| Objective:To investigate the effect of hyperbaric oxygen on the expression of extracellular matrix in the inflammatory condylar chondrocytes through PI3K/AKT signaling pathway, and to provide a theoretical basis for the prevention and treatment of TMJ-OA.Methods:Take 3-4 weeks SD rat mandibular condylar chondrocytes were cultured in vitro. Bilateral condylar incised under sterile conditions, the anti phosphate buffer solution (PBS) washing and stripping cartilage, repeated shear to 1mm3, first of all with the concentration of 0.25% trypsin,37℃ digestion for 30 minutes, then concentration of 0.2% collagenase type II, oscillation at 37℃ digestive 1-2 hours to digest, separation, primary chondrocytes culture, the growth, morphological changes and morphological changes of primary chondrocytes were observed by inverted microscope, and using the cell morphology, type II collagen immunohistochemistry staining, type II collagen immunofluorescence staining to identify the results of culture and cell activity. Take the second generation in the logarithmic growth phase of condylar cartilage cells were divided into four groups, respectively, with 0,0.1,1,10ng/ml IL-1 beta processing 24 hours, detected between type Ⅱ collagen and AGG protein and mRNA expression and screened can produce IL-1 beta optimal concentration of condylar cartilage cell inflammatory state; take the second generation in logarithmic growth period of condylar cartilage cells were divided into four groups: 1、normal control group, cells do not any treatment; 2、IL-1 group,10ng/ml IL-1 treating cells 24 hours; 3、HBO group, 10ng/ml interleukin-1 (IL-1) at 24 hours after HBO every 90 minutes, for a total of four days.4、Inhibitor+HBO group:10ng/ml IL-1 treatment 24 hours, an hour early to join 25um LY294002 (PI3K inhibitor) after HBO every 90 minutes, a total of four days respectively. CCK-8 assay was used to detect the cell proliferation activity; Western blotting, immune group or immunofluorescence detected between collagen Ⅱ, AGG, PI3K and p-pi3k, Akt and p-Akt protein expression; QRT-PCR detects mRNA expression of collagen Ⅱ, AGG, PI3K,and p-PI3K, AKT and p-AKT expression in each group.Results:(1) The primary mandibular condylar chondrocytes were digested and extracted, and the lens was small and round, after 24hours the majority of adherent cells were polygonal or irregular shape. Cultured for 5-6 days, the number of cells increased obviously, cells covered the bottom of about 75%, showing a cobblestone appearance. After passage, the cell adherence rate of 24h cells was significantly higher than that of the primary cells, and the distribution was relatively uniform. After 5 days, the cells were fused into pieces. The proliferation of chondrocytes in the first 3 generations was fast. After the fourth generation, the proliferation rate of condylar chondrocytes was significantly slowed down, and the appearance was long spindle shape, especially after the fifth generation. Type Ⅱ collagen immunocytochemistry staining showed that there were brownish yellow granules in cytoplasm, and immunofluorescence staining of type Ⅱ collagen cells were found in the cytoplasm of red fluorescent particles.(2) The effect of IL-1 beta 10ng/ml on the expression of type Ⅱ collagen and AGG in mandibular condylar chondrocytes was the most obvious.(3)After hyperbaric oxygen intervention, the cell proliferation activity was significantly increased, and the expression levels of collagen Ⅱ, AGG, p-PI3K and p-AKT were significantly increased; After hyperbaric oxygen intervention and PI3K inhibitors were added, the cell proliferation activity was decreased, and the expression levels of collagen Ⅱ, AGG, p-PI3K and p-AKT were decreased.Conclusion:Hyperbaric oxygen can promote the expression of extracellular matrix of inflammatory condylar chondrocytes through PI3K/AKT signaling pathway, and can improve the proliferation activity of condylar chondrocytes, and provide a new treatment method for TMJ-OA. |
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