辛温开窍中药对脑梗死大鼠NLRP3炎性反应通路的影响

The effect of pungent and warm natured resuscitation drugs on the inflammatory reaction of NLRP3 in rats with cerebral infarction

目的观察辛温开窍中药对脑梗死大鼠NOD样受体蛋白3(NLRP3)、半胱氨酸天冬氨酸蛋白酶1(Caspase-1)、白细胞介素-1β(IL-1β)、IL-18的影响,探讨脑卒中的炎性反应通道及辛温开窍中药对缺血大鼠脑组织炎症反应的影响。方法将健康成年雄性SD大鼠120只随机分为假手术组、缺血组、常规中药组和辛温开窍组,每组30只。除假手术组外,其余组均选择从颈总动脉插入线栓直达颈内动脉末端栓塞一侧大脑中动脉法制备脑梗死模型。在术前连续3 d(每天2次)及术后(每6 h 1次)给予各组大鼠灌胃,常规中药组灌胃常规中药方煎剂,辛温开窍组灌胃常规中药方煎剂和麝香0.5 g,假手术组和缺血组灌胃生理盐水。分别于术后24 h和48 h取缺血区脑组织,采用Western Blot检测NLRP3、Caspase-1表达水平,采用酶联免疫法(ELISA)检测IL-18、 IL-1β表达水平,采用组织免疫荧光染色法检测NLRP3表达情况。结果术后24 h和48 h,缺血组、常规中药组和辛温开窍组大鼠脑组织中NLRP3、Caspase-1、IL-18、 IL-1β表达水平均显著高于假手术组(P均<0.05),但常规中药组和辛温开窍组均显著低于缺血组(P均<0.05),且辛温开窍组均显著低于常规中药组(P均<0.05)。结论脑组织缺血后存在炎性因子的表达和NLRP3炎性通道反应;辛温开窍中药可显著下调缺血脑组织NLRP3、Caspase-1、IL-1β和IL-18蛋白表达水平,可能与麝香快速通过血脑屏障,抑制缺血后脑组织NLRP3炎症通道有关。

Objective It is to investigate the effect of pungent and warm natured resuscitation drugs on the nod-like receptor protein-3(NLRP3), Caspase-1, interleukin-1β(IL-1β), IL-18 in in rats with cerebral infarction, thus to explore the inflammatory response pathway of stroke and the effect of pungent and warm natured resuscitation drugs on the inflammatory response of ischemic rat brain tissue. Methods 120 healthy adult male Sprague-Dawley rats were randomly divided into sham operation group, ischemia group, conventional Chinese medicine group and pungent and warm natured resuscitation drug group, with 30 rats in each group. Except for the sham operation group, the cerebral infarction models were prepared from the common carotid artery insertion line to the end of the internal carotid artery embolization side of the middle cerebral artery in the other groups. The conventional Chinese medicine group was given conventional Chinese medicine decoction, and the drug group was given pungent and warm natured resuscitation decoction and musk 0.5 g, sham operation group and ischemia group were normal saline, all by intragastrically administration for 3 days before operation(2 times a day) and after operation (every 6 hours).The brain tissue of ischemic area was taken at 24 h and 48 h after operation. The expression levels of NLRP3 and Caspase-1 were detected by Western Blot. The expression levels of IL-18 and IL-1β were detected by enzyme-linked immunosorbent assay (ELISA). The expression of NLRP3 was detected by tissue immunofluorescence staining. Results At 24 h and 48 h after operation, the expression levels of NLRP3, Caspase-1, IL-18 and IL-1β in the ischemic group, conventional Chinese medicine group and drug group were significantly higher than those in the sham operation group ( P <0.05). However, the indexes in the conventional Chinese medicine group and drug group were significantly lower than those in the ischemic group ( P <0.05), and the indexes in the drug group was significantly lower than those in the conventional Chinese medicine group ( P <0.05). Conclusion There was expression of inflammatory factors and NLRP3 inflammatory channel reaction after cerebral ischemia;pungent and warm natured resuscitation drugs can significantly down-regulate the expression levels of NLRP3, Caspase-1, IL-1β and IL-18 in ischemic brain tissue, the mechanism may be related with musk qulickly passing through the blood-brain barrier and inhibiting of NLRP3 inflammatory channels in ischemic brain tissue.