蟾蜍灵通过调节细胞间隙连接通讯抑制PDGF-BB诱导的系膜细胞增殖

Bufalin inhibits PDGF-BB-induced mesangial cell proliferation via mediating gap junctional intercellular communication

目的探讨细胞间隙连接通讯(GJIC)在蟾蜍灵抑制肾小球系膜细胞(MC)增殖中的作用及可能的机制。方法以体外培养的系膜细胞为研究对象,血小板衍生生长因子(PDGF-BB)诱导系膜细胞增殖,蟾蜍灵干预。MTT法测定细胞增殖,Lucifer Yellow划痕标记染料示踪技术(SLDT)观察GJIC的改变,RT-PCR法测定间隙连接蛋白(Cx)43、45、40基因在转录水平的改变情况,激光共聚焦显微术结合Fura-3染色法测定细胞内游离钙离子([Ca2+]i)浓度。结果 MTT结果显示蟾蜍灵能够抑制PDGF-BB引起的系膜细胞增殖(P<0.01)。与正常组相比,PDGF-BB刺激组GJIC受到抑制,Cx45和Cx40 mRNA显著上调(P<0.01),而Cx43 mRNA无明显变化(P>0.05),同时伴有细胞[Ca2+]i浓度升高(P<0.01);蟾蜍灵干预则可改善PDGF-BB引起的GJIC的抑制,下调PDGF-BB引起的Cx45和Cx40 mRNA的表达(P<0.01),同时降低细胞[Ca2+]i浓度(P<0.01)。结论蟾蜍灵可能通过从转录水平调节系膜细胞Cx45和Cx40的表达,降低细胞[Ca2+]i浓度,改善GJIC从而抑制系膜细胞增殖。

Objective To study the role and possible mechanisms of gap junctional intercellular communication（GJIC） involved in mesangial cell（MC） proliferation which could be inhibited by bufalin.Methods Rat mesangial cells were cultured in vitro.The effect of bufalin on platelet-derived growth factor-BB（PDGF-BB）-induced MC proliferation was evaluated by MTT assay.The function of GJIC was detected by Lucifer Yellow scrape loading and dye transfer（SLDT）.mRNA levels of Cx43,Cx45 and Cx40 were measured by RT-PCR.Intracellular calcium concentrations（[Ca2＋]i） were examined in laser scanning confocal microscopy after loading by Fura-3/AM.Results MTT indicated that bufalin could inhibited PDGF-BB-induced MC proliferation（P〈0.01）.Compared with the hormal control group,PDGF-BB inhibited GJIC function,increased the expression of Cx45 and Cx40（P〈0.01） without altering the Cx43（P〈0.05） in gene level and also increased [Ca2＋]i.However,bufalin treatment enhanced GJIC function,decreased Cx45 mRNA and Cx40 mRNA expression（P〈0.01）,and reduced [Ca2＋]i（P〈0.01）.Conclusions Bufalin inhibits PDGF-BB-induced MC proliferation,and its possible mechanisms may be related to regulation of Cx45 and Cx40 expression in the gene level,reducion of [Ca2＋]i and enhancment of GJIC function.