基于P38MAPK信号通路的调控探讨活血通络解毒方对急性脑梗死大鼠的神经保护作用

Neuroprotective Effect of Huoxue Tongluo Jiedu Decoction(活血通络解毒方)on Acute Cerebral Infarction Rats by Regulating P38MAPK Signaling Pathway

目的:观察活血通络解毒方对急性脑梗死大鼠神经细胞凋亡的影响,并探讨其机制。方法:将Wistar大鼠随机分成5组,每组16只,分别为假手术组,模型组,活血通络解毒方低、中、高剂量组。除假手术组外,各组大鼠建立局灶性脑缺血再灌注模型,模型组、假手术组给予蒸馏水灌胃,中药组分别给予相应浓度的中药液灌胃。分别于造模的第1、3、7天采用Longa评分法进行神经功能损伤评分,7 d后处死大鼠并取材,采用TTC染色法测定大鼠脑梗死体积,Tunel染色法测定脑细胞凋亡率,Western blot法检测缺血区脑组织p38丝裂原活化蛋白激酶(p38MAPK)、激活转录因子2(ATF2)、磷酸化p38丝裂原活化蛋白激酶(p-p38MAPK)、磷酸化激活转录因子2(p-ATF2)、半胱氨酸天冬氨酸蛋白酶3(Caspase3)蛋白表达水平。结果:与假手术组相比,模型组大鼠神经功能缺损评分、脑梗死体积、脑细胞凋亡率均显著增加(P<0.05),模型组大鼠脑组织Caspase3、p38MAPK、ATF-2、p-p38MAPK、p-ATF2蛋白表达明显升高(P<0.05);与模型组相比,活血通络解毒方高剂量组可明显减少大鼠脑梗死体积(P<0.05),中、高剂量组均可明显减轻大鼠神经功能损伤和脑细胞凋亡(P<0.05),并下调Casepase3、p-P38MAPK蛋白表达量(P<0.05),各剂量组均可显著降低ATF2、p-ATF2蛋白表达水平(P<0.05)。结论:活血通络解毒方可能是通过抑制P38MAPK信号通路的激活和下游转录因子ATF2的活化、下调凋亡相关蛋白的表达,从而实现对脑梗死大鼠的神经保护作用。

Objective:To study the effect of Huoxue Tongluo Jiedu Decoction on apoptosis of nerve cells in acute cerebral infarction rat model and explore its mechanism.Methods:Wistar male rats were randomly divided into 5 groups:sham-operation group,model group,Huoxue Tongluo Jiedu Decoction high-dose,medium-dose and low-dose groups,with 16 rats in each group.Except for the sham-operation group,focal cerebral ischemia-reperfusion model of rats in other groups was established by modified Longa suture method.After operation,the model group and sham-operation group were given intragastric injection of distilled water,and others were given the corresponding concentrations of Huoxue Tongluo Jiedu Decoction.Longa’s scale was used to evaluate the neurological deficit of each group at 1st,3rd and 7th day.After 7 days,TTC staining was used to detect the cerebral infarct volume of rats,and the apoptosis of nerve cells was determined by Tunel staining;the protein expressions of P38MAPK,ATF2,p-P38MAPK,p-ATF2,Caspase3 were detected by Western Blot.Results:Compared with sham-operation group,neurological deficit score,cerebral infarct volume and apoptosis rate of nerve cells in model group were significantly increased(P<0.05).The protein expressions of Caspase3,P38MAPK,ATF2,p-P38MAPK and P-ATF2 of model group were also significantly increased(P<0.05).Compared with the model group,high-dose Huoxue Tongluo Jiedu Decoction coould reduce the cerebral infarct volume significantly(P<0.05),the medium and high dose groups of Huoxue Tongluo Jiedu could significantly down-regulate the neurological deficit score,apoptosis rate,protein expressions of Casepase3 and p-P38MAPK(P<0.05).Meanwhile the protein expressions of ATF2 and p-ATF2 were significantly reduced in all the 3 dose groups(P<0.05).Conclusion:Huoxue Tongluo Jiedu Decoction maybe inhibiting the activation of P38MAPK signaling pathway and downstream transcription factor ATF2,decreasing the expressions of apoptosis-related protein to protect apoptosis of nerve cells in acute cerebral infarction rat.