姜黄素对氯胺酮诱导胎鼠海马神经元凋亡的影响

Effects of curcumin on ketamine-induced apoptosis of hippocampal neurons of fetal rats

目的：探讨姜黄素对氯胺酮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）、氯胺酮组（K组）和不同浓度姜黄素组（J1-3组,浓度分别为5,10,20μmol·L）-1,K组加入氯胺酮（终浓度为100μmol·L-1）孵育3 h,J1-3组给予氯胺酮前2 h加入姜黄素,使各组姜黄素浓度分别为5,10,20 mol·L-1,C组加入等量生理盐水。采用MTT法检测发育期神经元活性,流式细胞仪检测线粒体膜电位（Ψm）,Western blot法测定p-CREB（ser133）、细胞色素c表达。结果：与C组相比,K组神经元活性降低,神经元线粒体膜电位（Ψm）明显降低（P〈0.05）p-CREB（ser133）蛋白及BDNF、Bcl-2 mRNA表达下调,细胞色素c表达上调（P〈0.05）;与K组相比,J3组神经元Ψm升高,J1-3组神经元活性增加,p-CREB（ser133）蛋白及BDNF、Bcl-2 mRNA表达上调,细胞色素c表达下调（P〈0.05）。结论：姜黄素通过调节p-CREB（ser133）表达,上调BDNF和Bcl-2,稳定Ψm,抑制线粒体内细胞色素c释放,进而抑制氯胺酮诱导胎鼠海马神经元凋亡。

OBJECTIVE To investigate effects of curcumin on ketamine-induced apoptosis of hippocampal neurons of fetal rats.METHODS Sixteen to eighteen day pregnant Sprague Dawley rats were anesthetized.Fetal rats were obtained under sterile conditions and decapitated.Hippocampal neurons were isolated and primarily cultured for 5 days.Primarily cultured neurons were randomly divided into 5 groups（n=6 each）：control group（group C）,ketamine group（group K）,and 5,10,20μmol·L-1 melatoningroups（groups J1-3,respectively）.Ketamine at final concentration of 100μmol·L-1 was added to culture medium and neurons were incubated for 3 h in group K.In groups J1-3,5,10 and 20 mmol·L-1 melatonin were added to culture medium,respectively,at 60 min before addition of ketamine,neurons were incubated for 3 h.Equal volume of normal saline was added instead in group C.Neuronal viability during developmental phase was assessed by MTT assay.Mitochondrial membrane potential（Ψm）was measured by flow cytometry.Expression of cAMP response element binding protein phosphorylation（pCREB（Ser133））and cytochrome C were detected by Western blot.The mRNA expression of BDNF and Bcl-2 were detected by real-time PCR.RESULTS Compared with group C,neuronal viability andΨm were significantly decreased,and expression of p-CREB,BDNF mRNA and Bcl-2 mRNA were down-regulated,expression of cytochrome C was up-regulated in group K（P〈0.05）.Compared with group K,Ψm was significantly increased in groups J3,and neuronal viability was significantly increased,expression of p-CREB,BDNF mRNA and Bcl-2 mRNA were up-regulated,expression of cytochrome C was down-regulated in groups J1-3（P〈0.05）.CONCLUSION Curcumin can protect hippocampal neurons of fetal rats from apoptosis triggered by ketamine via regulating expression of p-CREB,Bcl-2 and BDNF,stabilizing Ψm,inhibiting release of cytochrome C from mitochondria,and preventing apoptosis.