不同氮源对山羊回肠黏膜上皮细胞凋亡的影响

Effect of different nitrogen sources on apoptosis of mucosal epithelial cells in goat ileum

试验旨在研究以蛋氨酸(Met)、NH4Cl为不同氮源对山羊回肠黏膜上皮细胞凋亡的影响。试验采用细胞增殖试验、Hochest33342染色荧光显微镜检测、流式细胞术检测、线粒体膜电位的检测、检测凋亡关键蛋白的表达、观察通路关键蛋白表达等方法进行研究。结果显示,以无机氮源NH4Cl代替有机氮源Met诱导回肠上皮细胞发生细胞体积缩小、细胞核固缩及由此引起的荧光染色颗粒增亮等形态学变化,发生线粒体膜电位和凋亡关键蛋白的表达显著下调(P<0.05),细胞发生凋亡。凋亡的发生促使促凋亡蛋白半胱氨酸的天冬氨酸蛋白水解酶3(Caspase-3)、P53抑癌基因(p53)和BCL2-Associated X的蛋白质(Bax)的表达显著上调(P<0.05),而抑凋亡蛋白B淋巴细胞瘤-2(Bcl-2)和B细胞淋巴瘤因子2 xl(Bcl-xl)显著下调(P<0.05)。研究表明,Met缺失组和0.20 mmol/L NH4Cl组均通过磷脂酰肌醇3-激酶(PI3K)-蛋白激酶B(AKT)-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路和单磷酸腺苷激活的蛋白激酶(AMPK)-哺乳动物雷帕霉素靶蛋白(mTOR)信号通路诱导回肠上皮细胞凋亡,而NH4Cl对细胞凋亡具有一定程度上的缓解,对反刍动物肠道健康具有重要意义。

The study was to investigate the effect of methionine(Met)and NH4Cl on apoptosis of ileal mucosal epithelial cells in goats.Cell proliferation test,Hochest33342 staining fluorescence microscope detection,flow cytometry detection,mitochondrial membrane potential detection,detection of apoptosis key protein expression,observation of pathway key protein expression and other methods were used to study.The results showed that the replacement of Met with inorganic nitrogen source by inorganic nitrogen source NH4Cl induced morphological changes such as cell volume reduction,nuclear pyknotic,and the resulting brighten of fluorescent staining particles in the ileum epithelial cells.The mitochondrial membrane potential and the expression of key apoptosis proteins were significantly down-regulated(P<0.05),and the cells were apoptotic.The occurrence of apoptosis led to significant up-regulation of the expression of pro-apoptotic proteins Caspase-3,P53 tumor suppressor gene(p53)and BCL2-Associated X protein(Bax)(P<0.05),while the anti-apoptotic proteins Bcl-2 and Bcl-xl were significantly down-regulated(P<0.05).The study indicates that both Met-deficient group and 0.2 mmol/L NH4Cl group induced apoptosis of ileal epithelial cells through PI3K-Akt-mTOR and AMPK-mTOR signaling pathways,while the NH4Cl could alleviate apoptosis to a certain extent,which is of great significance for the health of ruminant intestine.