骨髓间充质干细胞通过前列腺素E2调控NLRP3抑制肺泡巨噬细胞相关炎症反应

Bone marrow mesenchymal stem cells alleviate lipopolysaccharide-induced inflammatory response through PGE2-regulated NLRP3 inflammasome pathway

目的探讨骨髓间充质干细胞(bone marrow mesenchymal stem cell,BMSC)对脂多糖(lipopolysaccharide,LPS)诱导的炎症反应中肺泡巨噬细胞相关炎症通路的作用及可能机制。方法采用慢病毒转染ptges及ptges shRNA至BMSC,建立稳定的ptges过表达BMSC细胞株[BMSC-PGE2(+)]及ptges沉默的BMSC细胞株[BMSC-PGE2(-)]。将巨噬细胞分为对照组、LPS组、LPS+BMSC组、LPS+BMSC-PGE2(+)组、LPS+BMSC-PGE2(-)组,收集各组细胞,Western blot检测核苷酸结合寡聚化结构域样受体3(nucleotide-bound oligomerized domain-like receptor 3,NLRP3)、半胱氨酸天冬氨酸蛋白酶1前体(precursor cysteinyl aspartate specific proteinase 1,pro-Caspase-1)、Caspase-1及白细胞介素1β前体(precursor interleukin-1β,pro-IL-1β)蛋白表达水平;RT-PCR测定NLRP3、Caspase-1 mRNA表达水平;ELISA检测细胞上清液中肿瘤坏死因子α(tumor necrosis factor-α,TNF-α)、IL-1β、IL-10、IL-18及前列腺素E2(prostaglandin E2,PGE2)的表达水平。结果LPS的干预显著升高了巨噬细胞内NLRP3、pro-Caspase-1、Caspase-1和pro-IL-1β的表达,与BMSC共培养后,各蛋白表达量明显减少。加入PGE2过表达后,蛋白表达差异进一步下降。LPS组NLRP3、Caspase-1 mRNA表达量明显升高,与BMSC共培养后表达显著降低,PGE2过表达可以加大这种差异,而PGE2沉默组无明显变化。ELISA结果显示细胞上清液中TNF-α、IL-1β及IL-18含量在LPS组中最高,加入BMSC及过表达PGE2后可使得相关炎症因子下降。LPS组IL-10和PGE2水平较对照组有升高趋势,在LPS+BMSC组和LPS+BMSC-PGE2(+)组中水平进一步升高且有显著差异。结论当LPS诱导的炎症反应发生时,BMSC能够显著减轻巨噬细胞中的炎症反应,这一过程可能是通过过表达PGE2从而抑制NLRP3介导的细胞焦亡通路来实现的。

Objective To explore the role and possible mechanisms of bone marrow mesenchymal stem cell(BMSC)in the lipopolysaccharide(LPS)-induced inflammatory response involving alveolar macrophages through the inflammatory pathways.Methods ptges and ptges shRNA were transfected into BMSC by lentivirus,and stable ptges overexpression BMSC(BMSC-PGE2(+))and PTGEs silencing BMSC(BMSC-PGE2(-))were established.Macrophages were divided into control group,LPS group,LPS+BMSC group,LPS+BMSC-PGE2(+)group and LPS+BMSC-PGE2(-)group.The expression levels of nucleotide-bound oligomerized domain-like receptor 3(NLRP3),precursor cysteinyl aspartate specific proteinase 1(pro-caspase-1),caspase-1 and pro-IL-1βproteins were detected by Western blot.The mRNA expression levels of NLRP3 and caspase-1 were determined by RT-PCR.The expression levels of tumor necrosis factor-α(TNF-α),interleukin-1β(IL-1β),IL-10,IL-18 and prostaglandin E2(PGE2)in cell supernatant were detected by ELISA.Results The intervention of LPS significantly increased the expression of NLRP3,pro-caspase-1,caspase-1 and pro-IL-1βin macrophages.After co-culture with BMSC,the expression of each protein decreased significantly.After the overexpression of PGE2,the difference of protein expression further decreased.The expression of NLRP3 and caspase-1 mRNA in LPS group increased significantly,but decreased significantly after co-culture with BMSC.Overexpression of PGE2 could increase this difference,but there was no significant change in PGE2 silent group.The results of ELISA showed that the contents of TNF-α,IL-1βand IL-18 in cell supernatant were the highest in LPS group.Adding BMSC and overexpressing PGE2 could decrease the related inflammatory factors.The levels of IL-10 and PGE2 in LPS group were higher than those in control group,and further increased in LPS+BMSC group and LPS+BMSC-PGE2(+)group with significant differences.Conclusions When inflammation is induced by LPS,BMSC can significantly mitigate the inflammatory response within macrophages.This process is likely mediated through the overexpression of PGE2,which inhibits the NLRP3-mediated pyroptosis pathway.