GDF-15沉默对脑梗死大鼠模型脑内神经元凋亡的影响及机制研究

The effect and mechanism of GDF-15 silencing on neuronal apoptosis in rat models with cerebral infarction

目的探讨生长分化因子-15(Growth differentiation factor-15,GDF-15)沉默对脑梗死大鼠模型脑内神经元凋亡的影响及作用机制。方法取40只大鼠分为脑梗死组、假手术组、沉默组、空载组,每组10只;脑梗死组采用颈内动脉线栓法制备脑梗死大鼠模型,沉默组、空载组在建模后立即经尾静脉注射携带GDF-15反义寡核苷酸的GDF-15质粒、空载体质粒,假手术组大鼠暴露颈内动脉、颈外动脉后不插线阻断血流,直接缝合皮肤;评估大鼠造模3、7 d后的神经功能,流式细胞术检测脑皮质神经元凋亡情况,HE染色法观察各组大鼠脑组织病理改变,采用逆转录-聚合酶链反应(Real-time polymerase chain reaction, RT-PCR)测定脑组织中GDF-15,母亲抗十肽同系物2(Mothers against decapentaplegic homolog 2,Smad2),Smad4、p21 mRNA相对表达水平,采用Western blot法测定GDF-15,Smad2,Smad4,p21蛋白水平。结果与假手术组比较,脑梗死组、沉默组、空载组造模3、7 d后神经功能评分均升高(P<0.05);与脑梗死组、空载组比较,沉默组造模7 d后神经功能评分降低(P<0.05);与造模3 d后比较,脑梗死组、沉默组、空载组造模7 d后的神经功能评分均下降(P<0.05)。HE染色发现,与假手术组比较,脑梗死组出现神经元变性、坏死、脱失,脑组织稀疏、胶质细胞大量增生等;与脑梗死组比较,沉默组造模7 d后的神经元坏死程度较低,且细胞间质水肿与胶质细胞增生程度较轻微;空载组神经元病理形态变化与脑梗死组相似。沉默组大鼠神经元凋亡率低于脑梗死组,但高于假手术组(P<0.05)。与脑梗死组、空载组比较,沉默组大鼠脑组织内GDF-15,Smad2,Smad4 mRNA和蛋白相对表达水平更低,p21 mRNA和蛋白相对表达水平更高(P<0.05)。结论 GDF-15沉默可抑制脑梗死大鼠模型脑内神经元凋亡,保护神经功能,作用机制可能与Smad通路及p21有关。

Objective To investigate the effect and mechanism of growth differentiation factor 15(GDF-15) silencing on neuronal apoptosis in rats with cerebral infarction. Methods 40 rats were divided into cerebral infarction group, sham operation group, silence group and empty load group, with 10 rats in each group. The neural function was evaluated after 3 and 7 days of modeling. The apoptosis of cerebral cortex neurons was detected by flow cytometry. The pathological changes of brain tissue were observed by HE staining. The GDF-15, Smad2, Smad4 and p21 mRNA levels in brain tissue were determined by reverse transcription polymerase chain reaction(RT-PCR), and the GDF-15, Smad2, Smad4 and p21 protein levels were detected by Western blot.Results Compared with the sham operation group, the neurological function scores of cerebral infarction group, silence group and no-load group increased after 3 and 7 days of modeling(P<0.05), and the neurological function scores of cerebral infarction group, silence group and no-load group increased at each time point(P<0.05). Compared with after 3 days of modeling, the neurological function scores of cerebral infarction group, silence group and no-load group were decreased after 7 days of modeling(P<0.05). HE staining showed that compared with the sham operation group, neuron degeneration, necrosis, loss, sparse brain tissue and large number of glial cell proliferation appeared in the cerebral infarction group. Compared with the cerebral infarction group, the degree of neuronal necrosis in the silence group was lower, and the degree of interstitial edema and glial cell proliferation was slight. The pathological changes of neurons in the no-load group were the same as those in the cerebral infarction group. The apoptosis rate of neurons in silence group was lower than that in cerebral infarction group, and higher than that in sham operation group(P<0.05). Compared with cerebral infarction group and empty load group, GDF-15, Smad2, Smad4 mRNA and protein levels in silent group were lower, and p21 mRNA and protein levels were higher(P<0.05).Conclusion GDF-15 silencing could inhibit neuronal apoptosis and protect neural function in rat models with cerebral infarction. The mechanism mihgt be related to Smad channel and its target gene p21.