基于TLR4/NLRP3信号通路探讨偏瘫口服液对缺血性脑卒中大鼠的改善作用

Ameliorative Effect of Hemiplegic Oral Solution on Ischemic Stroke Rats Based on TLR4/NLRP3 Signaling Pathway

目的探讨偏瘫口服液对缺血性脑卒中模型大鼠的改善作用及机制。方法将40只大鼠随机分为假手术组、模型组、银杏叶片组(阳性对照,5.76 mg/kg)和偏瘫口服液低、高剂量组(1.5、6.0 g/kg)。除假手术组外,其余各组均采用改良线栓法制备大鼠大脑中动脉阻塞(MCAO)模型。造模成功后,各组大鼠分别连续给予相应药物治疗5 d。实验期间,监测大鼠活动并进行神经功能评分;末次给药后,分析脏器指数;采用ELISA法检测脑组织白细胞介素-1β(IL-1β)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量;试剂盒法测定丙二醛(MDA)、超氧化物歧化酶(SOD)及谷胱甘肽(GSH)水平;以苏木素-伊红(HE)染色法比较缺血区脑组织病理学特征;通过Western blot法测定大鼠脑组织中凋亡相关蛋白及TLR4/NLRP3信号通路相关蛋白的表达水平。结果相较于假手术组,模型组大鼠神经功能评分、脑指数、脾指数以及脑组织IL-1β、IL-6、TNF-α、MDA含量均显著升高(P<0.01),而脑组织SOD、GSH水平均显著降低(P<0.01);脑组织出现结构松弛、大量排列不规则空洞、细胞核固缩等典型病变。与模型组比较,偏瘫口服液高剂量组和银杏叶片组大鼠神经功能缺损评分、脑指数、脾指数,以及脑组织IL-1β、IL-6、TNF-α、MDA水平均显著下降(P<0.05),而脑组织SOD、GSH含量均明显上升(P<0.01),脑组织病理明显改善。与假手术组相比,模型组脑组织中Bax、TLR4、p-NF-κB、NF-κB与NLRP3蛋白表达均显著上调(P<0.01),Bcl-2蛋白表达显著下调(P<0.01);与模型组相比,偏瘫口服液低、高剂量组可有效地逆转上述脑组织损伤的蛋白表达趋势。结论偏瘫口服液对缺血性脑卒中模型大鼠具有良好的治疗作用,其机制可能与调节TLR4/NLRP3信号通路、抑制神经炎症反应和氧化应激相关。

Objective To study the improvement effect and mechanism of hemiplegic oral solution on ischemic stroke model rats.Methods Totally 40 rats were randomly divided into sham operation group,model group,ginkgo leaf tablet group(positive control,5.76 mg/kg),low-dose group and high-dose group of hemiplegic oral solution(1.5,6.0 g/kg).The rat model of middle cerebral artery occlusion(MCAO)was duplicated with the improved suture method in other groups except the sham operation group.Rats in each group continuous administered for 5 days after modeling.During experiment,general condition of the rats and the neurological function score was examined and performed respectively.Organ index was calculated after the last administration.ELISA was applied to measure the levels of interleukin-1β(IL-1β),interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and kit method evaluate malondialdehyde(MDA),superoxide dismutase(SOD),glutathione(GSH)in brain tissue.HE staining was used to observe the histopathological changes in the ischemic area.Western blotting was employed to assay the expression levels of apoptosis-related and TLR4/NLRP3 signaling pathway-related proteins in the rat brain tissue.Results Compared with the sham operation group,neurological deficit score,brain tissue index,spleen tissue index,IL-1β,IL-6,TNF-αand MDA levels were significantly increased(P<0.01),and SOD,GSH levels were significantly lowered in the brain tissue of model group(P<0.01).The brain tissue showed structural relaxation,a large number of irregular arrangement of cavities,nuclear shrinkage and other typical lesions.Compared with model group,high-dose hemiplegic oral solution group and ginkgo leaf tablet group decreased the neurological deficit score,lowered the IL-1β,IL-6,TNF-αand MDA levels(P<0.05),and elevated the SOD and GSH levels in the brain tissue(P<0.01),the pathology of brain tissue was improved obviously.Compared with the sham operation group,the model group showed elevated protein levels of Bax,TLR4,p-NF-κB,NF-κB,NLRP3(P<0.01),and inhibited protein level of Bcl-2 in the brain tissue(P<0.01).Compared with the model group,the low and high dose groups of hemiplegia oral solution could effectively reverse the protein expression trend of the above-mentioned brain tissue injury.Conclusion Hemiplegia oral solution has powerful therapeutic effect on ischemic stroke model rats,the mechanism may be related to the regulation of TLR4/NLRP3 signaling pathway to inhibit neuroinflammatory response and oxidative stress.