维生素E对脂多糖诱导的大鼠系膜细胞增生的影响

Effects of Vitamin E on rat glomerular mesangial cells proliferation induced by lipopolysaccharide

目的 探讨维生素E(VitaminE ,VitE)对大鼠肾小球系膜细胞 (GMCs)增生的影响及可能机制。方法 应用不同剂量 ( 5 0mg/L、10 0mg/L、2 0 0mg/L)VitE在不同时间 ( 2 4h、48h、72h)对GMCs增生的影响 ;选定最佳剂量与最佳时间 ,将GMCs分为脂多糖 (LPS)组、对照组、VitE组、激素组与激素+VitE组共 5组分瓶培养 ,后三组加LPS诱导实验末 ,收集培养细胞 ,涂片 ,应用免疫组化法检测GMCs的增生细胞核抗原 (PCNA)阳性表达率 ,TUNEL法检测GMCs有效凋亡率 ;应用生化法对培养上清液进行羟自由基 ( OH)、总超氧化物歧化酶 (tSOD)、丙二醛 (MDA)的测定。结果 10 0mg/LVitE组在 48h( 3 3 3± 2 3 )对GMCs增生的抑制效果好于其他浓度与时间组 ;VitE组细胞上清中 OH( 2 0 5±3 8)、MDA( 5 7± 0 6)低于LPS组 ( 2 93± 42 ,19 3± 6 0 ) ,tSOD( 10 4± 3 1)活性高于LPS组 ( 15 6± 2 1) (P均 <0 0 1) ;VitE组培养细胞PCNA阳性率 [( 3 3 3± 8 8) % ]低于脂多糖组 [( 46 8± 5 3 ) % ],有效凋亡率高于脂多糖组 (P均 <0 0 1)。PCNA阳性率与 OH、MDA正相关 (r =0 880 ,0 70 2 ,P均 <0 0 1) ,与tSOD活性负相关 (r=- 0 682 ,P <0 0 1)。结论 VitE可以上调tSOD的活性 ,抑制系膜细胞

Objective Vitamin E (VitE) has antioxidant property which has been widely applied in treatment of kidney diseases; moreover it was shown that VitE could relieve proliferation of the renal intrinsic cells,its precise mechanism has not been clarified. The proliferation of glomerular mesangial cells (GMCs) was the most common pathologic feature, and it is important to inhibit the proliferation of GMCs during the treatment of hyperplastic renal diseases. The study aimed to observe the effect of VitE on GMCs proliferation in vitro, and to explore the possible mechanism of VitE on the proliferation of rat GMCs. Methods Different dosages of VitE (50 mg/L, 100 mg/L and 200 mg/L) were applied in GMCs culture for 24 hs, 48 hs and 72 hs, respectively in order to observe the effects of VitE on the proliferation of GMCs. The experiment was designed to involve the following five groups : control group, lipopolysaccharide (LPS) group, VitE group, dexamethasone (DXM) group and DXM+VitE group. After culturing for 24 h, 48 h and 72 h, respectively, the GMCs suspension was collected for detecting the positive rate of PCNA (immuno histo chemistry) and the rate of effective apoptosis (TUNEL method). Meanwhile, the culture supernatant was saved for testing the levels of -OH, MDA and tSOD activity (biochemical method). Results At the end of experiment, the inhibition effect on GMCs proliferation of VitE was lower than that of control group(all P<0.01),but higher than that of LPS group(all P<0.01). The inhibition rates of GMCs proliferation with VitE showed 72 h (19.6±2.0,31.8±2.4 and 41.6±3.8)>48h(18.4±1.3,33.3±2.3 and 38.2±1.6)>24 h(11.4±1.6,17.0±2.0 and 21.0±2.8). However, the inhibition rates of GMCs proliferation with VitE were not significantly different between 48 h and 72 h. For dose-response, the inhibition effects on GMCs proliferation with VitE at the dose of 100 mg(17.0±2.0,33.3±2.3 and 31.8±2.4)and 200 mg/L(21.0±2.8,38.2±1.6 and 41.6±3.8)were better than those at the dose of 50 mg/L(11.4±1.6,18.4±1.3 and 19.6±2.0, all P<0.01). While the inhibition rates of GMCs proliferation with VitE were not significantly different between 100 mg/L and 200 mg/L. The concentrations of -OH were 43±7 in control group,127±27 in DXM+VitE group, 183±31 in DXM group, 205±38 in VitE group, and 293±42 in LPS group. The concentrations of -OH were not significantly different between VitE group and DXM group,but showed extraordinarily significant difference between VitE+DXM group and DXM group (P<0.01). MDA concentrations were 2.3±0.7 in control group, 3.7±1.0 in VitE+DXM group, 5.7±0.6 in VitE group, 8.3±1.7 in DXM group, and 19.3±6.0 in LPS group. The concentrations in tSOD were 196±15 in control group, 181±25 in VitE+DXM group, 125±23 in DXM group, 104±31 in VitE group, and 19±7 in LPS group. The tSOD concentrations in VitE+DXM group, VitE group and DXM group were higher than that in LPS group(all P<0.01). There was no significant difference in tSOD concentrations between VitE group and DXM group. The positive rates of PCNA were 46.8±5.3 in LPS group, 33.3±8.8 in VitE group, 26.9±5.5 in DXM group, 17.6±4.3 in VitE+ DXM group, and 8.4±1.2 in control group. The effective apoptosis rates were 17±5 in LPS group, 49±8 in VitE group, 56±11 in DXM group, 70±12 in VitE+DXM group, and 79±5 in control group. The effective apoptosis rates in the other groups were higher than that in LPS group(all P<0.01). Compared with DXM group, the effective apoptosis rate in VitE+DXM group was higher(P<0.01). The positive rate of PCNA showed positive correlation with the concentrations of -OH and MDA(r=0.880,P<0.01). The concentration of -OH showed positive correlation to the MDA concentration (r=0.880,P<0.01), while negatively correlated to the concentrations of tSOD, -OH and MDA (r= -0.884,-0.816, all P<0.01). Conclusion VitE could up-regulate the activity of tSOD, inhibit the secretion of -OH and the accumulation of MDA, prevent GMCs from the abnormal proliferation, and promote the apoptosis of GMCs.