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小胶质细胞P2Y12受体在小鼠卒中后中枢痛中的作用和机制

Role and mechanism of P2Y12 receptor in microglia in central post-stroke pain in mice

    摘要
  • 摘要:
    目的 探讨小胶质细胞P2Y12受体在小鼠卒中后中枢痛中的作用及机制。
    方法 选取无特定病原体(SPF)级雄性C57BL/6J小鼠32只, 7~8周龄，体质量25~30 g, 采用随机数字表法分为4组: 假手术组(C组)、CPSP组、CPSP-M组(CPSP+P2Y12受体拮抗剂MRS2395)、C-M组(假手术+P2Y12受体拮抗剂MRS2395)，每组8只。采用右侧丘脑腹后内侧核和腹后外侧核内注射10 nL 0.001 U/nL Ⅳ型胶原酶制备CPSP模型。CPSP-M组和C-M组在CPSP模型制备前30 min时腹腔注射MRS 2395(1.5 mg/kg), 1次/d, 连续注射5 d; C组和CPSP组给予等量生理盐水。造模前1 d(T0)和造模后1、3、5 d(T1、T2、T3)时测定热缩足潜伏期(TWL)、冷缩足潜伏期(CWL)和机械缩足反应频率(PWF)。随后处死小鼠取脑组织，采用免疫荧光法观察P2Y12受体的表达部位，苏木精-伊红(HE)染色法观察脑组织病理改变，蛋白质印迹法检测脑组织P2Y12受体、Toll样受体4(TLR4)、磷酸化核转录因子κB-p65(p-NF-κB p65)和NF-κB p65的表达水平。
    结果 P2Y12受体在CPSP组脑组织内只表达于小胶质细胞中，星形胶质细胞和神经元中未表达。与C组比较, CPSP组和CPSP-M组T1~T3时TWL、CWL缩短, PWF升高，脑组织损伤程度加重, P2Y12受体、TLR4和p-NF-κB p65表达上调, C-M组T1~T3时以上各项指标与C组比较，差异无统计学意义(P>0.05); 与CPSP组比较, CPSP-M组T1~T3时TWL、CWL延长，PWF降低，脑组织损伤程度减轻, P2Y12受体、TLR4和p-NF-κB p65表达下调(P < 0.05)。
    结论 小胶质细胞P2Y12受体可能通过激活TLR4/NF-κB信号通路参与小鼠CPSP的形成。

     

    Abstract:
    Objective To evaluate the role of P2Y12 receptor in microglia in central post-stroke pain (CPSP) in mice and its mechanism.
    Methods Thirty-two specific pathogen-free(SPF)-grade male C57BL/6J mice were selected, aged 7 to 8 weeks, weighing 25 to 30 g, were divided into four groups using a random number table method: C group (sham operation group, n=8), CPSP group (n=8), CPSP-M group (CPSP plus P2Y12 receptor inhibitor MRS2395, n=8) and C-M group (sham operation plus P2Y12 receptor inhibitor MRS2395, n=8). CPSP model was induced by injecting 10 nL type Ⅳ collagenase(0.001 U/nL) into the right ventral posterior medial nucleus andventral posterior lateral nucleus. MRS2395(1.5 mg/kg) was injected intraperitoneally at 30 min before model preparation in CPSP-M group and C-M group and then injected once a day for 5 consecutive days, mice in C group and CPSP group were given saline at the same volume. Before the modeling (T0) and 3, 7 and 14 days (T1, T2, T3) after the establishment of the model, the thermal withdrawal latency (TWL), cold withdrawal latency (CWL) and paw withdrawal frequency (PWF) were measured. The mice were then sacrificed and the brain tissues were obtained for observing the location of P2Y12receptor by double immunofluorescence staining and the pathological changes by HE staining, and for determination of the expression levels of P2Y12 receptor, toll-like receptor 4 (TLR4), phospho-nuclear factor-κB p65 (p-NF-κB p65) and NF-κB p65 by western blot.
    Results P2Y12 receptor was co-localized only with microglia, but not with neurons or astrocytes in brain tissue of CPSP group. Compared with C group, the TWL and CWL were significantly reduced and the PWF was significantly increased at T1 to T3, cerebral injury was aggravated significantly and the expressions of P2Y12 receptor, TLR4 and p-NF-κB p65 in brain tissue were up-regulated in CPSP group and CPSP-M group (P < 0.05). There was no statistically significant difference between C group and C-M group in the above indicators (P>0.05). Compared with CPSP group, the TWL and CWL were significantly prolonged and the PWF was significantly reduced at T1 to T3, cerebral injury was relieved significantly and the expressions of P2Y12 receptor, TLR4 and p-NF-κB p65 in brain tissue were down-regulated in CPSP group and CPSP-M group (P < 0.05).
    Conclusion P2Y12 receptors in microglia may be involved in the development of CPSP by activating TLR4/NF-κB signaling pathway in mice.

     

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