Role and mechanism of low-dose lipopolysaccharide-treated human periodontal ligament stem cells on the expression of macrophage pro-inflammatory factors
10.3760/cma.j.cn112144-20210329-00146
- VernacularTitle:低剂量脂多糖诱导下人牙周膜干细胞对巨噬细胞促炎因子表达的影响及其机制研究
- Author:
Yazheng WANG
1
;
Dongdong FEI
;
Yang ZHANG
;
Xige ZHANG
;
Yang WANG
;
Qintao WANG
Author Information
1. 第四军医大学口腔医学院牙周病科 军事口腔医学国家重点实验室 口腔疾病国家临床医学研究中心 陕西省口腔生物工程技术研究中心，西安 710032
- Keywords:
Lipopolysaccharides;
Macrophages;
Nuclear factor (erythroid-derived 2)-like 2;
Human periodontal ligament stem cells;
Oxidative stress
- From:
Chinese Journal of Stomatology
2021;56(7):672-678
- CountryChina
- Language:Chinese
-
Abstract:
Objective:To investigate the effect of low dose lipopolysaccharide (LPS)-treated human periodontal ligament stem cells (HPDLSC) on the expression of macrophage pro-inflammatory factors and the mechanism involved.Methods:The primary HPDLSCs were obtained from healthy third molar periodontal ligament tissue. Phosphate buffer saline (PBS), 100 μg/L or 10 mg/L of LPS were used to treat HPDLSCs for 48 h, and their conditioned media were respectively co-cultured with THP-1-derived macrophages for 48 h. The corresponding experimental groups were PBS-treated HPDLSC-derived conditioned medium (CM-C) group, low dose LPS-treated HPDLSC-derived conditioned medium (CM-L) group, and high dose LPS-treated HPDLSC-derived conditioned medium (CM-H) group. Quantitative real-time PCR was performed to explore the mRNA expressions of macrophage interleukin (IL)-6, IL-8, IL-12, tumor necrosis factor-α (TNF-α) in the CM-C, CM-L and CM-H groups, and the expressions of nuclear factor (erythroid-derived 2)-like 2 (NRF2), glutamate-cysteine ligase catalytic subunit (GCLC), NAD(P)H quinone dehydrogenase 1 (NQO1) and heme oxygenase 1 (HO-1) in the CM-C and CM-L groups. Meanwhile, Western blotting was used to detect the change of nuclear and cytoplasmic NRF2 and the levels of GCLC and HO-1 in the CM-C and CM-L groups. The 2′, 7′-dichlorofluorescein probe was adopted to detect the reactive oxygen species (ROS) levels of macrophages in the CM-C and CM-L groups and the data were characterized by the mean fluorescent intensity (MFI).Results:The mRNA expressions of macrophage pro-inflammatory factors IL-6, IL-8, IL-12 and TNF-α in the CM-H group (2.332±0.594, 3.601±0.639, 2.120±0.677 and 2.468±0.236) were significantly upregulated compared with those in the CM-C group (1.000±0.321, 1.000±0.151, 1.000±0.059 and 1.000±0.095) ( P<0.05); while the relative mRNA levels of IL-6, IL-12 and TNF-α in the CM-L group (0.056±0.002, 0.215±0.024 and 0.567±0.071) were much lower than those in the CM-C group (1.000±0.209, 1.000±0.220 and 1.000±0.220) ( P<0.05). At the mRNA level, the expression of NRF2 was significantly increased in the CM-L group (1.864±0.198) compared with that in the CM-C group (1.000±0.094) ( P<0.05). At the protein level, the cytoplasmic NRF2 and nuclear NRF2 were increased in CM-L group (1.175±0.104 and 1.308±0.082) compared with those in the CM-C group (1.000±0.025 and 1.000±0.049) ( P<0.05). Furthermore, the antioxidative genes, i.e. GCLC and NQO1, localized in NRF2 downstream, were significantly upregulated in the CM-L group (1.786±0.278 and 1.444±0.078) compared with the CM-C group (1.000±0.139 and 1.000±0.226) ( P<0.05). The protein levels of GCLC and HO-1 were augmented in the CM-L group (1.159±0.036 and 1.412±0.075) in contrast with those in the CM-C group (1.000±0.050 and 1.000±0.013) ( P<0.05). In addition, the MFI in the CM-L group (123 419±1 302) was significantly lower than that in the CM-C group (139 193±1 241) ( P<0.05). Conclusions:Low-dose LPS-treated HPDLSCs could regulate oxidative stress response through activating the NRF2 signaling pathway of macrophages and further downregulating the expressions of macrophage pro-inflammatory factors.
