乙酰紫堇灵促进大鼠脊髓损伤后的功能恢复:基于调控EGFR/MAPK信号通路抑制小胶质细胞活化

Acetylcorynoline inhibits microglia activation by regulating EGFR/MAPK signaling to promote functional recovery of injured mouse spinal cord

目的探讨乙酰紫堇灵(Ace)对大鼠脊髓损伤(SCI)后功能恢复的作用和机制。方法使用脊髓撞击仪制备SD大鼠中度挫伤性SCI模型(SCI组),给予不同浓度Ace(10、20、40 mg/kg)腹腔注射干预,以假手术大鼠(Sham组)为对照。通过BBB评分评估SCI大鼠运动功能恢复情况;利用HE染色观察脊髓组织损伤面积变化;采用PCR、ELISA与免疫荧光染色评估脊髓损伤后体内炎症水平(TNF-α、IL-6、IL-1β)变化和小胶质细胞的活化情况(CD11b/CD68)。使用脂多糖(LPS组,100 ng/mL)诱导小胶质细胞BV2活化,分别给予不同浓度Ace处理(Ace组,1、2、4μmol/L),Control组给予等体积DMSO处理。免疫荧光和PCR验证Ace对BV2活化及炎症因子分泌的作用;利用网络药理学预测Ace抑制小胶质细胞活化的靶点蛋白和信号机制,采用AutoDock软件对Ace与预测的靶点蛋白进行分子对接。使用信号通路阻断剂(Osimertinib),在体干预SCI大鼠模型,体外干预LPS诱导的BV2细胞模型,以验证信号机制。结果大鼠体内实验显示,与SCI组相比,Ace组大鼠BBB评分增高、脊髓损伤面积减小、小胶质细胞活化数量和促炎因子水平减少(P<0.05);体外LPS刺激BV2细胞的研究结果显示,Ace组BV2活化及炎症因子水平显著低于对照组(P<0.05)。网络药理学预测EGFR是Ace的主要作用靶点,分子对接技术显示EGFR与Ace的结合能为-8.9 kJ/mol,二者之间存在连接氢键;免疫印迹证实Ace可抑制脊髓组织及BV2细胞中EGFR/MAPK信号通路活化,加入EGFR抑制剂Osimertinib干预后,其抑制效能与Ace相当。结论Ace可能通过调控EGFR/MAPK通路抑制小胶质细胞介导的炎症反应,从而促进脊髓损伤后组织修复和运动功能恢复,为SCI治疗药物选择提供新的方向。

Objective To investigate the effect of acetylcorynoline(Ace)for promoting functional recovery of injured spinal cord in rats and explore the underlying mechanism.Methods Rat models of spinal cord injury(SCI)were treated with intraperitoneal injection of different concentrations of Ace,with the sham-operated rats as the control group.After the treatment,the changes in motor function of the rats and the area of spinal cord injury were assessed with BBB score and HE staining,and the changes in pro-inflammatory cytokine levels and microglial activation were determined using PCR,ELISA and immunofluorescence staining.In a lipopolysaccharide(LPS)-treated BV2 cell model,the effects of different concentrations of Ace or DMSO on microglial activation and inflammatory cytokine production were observed.Network pharmacology analysis was performed to predict the target protein and signaling mechanism that mediated the inhibitory effect of Ace on microglia activation,and AutoDock software was used for molecular docking between Ace and the target protein.A signaling pathway blocker(Osimertinib)was used to verify the signaling mechanism in rat models of SCI and LPS-treated BV2 cell model.Results In rat models of SCI,Ace treatment significantly increased the BBB score,reduced the area of spinal cord injury,and lowered the number of activated microglia cells and the levels of pro-inflammatory cytokines(P<0.05).The cell experiments showed that Ace treatment significantly lower the level of cell activation and the production of inflammatory cytokines in LPS-treated BV2 cells(P<0.05).Network pharmacology analysis suggested that EGFR was the main target of Ace,and they bound to each other via hydrogen bonds as shown by molecular docking.Western blotting confirmed that Ace inhibited the activation of the EGFR/MAPK signaling pathway in injured mouse spinal cord tissue and in LPS-treated BV2 cells,and its inhibitory effect was comparable to that of Osimertinib.Conclusion In rat models of SCI,treatment with Ace can inhibit microglia-mediated inflammatory response by regulating the EGFR/MAPK pathway,thus promoting tissue repair and motor function recovery.