炎性牙周膜干细胞通过调控巨噬细胞内质网应激介导白介素-1β分泌的研究

Inflammatory periodontal stem cells mediate interleukin-1βsecretion of macrophage by regulating macrophage endoplasmic reticulum stress

目的探讨炎性牙周膜干细胞(periodontal ligament stem cell,PDLSC)对巨噬细胞分泌白介素-1β(interleukin-1β,IL-1β)的影响及其机制。方法使用脂多糖(lipopolysaccharide,LPS)刺激PDLSC模拟炎性环境。收集健康PDLSC培养基和模拟炎性环境条件PDLSC培养基,分别作用于人单核细胞系THP-1细胞,以此分为健康PDLSC条件培养基(conditioned medium of health PDLSC,CM-H)组和炎性PDLSC条件培养基(conditioned medium of LPS-PDLSC,CM-LPS)组。条件培养24 h后,弃条件培养基,正常培养THP-1细胞24 h。酶联免疫吸附测定(enzyme linked immunosorbent assay,ELISA)法检测THP-1细胞上清液中IL-1β的表达量,实时荧光定量PCR检测THP-1细胞内质网应激(endoplasmic reticulum stress,ERS)相关基因葡萄糖调节蛋白78(glucose regulated protein 78,GRP78)、活化转录因子6(activating transcription factor-6,ATF6)、需肌醇酶1(inositol requiring enzyme 1,IRE1)、蛋白激酶R样内质网激酶(protein kinase R-like endoplasmic reticulum kinase,PERK)、CCAAT增强子结合蛋白同源蛋白(CCAAT enhancer binding protein homologous protein,CHOP)、活化转录因子4(activating transcription factor-4,ATF4)、剪切型X-盒结合蛋白1(X box binding protein 1 spliced,XBP1s)的表达,蛋白质印迹法检测GRP78、CHOP蛋白表达。使用ERS抑制剂4-苯基丁酸(4-phenylbutyrate,4-PBA)预处理THP-1细胞进行干预实验,以不同浓度进行分组,包括0(对照组)、1、10和20 mmol/L组,检测ERS相关基因mRNA表达和IL-1β分泌的变化。结果ELISA结果显示,CM-LPS组THP-1细胞IL-1β表达量[(31.35±2.11)ng/L]显著高于CM-H组[(8.19±1.51)ng/L](t=12.60,P<0.01)。实时荧光定量PCR结果显示,与CM-H组相比,CM-LPS组GRP78、ATF6、IRE1、PERK、CHOP、ATF4、XBP1s表达(分别为1.782±0.070、1.387±0.204、1.404±0.119、1.777±0.187、1.325±0.156、1.295±0.066、1.137±0.149)均显著升高(P<0.05)。4-PBA干预实验中,1、10、20 mmol/L组中GRP78、IRE1、ATF6、PERK、CHOP的表达均显著低于对照组(P<0.05)。与对照组[(31.23±1.98)ng/L]相比,10和20 mmol/L组THP-1细胞IL-1β分泌水平[分别为(21.20±0.37)、(23.85±1.80)ng/L]均显著下降(P<0.05)。结论炎性PDLSC可以通过上调巨噬细胞的ERS促进巨噬细胞分泌IL-1β。

Objective To investigate the effect and mechanism of periodontal ligament stem cell(PDLSC)from inflammatory environment on the secretion of interleukin-1β(IL-1β)by macrophages.Methods PDLSCs were pretreated with lipopolysaccharide(LPS)in order to simulate the inflammatory environment.Human monocyte cell line(THP-1)cells were treated with conditioned media collected from healthy and inflammatory PDLSCs respectively and divided into conditioned medium of health PDLSC(CM-H)group and conditioned medium of LPS-PDLSC(CM-LPS)group.After 24 h of co-culture,the condition media were abandoned and THP-1 cells were then cultured for another 24 h.The expression of IL-1βin THP-1 cells supernatant was detected by enzyme-linked immunosorbent assay(ELISA).Quantitative real time-PCR(qRT-PCR)was used to detect the expression of glucose regulated protein 78(GRP78),activating transcription factor-6(ATF6),inositol requiring enzyme 1(IRE1),protein kinase R-like endoplasmic reticulum kinase(PERK),CCAAT enhancer binding protein homologous protein(CHOP),activating transcription factor-4(ATF4)and X box binding protein 1 spliced(XBP1s),which were all related with endoplasmic reticulum stress(ERS),in THP-1 cells.The expressions of proteins GRP78 and CHOP were detected by Western blotting.Furthermore,THP-1 cells,which pretreated with ER inhibitor 4-phenylbutyrate(4-PBA)for intervention experiments were grouped by various concentrations of 4-PBA including groups 0(control group),1,10 and 20 mmol/L and treated with condition medium of inflammatory PDLSC.ELISA was used to detect IL-1βexpression and qRT-PCR to detect expression of ERS related genes.Results ELISA results showed that the expression of IL-1βin THP-1 cells of group CM-LPS[(31.35±2.11)ng/L]was significantly higher than group CM-H[(8.19±1.51)ng/L](t=12.60,P<0.01).qRT-PCR results showed that the relative expressions of GRP78,ATF6,IRE1,PERK,CHOP,ATF4 and XBP1s genes in THP-1 cells of group CM-LPS(1.782±0.070,1.387±0.204,1.404±0.119,1.777±0.187,1.325±0.156,1.295±0.066 and 1.137±0.149,respectively)were significantly higher than those in group CM-H(P<0.05).In the 4-PBA intervention experiment,compared with group 0 mmol/L,the expressions of GRP78,IRE-1,ATF-6,PERK and CHOP were significantly lower in group 1,10 and 20 mmol/L(P<0.05).Moreover,compared with control group[(31.23±1.98)ng/L],the expression of IL-1βin THP-1 cells were significantly lower in group 10 mmol/L[(21.20±0.37)ng/L]and group 20 mmol/L[(23.85±1.80)ng/L](P<0.05)with ERS inhibited.Conclusions PDLSC from inflammatory environment could promote IL-1βsecretion of macrophages through upregulating macrophages ERS.