毛冬青皂酮胶囊对急性脑梗死模型大鼠神经保护作用

Neuroprotective Effect of Mao Dongqing Zaotong Capsule on Rats with Acute Cerebral Infarction

目的:分析毛冬青皂酮胶囊对急性脑梗死模型大鼠神经保护作用并初步探索其保护机制。方法:从60只SPF级SD大鼠中随机选取50只,建立大鼠动脉闭塞模型(middle cerebral artery occlusion,MCAO),余下10只记作假手术组。将成功建模大鼠随机分为模型组,对照组,毛冬青皂酮胶囊高剂量组、中剂量组、低剂量组。对照组给予0.03 g·kg^-1莫尼地平溶于2 mL生理盐水灌胃,毛冬青皂酮胶囊高剂量组、中剂量组、低剂量组分别使用0.2 g·kg^-1、0.1 g·kg^-1、0.05 g·kg^-1毛冬青皂酮胶囊颗粒溶于2 mL生理盐水灌胃,假手术组和模型组给予2 mL生理盐水灌胃,连续用药6 d,每天1次。治疗6 d后,在第7天时,按照Longa评分标准对大鼠进行神经功能评分,并取大鼠眼底静脉丛血,收集血清,通过酶联免疫吸附实验法检测大鼠血清炎性因子白细胞介素-1β(interleukin-1β,IL^-1β)和肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)水平;断头法处死大鼠后取模型大鼠大脑皮层梗死部位组织和假手术组大鼠相同位置的大脑皮层组织,通过HE染色观察大脑皮层病理变化;通过RT-qPCR检测大鼠大脑皮层组织中IκBα、核因子κBp65(nuclear factorκ-Bp65,NF-κBp65)、κB抑制蛋白激酶β(inhibitor of nuclear factorκB kinaseβ,IKKβ)mRNA表达水平;通过蛋白免疫印迹法检测大鼠大脑皮层组织中κB抑制蛋白α(inhibitor of NF-κBα,IκBα)、NF-κBp65、IKKβ、磷酸化κB抑制蛋白激酶β(phosphorylation inhibitor of nuclear factorκB kinaseβ,p-IKKβ)水平。结果:神经功能评分结果显示:对照组、毛冬青皂酮胶囊高剂量组、毛冬青皂酮胶囊中剂量组、毛冬青皂酮胶囊低剂量组在治疗后评分均降低(P<0.05),给药前模型组、对照组、毛冬青皂酮胶囊高剂量组、毛冬青皂酮胶囊中剂量组、毛冬青皂酮胶囊低剂量组均高于假手术组,给药后模型组高于假手术组(P<0.05),对照组和毛冬青皂酮胶囊高剂量组、中剂量组、低剂量组均低于模型组(P<0.05),毛冬青皂酮胶囊高剂量组和毛冬青皂酮胶囊中剂量组均低于毛冬青皂酮胶囊低剂量组和对照组(P<0.05),毛冬青皂酮胶囊高剂量组低于中剂量组(P<0.05);血清IL^-1β和TNF-α水平模型组高于假手术组(P<0.05),对照组和毛冬青皂酮胶囊高剂量组、中剂量组、低剂量组均低于模型组(P<0.05),毛冬青皂酮胶囊高剂量组和中剂量组均低于毛冬青皂酮胶囊低剂量组和对照组(P<0.05),毛冬青皂酮胶囊高剂量组低于中剂量组(P<0.05);大脑皮层组织HE染色结果显示,模型组细胞水肿严重,对照组和毛冬青皂酮胶囊高剂量组、中剂量组、低剂量组均有所减轻,高剂量组病理变化与假手术组最接近;大脑皮层组织IκBα和IKKβmRNA和蛋白表达水平比较,差异无统计学意义(P>0.05);NF-κBp65 mRNA和蛋白表达水平及p-IκBα、p-IKKβ水平比较:模型组显著高于假手术组(P<0.05),对照组和毛冬青皂酮胶囊高剂量组、中剂量组、低剂量组均低于模型组(P<0.05),毛冬青皂酮胶囊高剂量组和中剂量组低于毛冬青皂酮胶囊低剂量组和对照组(P<0.05),毛冬青皂酮胶囊高剂量组低于中剂量组(P<0.05)。结论:毛冬青皂酮可显著降低急性脑梗死大鼠神经功能评分,减轻炎症反应,减轻神经细胞水肿,其减轻大鼠神经炎性损伤的机制为下调NF-κBp65、IκBα基因、蛋白表达水平,抑制p-IκBα、p-IKKβ水平,从而对急性脑梗死大鼠神经具有保护作用。

