TRIB3沉默对缺氧-缺血新生大鼠海马神经元细胞凋亡的影响

Effect of TRIB3 silencing on the proliferation and cell apoptosis of newborn rat hippocampal neuron cells after after hypoxia-ischemia

目的:研究Tribble同源蛋白3(TRIB3)对新生大鼠海马神经元细胞缺氧缺血后细胞增殖和凋亡的影响,为TRIB3在新生儿缺氧缺血性脑损伤治疗方面提供初步的实验依据。方法:无血清法培养新生大鼠海马神经元细胞,氧糖剥夺(OGD)法体外模拟新生儿缺氧缺血性脑损伤细胞模型,Western blot和qRT-PCR检测OGD后海马神经元细胞中TRIB3蛋白和mRNA表达情况;TRIB3 siRNA或毒胡萝卜素(Tg)处理OGD海马神经元细胞并培养24 h,Western blot检测TRIB3的蛋白表达水平。采用MTT检测细胞存活率,Western blot检测凋亡相关基因(Bax、Caspase-3和Bcl-2)以及NF-κB的蛋白表达水平。结果:与正常对照组(Control)比较,OGD后海马神经元细胞TRIB3蛋白和mRNA表达水平显著升高(P<0.05);TRIB3 siRNA显著降低TRIB3蛋白表达水平,并促进OGD后海马神经元细胞增殖;TRIB3 siRNA显著增加Bcl-2/Bax比值,而显著降低Caspase-3蛋白表达水平(P<0.05);TRIB3低表达显著抑制NF-κB表达(P<0.05);另外,Tg逆转TRIB3 siRNA对NF-κB蛋白表达的抑制作用。结论:TRIB3沉默促进缺氧缺血后海马神经元细胞增殖、抑制细胞凋亡,其机制与抑制NF-κB蛋白表达水平相关。

Objective:To investigate the effect of TRIB3 on the cell proliferation and cell apoptosis of newborn rat hippocampal neuron cells after after hypoxia ischemia,and to provide preliminary experimental basis for treatment of infant hypoxic-ischemic brain damage(HIBD)by TRIB3.Methods:Newborn rat hippocampal neuron cells were cultured by serum-free media and the oxygen-glucose deprivation(OGD)model was established to mimic HIBD.The protein and mRNA expression of TRIB3 was detected by Western blot and qRT-PCR in hippocampal neuron cells after OGD.Cells were treated with TRIB3 siRNA or thapsigargin(Tg)and were cultured for 24 h in vitro,and the TRIB3 protein level was detected by Western blot.Cell proliferation was examined by MTT assay.The protein expression level of apoptosis-related genes(Bax,Caspase-3 and Bcl-2)and NF-κB was detected by Western blot.Results:Compared with that of the control group,the protein and mRNA expression level of TRIB3 was remarkably increased in hippocampal neuron cells after OGD.TRIB3 siRNA significantly decreased.TRIB3 protein expression level,and promoted cell proliferation in hippocampal neuron cells after OGD(P<0.05).Moreover,TRIB3 siRNA notably increased the rate of Bcl-2/Bax,but decreased the protein expression level of Caspase-3(P<0.05).TRIB3 down-expression inhibited the NF-κB protein expression level.In addition,Tg abrogates the suppressive effect of TRIB3 silencing on NF-κB protein expression.Conclusion:TRIB3 silencing can promote cell proliferation and inhibit cell apoptosis in hippocampal neuron cells after OGD,and its potential mechanism is related with the suppression of NF-κB protein expression.