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

doi:10.12307/2025.283

Abstract

Abstract:
BACKGROUND: Rev-erbα is involved in the regulation of inflammation, but pharmacological activation of Rev-erbα increases the risk for cardiovascular diseases. To reduce the relevant risk, an exploration on SR9009, a Rev-erbα agonist, 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 (1 μg/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 (1 μg/mL) group, SR9009 (10 μmol/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 factor α, 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-erbα was knocked down by siRNA, knockdown efficiency was assessed by RT-qPCR. And mRNA levels of interleukin-6 and tumor necrosis factor α were 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 factor α were 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 lipopolysaccharide. Indolepropionic acid exhibited significant suppression of C2C12 myoblasts viability when its concentration exceeded 80 μmol/L. Indolepropionic acid and SR9009 inhibited the activation of NF-κB signaling pathway, thereby played an anti-inflammatory role, and suppressed the mRNA expression levels of interleukin-6, tumor necrosis factor α, TLR4 and CD14. Compared with the lipopolysaccharide group, the ratio of p-NF-κB p65/NF-κB p65 protein expression were downregulated. SR9009 combined with indolepropionic acid notably reduced lipopolysaccharide-induced inflammation, further downregulated the mRNA expression levels of interleukin-6, tumor necrosis factor α, TLR4 and CD14. The ratio of p-NF-κB p65/NF-κB p65 protein expression was significantly lower than that in the SR9009+lipopolysaccharide group or indolepropionic acid+lipopolysaccharide group. Rev-erbα increases time-dependently with lipopolysaccharide induction. The knockdown efficiency of Rev-erbα by siRNA reached over 58%, and lipopolysaccharide was added after Rev-erbα was successfully knocked down. Compared with the lipopolysaccharide group, the mRNA expression levels of interleukin-6 and tumor necrosis factor α were significantly up-regulated. These results conclude that Rev-erbα may act as a promising pharmacological target to reduce inflammation. SR9009 targeted activation of Rev-erbα combined with indolepropionic acid significantly inhibits the nuclear factor-κB signaling pathway and attenuates the inflammatory response of C2C12 myofibroblasts. Moreover, the combined anti-inflammatory effect is superior to that of the intervention alone.

Key words: Rev-erbα, SR9009, Indolepropionic acid, lipopolysaccharide, nuclear factor-κB signaling pathway, C2C12 myoblast

CLC Number:

Ji Huihui, Jiang Xu, Zhang Zhimin, Xing Yunhong, Wang Liangliang, Li Na, Song Yuting, Luo Xuguang, Cui Huilin, Cao Ximei. SR9009 combined with indolepropionic acid alleviates inflammation in C2C12 myoblasts through the nuclear factor-kappa B signaling pathway[J]. Chinese Journal of Tissue Engineering Research, 2025, 29(6): 1220-1229.

