谷胱甘肽对糖基化终产物诱导平滑肌细胞增殖的影响

Study on the effects of glutathione on the proliferation of rat vascular smooth muscle cells induced by advanced glycation end products

目的探讨糖基化终产物(AGEs)对大鼠胸主动脉平滑肌细胞的增殖作用和还原型谷胱甘肽(GSH)对AGEs介导的动脉平滑肌细胞增殖作用的抑制机制。方法以体外培养的大鼠胸主动脉平滑肌细胞为实验模型,分为8组,每组12孔,每孔细胞密度1×105/ml,与不同浓度的AGEs共同培养,并用不同浓度的GSH干预。分别用噻唑蓝(MTT)比色法测定血管平滑肌细胞(VSMC)增殖和用ELISA测定细胞的磷酸化p38丝裂原蛋白激酶(p-p38 MAPK)的浓度来探讨平滑肌增殖的机制和干预因素。结果 (1)AGEs对VSMC MTT吸光度的影响:经过AGEs干预,B、C、D组(吸光度值分别为0.43±0.15,0.49±0.16,0.48±0.18)与对照组比较,VSMC MTT的吸光度值增高(P<0.01)。但随着AGEs浓度增加,B、C、D组间MTT吸光度值差异无统计学意义(P>0.05)。(2)GSH对AGEs干预的VSMC MTT吸光度的影响:在加入GSH的AGEs干预组,F、G、H组(吸光度值分别为0.35±0.10,0.33±0.11,0.28±0.08)与AGEs(25 mg/L)对照组比较,MTT吸光度值下降(P<0.01)。但随着GSH浓度增加,F、G、H组组间差异无统计学意义(P>0.05)。(3)AGEs对VSMC内p-p38 MAPK的影响:经过AGEs干预后,p-p38 MAPK吸光度值增加(P<0.01),B组(0.65±0.17)、C组(0.85±0.26)、D组(0.94±0.17)分别为对照组(0.26±0.06)的2.5、3.2和3.6倍(P<0.01)。随AGEs浓度增加,C、D组与B组相比,p-p38 MAPK显著增加(P<0.01)。(4)GSH对AGEs干预的VSMC内p-p38 MAPK的影响:在加入GSH的AGEs干预组,F、G、H组(吸光度值分别为0.36±0.09,0.28±0.07,0.26±0.07)与B组即AGEs对照组比较,p-p38 MAPK吸光度降低(P<0.01),与对照组相比分别下降45%、56%和60%(P<0.01)。随着GSH浓度增加,G、H组与F组相比,p-p38 MAPK逐渐降低(P<0.01)。结论 (1)AGEs具有促进VSMC增殖的作用,其作用机理可能与激活了p-p38 MAPK信号传导通路有关。(2)GSH对AGEs介导的VSMC增殖具有抑制作用,其作用机制可能与抑制了p-p38 MAPK信号传导通路有关。

Objective To confirm the proliferation of vascular smooth muscle cell(VSMC) lead by advanced glycation end products(AGEs) and investigate weather the mechanism is work through MAPK pathway.To investigate weather the proliferation of VSMC lead by AGEs can be inhibited by reduced glutathione(GSH) and the mechanism.Methods VSMC of rats were isolated and cultivated,separated in 8 groups,each group contained 12 samples.Density of cell was 1×105 /ml in each sample,cultivated with AGEs at different concentrations and intervened with GSH at different concentrations.In order to determine the mechanism and interventional factors of VSMC,sandwich ELISA method was used to test the concentration of p-p38 MAPK and MTT colorimetry was adopted to evaluate the amount of VSMC.Results(1) Effect of AGEs on the OD value of MTT in VSMC: with stimulation of AGEs,OD value of p-p38 MAPK in VSMC increased simultaneously(0.43±0.15,0.49±0.16,0.48±0.18,respectively,P<0.01).With the increase of the dose of AGEs,there was no significant difference between groups B and C in the MTT OD value(P<0.05).(2)Effect of GSH on the OD value of MTT in VSMC stimulated by AGEs:OD value of MTT(0.35±0.10,0.33±0.11,0.285±0.08,respectively) decreased with the increase of GSH concentration,decreased by 45%,56%,60% compared with the value of the AGEs control group(P<0.01).With the increasing of the dose of GSH,the MTT OD value had no significant difference between groups F,G and H(P>0.05).(3)Effect of AGEs on the OD value of p-p38 MAPK in VSMC:with stimulation of AGEs,OD valued of p-p38 MAPK(0.65±0.17,0.85±0.26,0.94±0.17) in VSMC increased obviously(P<0.01).With the increasing of the dose of AGEs,the p-p38 MAPK OD value increased simultaneously(P<0.05).(4) Effect of GSH on the OD value of p-p38 MAPK in VSMC stimulated by AGEs:OD value of p-p38 MAPK(0.36±0.09,0.28±0.07,0.26±0.07,respectively) decreased with the increasing of GSH concentration,decreased by 45%,56%,60% compared with the value of control group(P<0.01).With the increasing of the dose of GSH,the p-p38 MAPK OD value of groups G,H were decreased gradually compared with group F(P<0.01).Conclusions(1)AGEs has the function of inducing the proliferation of VSMC,the activation of the P-p38 MAPK signal pathway may be the mechanism of the proliferation of VSMC.(2)GSH can inhibit the proliferation of VSMC lead by AGEs,The p-p38 MAPK pathway is being blocked by GSH,which is the mechanism of inhibiting the proliferation of VSMC lead by AGEs.