蒙药额日敦·乌日勒对急性脊髓损伤大鼠神经元凋亡的影响及机制研究

Mechanism and effect of Mongolian medicine Eridun·Wurile on neuronal apoptosis in rats with acute spinal cord injury

  • 摘要:
    目的 探讨蒙药额日敦·乌日勒(EW)对急性脊髓损伤(SCI)大鼠神经元凋亡的影响及机制。
    方法 将90只雄性SD大鼠随机分为假手术组(sham组)、SCI组、EW低剂量组、EW中剂量组、EW高剂量组和EW高剂量+AG490组,每组15只,采用Allen法建立大鼠SCI模型。EW低剂量组、EW中剂量组、EW高剂量组术后分别给予EW混悬液0.32、0.63、1.26 g/kg灌胃,EW高剂量+AG490组术前给予蛋白酪氨酸激酶2(JAK2)抑制剂AG490(5 mg/kg)灌胃,术后给予EW(1.26 g/kg)灌胃,sham组、SCI组给予等量蒸馏水,2次/d,连续给药2周。术后1、3、7、14 d采用脊髓损伤运动评分法(BBB)评估大鼠后肢运动功能;采用苏木素-伊红(HE)染色法观察大鼠脊髓组织病理学变化;采用原位缺口末端标记法(TUNEL)检测脊髓神经元细胞凋亡情况;采用蛋白质印迹法(Western blot)检测脊髓组织Cleaved caspase-3、B淋巴细胞瘤-2(Bcl-2)、Bcl-2关联X蛋白(Bax)、磷酸化(p)-JAK2和p-信号转导和转录激活因子3(STAT3)蛋白表达。
    结果 与sham组比较,SCI组大鼠BBB评分降低,脊髓组织的病理损伤严重,脊髓组织凋亡细胞数量增加,Cleaved caspase-3、Bax、p-JAK2和p-STAT3蛋白表达上调,Bcl-2蛋白表达下调,差异有统计学意义(P < 0.05或P < 0.01);与SCI组比较,EW低剂量组、EW中剂量组、EW高剂量组BBB评分逐渐升高,脊髓组织的病理损伤程度减轻,脊髓组织凋亡细胞数量减少,Cleaved caspase-3、Bax、p-JAK2和p-STAT3蛋白表达逐渐下调,Bcl-2蛋白表达逐渐上调,差异有统计学意义(P < 0.05或P < 0.01);与EW高剂量组相比,EW高剂量+AG490组脊髓组织p-JAK2、p-STAT3、Bax、Cleaved caspase-3蛋白表达下调,Bcl-2蛋白表达上调,差异有统计学意义(P < 0.01)。
    结论 EW能够抑制JAK2/STAT3信号通路激活,减少急性SCI大鼠脊髓神经元细胞凋亡,进而发挥神经保护作用。

     

    Abstract:
    Objective To explore the effects and possible mechanism of Mongolian medicine Eridun·Wurile (EW) on neuronal apoptosis in rats with acute spinal cord injury(SCI).
    Methods The male SD rats were randomly divided into sham-operated group(sham group), SCI group, EW low-dose group, EW medium-dose group, EW high-dose group and EW high dose+AG490 group, with 15 rats in each group, and the SCI model of rats was established by Allen's method. EW low-dose group, EW medium-dose group and EW high-dose group were given EW suspension at a dosage of 0.32, 0.63 and 1.26 g/kg for gavage after surgery, respectively; the EW high-dose+AG490 group was given Janus kinase 2 (JAK2) inhibitor AG490 (5 mg/kg) by gavage before surgery and EW (1.26 g/kg) by gavage after surgery; and the sham and SCI groups were given equal volume of distilled water, twice a day for 2 weeks. The hind limb motor function of each group was assessed by BBB grading score at 1 day, 3, 7 and 14 days postoperatively; the rats were executed 12 h after the last administration and the spinal cord water content was examined; the histopathological changes were observed by Hematoxylin-Eosin (HE) staining in the spinal cord of each group; the apoptosis of spinal neurons was detected by in situ notch end labeling (TUNEL). The expression of Cleaved caspase-3, B-lymphoblastoma-2 (Bcl-2), Bcl-2 associated X protein (Bax), phosphorylated (p)-JAK2, and p-signal transduction and transcriptional activator 3 (STAT3) proteins in spinal cord were detected by Western blot.
    Results Compared with the sham group, the BBB scores of rats in the SCI group were significantly lower, spinal cord edema and pathological structural damage were severe, the number of apoptotic cells and the expression of Cleaved caspase-3, Bax, p-JAK2 and p-STAT3 proteins in the spinal cord were significantly increased, while the expression of Bcl-2 was decreased (P < 0.05 or P < 0.01); compared with the SCI group, the BBB scores of EW low-dose group, EW medium-dose group and EW high-dose group gradually increased, the pathological injury degree of spinal cord tissue was relieved, and the number of apoptotic cells in spinal cord tissue was reduced, Cleaved caspase-3, Bax, p-JAK2, and p-STAT3 protein expressions were gradually down-regulated, while Bcl-2 protein expression was gradually up-regulated (P < 0.05 or P < 0.01); compared with EW high-dose group, the expression of P-JAK2, p-STAT3, Bax, Cleaved caspase-3 proteins down-regulated in the spinal cord tissue of EW high dose+AG490 group, and Bcl-2 protein were up-regulated, and the differences were statistically significant (P < 0.01).
    Conclusion EW plays a neuroprotective role via inhibiting JAK2/STAT3 signaling pathway and reducing apoptosis in acute spinal cord injury rats.

     

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