SR9009联合吲哚丙酸通过核因子κB信号通路减轻C2C12成肌细胞的炎症反应  

SR9009 combined with indolepropionic acid alleviates inflammation in C2C12 myoblasts through the nuclear factor-kappa B signaling pathway

作　　者：姬慧慧 蒋旭 张志敏[1] 邢运虹 王亮亮 李娜[2] 宋雨庭 罗旭光[3] 崔慧林[1] 曹锡梅[1,4] Ji Huihui;Jiang Xu;Zhang Zhimin;Xing Yunhong;Wang Liangliang;Li Na;Song Yuting;Luo Xuguang;Cui Huilin;Cao Ximei(Department of Histology and Embryology,Shanxi Medical University,Jinzhong 030604,Shanxi Province,China;School of Forensic Medicine,Shanxi Medical University,Jinzhong 030604,Shanxi Province,China;Department of Microbiology and Immunology,Shanxi Medical University,Jinzhong 030604,Shanxi Province,China;The Transformation Pilot-Base of Shanxi Clinical Cell Therapy,Shanxi Medical University,Jinzhong 030604,Shanxi Province,China)

机构地区：[1]山西医科大学组织学与胚胎学教研室,山西省晋中市030604 [2]山西医科大学法医学院,山西省晋中市030604 [3]山西医科大学微生物免疫学教研室,山西省晋中市030604 [4]山西医科大学山西省临床级细胞治疗转化中试基地,山西省晋中市030604

出　　处：《中国组织工程研究》2025年第6期1220-1229,共10页Chinese Journal of Tissue Engineering Research

基　　金：山西省回国留学人员科研资助项目(2023-095),项目负责人:曹锡梅;山西省自然科学基金面上项目(202303021211113),项目负责人:曹锡梅。

摘　　要：背景:钟基因Rev-erbα参与调节炎症,但激活Rev-erbα会增加心脑血管疾病风险。为降低相关风险,探索Rev-erbα激动剂SR9009联合其他药物来减轻骨骼肌成肌细胞炎症,奠定治疗炎症相关性骨骼肌萎缩的理论基础。目的:探讨脂多糖刺激C2C12成肌细胞时吲哚丙酸、SR9009与核因子κB信号通路的关系。方法:①1μg/mL脂多糖刺激C2C12成肌细胞,RNA转录组测序结合KEGG通路富集分析信号通路。②CCK-8法检测C2C12成肌细胞活性,筛选吲哚丙酸的最佳给药浓度;然后将细胞分为空白对照组、脂多糖(1μg/mL)组、SR9009(10μmol/L)+脂多糖组、吲哚丙酸(80μmol/L)+脂多糖组、吲哚丙酸+SR9009+脂多糖组,ELISA检测细胞上清液中白细胞介素6水平,RT-qPCR检测白细胞介素6、肿瘤坏死因子α、Toll样受体4、CD14 mRNA表达,Western blot检测NF-κB p65、p-NF-κB p65蛋白表达。③siRNA敲减Rev-erbα,RT-qPCR评估敲减效率,检测白细胞介素6、肿瘤坏死因子αmRNA表达。结果与结论:①与空白对照组比较,脂多糖时间依赖性抑制成肌细胞融合形成肌管,白细胞介素6、肿瘤坏死因子αmRNA表达水平升高,细胞上清液中白细胞介素6水平显著升高;KEGG通路分析支持脂多糖刺激激活核因子κB信号通路。②吲哚丙酸浓度>80μmol/L时抑制C2C12成肌细胞活性;吲哚丙酸和SR9009通过抑制核因子κB信号通路发挥抗炎作用,降低白细胞介素6、肿瘤坏死因子α、Toll样受体4、CD14 mRNA表达水平,p-NF-κB p65/NF-κB p65蛋白表达比值低于脂多糖组。SR9009联合吲哚丙酸显著降低脂多糖诱导的炎症,Toll样受体4、CD14、白细胞介素6和肿瘤坏死因子αmRNA表达水平进一步下调,p-NF-κB p65/NF-κB p65蛋白表达比值显著低于吲哚丙酸+脂多糖组和SR9009+脂多糖组。③Rev-erbα随脂多糖刺激时间依赖性升高;siRNA敲减Rev-erbα效率达58%以上,成功敲减Rev-erbα后添加脂多糖,白细胞介素6和肿瘤坏死因子αmBACKGROUND:Rev-erbαis involved in the regulation of inflammation,but pharmacological activation of Rev-erbαincreases the risk for cardiovascular diseases.To reduce the relevant risk,an exploration on SR9009,a Rev-erbαagonist,combined with other drugs to relieve inflammation in skeletal myoblasts was conducted,laying the theoretical foundation for the treatment of inflammation-associated skeletal muscle atrophy.OBJECTIVE:To investigate the relationship of SR9009,indolepropionic acid and nuclear factor-κB signaling pathways in lipopolysaccharide-induced C2C12 myoblasts.METHODS:(1)C2C12 myoblasts were induced to differentiate in the presence of lipopolysaccharide(1μg/mL).RNA-seq and KEGG pathway analysis were used to study signaling pathways.(2)C2C12 myoblast viability was assessed using the cell counting kit-8 assay to determine optimal concentrations of indolepropionic acid.Subsequently,cells were categorized into control group,lipopolysaccharide(1μg/mL)group,SR9009(10μmol/L)+lipopolysaccharide group,indolepropionic acid(80μmol/L)+lipopolysaccharide group,and SR9009+indolepropionic acid+lipopolysaccharide group.ELISA was employed to measure protein expression levels of interleukin-6 in the cultured supernatant.Real-time quantitative PCR were employed to measure mRNA expression levels of interleukin-6,tumor necrosis factorα,TLR4 and CD14.Western blot assay were employed to measure protein expression levels of NF-κB p65 and p-NF-κB p65.(3)After Rev-erbαwas knocked down by siRNA,knockdown efficiency was assessed by RT-qPCR.And mRNA levels of interleukin-6 and tumor necrosis factorαwere also measured.RESULTS AND CONCLUSION:Compared with the blank control group,lipopolysaccharide time-dependently inhibited myofibroblast fusion to form myotubes,the mRNA expression levels of interleukin-6 and tumor necrosis factorαwere elevated,and the level of interleukin-6 in the cell supernatant was significantly increased.The results of KEGG pathway showed that the nuclear factor-κB signaling pathway was activated by l

关 键 词：Rev-erbα SR9009 吲哚丙酸 脂多糖 核因子ΚB信号通路 C2C12成肌细胞 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R446