补肾抗衰片对乳兔来源内皮细胞HO-1相关氧化应激水平的影响

Effects of Bushen Kangshuai Tablet on HO-1-related oxidative stress level in endothelial cells derived from new born rabbit

目的:研究补肾抗衰片含药血清调节乳兔主动脉内皮细胞(RaECs)血红素氧合酶-1(HO-1)相关信号通路的机制。方法:分离培养RaECs,分别以1、10、100μmol/L浓度的HO-1抑制剂Znpp-IX孵育RaECs 24h,再以10%补肾抗衰片含药血清干预72h。采用ELISA方法检测HO-1、单核细胞趋化蛋白-1(MCP-1)、基质金属蛋白酶-2(MMP-2)、基质金属蛋白酶-9(MMP-9)、P-选择素(P-selection)水平,荧光免疫细胞化学染色检测HO-1蛋白水平。结果:补肾抗衰片含药血清对RaECs未显示毒性作用,10%含药血清显示出最佳促细胞增殖效果;HO-1的特异性抑制剂Znpp-IX预处理RaECs 24h后可以使RaECs HO-1水平明显降低,而补肾抗衰片上调了HO-1水平(P<0.05);不同浓度含药血清作用72h后显示,与对照组比较,补肾抗衰片降低了10μmol/L组MMP-2水平(P<0.05),同时升高了MCP-1水平(P<0.05),而补肾抗衰片对10μmol/L与100μmol/L Znpp-IX处理组的P-selectin、MMP-9水平没有明显影响。结论:补肾抗衰片可诱导乳兔来源的主动脉内皮细胞MMP-2、MCP-1水平,这可能与调控其HO-1/CO信号通路有关。

Objective:To study the mechanism of the serum containing Bushen Kangshuai Tablet on regulation of Heme Oxygenase-1(HO-1) signal pathway in endothelial cells derived from neonatal rabbit aortic endothelial cells(RaECs).Methods:RaECs were isolated and incubated with HO-1 inhibitor Znpp-IX(1,10,100μmol/L) for 24 h,and then treated with 10% serum containing Bushen Kangshuai Tablet for 72 h.Elisa method was used to test the levels of HO-1,monocyte chemoattractant protein-1(MCP-1),matrix metalloproteinase-2(MMP-2),matrix metalloproteinase-9(MMP-9),and P-selection level(P-selection).Fluorescence immunocytochemical staining was used to detect HO-1 protein levels.Results:Serum containing Bushen Kangshuai Tablet did not show toxic effect on RaECs,and 10% drug-containing serum showed optimal cell proliferation.Znpp-IX could decrease the level of HO-1 significantly,and serum containing Bushen Kangshuai Tablet could increase the level of HO-1 significantly(P<0.05).Compared with control group,after treating with serum containing Bushen Kangshuai Tablet for 24 h,the levels of MMP-2 of Znpp-IX 10 μmol/L group decreased,and the level of MCP-1 increased(P<0.05).There was no significant influence of Bushen Kangshuai Tablet on P-selectin and MMP-9 level of Znpp-IX 10 μmol/L and 100 μmol/L groups.Conclusion:Bushen Kangshuai Tablet could regulate the level of MMP-2 and MCP-1 in RaECs,which was related with the regulatory effect on HO-1/CO signal pathway.