Zhang Y, Wang Y Z, Fei D D, Zhang X G, Liao Z X, Liu L X, Wang Q T
Department of Periodontology, School of Stomatology, The Fourth Military Medical University & State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Engineering Research Center for Dental Materials and Advanced Manufacture, Xi'an 710032, China.
Department of Prosthodontics, School of Stomatology, The Fourth Military Medical University & State Key Laboratory of Military Stomatology & National Clinical Research Center for Oral Diseases & Shaanxi Key Laboratory of Stomatology, Xi'an 710032, China.
Zhonghua Kou Qiang Yi Xue Za Zhi. 2021 Apr 9;56(4):329-334. doi: 10.3760/cma.j.cn112144-20201105-00553.
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. 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-1β in 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-1β expression and qRT-PCR to detect expression of ERS related genes. ELISA results showed that the expression of IL-1β in 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] (12.60, 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 (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 (0.05). Moreover, compared with control group [(31.23±1.98) ng/L], the expression of IL-1β in 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] (0.05) with ERS inhibited. PDLSC from inflammatory environment could promote IL-1β secretion of macrophages through upregulating macrophages ERS.
探讨炎症环境下牙周膜干细胞（PDLSC）对巨噬细胞分泌白细胞介素-1β（IL-1β）的影响及机制。用脂多糖（LPS）预处理PDLSCs以模拟炎症环境。将人单核细胞系（THP-1）细胞分别用从健康和炎症性PDLSCs收集的条件培养基处理，分为健康PDLSC条件培养基（CM-H）组和LPS-PDLSC条件培养基（CM-LPS）组。共培养24小时后，弃去条件培养基，然后将THP-1细胞再培养24小时。采用酶联免疫吸附测定（ELISA）检测THP-1细胞上清液中IL-1β的表达。采用定量实时聚合酶链反应（qRT-PCR）检测THP-1细胞中与内质网应激（ERS）相关的葡萄糖调节蛋白78（GRP78）、活化转录因子-6（ATF6）、肌醇需求酶1（IRE1）、蛋白激酶R样内质网激酶（PERK）、CCAAT增强子结合蛋白同源蛋白（CHOP）、活化转录因子-4（ATF4）和X盒结合蛋白1剪接体（XBP1s）的表达。采用蛋白质印迹法检测GRP78和CHOP蛋白的表达。此外，用内质网抑制剂4-苯基丁酸（4-PBA）预处理进行干预实验的THP-1细胞，按不同浓度的4-PBA分为0（对照组）、1、10和20 mmol/L组，并用炎症性PDLSC的条件培养基处理。用ELISA检测IL-1β表达，用qRT-PCR检测ERS相关基因的表达。ELISA结果显示CM-LPS组THP-1细胞中IL-1β的表达[（31.35±2.11）ng/L]明显高于CM-H组[（8.19±1.51）ng/L]（t = 12.60，P = 0.01）。qRT-PCR结果显示CM-LPS组THP-1细胞中GRP78、ATF6、IRE1、PERK、CHOP、ATF4和XBP1s基因的相对表达（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和1.137±0.149）明显高于CM-H组（P < 0.05）。在4-PBA干预实验中，与0 mmol/L组相比，1、10和20 mmol/L组中GRP78、IRE-1、ATF-6、PERK和CHOP的表达明显降低（P < 0.05）。此外，与对照组[（31.23±1.98）ng/L]相比，10 mmol/L组[（21.20±0.37）ng/L]和20 mmol/L组[（23.85±1.80）ng/L]中THP-1细胞中IL-1β的表达在ERS被抑制时明显降低（P < 0.05）。炎症环境下PDLSC可通过上调巨噬细胞ERS促进巨噬细胞分泌IL-1β。