Objective:To analyze the neuroprotective effect of Mao Dongqing Zaotong Capsule on rats with acute cerebral infarction and explore its protective mechanism.Method:Fifty rats were randomly selected from 60 SPF SD rats to establish middle cerebral artery occlusion(MCAO)model,and the remaining 10 rats were recorded as sham operation group.The rats were randomly divided into model group,control group,high-dose group,medium-dose group and low-dose group.The control group was given 0.03 g·kg^-1 monidipine dissolved in 2 mL normal saline,the high dose group,the middle dose group and the low dose group were given 0.2 g·kg^-1,0.1 g·kg^-1 and 0.05 g·kg^-1 capsule particles dissolved in 2 mL normal saline,and the sham operation group and the model group were given 2 mL normal saline once a day for 6 days.After 6 days of treatment,on the 7th day,the rats were scored for neurological function according to Longa scoring standard,and the blood of fundus venous plexus was collected,and the serum levels of interleukin-1β(IL-1β)and tumor necrosis factor-α(TNF-α)were detected by enzyme-linked immunosorbent assay.After the rats were killed by decapitation,the cerebral cortex infarction tissues of the model rats and the cerebral cortex tissues of the rats in the sham operation group were taken,and the pathological changes of the cerebral cortex were observed by HE staining.The mrna expression levels of IκBα,nuclear factorκBp65(NF-κBp65)andκB inhibitory protein kinaseβ(IKKβ)mRNA in rat cerebral cortex were detected by RT-qPCR.Western blot method(WB)was used to detect the inhibitor of NF-κBα(IκBα),nuclear factorκ-Bp65(NF-κBp65),inhibitor of nuclear factorκB kinaseβ(IKKβ)and phosphorylation inhibitor of nuclear factorκB kinaseβ(p-IKKβ).Result:The results of neurological function score showed that the scores of control group,high dose group,middle dose group and low dose group were all decreased after treatment(P<0.05),and the scores of model group,control group,high dose group,middle dose group and low dose group were higher than those of sham operation group before administration,but higher than those of model group after administration.The control group,high-dose,medium-dose and low-dose groups of Mao Dongqing Zaotong Capsule were all lower than the model group(P<0.05);Serum levels of IL-1βand TNF-αin model group were higher than those in sham operation group(P<0.05),while those in control group and high,medium and low dose groups were lower than those in model group(P<0.05);HE staining results of cerebral cortex tissue showed that the cell edema in model group was serious,and it was alleviated in control group,high dose group,middle dose group and low dose group of Mao Dongqing Zaotong Capsule.The pathological changes in high dose group were closest to those in sham operation group;there was no significant difference in the mRNA and protein expression levels of IκBαand IKKβin cerebral cortex among the three groups(P>0.05).The expression levels of NF-κBp65 mRNA and protein,p-IκBαand p-IKKβin the model group were significantly higher than those in the sham operation group(P<0.05).The control group,high-dose group,middle-dose group and low-dose group of Mao Dongqing Zaotong Capsule were all lower than the model group(P<0.05).The high-dose and middle-dose groups of Mao Dongqing Zaotong Capsule were lower than those of Mao Dongqing Zaotong Capsule and control group(P<0.05).The high-dose group of Dongqing Zaotong Capsule was lower than the middle dose group(P<0.05).Conclusion:Mao Dongqing Zaotong Capsule can significantly reduce the neurological function score,inflammatory reaction and nerve cell edema in rats with acute cerebral infarction.The mechanism of relieving nerve inflammatory injury in rats is to down-regulate the expression levels of NF-κBp65,IκBαgene and protein,and inhibit the levels of p-IκBαand p-IKKβ,thus protecting the nerve of rats with acute cerebral infarction.