Z-没药甾酮基于PI3K/AKT通路化瘀解毒抗缺血性脑卒中的作用及机制

Effect and mechanism of removing blood stasis and detoxification of Z-guggulsterone on ischemic stroke based on PI3K/Akt pathway

目的基于磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)通路,从血管活性物质和氧化应激角度研究Z-没药甾酮化瘀解毒抗缺血性脑卒中的作用及机制。方法将60只大鼠随机分为假手术组、模型组和Z-没药甾酮低剂量组、Z-没药甾酮中剂量组、Z-没药甾酮高剂量组,每组12只。除假手术组外,其余组大鼠采用大脑中动脉阻闭术建立缺血性脑卒中模型,Z-没药甾酮低、中、高剂量组大鼠分别于术前30 min给予25 mg/kg、50 mg/kg、100 mg/kg Z-没药甾酮腹腔注射,术后继续干预3 d。干预结束后评估各组大鼠神经功能,测定脑梗死体积和脑含水量;取各组大鼠腹主动脉血,检测全血黏度、凝血参数[凝血酶原时间(PT)、凝血酶时间(TT)、活化部分凝血活酶时间(APTT)、纤维蛋白原(FIB)]、血管活性物质[内皮素-1(ET-1)、血栓素B_(2)(TXB_(2))、血栓调节蛋白(TM)、6-酮-前列环素1α(6-keto-PGF1α)]和氧化应激物质[超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)、过氧化氢酶(CAT)、活性氧簇(ROS)、丙二醛(MDA)]水平;免疫组化法检测各组大鼠脑组织中PI3K、磷酸化蛋白激酶B(p-Akt)、Nrf2和磷酸化内皮型一氧化氮合酶(p-eNOS)蛋白表达情况。结果模型组大鼠神经功能评分、脑梗死体积和脑含水量均显著高于假手术组(P均<0.05),Z-没药甾酮中、高剂量组大鼠各指标及Z-没药甾酮低剂量组大鼠神经功能评分均显著低于模型组(P均<0.05)。模型组大鼠不同切变率下全血黏度均显著高于假手术组(P均<0.05),Z-没药甾酮中、高剂量组大鼠不同切变率下全血黏度及Z-没药甾酮低剂量组1/s和30/s切变率下全血黏度均显著低于模型组(P均<0.05)。与假手术组比较,模型组大鼠TT、PT、APTT均显著缩短(P均<0.05),血清FIB、ET-1、TXB_(2)、ROS、MDA水平均显著升高(P均<0.05),血清TM、6-keto-PGF1α、SOD、GSH-Px、CAT水平均显著降低(P均<0.05);与模型组比较,Z-没药甾酮中、高剂量组大鼠TT、PT、APTT及Z-没药甾酮低剂量组大鼠APTT均显著延长(P均<0.05),Z-没药甾酮各剂量组大鼠血清FIB、ROS、MDA水平和Z-没药甾酮中、高剂量组大鼠血清ET-1和TXB_(2)水平均显著降低(P均<0.05),Z-没药甾酮中、高剂量组大鼠血清TM、6-keto-PGF1α、SOD、GSH-Px、CAT水平及Z-没药甾酮低剂量组大鼠血清SOD水平均显著升高(P均<0.05)。模型组大鼠脑组织中PI3K、p-Akt、Nrf2和p-eNOS蛋白表达光密度值均显著低于假手术组(P均<0.05),Z-没药甾酮各剂量组各蛋白表达光密度值均显著高于模型组(P均<0.05)。结论Z-没药甾酮可通过调控血管活性物质和氧化应激指标水平发挥化瘀解毒的脑保护作用,其机制可能与活化PI3K/Akt通路及其下游的eNOS和Nrf2途径有关。

Objective It is to explore the effect and mechanism of removing blood stasis and detoxification of Z-guggulsterone(Z-GS)on ischemic stroke from the perspective of vasoactive substances and oxidative stress and based on phosphatidylinositol 3-kinase(PI3K)/protein kinase B(Akt)pathway.Methods Sixty rats were randomly divided into sham operation group,model group and Z-GS low-dose group,Z-GS medium-dose group and Z-GS high-dose group,each group had 12 rats.Except for the sham operation group,the rats in other groups were treated with middle cerebral artery occlusion to establish ischemic stroke models.The rats in the Z-GS low-dose,medium-dose,and high-dose groups were respectively given 25 mg/kg,50 mg/kg and 100 mg/kg Z-GS by intraperitoneal injection,and the intervention continued for 3 days after the operation.After the intervention,the neurological function of the rats in each group was evaluated,and the volume of cerebral infarction and cerebral water content were detected;abdominal aortic blood of the rats in each group was collected to detect the levels of whole blood viscosity and coagulation parameters[prothrombin time(PT),thrombin time(TT),activated partial thromboplastin time(APTT),fibrinogen(FIB)],vasoactive substances[endothelin-1(ET-1),thromboxane B_(2)(TXB_(2)),thrombomodulin(TM),6-keto-prostacyclin 1α(6-keto-PGF1α)]and oxidative stress substances[superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),catalase(CAT),reactive oxygen species(ROS),malondialdehyde(MDA)];the protein expressions of PI3K,phosphorylated protein kinase B(p-Akt),Nrf2 and phosphorylated endothelial nitric oxide synthase(p-eNOS)in the brain tissue of the rats in each group were detected by immunohistochemistry.Results The neurological function scores,cerebral infarction volume and cerebral water content of the rats in the model group were significantly higher than those in the sham operation group(all P<0.05),these indexes of the rats in the Z-GS medium and high dose groups and the neurological function scores in the Z-GS low dose group were significantly lower than those in the model group(all P<0.05).The whole blood viscosity at different shear rates of the rats in the model group were significantly higher than those in the sham operation group(all P<0.05),the whole blood viscosity at different shear rates of the rats in the Z-GS medium and high dose groups and the whole blood viscosity at 1/s and 30/s shear rates of Z-GS low dose group were significantly lower than those of the model group(all P<0.05).Compared with the sham operation group,the TT,PT,and APTT of the model group were significantly shortened(all P<0.05),and the levels of serum FIB,ET-1,TXB_(2),ROS,and MDA were significantly increased(all P<0.05),the levels of TM,6-keto-PGF1α,SOD,GSH-Px and CAT were significantly decreased(all P<0.05);compared with the model group,the TT,PT,APTT of the Z-GS medium and high dose groups and the APTT of the Z-GS low dose group were significantly prolonged(all P<0.05),the levels of serum FIB,ROS and MDA of the rats in every Z-GS group and the levels of serum ET-1 and TXB_(2) of the rats in the Z-GS medium and high dose groups were significantly decreased(all P<0.05),the levels of serum TM,6-keto-PGF1α,SOD,GSH-Px,CAT of the rats in the Z-GS medium and high dose groups,and the level of serum SOD in the Z-GS low-dose group were significantly increased(all P<0.05).The optical density values of PI3K,p-Akt,Nrf2 and p-eNOS protein expression in the brain tissue of the model group were significantly lower than those in the sham operation group(all P<0.05).The optical density values of PI3K,p-Akt,Nrf2 and p-eNOS protein expression in the brain tissue of the rats in the model group were significantly lower than those in the sham operation group(all P<0.05),the optical density of each protein expression in every Z-GS group was significantly higher than that in the model group(all P<0.05).Conclusion Z-GS can exert the brain protective effect of removing blood stasis and detoxification by regulating the levels of vasoactive substances and oxidative stress indexes,and its mechanism may be related to the activation of PI3K/Akt pathway and its downstream eNOS and Nrf2 pathways.