Figures/Tables 6

2.1 脂多糖刺激C2C12成肌细胞分泌白细胞介素6和肿瘤坏死因子α，并激活NF-κB信号通路 C2C12成肌细胞分化过程中添加1 μg/mL脂多糖诱导炎症反应，与对照组相比，C2C12成肌细胞生长减慢、细胞融合和肌管的形成被抑制。实验观察期内，脂多糖时间依赖性地抑制肌管形成(图1A)。RNA转录组测序结合KEGG通路分析结果显示，脂多糖刺激C2C12成肌细胞分化过程中的炎症反应与肿瘤坏死因子α、NF-κB及其相关信号通路的激活相关(图1B)。验证实验结果显示，与对照组相比，C2C12成肌细胞分化过程中添加脂多糖上调NF-κB p65和p-NF-κB p65蛋白表达，且p-NF-κB p65/NF-κB p65的比值随脂多糖刺激时间延长显著升高(图1C，D)，提示脂多糖刺激C2C12成肌细胞条件下，NF-κB信号通路被激活。RT-qPCR结果显示，脂多糖刺激组细胞中白细胞介素6 mRNA表达较对照组显著升高，且随时间依赖性升高(图1E)，肿瘤坏死因子α mRNA表达随脂多糖刺激时间依赖性升高，72 h达到小高峰后略有下降(图1F)。ELISA检测细胞上清液中白细胞介素6水平，其与mRNA的表达呈现相同升高趋势(图1G)。以上结果支持脂多糖刺激C2C12成肌细胞条件下，NF-κB信号通路被激活，诱发炎症反应。 2.2 吲哚丙酸抑制脂多糖刺激的C2C12成肌细胞炎症反应已知吲哚丙酸能通过NF-κB信号通路发挥抗炎作用[14-16]。为进一步验证C2C12成肌细胞分化过程中脂多糖刺激的炎症反应与NF-κB信号通路有关，在生长培养基中添加吲哚丙酸培养18 h后，转入含1 μg/mL脂多糖的分化培养基，并维持吲哚丙酸干预剂量培养72 h。CCK-8检测吲哚丙酸的细胞毒性，筛选最佳给药浓度。结果显示，随着吲哚丙酸作用时间延长，吲哚丙酸浓度> 80 μmol/L对C2C12成肌细胞造成明显毒性损伤；吲哚丙酸浓度< 80 μmol/L时未对C2C12成肌细胞活性造成显著性影响，且低浓度吲哚丙酸表现出促增殖作用，以10 μmol/L最显著(图2A-C)。结合利用吲哚丙酸抑炎的实验目的，选择80 μmol/L作为吲哚丙酸的使用浓度进行后续实验。与脂多糖组相比，吲哚丙酸+脂多糖组干预72 h后白细胞介素6、肿瘤坏死因子α mRNA表达水平显著降低(图2D，E)；NF-κB p65和p-NF-κB p65蛋白表达水平下调，尤以p-NF-κB p65下调显著，p-NF-κB p65/NF-κB p65比值较脂多糖组显著降低(图2F，G)。 2.3 靶向抑制或激活Rev-erbα对炎性细胞因子白细胞介素6和肿瘤坏死因子α的影响 KEGG通路分析结果显示昼夜节律钟基因参与调节炎症反应(图1B)。研究表明，Rev-erbα参与调节巨噬细胞的炎症反应[17]。靶向Rev-erbα是否参与调节脂多糖刺激C2C12成肌细胞的炎症反应值得进一步研究。脂多糖组Rev-erbα mRNA的表达水平呈时间依赖性升高(图3A)。通过Rev-erbα siRNA有效敲减Rev-erbα，并用RT-qPCR确定其抑制效率达58%以上(图3B)。Rev-erbα敲减组白细胞介素6和肿瘤坏死因子α mRNA表达水平较脂多糖组显著上调(图3C，D)。提示，敲减Rev-erbα加重脂多糖刺激C2C12成肌细胞的炎症反应。吡咯衍生物SR9009是Rev-erbα的激动剂，可以增强Rev-erbα依赖性阻遏因子活性[18]。10 μmol/L SR9009预处理C2C12成肌细胞后给予脂多糖刺激，白细胞介素6和肿瘤坏死因子α mRNA表达水平较脂多糖组呈现下降趋势(图3E，F)；细胞上清液中白细胞介素6水平的表达趋势与其mRNA水平基本一致(图3G)。提示，药理性激活Rev-erbα能够发挥抗炎效应。 2.4 Rev-erbα激动剂SR9009抑制NF-κB p65磷酸化，减轻脂多糖刺激的炎症反应 TLR4是与脂多糖结合的主要受体，TLR4/CD14信号通路是介导脂多糖刺激引发炎症反应的重要通路[19-20]。随脂多糖刺激时间延长，TLR4、CD14 mRNA的表达水平依赖性增加。当SR9009药理性激活Rev-erbα后，SR9009+脂多糖组较脂多糖组TLR4、CD14 mRNA的表达水平下调，尤以72 h以后显著(图4A，B)。NF-κB是TLR4介导的信号通路中的关键转录因子。蛋白免疫印迹实验进一步检测脂多糖刺激C2C12成肌细胞分化过程中NF-κB信号通路的变化，结果显示p-NF-κB p65/NF-κB p65蛋白比值较对照组时间依赖性升高。激动剂SR9009预处理后，SR9009+脂多糖组p-NF-κB p65/NF-κB p65蛋白比值逐渐下调且显著低于脂多糖组(图4C，D)。同一时间点NF-κB p65的表达水平在脂多糖组和SR9009+脂多糖组之间无显著差异。提示，SR9009预处理通过下调TLR4、CD14 mRNA的表达，抑制NF-κB p65激活，以72 h后显著，进而减轻脂多糖刺激的炎症反应。 2.5 Rev-erbα激动剂SR9009联合吲哚丙酸具有更强的抗炎作用为评价脂多糖刺激C2C12成肌细胞分化过程中Rev-erbα激动剂SR9009联合吲哚丙酸共培养条件下NF-κB信号通路的变化，检测脂多糖刺激72 h时TLR4、CD14、炎性细胞因子白细胞介素6、肿瘤坏死因子α mRNA的表达，结果显示SR9009+脂多糖组和吲哚丙酸+脂多糖组TLR4、CD14、白细胞介素6、肿瘤坏死因子α mRNA的表达均显著低于脂多糖组；SR9009和吲哚丙酸联合后，上述因子的表达水平进一步下降，尤以白细胞介素6的下降更为显著(图5A-D)。SR9009和吲哚丙酸联合后，p-NF-κB p65/NF-κB p65蛋白比值较SR9009+脂多糖组或吲哚丙酸+脂多糖组均显著下调(图5E，F)。提示，Rev-erbα激动剂SR9009联合吲哚丙酸具有更强的抗炎作用。"

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