phenotypes and efferocytosis in an in vitro co-culture system. A: Immunofluorescence detection of macrophage phenotypes in the in vitro co-culture system. B: Immunofluorescence detection and flow cytometry analysis of macrophage efferocytosis in the in vitro co-culture system. *P<0.05; Scale bar=50 μm.
图8 肌纤维E2信号调控损伤骨骼肌内肌纤维再生修复
Fig.8 E2 signaling in myofibers regulates regeneration and repair of intramuscular myofibers in injured mouse skeletal muscles of female mice after different treatments (Immunofluorescence staining, scale bar=50 μm; *P<0.05).
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