半枝莲总黄酮调控RIP1/RIP3通路减轻急性脑梗死缺血再灌注大鼠大脑皮层神经元的损伤

Scutellaria Barbata Total Flavonoids Regulating RIP1/RIP3 Pathway to Reduce the Damage of Cerebral Cortex Neurons in Rats with Acute Cerebral Infarction and Ischemia Reperfusion

目的:分析半枝莲总黄酮通过调控受体相互作用蛋白激酶-1(receptor interaction protein kinase-1,RIP1)/受体相互作用蛋白激酶-3(receptor interaction protein kinase-3,RIP3)通路减轻急性脑梗死缺血再灌注大鼠大脑皮层神经元损伤的机制。方法:从48只SPF级SD大鼠中随机挑出40只建立大鼠中脑动脉闭塞(middle cerebral artery occlusion,MCAO)模型,剩余8只记为阴性对照组。将MCAO建模成功的大鼠随机分为模型组,阳性对照组,半枝莲总黄酮高剂量组、中剂量组、低剂量组。阳性对照组以30 mg·kg^-1尼莫地平溶于2 mL生理盐水中灌胃;半枝莲总黄酮高剂量组、中剂量组、低剂量组分别使用200 mg·kg^-1、100 mg·kg^-1、50 mg·kg^-1半枝莲总黄酮溶于2 mL生理盐水中灌胃;模型组和阴性对照组使用2 mL生理盐水灌胃,每天1次,连续给药10 d。给药前后分别使用Longa评分标准对大鼠神经功能进行评分;治疗前后分别通过酶联免疫吸附实验检测血清白细胞介素-1β(interleukin-1β,IL-1β)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、IL-6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阴性对照组大鼠大脑皮层相同部位组织,通过脱氧核糖核苷酸末端转移酶介导的缺口末端标记法观察大脑皮层神经细胞凋亡情况;采用HE染色观察大脑皮层组织病理变化;使用RT-qPCR检测大脑皮层组织中RIP1、RIP3和MLKL mRNA相对表达量;通过蛋白免疫印记法检测大脑皮层组织RIP1、RIP3和MLKL蛋白表达量及p-RIP1、p-RIP3、p-MLKL水平。结果:神经功能评分比较显示:模型组,阳性对照组和半枝莲总黄酮不同剂量组均低于治疗前(P<0.05)。给药后,模型组高于阴性对照组(P<0.05),阳性对照组和半枝莲总黄酮高剂量组、中剂量组、低剂量组低于模型组(P<0.05),半枝莲总黄酮高剂量组和中剂量组低于阳性对照组和半枝莲总黄酮低剂量组(P<0.05),半枝莲总黄酮高剂量组低于半枝莲总黄酮中剂量组(P<0.05)。大鼠血清中IL^-1β、TNF-α和IL-6水平显示:模型组给药后以上指标高于给药前(P<0.05),阳性对照组和半枝莲总黄酮高剂量组、中剂量组、低剂量组给药后均低于给药前(P<0.05);给药后,模型组高于阴性对照组(P <0. 05),阳性对照组和半枝莲总黄酮高剂量组、中剂量组、低剂量组低于模型组(P <0. 05),半枝莲总黄酮高剂量和中剂量组低于阳性对照组和半枝莲总黄酮低剂量组(P <0. 05),半枝莲总黄酮高剂量组低于半枝莲总黄酮中剂量组(P <0. 05)。脑神经细胞凋亡TUNEL染色结果显示:阳性对照组和半枝莲总黄酮高剂量组、中剂量组、低剂量组均低于模型组(P <0. 05),半枝莲总黄酮高剂量组、中剂量组均低于阳性对照组和半枝莲总黄酮低剂量组(P <0. 05),半枝莲总黄酮高剂量组低于半枝莲总黄酮中剂量组(P <0. 05)。HE染色结果显示:模型组大脑皮层组织出现严重水肿,空泡样变,阳性对照组和半枝莲总黄酮高剂量组、中剂量组、低剂量组均减轻,其中半枝莲总黄酮高剂量组病变程度最轻;RIP1、RIP3和MLKL蛋白表达量各组间比较,差异无统计学意义(P> 0. 05)。RIP1、RIP3和MLKL m RNA相对表达量和p-RIP1、p-RIP3、p-MLKL水平结果显示:阳性对照组和半枝莲总黄酮高剂量组、中剂量组、低剂量组均低于模型组(P <0. 05),半枝莲总黄酮高剂量组和中剂量组低于阳性对照组和半枝莲总黄酮低剂量组(P <0. 05),半枝莲总黄酮高剂量组低于半枝莲总黄酮中剂量组(P <0. 05)。结论:半枝莲总黄酮能降低急性脑梗死缺血再灌注大鼠大脑皮层神经功能损伤,减轻炎性反应,减轻神经元损伤,推测其作用机制与下调RIP1、RIP3和MLKL m RNA表达,降低p-RIP1、pRIP3、p-MLKL水平,抑制RIP1/RIP3通路对细胞程序性凋亡的调控有关。

