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Objective@#To explore the potential role of alpinumisoflavone (AIF) in the treatment of temporomandibular joint osteoarthritis (TMJOA) cell model through network pharmacology and molecular docking and to provide a research basis for AIF in the treatment of TMJOA.@*Methods@#GeneCards, OMIM, DisGeNET, and PharmGKB databases were used to screen TMJOA disease targets, and PharmMapper and HERB were used to retrieve AIF-related targets. The intersection targets of the compounds and diseases were uploaded to the STRING database to obtain the key targets for GO and KEGG enrichment analysis, while the key targets in related signaling pathways were evaluated through molecular docking. Approval was obtained from the Ethics Committee to extract condylar chondrocytes from 3-week-old SD rats. The CCK-8 assay was used to detect AIF cytotoxicity on condylar chondrocytes. Condylar chondrocytes were induced with 10 ng/mL interleukin 1β (IL-1β) for 24 h to construct a TMJOA cell model. The experiment was divided into three groups: control group, comprising condylar chondrocytes cultured in DMEM for 48 h; IL-1β group, comprising condylar chondrocytes pre-cultured in DMEM for 24 h, after which IL-1β was added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h; and the IL-1β+10 μmol/L AIF group, comprising condylar chondrocytes pre-cultured in DMEM medium containing 10 μmol/L AIF for 24 h, after which IL-1β was added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h. The effect of AIF on condylar chondrocyte apoptosis in the TMJOA cell model was detected by flow cytometry. The experiment was divided into four groups: control group, IL-1β group, IL-1β+10 μmol/L AIF group, and IL-1β+30 μmol/L AIF group. The IL-1β+30 μmol/L AIF group was pre-cultured in DMEM containing 30 μmol/L AIF for 24 h, after which IL-1β was added to the original culture medium to obtain a medium concentration of 10 ng/mL and allowed to culture for 24 h. The remaining three groups were cultured in the same manner as before. The mRNA and protein expression of apoptosis-associated B-cell leukemia/lymphoma-2 (Bcl2), cysteinyl aspartate specific protease 3 (caspase-3), matrix degradation-associated a disintegrin and metalloproteinase with thrombospondin motifs 4 (ADAMTS4), matrix metalloproteinase 3 (MMP3), and matrix metalloproteinase 13 (MMP13) were detected by qPCR and western blot, by AIF in the TMJOA cell model.@*Results@#The PharmMapper and HERB database search yielded 300 AIF compound targets. The GeneCards, OMIM, DisGeNET, and PharmGKB databases yielded 378 TMJOA disease targets. Thirty-three potential common targets were obtained by intersecting compounds with disease targets. The common targets were uploaded into the STRING database to obtain 31 key targets that were mainly associated with apoptosis and extracellular matrix degradation. This process may be associated with the MAPK, estrogen, and TNF signaling pathways. The molecular docking results showed that AIF has good binding activity with extracellular signal-regulated kinase 1/2 (ERK1/2) and estrogen receptor gene 1/2 (ESR1/2), which are key targets in the MAPK and estrogen signaling pathways. The CCK-8 assay showed that AIF had no obvious cytotoxicity to condylar chondrocytes. The cell experiments showed that AIF inhibited apoptosis in the IL-1β+10 μmol/L AIF group compared to the IL-1β group. Compared to the IL-1β group in the IL-1β+10 μmol/L AIF group and the IL-1β+30 μmol/L AIF group, AIF upregulated Bcl2 and downregulated caspase-3 mRNA and protein expression and inhibited ADAMTS4, MMP3, and MMP13 mRNA and protein expression.@*Conclusion@#AIF inhibited apoptosis in the TMJOA cell model by upregulating Bcl2 and downregulating caspase-3 mRNA and protein expression, and inhibited extracellular matrix degradation induced by IL-1β, thereby delaying TMJOA progression.
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Objective:To investigate the effects of the different static compressive stress on the proliferation and apoptosis of condylar chondrocytes in vitro.Methods: 0, 12, 24, 36 kPa static compressive stress were applied to the third passage of mandibular condylar chondrocytes for 1 hour respectively. The changes of proliferation and apoptosis of condylar chondrocytes were evaluated by flow cytometry analysis. Results: The index of proliferation and apoptosis of cells decreased with the magnitude value of static compressive stress except 24 kPa group. The most significant decrease of proliferative index and apoptosis index was found in 36 kPa group and 12 kPa group respectively. Conclusion: There might be some corelationships between magnitude of static compressive stress and the proliferation and apoptosis of condylar chondrocytes.
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Objective: To investigate the expression and distribution of G protein and protein kinase C (PKC) under the mechanical pressure in rabbit mandibular condylar chondrocytes (MCCs) and to study the role of G protein in PKC signalling pathway. Methods:MCCs from two-week-old New Zealand rabbits were cultured. After treatment under continuous pressure of 90 kPa for 60 min or 360 min by hydraulic pressure controlled cellular strain unit, the expression of G?q/11 protein was examined by Western Blot. The expression and distribution of PKC was observed by immunocytochemical staining. Results:Gaq/11 protein in MCCs treated by 90 kPa for 60 min and 360 min was increased by 163.7% and 65.8% respectively(P0.05). PKC in control cells distributed uniformly in the cytoplasm. After been pressed under 90 kPa for 60 min,PKC translocated to the membrane and, partly,into nuclei. When the pressure prolonged to 360 min, PKC distributed uniformly again in cytoplasm. By treatment of G protein inhibitor, the translocation of PKC under 90 kPa of 60 min was not observed. Conclusion:Feasible pressure may promote G protein expression and activate PKC. The activation of PKC signalling pathway is mediated by G protein.