Mdivi-1减轻异丙酚诱导的发育期神经元凋亡

Mdivi-1 mitigates propofol-induced developmental hippocampal neuroapoptosis in vitro

目的:探讨mdivi-1对异丙酚诱导胎鼠海马神经元凋亡的影响。方法:原代培养孕16～18 d SD大鼠胎鼠海马神经元5 d,采用随机数字表法,将神经元随机分为5组(n=6):对照组(C组)、异丙酚组(P组)和不同浓度mdivi-1组(M1～M3组,浓度分别为1,2. 5,5μmol·L-1,P组加入异丙酚(终浓度为30μmol·L-1)孵育3 h,M1～3组给予异丙酚前4 h加入mdivi-1,使各组mdivi-1浓度分别为1、2. 5和5μmol·L-1,C组加入等量生理盐水。检测发育期神经元活性和caspase3活性; Western blot法测定线粒体p-drp1 (ser616)、Bax表达;采用实时定量PCR检测BDNF及Bcl-2 mRNA的表达。结果:与C组相比,P组神经元活性降低(P <0. 05),Caspase3活性增加(P <0. 05);与P组比较,M1～M3组神经元活性增高(P <0. 05)、Caspase3活性降低(P <0. 05);与M1组比较,M2-3组神经活性增高(P <0. 05)、Caspase3活性降低(P <0. 05); M2与M3比较,神经元活性及Caspase3活性差异无显著性(P> 0. 05),因此后续实验选择M2浓度处理,称为M组。与C组比较,P组线粒体p-drp1(ser616)及Bax表达升高(P <0. 05)、BDNF和Bcl-2 mRNA表达下调(P<0. 05);与P组比较,M组线粒体p-drp1 (ser616)及Bax表达降低(P <0. 05),BDNF和Bcl-2 mRNA表达下调(P <0. 05)。结论:Mdivi-1通过减少p-drp1 (ser616)和Bax向神经元移位,抑制线粒体过度分裂及降低线粒体通透性并且上调BDNF和Bcl-2,从而减轻异丙酚诱导发育期海马神经元凋亡。

OBJECTIVE To investigate the effect of mdivi-1 on propofol-induced hippocampal neuroapoptosis in vitro.METHODS The fetal rats were obtained from 16 to 18-day gestational Sprague Dawley rats under sterile conditions and decapi-tated to dissect the hippocampal parts.The hippocampal neurons were isolated and primarily cultured in vitro for 5 days,thenrandomly divided into 5 groups(n=6 each):control group(group C),propofol group(group P),and 1,2.5,5μmol·L-1 mdivi-1 groups(groups M1-M3,respectively).Neurons were incubated with culture medium containing 30μmol·L-1 propofolfor 3 h in group P.In groups M1-M3,neurons were pretreated with 1,2.5,5μmol·L-1 mdivi-1,respectively 4 h before treatedwith propofol and incubated for 4 h,while the equal volume of normal saline was added in group C.The neuronal viability and ac-tivity of caspase 3 were detected by Microplate reader.The expression of dynamin related protein 1 phosphorylation(p-drp1(Ser616)),and Bax was detected by Western blot.The mRNA expressions of BDNF and Bcl-2 were examined by real-timePCR.RESULTS Compared with group C,the neuronal activity in group P was decreased(P<0.05)and the activity ofcaspase 3 was increased(P<0.05).Compared with group P,the activity of neurons was increased in group M1-M3(P<0.05).Compared with M1 group,the activity of neurons was further upregulated in M2-M3 group(P<0.05)and the activityof Caspase3 was downregulated(P<0.05).There was no significant difference of neuronal activity and Caspase3 activity be-tween group M2 and group M3(P>0.05).The concentration of group M2(μmol·L-1)therefore was selected as targeted con-centration of intervention(called group M).Compared with group C,the mitochondrial expressions of p-drp1(ser616)and Baxwere increased(P<0.05)and the mRNA expressions of BDNF and Bcl-2 were decreased in group P.Compared with group P,the mitochondrial expressions of p-drp1(ser616)and Bax were reduced in group M(P<0.05)while the mRNA expressions ofBDNF and Bcl-2 were enhanced in group P(P<0.05).CONCLUSION Mdivi-1 attenuates propofol-induced hippocampalneuroapoptosis by reducing the translocation of drp1 and Bax to mitochondria,preventing mitochondrial over-fission and incrementin mitochondrial permeability and up-regulating pro-survival factors of BDNF and Bcl-2.