晚期糖基化终末产物抑制大鼠外周血单个核细胞及成骨细胞增殖的作用机制

Mechanism of advanced glycation end products inhibiting the proliferation of peripheral blood mononuclear cells and osteoblasts in rats

目的:探索核因子κB(nuclear factor-kappa B,NF-κB)、磷脂酰肌醇3-激酶(phosphatidylinositol 3-kinase,PI3K)/蛋白激酶B(protein kinase B,PKB/Akt)以及丝裂原活化蛋白激酶(mitogen-activated protein kinase,MAPK)信号通路在晚期糖基化终末产物(advanced glycosylation end products,AGEs)干预大鼠外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)及下颌成骨细胞(osteoblasts,OB)后的作用机制,为进一步研究糖尿病所致牙周组织炎症的临床治疗提供一定的实验基础和理论依据。方法:采用经典的制备方法获得AGEs,体外分离培养大鼠OB、PBMCs。应用CCK-8活力测试检测AGEs不同浓度、不同时间对细胞活力的影响。采用Western blot及qRT-PCR检测NF-κB、PI3K/PKB以及MAPK信号通路相关基因的表达变化。结果:体外成功分离并培养出PBMCs和OB,PBMCs和OB的细胞活力与AGEs的作用浓度、时间以及二者交互作用均显著相关,随着AGEs刺激浓度及时间的增加,PBMCs和OB活力显著降低(P<0.001)。AGEs刺激可显著增加PBMCs中NF-κB的表达,并使肿瘤坏死因子α(tumor necrosis factorα,TNF-α)、白细胞介素-1β(interleukin-1β,IL-1β)含量增加(P<0.01);抑制NF-κB通路后,TNF-α、IL-1β含量显著降低(P<0.01)。AGEs刺激OB后,随着作用时间的延长,NF-κB p65、JNK、p38磷酸化和非磷酸化蛋白显著升高(P<0.05),而IκB磷酸化蛋白表达显著升高的同时还伴有非磷酸化蛋白表达下降(P<0.01)。在mRNA水平,NF-κB p65、JNK、p38的表达显著升高,IκB mRNA表达显著下降(P<0.05);而Akt不论是磷酸化及非磷酸化蛋白表达,还是在mRNA水平的表达,差异均无统计学意义(P>0.05);加入MAPK信号通路抑制剂后,NF-κB p65、p38、JNK磷酸化和非磷酸化蛋白表达较只加了AGEs组均显著降低,IκB磷酸化蛋白显著降低,非磷酸化蛋白显著升高(P<0.05);qRT-PCR结果发现,与只加了AGEs组相比,加入JNK通路阻断剂后IκB表达显著升高(P<0.05),NF-κB p65、p38、JNK的表达呈下降趋势,但差异不具有统计学意义(P>0.05);与只加了AGEs组相比,加入p38信号通路阻断剂后NF-κB p65、p38、JNK的表达显著降低(P<0.05),IκB表达显著升高(P<0.05);而Akt改变在蛋白水平和mRNA水平依然未见统计学意义(P>0.05)。结论:AGEs能够抑制PBMCs和OB的增殖,NF-κB和MAPK信号通路很有可能参与调控过程,但与PI3K/PKB信号通路无关。

Objective:To explore the mechanism of nuclear factor-kappa B(NF-κB),phosphatidylinositol 3-kinase(PI3K)/protein kinase B(PKB/Akt)and mitogen-activated protein kinase(MAPK)signaling pathways after intervention of advanced glycosylation end products(AGEs)in peripheral blood mononuclear cells(PBMCs)and osteoblasts(OB)in rats,so as to provide certain experimental basis and theoretical basis for further research on the clinical treatment of periodontal tissue inflammation caused by diabetes mellitus.Methods:AGEs were prepared,PBMCs and OB were isolated and cultured in vitro.CCK-8 was used to detect the cell viability intervened by different concentrations and time of AGEs.Western blot and qRT-PCR were used to detect the expression changes of genes related to NF-κB,PI3K/PKB and MAPK signaling pathways.Results:OB and PBMCs were successfully isolated and cultured in vitro.The activity of PBMCs and OB cells was significantly correlated with the concentration,time and interaction of AGEs.With the increase of AGEs concentration and time,the activity of PBMCs and OB cells significantly decreased(P<0.001).AGEs stimulation significantly increased the expression of NF-κB in PBMCs and the contents of tumor necrosis factorα(TNF-α),interleukin-1β(IL-1β)(P<0.01).TNF-α,IL-1βlevels were significantly reduced after inhibition of NF-κB pathway(P<0.01).NF-κB p65,JNK,and p38 phosphorylated and non-phosphorylated proteins increased significantly after AGEs stimulation of OB(P<0.05).The phosphorylated protein expression of IκB was significantly increased,while the expression of non-phosphorylated protein was decreased(P<0.01).The expressions of NF-κB p65,JNK,and IκB were significantly increased at the mRNA levels,and the expressions of IκB mRNA were significantly decreased(P<0.05).There was no difference in the expression of Akt in either phosphorylated or non-phosphorylated proteins or at the mRNA level(P>0.05).With the addition of MAPK signaling pathway inhibitors,the phosphorylation and non-phosphorylated protein expressions of NF-κB p65,p38 and JNK were significantly reduced,and the phosphorylated protein of IκB was significantly decreased and the non-phosphorylated protein was significantly increased compared with the group with AGEs alone(P<0.05).The results of qRT-PCR showed that the expression of IκB increased significantly after the addition of the JNK pathway blocker(P<0.05),and the expression of NF-κB p65,p38 and JNK decreased,but the difference was not significant(P>0.05).While NF-κB p65,p38 and JNK were significantly decreased and IκB was significantly increased in the AGEs group after the addition of the p38 pathway blocker(P<0.05).At this time,there was still no significant change in the expression of Akt at the protein level and mRNA level(P>0.05).Conclusion:AGEs inhibit the proliferation of PBMCs and OB,and the NF-κB and MAPK pathways are likely involved in regulating this process,but not the PI3K/PKB pathway.