EGCG Regulates the Effect of HDAC6 on Oxidative Stress of Human Periodontal Ligament Fibroblasts Induced by Lipopolysaccharide

Abstract

Background: Epigallocatechin gallate (EGCG) has anti-inflammatory and antioxidative stress effects in periodontitis. However, the specific mechanisms involved remain unclear. Our study explored whether the mechanism by which EGCG on alleviates inflammation and oxidative stress in human periodontal ligament fibroblasts (hPDLCs) involves HDAC6.

Methods: We treated hPDLCs with lipopolysaccharide (LPS) and EGCG, and detected the resultant effects on cell proliferation by the CCK-8 method. Cells were divided into three groups: control, LPS, and EGCG + LPS. The expression of tumor necrosis factor α (TNF-α) and interleukin-1β (IL-1β) was detected by enzyme-linked immunosorbent assay (ELISA), and the expression of reactive oxygen species (ROS) was detected using 2',7'-dichlorofluorescein diacetate. The expression of histone deacetylase 6 (HDAC6), p62, heat shock protein 70 (Hsp70), Kelch-like ECH-associating protein (Keap1), nuclear factor E2-related factor 2 (Nrf2), and heme oxygenase-1(HO-1) mRNA was detected by real-time quantitative polymerase chain reaction (RT-qPCR). The protein expression of HDAC6, Nrf2, and nod-like receptor protein 3 (NLRP3) was detected by western blotting.

Results: At concentrations of less than 100 μmol/L, EGCG can promote cell proliferation and significantly inhibit the levels of TNF-α and IL-1β. Moreover, EGCG can activate the Nrf2 pathway and inhibit ROS production. Furthermore, EGCG inhibited the expression of HDAC6 and promoted the expression of p62 and Hsp70, indicating that the anti-inflammatory and antioxidant effects of EGCG are closely related to HDAC6.

Conclusions: EGCG can regulate LPS-induced oxidative stress levels of hPDLCs through the Keap1/Nrf2/HO-1 pathway and reduce the expression of HDAC6-related factors. Therefore, HDAC6 may be a potential target for EGCG in the treatment of periodontal inflammation and oxidative stress.

Keywords: HDAC6; Keap1/Nrf2/HO‐1; epigallocatechin gallate; periodontitis.

Figure 1

At a concentration of 5 μg/mL, LPS had no inhibitory effect on the cell viability of hPDLCs; however, at 10 and 20 μg/mL, LPS significantly inhibited the cell viability of hPDLCs (A). At 5–100 μmol/L, EGCG promoted the proliferation of hPDLCs at 12 and 24 h, and there was no significant difference in cell viability between 12 and 24 h (B).

Figure 2

The expression of IL‐1β and TNF‐α increased after 24 h of hPDLCs treated by LPS (A and B). Compared with the LPS (5 μg/mL) group, the expression of IL‐1β and TNF‐α was significantly inhibited after the addition of EGCG, and the inhibitory effect was most obvious when the EGCG concentration was 100 μmol/L (C and D). When EGCG concentration was 100 μmol/L, LPS‐induced ROS expression was significantly inhibited (E). **p < 0.01, *p < 0.05, ns, not significant.

Figure 3

RT‐qPCR showed that EGCG could significantly promote the expression of HO‐1 and Nrf2 in hPDLCs (A and B) and inhibit the expression of Keap1 (C). EGCG inhibits HDAC6 expression; (D) promotes the expression of p62, which regulates the HDAC6 ubiquitination protease system (E); and promotes Hsp70 expression, which is regulated by HDAC6 (F). *Compared with the LPS group, p < 0.05; **compared with the LPS group, p < 0.01; ^##compared with the control group, p < 0.01.

Figure 4

EGCG significantly promoted Nrf2 nuclear translocation in hPDLCs (A and B) and LPS‐induced NLRP3 expression was inhibited (C). EGCG also inhibits HDAC6 protein expression (D). **p < 0.01, *p < 0.05 vs. LPS group.