温肾通督方调控Akt/mTOR信号通路激活自噬调节小胶质细胞极化促进脊髓损伤大鼠恢复

Wenshen Tongdu Recipe Promotes the Recovery of Rats with Spinal Cord Injury by Activating Autophagy through the Akt/mTOR Signaling Pathway and Regulating Microglial Polarization

  • 摘要:
    目的 研究温肾通督方促进脊髓损伤大鼠运动功能恢复的作用机制。
    方法 将144只SD大鼠随机分为假手术组、模型组、强的松组、温肾通督方低剂量组、温肾通督方中剂量组、温肾通督方高剂量组、3BDO组以及3BDO+温肾通督方(中剂量)组。通过改良Allen's法构建大鼠脊髓损伤模型,造模1 d后开始干预,连续给药14 d。给药期间,通过BBB评分和斜板试验评估各组大鼠运动功能。造模第3、7、14天,采用HE染色观测各组大鼠脊髓组织病理变化; ELISA法检测血清中炎症因子IL-1β、IL-6、IL-4、IL-10的表达水平; 免疫组织化学技术检测脊髓组织Beclin1、LC3B、p62蛋白表达; 免疫荧光染色法检测脊髓组织CD16、CD206蛋白表达; Western blot法检测脊髓组织Beclin1、LC3B及Akt/mTOR通路相关蛋白表达。
    结果 与模型组比较,温肾通督方中剂量组大鼠BBB评分和斜板试验角度升高,脊髓组织排列紊乱、脊髓空泡和炎性浸润现象显著改善,尤以第14天差异最为显著。与假手术组相比,模型组血清IL-1β、IL-6表达水平增加(P<0.01),IL-4、IL-10表达水平降低(P<0.05,P<0.01);与模型组相比,温肾通督方组IL-1β、IL-6水平明显降低(P<0.01),IL-4、IL-10水平显著增加(P < 0.05,P<0.01);与温肾通督方组相比,3BDO组小鼠血清IL-1β、IL-6浓度上升(P<0.01),IL-10水平下降(P<0.05)。与假手术组比较,模型组大鼠脊髓组织M1/M2比例、P62蛋白表达、p-Akt/Akt及p-mTOR/mTOR比值均显著增加(P<0.05,P<0.01),Beclin1相对蛋白表达量降低(P<0.01);与模型组比较,温肾通督方组脊髓组织M1/M2比例、p-Akt/Akt及p-p70S6K/p70S6K比值均显著降低(P<0.01)。与模型组比,3BDO组Beclin1蛋白水平下降,p-Akt/Akt、p-mTOR/mTOR比值增加(P < 0.05,P<0.01)。与温肾通督方组比较, 3BDO组和3BDO+温肾通督方组M1/M2比例增加(P < 0.01),3BDO组Beclin1、LC3B蛋白阳性率显著下降(P < 0.01),3BDO+温肾通督方组p-p70S6K/p70S6K的比值显著增加(P < 0.01)。
    结论 温肾通督方可促进脊髓损伤修复,其机制可能为通过调控Akt/mTOR信号通路,激活自噬调节小胶质细胞极化,从而减轻脊髓神经炎症反应。

     

    Abstract:
    OBJECTIVE To study the mechanism of Wenshen Tongdu Recipe in promoting the recovery of motor function in rats with spinal cord injury.
    METHODS A total of 144 SD rats were randomly divided into sham operation group, model group, prednisone group, low-dose Wenshen Tongdu Recipe group, medium-dose Wenshen Tongdu Recipe group, high-dose Wenshen Tongdu Recipe group, 3BDO group, and 3BDO + Wenshen Tongdu Recipe group (medium dose). The rat spinal cord injury model was established by modified Allen's method. Intervention began 1 day after modeling, and the drug was administered continuously for 14 days. During the drug administration period, the motor function of the rats in each group was evaluated by Basso-Beattie-Bresnahan (BBB) score and inclined plane test. On the 3rd, 7th and 14th days after modeling, the pathological changes of the spinal cord tissues of the rats in each group were observed by HE staining; the expression levels of inflammatory factors IL-1β, IL-6, IL-4 and IL-10 in the serum of the rats in each group were detected by ELISA; the expression of Beclin 1, LC3B and p62 proteins was detected by immunohistochemistry; the expression of CD16 and CD206 proteins was detected by immunofluorescence staining; the expression of autophagy-related molecules (Beclin 1, LC3B) and Akt/mTOR pathway-related proteins in the spinal cord tissues was detected by Western blot.
    RESULTS Compared with the model group, the BBB score and angle of the inclined board test of rats in the medium-dose Wenshen Tongdu Recipe group were increased, and the disordered arrangement of spinal cord tissue, spinal cord vacuoles and inflammatory infiltration were significantly improved, especially on the 14th day. Compared with the sham operation group, the expression levels of serum IL-1β and IL-6 in the model group increased (P < 0.01), and the expression levels of IL-4 and IL-10 decreased (P < 0.05, P < 0.01); compared with the model group, the levels of IL-1β and IL-6 in the Wenshen Tongdu Recipe group were significantly decreased (P < 0.01), and the levels of IL-4 and IL-10 were significantly increased (P < 0.05, P < 0.01); compared with the Wenshen Tongdu Recipe group, the serum IL-1β and IL-6 concentrations of mice in the 3BDO group increased (P < 0.01), and the level of IL-10 decreased (P < 0.05). Compared with the sham operation group, the M1/M2 ratio, P62 protein expression, p-Akt/Akt and p-mTOR/mTOR ratios in the spinal cord tissue of the rats in the model group were significantly increased (P < 0.05, P < 0.01), and the relative protein expression of Beclin1 was decreased (P < 0.01); compared with the model group, the M1/M2 ratio, p-Akt/Akt and p-p70S6K/p70S6K ratios in the Wenshen Tongdu Recipe group were significantly decreased (P < 0.01). Compared with the model group, the Beclin1 protein level in the 3BDO group was decreased, and the p-Akt/Akt and p-mTOR/mTOR ratios were increased (P < 0.05, P < 0.01). Compared with the Wenshen Tongdu Recipe group, the M1/M2 ratio in the 3BDO group and the 3BDO + Wenshen Tongdu Recipe group increased (P < 0.01), the positive rates of Beclin1 and LC3B proteins in the 3BDO group decreased significantly (P < 0.01), and the p-p70S6K/p70S6K ratio in the 3BDO + Wenshen Tongdu Recipe group increased significantly (P < 0.01).
    CONCLUSION Wenshen Tongdu Recipe may regulate microglial polarization through Akt/mTOR signaling pathway to activate autophagy, promote the secretion of anti-inflammatory factors, reduce the release of pro-inflammatory factors, alleviate neuroinflammatory response, and thus promote spinal cord injury repair.

     

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