Objective:To analyze the mechanism of scutellaria barbata total flavonoids in alleviating cerebral cortex neuron injury in rats with acute cerebral infarction and ischemia reperfusion by regulating receptor interaction protein kinase-1(RIP1)/receptor interaction protein kinase-3(RIP3)pathway.Method:Forty of 48 SPF SD rats were randomly selected to establish a middle cerebral artery occlusion(MCAO)model of middle cerebral artery occlusion in rats,and the remaining 8 rats were recorded as negative control groups.Rats successfully modeled by MCAO were randomly divided into model group,positive control group,scutellaria barbata total flavone high dose group,medium dose group and low dose group.In the positive control group,nimodipine 30 mg·kg^-1 was dissolved in 2 mL of normal saline for gastric perfusion.The high dose group,medium dose group and low dose group of scutellaria barbata total flavonoids were respectively dissolved in 2 mL of normal saline and gavage with 200 mg·kg^-1,100 mg·kg^-1 and 50 mg·kg^-1 of scutellaria barbata total flavonoids.The model group and negative control group were fed with 2 mL of normal saline once a day for 10 days.Before and after administration,the nerve function of rats was scored by Longa scoring standard.The levels of serum interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and IL-6 were detected by enzymelinked immunosorbent assay before and after treatment.After the rats were killed by decapitation,the cerebral cortex infarct tissues of the model rats and the same parts of the cerebral cortex tissues of the negative control rats were taken,and the apoptosis of cerebral cortex nerve cells was observed by the nick end labeling method mediated by deoxynucleotide terminal transferase.Histopathological changes of cerebral cortex were observed by HE staining.RT-qPCR was used to detect the relative expression of RIP1,RIP3 and MLKL mRNA in cerebral cortex tissue.The expression of RIP1,RIP3 and MLKL protein and the levels of p-RIP1,p-RIP3 and p-MLKL were detected by protein immunoblotting.Results:The comparison of neurological function scores showed that in model group,positive control group and scutellaria barbata total flavonoids of different dosage groups were lower than before treatment(P<0.05).After administration,the model group was higher than the negative control group(P<0.05),the positive control group and scutellaria barbata total flavonoids high dose group,medium dose group and low dose group were lower than the model group(P<0.05),the scutellaria barbata total flavonoids high dose and medium dose group were lower than the positive control group and scutellaria barbata total flavonoids low dose group(P<0.05),and the scutellaria barbata total flavonoids high dose group was lower than the scutellaria barbata total flavonoids medium dose group(P<0.05).The levels of IL-1β,TNF-αand IL-6 in rat serum showed that the above indexes in the model group were higher than those before administration(P<0.05),and the positive control group and scutellaria barbata flavonoids high dose group,medium dose group and low dose group were lower than those before administration(P<0.05).After administration,the model group was higher than the negative control group(P<0.05),the positive control group and scutellaria barbata total flavonoids high dose group,medium dose group and low dose group were lower than the model group(P<0.05),the scutellaria barbata total flavonoids high dose and medium dose group were lower than the positive control group and scutellaria barbata total flavonoids low dose group(P<0.05),and the scutellaria barbata total flavonoids high dose group was lower than the scutellaria barbata total flavonoids medium dose group(P<0.05).TUNEL staining showed that the positive control group,high dose group,medium dose group and low dose group of scutellaria barbata total flavonoids were lower than that in the model group(P<0.05).The high dose group and medium dose group of scutellaria barbata total flavonoids were lower than that in the positive control group and low dose group of scutellaria barbata total flavonoids(P<0.05).The high dose group of scutellaria barbata total flavonoids was lower than that in the medium dose group of scutellaria barbata total flavonoids(P<0.05).HE staining results showed that the cerebral cortex tissue in the model group showed severe edema and vacuolar degeneration,and the positive control group,high dose group,medium dose group and low dose group of scutellaria barbata total flavonoids all reduced,among which the lesion degree in high dose group of scutellaria barbata total flavonoids was the lightest.There was no significant difference in the expression of RIP1,RIP3 and MLKL protein among the groups(P>0.05).The relative expression of RIP1,RIP3 and MLKL mRNA and the levels of p-RIP1,p-RIP3 and p-MLKL showed that the positive control group,scutellaria barbata total flavone high dose group,medium dose group and low dose group were lower than the model group(P<0.05),the scutellaria barbata total flavone high dose group and medium dose group were lower than the positive control group and scutellaria barbata total flavone low dose group(P<0.05),and the scutellaria barbata total flavone high dose group was lower than the scutellaria barbata total flavone medium dose group(P<0.05).Conclusion:Scutellaria barbata total flavonoids can reduce cerebral cortex nerve function damage,alleviate inflammatory reaction and reduce neuronal damage in acute cerebral infarction ischemia-reperfusion rats.It is speculated that its mechanism of action is related to down-regulation of RIP1,RIP3 and MLKL mRNA expression,reduction of p-RIP1,p-RIP3 and p-MLKL levels,and inhibition of RIP1/RIP3 pathway regulation on programmed cell apoptosis.