眼镜蛇毒因子耗竭补体对大鼠Thy-1肾炎凋亡病变及其Gadd45α基因表达的影响

CVF depleted complement effect on apoptosis of glomerular mesangial cells(GMCs) and expression of Gadd45α gene in rats with Thy-1 nephritis

目的:研究眼镜蛇毒因子(CVF)耗竭体内补体对Thy-1肾炎大鼠肾小球系膜细胞(GMCs)凋亡病变及其生长阻滞和DNA损伤诱生蛋白45α(Gadd45α)基因表达的影响。方法:利用抗胸腺细胞抗血清(ATS)复制大鼠Thy-1肾炎(Thy-1 N)模型。大鼠随机分为正常对照组、Thy-1 N组和CVF+Thy-1 N组。取各组大鼠血清检测补体CH50活性。另取各组大鼠肾皮质,行电镜和TUNEL染色检查判断其肾组织细胞凋亡的病理变化;用RT-PCR和real-time PCR检测大鼠肾组织凋亡相关基因,即Gadd45α mRNA丰度;用Western blotting检测Gadd45α蛋白表达水平。结果:CVF+Thy-1 N组补体CH50活性明显下降,与Thy-1 N组和正常对照组相比差异明显(P<0.01)。电镜和TUNEL染色观察到Thy-1 N组大鼠GMCs有凋亡病变,而CVF+Thy-1 N组凋亡病变则显著减轻。Thy-1 N组大鼠肾皮质Gadd45α mRNA 40min开始上调,3h达到峰值,而用CVF处理的Thy-1 N大鼠,Gadd45α mRNA丰度则明显低于Thy-1N组(P<0.01);Thy-1 N组Gadd45α蛋白水平3h时显著增多,而CVF+Thy-1 N Gadd45α蛋白水平明显低于Thy-1 N组(P<0.01)。结论:CVF耗竭补体可显著减轻大鼠Thy-1肾炎凋亡病变并抑制Gadd45α基因的表达。

Objective：To explore the effect of complement depleting with cobra venom factor （CVF） on apoptosis of glomerular mesangial cells（GMCs） and expression of growth arrest and DNA damage inducible protein 45 α（Gadd45α） in rats with Thy-1 nephritis （Thy-1 N）. Methods：The rat model of Thy-1 N was reproduced with rabbit anti-thymocyte serum（ATS）. The rats were divided into 3 groups, control group,Thy-1 N group and CVF＋Thy-1 N group. The complement hemolytic activity （CH50） was determined by red cell hemolytic test. Apoptosis in GMCs was examined by electron microscope （EM） and Tdt-mediated X-dUTP nick end labeling （TUNEL）. The mRNA of Gadd45α was measured by RT-PCR and real-time PCR at 40 min,3,6,12 and 24 h, meanwhile the expression of Gadd45α protein was determined by Western blot at 3 h. Results：CH50 activity in CVF＋Thy-1 N group at 40 min, 3,6, 12,24 h and 5 d was significantly lower than that in Thy-1 N group and control group（P 〈 0.01 ）. In Thy-1 N group, some GMCs began to undergo apoptosis at 40 min, more prominent at 3 h, and decreased gradually. But, in CVF＋Thy-1 N group, the number of apoptosis cells was markedly less than that in Thy-1 N group at above-mentioned time. The expression of Gadd45α gene from renal tissues in Thy-1 N group was up-regulated at 40 min, reached peak level at 3 h, but the expression level of Gadd45α mRNA in CVF＋Thy-1 N group was significantly less than that in Thy-1 N group（P 〈 0.01 ）. The expression of Gadd45α protein in Thy-1 N group was higher than that in control group at 3 h, but the protein expression in CVF＋Thy-1 N group was markedly less than that in Thy-1N group（P 〈 0.01 ）. Conclusion： Complement depleting with CVF could obviously decrease the apoptosis of GMCs and inhibit the expression of Gadd45α gene in rats with Thy-1 nephritis.