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

Inhibitory effect of dipeptide peptidase inhibitors analogues on LPS-induced inflammatory response on microglia

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。

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 mieroglia in different concentrations. The interleukin113 （ 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-KB 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 IC50 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-α and IL-1 significantly（P 〈0.01 ） ,and it decreased the expression of TLR4 and NF-KB signif- icantly（P 〈0. 01 ）. Condusion 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.