蛋白激酶G对THP-1巨噬细胞源性泡沫细胞炎症因子分泌的影响

Effect of PKG on the secretion of inflammatory cytokines in THP-1 macrophage-derived foam cells

目的:探讨蛋白激酶G(PKG)对THP-1巨噬细胞源性泡沫细胞中炎症因子[白介素-6(IL-6)、白介素-10(IL-10)、肿瘤坏死因子-α(TNF-α)]分泌的影响。方法:THP-1单核细胞用160 nmol/L的佛波酯诱导分化成巨噬细胞,再以50 mg/L氧化低密度脂蛋白(ox-LDL)处理形成泡沫细胞,油红"O"染色,镜下观察细胞形态。以PKG激动剂100μmol/L 8-Br-cGMP和抑制剂2μmol/L KT-5823分别处理巨噬细胞和泡沫细胞,同时设立巨噬细胞正常对照组和泡沫细胞模型对照组,24 h后收集细胞上清液,ELISA检测各组细胞IL-6、IL-10、TNF-α的分泌。结果 :ox-LDL处理巨噬细胞48 h后,成功构建为泡沫细胞。泡沫细胞中IL-6、TNF-α分泌显著增加(P<0.05)。8-Br-cGMP处理巨噬细胞后,IL-6分泌显著减少(P<0.05),IL-10分泌显著增加(P<0.05);KT-5823处理巨噬细胞后,IL-10分泌显著减少(P<0.05)。8-Br-cGMP处理泡沫细胞后,IL-6和TNF-α分泌显著减少(P<0.05),IL-10分泌显著增加(P<0.05);KT-5823处理泡沫细胞后,IL-6和TNF-α分泌显著减少(P<0.05)。结论:PKG可能通过上调抗炎因子IL-10的表达、下调促炎因子IL-6和TNF-α的表达,抑制炎症的发生发展;PKG可作为潜在的抗动脉粥样硬化的新思路和新靶点。

Objective To investigate the effect of PKG on the secretion of IL-6, IL-10, and TNF-α in THP-1 macrophage-derived foam cells. Methods THP-1 monocytes were induced to construct macrophages by treating with 160 nmol/L TPA. Then the macrophages were further treated with 50 mg/L ox-LDL to become foam cells. Four groups were set in this study, including the macrophage group, the foam cell group, the group of foam cell treated with PKG agonist 8-Br-cGMP, and the group of foam cell treated with PKG inhibitor KT-5823. The morphology of THP-1 cells, macrophages and foam cells were observed under microscope. The cellular lipid accumulation was detected by oil red ostaining. The secretion of IL-6, IL-10, and TNF-α into the supernatant was detected by ELISA assay. Results The foam cell was obtained after macrophage incubated with α-LDL for 48 hours. The secretion of IL-6 and TNF-α increased significantly from the foam cells than that from the macrophages （P 〈 0.05）. After the THP-1 monocyte-derived macrophages were incubated with 8-Br-cGMP, the secretion of IL-6 in the superuatant decreased significantly （P 〈 0.05 ） and IL-10 level in the supernatant increased significantly （P 〈 0.05）. After the macrophages were incubated with KT-5823, the secretion of IL-10 decreased significantly （P 〈 0.05）, but the secretion of IL-6 was not significantly changed （P 〉 0.05）. After incubation with 8-Br-cGMP, the secretion of IL-6 and TNF-α from the macrophage-derived foam cells decreased significantly （P 〈 0.05 ）, but IL-10 increased significantly （P 〈 0.05）. After the foam cells were treated with KT-5823, the secretion of IL-6 and TNF-α were also decreased significantly （P 〈 0.05）, with no significant change of IL-10 secretion （P 〉 0.05）. Conclusions PKG may enhance the expression of anti-inflammatory cytokine IL-10, and inhibit the expression of inflammatory cytokine IL-6 and TNF-α, contributing to prevent the development of inflammation. PKG might have a potential anti-atherosclerosis effect.