Oridonin alleviates the inhibitory effect of lipopolysaccharide on the proliferation and osteogenic potential of periodontal ligament stem cells by inhibiting endoplasmic reticulum stress and NF-κB/NLRP3 inflammasome signaling

Abstract

Background: The aim of this study was to investigate the protective effect and mechanism of oridonin in an in vitro lipopolysaccharide (LPS)-induced human periodontal ligament stem cells (hPDLSCs) model of periodontitis.

Methods: Primary hPDLSCs were isolated and cultured, and then the expression of surface antigens CD146, STRO-1 and CD45 of hPDLSCs was detected by flow cytometry. The mRNA expression level of Runx2, OPN, Col-1, GRP78, CHOP, ATF4 and ATF6 in the cells was tested by qRT-PCR. MTT was taken to determine the cytotoxicity of oridonin at different concentrations (0-4 μM) on hPDLSCs. Besides, ALP staining, alizarin red staining and Oil Red O staining were utilized to assess the osteogenic differentiation (ALP concentration, mineralized calcium nodule formation) and adipogenic differentiation abilities of the cells. The proinflammatory factors level in the cells was measured by ELISA. The protein expression level of NF-κB/NLRP3 pathway-related proteins and endoplasmic reticulum (ER) stress-related markers in the cells were detected by Western blot.

Results: hPDLSCs with positive CD146 and STRO-1 expression and negative CD45 expression were successfully isolated in this study. 0.1-2 μM of oridonin had no significant cytotoxicity on the growth of hPDLSCs, while 2 μM of oridonin could not only greatly reduce the inhibitory effect of LPS on the proliferation and osteogenic differentiation of hPDLSCs cells, but also inhibit LPS-induced inflammation and ER stress in hPDLSCs cells. Moreover, further mechanism research showed that 2 μM of oridonin suppressed NF-κB/NLRP3 signaling pathway activity in LPS-induced hPDLSCs cells.

Conclusions: Oridonin promotes proliferation and osteogenic differentiation of LPS-induced hPDLSCs in an inflammatory environment, possibly by inhibiting ER stress and NF-κB/NLRP3 pathway. Oridonin may have a potential role in the repair and regeneration of hPDLSCs.

Keywords: Endoplasmic reticulum (ER) stress; Human periodontal ligament stem cells (hPDLSCs); NF-κB/NLRP3 inflammasome; Oridonin; Osteogenic potential.

Fig. 1

Isolation and identification of human periodontal ligament stem cells (hPDLSCs). A Expression of surface antigens CD146, CD90, STRO-1, and CD45 on hPDLSCs by flow cytometry; B Alizarin red staining for assessment of mineralized nodules formation in osteogenic differentiation of hPDLSCs; C Oil Red O staining for assessment of lipid droplet formation in adipogenic differentiation of hPDLSCs

Fig. 2

Low concentrations of oridonin were not cytotoxic to hPDLSCs. MTT was taken to detect the proliferation rate of hPDLSCs cells treated with oridonin at different concentrations (0, 0.1, 0.5, 1, 2, 3 and 4 μM), **P < 0.01 versus 0 concentrations

Fig. 3

Oridonin alleviated the inhibitory effect of lipopolysaccharide (LPS) on the proliferation and osteogenic differentiation of hPDLSCs. A MTT to detect the proliferation rate of hPDLSCs cells in each group; B-D, ALP staining (B/D) and alizarin red staining (C/E) to assess ALP concentration and calcium nodule formation in hPDLSCs cells, respectively; F–H, qRT-PCR to detect the expression level of osteogenesis-related genes Runx2 (F), OPN (G) and Col-1 (H) in hPDLSCs cells in each group, **P < 0.01 versus CTRL group, #P < 0.05 and ##P < 0.01 versus LPS group

Fig. 4

Oridonin attenuates inflammation and ER stress in LPS-induced hPDLSCs. A–C, the level of proinflammatory cytokines TNF-α (A), IL-6 (B) and IL-1β (C) in hPDLSCs cells of each group was measured by ELISA; D–G, the expression level of GRP78 (D), CHOP (E), ATF4 (F) and ATF6 (G) in hPDLSCs cells of each group was detected by qRT-PCR; H/I, the protein expression level of GRP78, CHOP, ATF4 and ATF6 in cells was tested by Western blot and the relative protein expression level was analyzed, **P < 0.01 versus CTRL group, ##P < 0.01 versus LPS group

Fig. 5

Oridonin inhibits NF-κB/NLRP3 pathway in LPS-induced hPDLSCs cells. A/B, The protein expression level of p65, p-p65, NLRP3 and Caspase-1 in hPDLSCs of each group were detected by Western blot (A), the p-p65/p65 ratio and the relative protein expression level of NLRP3 and Caspase-1 were analyzed (B), **P < 0.01 versus CTRL group, ##P < 0.01 versus LPS group

Fig. 6

The schematic diagram shows the mechanism of LPS-induced cell injury in hPDLSCs regulated by oridonin. hPDLSCs, human periodontal ligament stem cells; LPS, lipopolysaccharide