二肽基肽酶抑制剂类似物对LPS诱导的小胶质细胞炎症反应的抑制作用

摘要

Objective To study the inhibitory effect and mechanism of dipeptide peptidase inhibitors analogues on LPS -induced inflam-matory response on microglia .Methods Microglia cells were cultured ,isolated and purified from the neonatal Sprague-Dawley rats.Divided them into blank group ,negative control ,LPS group and medicine group ( parallel determination for 3 times each group ) after pharmacological preconditioning for 48 hours.The optimal concentration of microglia proliferation induced by LPS were measured by MTT assay .Observed the role of dipeptide peptidase inhibitors analogues on LPS-induced microglia in different concentrations .The interleukin1β( IL-1β) ,tumor necro-sis factor alpha ( TNF-α) were assayed by enzyme-linked immunorrbent assay ( ELISA ) .The expression of TLR-4 was detected by Western blotting and the expression of NF-κB was detected by RT-PCR.Results LPS induced the proliferation of microglia and significantly in-creased the release of inflammatory cytokines in LPS-stimulated primary microglia .Compared with the blank group ,dipeptide peptidase inhibi-tors analogues could inhibit this effect and the IC 50 values was 1.014 ×10 -2 mol/L to MG after pretreatment for 48 hours.Dipeptide peptidase inhibitors analogues could inhibit the release of TNF-αand IL-1 significantly(P<0.01),and it decreased the expression of TLR4 and NF-κB signif-icantly(P<0.01).Conclusion This research suggests that dipeptide peptidase inhibitors analogues restrain cell proliferation and inflammatory re-sponse of LPS-stimulated microglia,and the possible mechanism may be related to the inhibition of the expression of TLR-4 and NF-κB.%目的：探讨二肽基肽酶抑制剂类似物对脂多糖（ LPS）诱导的小胶质细胞炎症反应的抑制作用及机制。方法取新生SD大鼠进行小胶质细胞的原代培养并分离纯化。本研究分为空白组、阴性对照组、LPS组、药物组（每组平行测定3次），药物提前48 h预处理。使用MTT法筛选LPS诱导小胶质细胞增殖的最佳浓度，观察不同浓度下二肽基肽酶抑制剂类似物对LPS刺激小胶质细胞的作用。使用ELISA检测细胞上清液中白细胞介素1β（ IL-1β）、肿瘤坏死因子α（ TNF-α）的含量，Western blotting 法检测Toll样受体4（TLR-4）蛋白的表达，RT-PCR法检测NF-κB mRNA的表达。结果经LPS刺激后，SD大鼠小胶质细胞生长迅速，并可检测到大量炎性因子释放。与空白组相比，加入二肽基肽酶抑制剂类似物后，LPS诱导的小胶质细胞增殖被明显抑制，处理48 h后对小胶质细胞的IC50为1．014×10－2μmol／L。二肽基肽酶抑制剂类似物能明显抑制小胶质细胞TNF-α和IL-1β的释放（P＜0．01），并降低小胶质细胞TLR-4和NF-κB的表达。结论二肽基肽酶抑制剂类似物对LPS刺激的小胶质细胞增殖及炎症反应具有抑制作用，可能与抑制小胶质细胞TLR-4、NF-κB表达有关。