[Role and mechanism of low-dose lipopolysaccharide-treated human periodontal ligament stem cells on the expression of macrophage pro-inflammatory factors].

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.

[Inflammatory periodontal stem cells mediate interleukin-1ß secretion of macrophage by regulating macrophage endoplasmic reticulum stress].

Objective: To investigate the effect and mechanism of periodontal ligament stem cell (PDLSC) from inflammatory environment on the secretion of interleukin-1ß (IL-1ß) 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-1ß 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-1ß expression and qRT-PCR to detect expression of ERS related genes. Results: ELISA results showed that the expression of IL-1ß 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] (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-1ß 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] (P<0.05) with ERS inhibited. Conclusions: PDLSC from inflammatory environment could promote IL-1ß secretion of macrophages through upregulating macrophages ERS.

[Effects of cell-to-cell communication and histone acetyltransferase on the change of osteogenic differentiation ability among single-cell clones from healthy periodontium with heterogeneity of osteogenic differentiation abilities].

Objective: To investigate the effect of cell-to-cell communication amongst single-cell clones from healthy periodontium with different osteogenic differentiation potentials on change of osteogenic differentiation capabilities and the role histone acetyltransferase partaken in this process. Methods: In order to research the change of osteogenic differentiation ability via cell-to-cell communication, indirect co-culture method was used by placing two single-cell clones with different osteogenesis potentials in each of the 6-well plates. Blank control, weak and strong osteogenic groups were set up, corresponding to Transwell chambers with blank, cells of weak osteogenesis ability and cells of strong osteogenesis ability, respectively. Each group was made in triplicate. After co-culture for four days, Transwell chamber was removed. Quantitative real-time PCR (qPCR) and alizarin red staining were employed to detect the change of osteogenic differentiation ability. The acetylation level of H3 was measured by using Western blotting. Histone acetyltransferases were detected by qPCR. Results: Single-cell clones were ensured from mesenchymal stem cells by flow cytometer, the positive expression of CD29, CD90, CD105, CD146 was (99.80±0.02)%, (99.36±0.18)%, (99.41±0.05)% and (95.10±2.11)%, respectively. And CD31 and CD34 expression were (0.29±0.11)% and (0.22±0.13)%, respectively. Alizarin red and oil red O staining confirmed that single-cell clones had the abilities of adipogenesis and osteogenesis. Alkaline phosphatase (ALP) and alizarin red staining indicated that different single-cell clones were heterogeneity in osteogenesis differentiation. Indirect co-culture indicated that the mRNA expression of osteocalcin (OCN) were 14.24±5.60 and 4.78±2.90, respectively and Runt-related transcription factor 2 (RUNX2) were 2.75±1.44 and 1.61±0.44, respectively, in strong and weak osteogenic groups. They were significantly higher compared to the blank group (the mRNA expression of OCN and RUNX2 were 1.00±0.47 and 1.00±0.39, respectively). The expression of OCN and RUNX2 were also higher in strong osteogenic group than that in weak osteogenic group (P<0.05). The mean gray level of the acetylation of H3 in strong osteogenic group (0.76±0.09) and weak osteogenic group (0.54±0.12) were also higher than that in the blank group (0.30±0.04)(P<0.05). qPCR results showed that KAT6A in strong osteogenic group exhibiting higher expression (P<0.05) compared to weak osteogenic group and the blank group, which were corresponding to the changes of acetylation levels. Conclusions: Single-cell clones from healthy periodontium showed heterogeneity in osteogenic differentiation abilities. Single-cell clones with strong osteogenesis abilities had an advantage over others by promoting others' osteogenesis differentiation and this change mediated by cell-to-cell communication might be caused by modulating KAT6A to affect the acetylation level of histone.