栝楼桂枝颗粒对脑缺血再灌注损伤模型大鼠缺血区脑组织内质网应激及细胞凋亡的影响

Effects of Gualou Guizhi Granules(栝楼桂枝颗粒)on Endoplasmic Reticulum Stress and Apoptosis in Brain Tissue of the Cerebral Ischemic Area of Cerebral Ischemia-reperfusion Injury Model Rats

目的探讨栝楼桂枝颗粒治疗脑缺血再灌注损伤的可能作用机制。方法54只SD大鼠随机分为假手术组、模型组、栝楼桂枝颗粒组,每组18只。模型组、栝楼桂枝颗粒组大鼠采用线栓法致大脑中动脉栓塞建立脑缺血再灌注损伤模型,假手术组除不插入线栓外其余操作同模型组。各组于造模后2 h开始干预,假手术组和模型组给予生理盐水10 ml/kg灌胃,栝楼桂枝颗粒组给予栝楼桂枝汤颗粒剂3.6 g/(kg·d)灌胃。各组均每日1次,连续7天。采用改良的神经功能缺损评分(mNSS)、转角实验对大鼠神经损害程度进行评价;采用TUNEL法检测大鼠缺血区脑组织细胞凋亡情况,采用Western blot法检测脑组织中Bcl-2相关X蛋白(Bax)、B淋巴细胞瘤-2基因(Bcl-2)、神经元特异核蛋白(NeuN)的相对表达量;Real-Time PCR法检测缺血区脑组织内质网应激相关因子真核生物起始因子2α(elF2α)、蛋白激酶RNA样内质网激酶(PERK)、内质网源性转录因子(CHOP)、转录激活因子4(ATF4)、葡萄糖调节蛋白78(GRP78)的mRNA表达;Western blot法检测缺血区脑组织ATF4、CHOP、PERK、elF2α、磷酸化真核生物起始因子2α抗体(p-elF2α)、磷酸化蛋白激酶RNA样内质网激酶(p-PERK)蛋白的表达水平。结果与假手术组比较,模型组大鼠mNSS评分、转角百分率升高,缺血区脑组织细胞凋亡率、Bax蛋白表达升高,Bcl-2及NeuN蛋白表达降低(P<0.01);缺血区脑组织中GRP78、PERK、eIF2α、CHOP、ATF4 mRNA升高,CHOP、ATF4蛋白表达及p-PERK/PERK、p-eIF2α/eIF2α值亦升高(P<0.01)。与模型组比较,栝楼桂枝颗粒组大鼠上述各指标均明显改善(P<0.05或P<0.01)。结论栝楼桂枝颗粒可通过抑制内质网应激,从而抑制脑缺血损伤后神经细胞凋亡,进而发挥抗脑缺血再灌注损伤作用。

Objective To investigate the possible mechanism of Gualou Guizhi Granules(栝楼桂枝颗粒,GGG)in the treatment of cerebral ischemia-reperfusion injury(CI-RI).MethodsFifty-four SD rats were randomized into sham-operation group,model group and GGG group,with 18 rats in each group.The CI-RI model was established in the model group and the CI-RI group with thread occlusion to induce middle cerebral artery occlusion(MCAO),while thread occlusion was not performed in the sham operation group.Two hours after the modelling,the sham operation group and the model group were given normal saline(10 ml/kg)by gavage,and the GGG group was administered with GGG 3.6 g/(kg·d)by gavage;the interventions in all the groups were given once daily for seven consecutive days.The modified neurological severity score(mNSS)and rotation angle test were used to assess the neurological damage in rats.TUNEL method was used to detect cell apoptosis in the brain tissue of the ischemic area,and Western blot was used to detect the relative expression of B lymphoma-2 gene(Bcl-2)related X protein(Bax),Bcl-2 and neuron specific nuclear protein(NeuN)in brain tissue.Real-Time PCR method was used to detect mRNA expressions of endoplasmic reticulum stress-related factors in the ischemic zone including eukaryotic initiation factor 2α(elF2α),protein kinase RNA-like endoplasmic reticulum kinase(PERK),endoplasmic reticulum-derived transcription factor(CHOP),activator of transcription 4(ATF4)and glucose regulatory protein 78(GRP78).Western blot was used to detect the protein expressions of ATF4,CHOP,PERK,elF2α,phosphorylated elF2α(p-elF2α)and phosphorylated PERK(p-PERK)in the ischemic brain tissue of rats.ResultsCompared to those of the sham operation group,the mNSS score and the rotation angle rate of the model group increased;the apoptosis rate and the Bax protein expression in the brain tissue of the ischemic area increased;the Bcl-2 and NeuN protein expression decreased(P<0.01);the mRNA expressions of GRP78,PERK,eIF2α,CHOP,ATF4,the protein expressions of CHOP and ATF4,and the value of p-PERK/PERK and p-elF2α/elF2αall increased(P<0.01).Compared to those of the model group,all the above indicators of the GGG group were significantly improved(P<0.05 or P<0.01).ConclusionGGG can inhibit the endoplasmic reticulum stress,thereby inhibiting neuronal apoptosis after cerebral ischemia injury and then preventing the occurrence of CI-RI.