Salvianolic Acid C Attenuates LPS-Induced Inflammation and Apoptosis in Human Periodontal Ligament Stem Cells via Toll-Like Receptors 4 (TLR4)/Nuclear Factor kappa B (NF-κB) Pathway

Abstract

BACKGROUND Periodontitis is a chronic inflammatory disease that causes gingival detachment and disintegration of alveolar bone. Salvianolic acid C (SAC) is a polyphenol compound with anti-inflammatory and antioxidant activities that is isolated from Danshen, a traditional Chinese medicine made from the roots of Salvia miltiorrhiza Bunge. The aim of this study was to investigate the mechanisms of underlying its protective effects and its inhibition effect on inflammation and apoptosis in human periodontal ligament stem cells (hPDLSCs). MATERIAL AND METHODS LPS-induced hPDLSCs, as a model mimicking an inflammatory process of periodontitis in vivo, were established to investigate the therapeutic effect of SAC in periodontitis. The inflammatory cytokines secretion and oxidative stress status were measured by use of specific commercial test kits. The hPDLSCs viability was analyzed by Cell Counting Kit-8 assay. The cell apoptosis and cell cycle were assayed with flow cytometry. Expressions levels of proteins involved in apoptosis, osteogenic differentiation, and TLR4/NF-kappaB pathway were evaluated by Western blotting. Alkaline phosphatase (ALP) activity was detected by ALP assay kit and ALP staining. The mineralized nodules formation of hPDLSCs was checked by Alizarin Red S staining. RESULTS Our results showed that LPS induced increased levels of inflammatory cytokines and oxidative stress and mediated the phosphorylation and nuclear translocation of NF‑kappaB p65 in hPDLSCs. SAC reversed the abnormal secretion of inflammatory cytokines and inhibited the TLR4/NF‑kappaB activation induced by LPS. SAC also upregulated cell viability, ALP activity, and the ability of osteogenic differentiation. The anti-inflammation and TLR4/NF‑kappaB inhibition effects of SAC were reversed by TLR4 overexpression. CONCLUSIONS Taken together, our results revealed that SAC effectively attenuates LPS-induced inflammation and apoptosis via the TLR4/NF-kappaB pathway and that SAC is effective in treating periodontitis.

Figure 1

Effects of SAC on inflammatory cytokines and oxidative stress in LPS-stimulated hPDLSCs. (A) The chemical structure of SAC. Inflammatory cytokines levels were detected by ELISA kits. (B) TNFα; (C) IL-6; (D) IL-1β. The oxidative stress status was analyzed by commercial test kits. (E) ROS; (F) NO; (G) iNOS. The data are expressed as means±SD; *** P<0.001 vs. Control. ^# P<0.05, ^## P<0.01, ^### P<0.001 vs. LPS.

Figure 2

SAC protected hPDLSCs against injury. (A) hPDLSCs viability was analyzed by Cell Counting Kit-8 assay. (B, C) The cell cycle was assayed with flow cytometry. (D) Expressions levels of CyclinD1 and CDK2 proteins were evaluated by Western blotting. The data are expressed as means±SD; ** P<0.01, *** P<0.001 vs. Control. ^# P<0.05, ^## P<0.01, ^### P<0.001 vs. LPS.

Figure 3

SAC had protective effect on cell apoptosis. (A, B) Cell apoptosis was assayed with flow cytometry. (C, D) Expressions levels of Bcl-2, Bax, cleaved caspase3, and caspase3 proteins were evaluated by Western blotting. The data are expressed as means±SD; *** P<0.001 vs. Control. ^# P<0.05, ^## P<0.01 vs. LPS.

Figure 4

Effects of SAC on ALP activity, mineralization, and osteogenic differentiation ability of hPDLSCs. Alkaline phosphatase (ALP) activity was checked by ALP assay kit (A) and ALP staining, 400× magnification (B). (C) The mineralized nodules formation of hPDLSCs was detected by Alizarin Red S staining, 200× magnification. (D) Expression levels of proteins involved in osteogenic differentiation, including bmp2, Oct4, Sox2, and Runx2, were detected by Western blotting. The data are expressed as means±SD; *** P<0.001 vs. Control. ^# P<0.05, ^## P<0.01 vs. LPS.

Figure 5

SAC attenuate LPS-induced inflammation via TLR4/NF-κB pathway in hPDLSCs. (A–C) Expressions levels of MyD88, p-NF-κB-p65, NF-κB-p65, and TLR4 proteins were evaluated by Western blotting. Inflammatory cytokines levels were detected by ELISA kits. (D) TNFα; (E) IL-1β; (F) IL-6. The data are expressed as means±SD; *** P<0.001 vs. Control. ^### P<0.001 vs. LPS. ^$$$ P<0.001 vs. LPS+SAC. ^@@@ P<0.001 vs. LPS+SAC. ^&&& P<0.001 vs. LPS+SAC.