RESUMEN
Introduction: Diabetes has been recognized as an independent risk factor for periodontitis. Increasing evidences indicate that hyperglycemia aggravates inflammatory response of human periodontal ligament cells (hPDLCs). Carbon monoxide-releasing molecule-3 (CORM-3) is a water-soluble compound that can release carbon monoxide (CO) in a controllable manner. CORM-3 has been shown the anti-inflammatory effect in different cell lineages. Methods: We stimulated periodontal ligament cells with LPS and high glucose. The expression of inflammatory cytokine was detected by ELISA. RT-qPCR, Western blot and immunofluorescence were used to detect the expression of TLR2, TLR4, RAGE and the activation of NF-κB pathway. We performed silencing and overexpression treatment of RAGE targeting the role of RAGE. We performed the immunostaining of paraffin sections of the periodontitis model in diabetes rats. Results: The results showed that CORM-3 significantly inhibited the expression of inflammatory cytokine in hPDLCs stimulated with LPS and high glucose. CORM-3 also inhibited LPS and high glucose-induced expression of RAGE/NF-κB pathway and TLR2/TLR4/NF-κB pathway. Silence of RAGE resulted in significantly decreased expression of proteins above. Overexpression of RAGE significantly enhanced the expression of these factors. CORM-3 abrogated the effect of RAGE partially. In animal model, CORM-3 suppressed the inflammatory response of periodontal tissues in experimental periodontitis of diabetic rats. Discussion: Our research proved CORM-3 reduced the inflammatory response via RAGE/NF-κB pathway and TLR2/TLR4/NF-κB pathway in the process of high glucose exacerbated periodontitis. These findings demonstrated the role of RAGE in the process of high glucose exacerbated periodontitis and suggested that CORM3 be a potential therapeutic strategy for the treatment of diabetes patients with periodontitis.
RESUMEN
PURPOSE: Limited intrinsic regeneration capacity following bone destruction remains a significant medical problem. Multiple regulatory effects of carbon monoxide releasing molecule-3 (CORM-3) have been reported. The aim of this study was to investigate the effect of CORM-3 on the osteogenic differentiation of human periodontal ligament stem cells (hPDLSCs) during osteogenesis. PATIENTS AND METHODS: hPDLSCs obtained from healthy periodontal ligament tissues were cultured and identified with specific surface antigens by flow cytometry. Effect of CORM-3 on the proliferation of hPDLSCs was determined by CCK-8 assay. Alizarin red staining and alkaline phosphatase (ALP) activity were used to assess the osteogenic differentiation of hPDLSCs. Real-time quantitative polymerase chain reaction (RT-qPCR) and Western blot analysis were used to detect the expression of the indicated genes. Critical-sized skull defect was made in Balb/c-nude mice, microcomputed tomography (Micro-CT) and Masson trichrome staining were used to assess the new bone regeneration in mice. RESULTS: CORM-3 (400 µmol/l) significantly promoted the proliferation of hPDLSCs. CORM-3 pretreatment not only notably enhanced the mRNA and protein expression of osteo-specific marker OPN, Runx2 and ALP, but also increased mineral deposition and ALP activity by the release of CO on day 3, 7 and 14 (P<0.05). Degassed CORM-3 did not show the same effect as CORM-3. In animal model, application of CORM-3 with hPDLSCs transplantation highly increased new bone formation in skull defect region. CONCLUSION: CORM-3 promoted osteogenic differentiation of hPDLSCs, and increased hPDLSCs-induced new bone formation in mice with critical-sized skull defect, which suggests an efficient and promising strategy in the treatment of disease with bone defect.