ERK1/2/CREB/BDNF信号通路在右美托咪定抑制丙泊酚致胎鼠离体海马神经元凋亡中的作用

Role of ERK1/2/CREB/BDNF signaling pathway in dexmedetomidine-induced inhibition of propofol-caused apoptosis in isolated hippocampal neurons of fetal rats

目的评价细胞外信号调节激酶(ERK1/2)/环磷腺苷反应元件结合蛋白(CREB)/脑源性神经营养因子(BDNF)信号通路在右美托咪定抑制丙泊酚致胎鼠离体海马神经元凋亡中的作用。方法将孕16 d SD大鼠处死,取胎鼠海马神经元,体外培养原代海马神经元至第7天,采用随机数字表法分为9组(n=12):对照组(C组)、脂肪乳剂组(I组)、二甲基亚砜组(DMSO组)、右美托咪定组(D组)、丙泊酚组(P组)、丙泊酚+右美托咪定组(PD组)、PD98059+丙泊酚+右美托咪定组(PDP组)、MH89+丙泊酚+右美托咪定组(HDP组)和KG501+丙泊酚+右美托咪定组(KDP组)。C组不做任何处理;I组加入20%脂肪乳剂,孵育30 min;DMSO组加入0.25%DMSO,孵育30 min;D组加入右美托咪定,终浓度10μmol/L,孵育30 min;P组加入丙泊酚,终浓度100μmol/L,孵育3 h;PD组加入右美托咪定,终浓度10μmol/L,孵育30 min,再加入丙泊酚,终浓度100μmol/L,继续孵育3 h;PDP组、HDP组和KDP组分别加入25μmol PD98059(p-ERK1/2抑制剂)、10μmol H89(p-CREB抑制剂)、25μmol KG501(CREB抑制剂)孵育30 min,再加入右美托咪定,终浓度10μmol/L,孵育30 min,最后加入丙泊酚,终浓度100μmol/L,继续孵育3 h。透射电镜下观察细胞超微结构,采用流式细胞术检测神经元凋亡情况,qRT-PCR法检测ERK1/2、CREB和BDNF mRNA的表达,Western blot法检测p-ERK1/2、CREB、p-CREB、BDNF及cleaved-caspase-3的表达。结果与C组比较,P组、PD组、PDP组、HDP组和KDP组神经元凋亡率升高,p-ERK 1/2和p-CREB表达下调,cleaved-caspase-3表达上调,P组、PDP组、HDP组和KDP组BDNF表达下调(P<0.05);与P组比较,PD组神经元凋亡率降低,p-ERK1/2、p-CREB和BDNF表达上调,cleaved-caspase-3表达下调(P<0.05);与PD组比较,PDP组、HDP组和KDP组神经元凋亡率升高,p-ERK1/2、p-CREB和BDNF表达下调,cleaved-caspase-3表达上调(P<0.05)。结论ERK1/2/CREB/BDNF信号通路参与了右美托咪定抑制丙泊酚致胎鼠离体海马神经元凋亡的过程。

Objective To evaluate the role of extracellular signal-regulated kinase 1/2(ERK1/2)/cyclic adenosine monophosphate response element-binding protein(CREB)/brain-derived neurotrophic factor(BDNF)signaling pathway in dexmedetomidine-induced inhibition of propofol-caused apoptosis in isolated hippocampal neurons of fetal rats.Methods Pregnant Sprague-Dawley rats at 16 days of gestation were sacrificed,and the fetal rats were taken out,and hippocampal neurons of fetal rats were obtained and primarily cultured in vitro for 7 days.The neurons were divided into 9 groups(n=12 each)using a random number table method:control group(group C),fat emulsion group(group I),dimethyl sulfoxide(DMSO)group,dexmedetomidine group(group D),propofol group(group P),propofol plus dexmedetomidine group(group PD),PD98059 plus propofol plus dexmedetomidine group(group PDP),MH89 plus propofol plus dexmedetomidine group(group HDP),and KG501 plus propofol plus dexmedetomidine group(group KDP).Group C received no treatment.In group I,20%fat emulsion was added,and the neurons were incubated for 30 min,and 0.25%DMSO was added in group DMSO,and the neurons were incubated for 30 min.Dexmedetomidine at a final concentration of 10μmol/L was added,and the neurons were incubated for 30 min in group D.Propofol at a final concentration of 100μmol/L was added,and the neurons were incubated for 3 h in group P.In group PD,dexmedetomidine at a final concentration of 10μmol/L was added,the neurons were incubated for 30 min,propofol at a final concentration of 100μmol/L was added,and the neurons were incubated for 3 h.In PDP,HDP and KDP groups,25μmol PD98059(p-ERK1/2 inhibitor),10μmol H89(p-CREB inhibitor)and 25μmol KG501(CREB inhibitor)were added,respectively,the neurons were incubated for 30 min,dexmedetomidine at a final concentration of 10μmol/L was added,the neurons were incubated for 30 min,and propofol at a final concentration of 100μmol/L was added,and the neurons were incubated for 3 h.The cell ultrastructure was observed with the transmission electron microscope,the apoptosis in neurons was detected by flow cytometry,the expression of ERK1/2,CREB and BDNF mRNA was detected by quantitative real-time polymerase chain reaction,and the expression of p-ERK1/2,CREB,p-CREB,BDNF and cleaved caspase-3 was detected by Western blot.Results Compared with group C,the apoptosis rate was significantly increased,the expression of p-ERK1/2 and p-CREB was down-regulated,and the expression of cleaved caspase-3 was up-regulated in P,PD,PDP,HDP and KDP groups,and the expression of BDNF was significantly down-regulated in P,PDP,HDP and KDP groups(P<0.05).Compared with group P,the apoptosis rate was significantly decreased,the expression of p-ERK1/2,p-CREB and BDNF was up-regulated,and the expression of cleaved caspase-3 was down-regulated in group PD(P<0.05).Compared with group PD,the apoptosis rate was significantly increased,the expression of p-ERK1/2,p-CREB and BDNF was down-regulated,and the expression of cleaved caspase-3 was up-regulated in PDP,HDP and KDP groups(P<0.05).Conclusion The ERK1/2/CREB/BDNF signaling pathway is involved in dexmedetomidine-induced inhibition of propofol-caused apoptosis in isolated hippocampal neurons of fetal rats.