基于雌激素受体介导的神经营养因子通路探讨左归丸对脑缺血再灌注损伤的保护作用

Based on estrogen receptor-mediated neurotrophic factor pathway to explore the protective effect of Zuogui Pills on cerebral ischemia-reperfusion injury

目的探讨左归丸是否能通过调节雌激素受体途径发挥神经保护作用。方法雌性SD大鼠108只,8~10周龄,SPF级。采用随机数字表法将大鼠分为六组:假手术组(只切口不插栓线)、I/R组[Longa线栓法行大脑中动脉阻塞(MCAO)手术]、OVX+I/R组(卵巢摘除+MCAO手术)、OVX+I/R+Zuogui Pills组(卵巢摘除+MCAO手术+左归丸1.62g/kg,灌胃)、OVX+I/R+Zuogui Pills+ICI182780组(卵巢摘除+MCAO手术+左归丸灌胃+ICI1827803 mg/kg,腹腔注射)、OVX+I/R+17β-estradiol组(卵巢摘除+MCAO手术+17β-estradiol 100μg/kg,腹腔注射),每组18只。神经功能缺损评分评估各组大鼠感觉运动功能,新奇物体识别实验观察大鼠的记忆力,观察雌激素受体(ER)α、ERβ、脑源性神经营养因子(BDNF)、神经生长因子(NGF)、酪氨酸激酶(Trk)A、TrkB、磷酸化cAMP反应元件结合蛋白(p-CREB)和CREB蛋白及mRNA的表达。结果I/R组大鼠的运动功能和记忆力低于假手术组(P<0.05);OVX+I/R组大鼠的运动功能和记忆力低于I/R组(P<0.05);OVX+I/R+Zuogui Pills组大鼠的运动功能和记忆力高于OVX+I/R组(P<0.05);OVX+I/R+Zuogui Pills+ICI182780组运动功能和记忆力低于OVX+I/R+Zuogui Pills组(P<0.05);OVX+I/R+Zuogui Pills组运动功能与记忆力与OVX+I/R+17β-estradiol组比较,差异无统计学意义(P>0.05)。I/R组ERα、ERβ、BDNF、NGF、TrkA、TrkB、p-CREB蛋白及mRNA(除BDNF外)表达高于假手术组(P<0.05或P<0.01);OVX+I/R组上述指标表达低于I/R组(P<0.05或P<0.01);OVX+I/R+Zuogui Pills组上述指标表达高于OVX+I/R组,OVX+I/R+Zuogui Pills+ICI182780组上述指标表达低于OVX+I/R+Zuogui Pills组(P<0.05或P<0.01)。OVX+I/R+Zuogui Pills组各指标表达与OVX+I/R+17β-estradiol组比较,差异无统计学意义(P>0.05)。结论左归丸可能通过ER介导的BDNF-TrkB和NGF-TrkA信号转导改善OVX大鼠的大脑I/R损伤。

Objective To explore whether Zuogui Pills can play a neuroprotective role by regulating estrogen receptor pathway.Methods A total of 108 female SD rats were eight-ten weeks old and SPF grade level.They were randomly divided into six groups:sham operation group(incision without thread insertion),I/R group(middle cerebral artery occlusion[MCAO]operation by longa thread bolt method),OVX+I/R group(ovariectomy+MCAO operation),OVX+I/R+Zuogui Pills group(ovariectomy+MCAO operation+Zuogui Pills 1.62 g/kg gavage),OVX+I/R+Zuogui Pills+ICI182780 group(ovariectomy+MCAO operation+Zuogui pills gavage+ICI1827803 mg/kg intraperitoneal injection),OVX+I/R+17β-estradiol group(ovariectomy+MCAO+17β-estradiol 100μg/kg intraperitoneal injection),18 rats in each group.The neurological deficit score was used to evaluated the sensory motor function of rats of each group,the novel object recognition experiment was used to observe the memory of rats,and the protein and mRNA expressions of estrogen receptor(ER)α,ERβ,brain-derived neurotrophic factor(BDNF),nerve growth factor(NGF),tyrosine kinase(Trk)A,TrkB,phosphorylated cAMP response element binding(p-CREB)and CREB were observed.Results The motor function and memory of the rats in the I/R group were lower than those in the sham operation group(P<0.05);the motor function and memory of the rats in the OVX+I/R group were lower than those in the I/R group(P<0.05);the motor function and memory of rats in the OVX+I/R+Zuogui Pills group were higher than those of the OVX+I/R group(P<0.05);the motor function and memory of the OVX+I/R+Zuogui Pills+ICI182780 group were lower than that of OVX+I/R+Zuogui Pills group(P<0.05);OVX+I/R+Zuogui Pills group motor function and memory compared with OVX+I/R+17β-estradiol group,and the differences were not statistically significant(P>0.05).The protein and mRNA(except BDNF)expressions of ERα,ERβ,BDNF,NGF,TrkA,TrkB,and P-CREB in the I/R group were higher than those in the sham operation group(P<0.05 or P<0.01);the expression of above indexes in the OVX+I/R group were lower than that in the I/R group(P<0.05 or P<0.01);the expression of above indexes in the OVX+I/R+Zuogui Pills group were higher than that in the OVX+I/R group;the expression of above indexes in the OVX+I/R+Zuogui Pills+ICI182780 group were lower than that in the OVX+I/R+Zuogui Pills group(P<0.05 or P<0.01).Compared with the OVX+I/R+17β-estradiol group,the expression of each indexes in the OVX+I/R+Zuogui Pills group were not statistically differences(P>0.05).Conclusion Zuogui Pills may improve the brain I/R damage of OVX rats through ER-mediated BDNF-TrkB and NGF-TrkA signal transduction.