Effect Of TLR4/PI3K Pathway On The Synovitis In Temporomandibular Joint In Rat

Part one:Effect of TLR4 on Expression of IL-1β and TNF-α in Synovial Fibroblasts from Temporomandibular Joint in rat Exposed to LipopolysaccharideObjectives:To investigate the effect of TLR4 on expression of IL-1β and TNF-α in Synovial Fibroblasts of Temporomandibular Joint in rats induced by lipopolysaccharide, and then to explore the role of TLR4 in the temporomandibular joint disease pathogenesis.Methods:To separate training primary synovial cells from the rat temporomandibular joint synovial tissue of double plate area by the enzyme digestion method, and observe cell morphological changes. To identify synovial cells by detecting the expression of vimentin and CD68 using immunofluorescence cytochemistry methods. Stimulate synovial fibroblasts using LPS with concentration of 0,0.1,1,10,100 ng/mL for 24 h, or with final concentration of 100ng/mL for 0,1,6,12,24h. Detecting the correlation between the expression of IL-1β, TNF-α and the stimulation time and LPS concentration. To detect the expression of TLR4 and MyD88 by Western blot, and detect the expression of TLR4 and MyD88 mRNA by real time PCR. To treat the cells with TLR4 inhibitor TAK-242 before the LPS stimulating, and detect the expression IL-1β and TNF-α using enzyme-linked immunoassay (ELISA), and detect the expression of IL-1β and TNF-α mRNA by Real time PCR.Results:The synovial cell is spindle and regular arrangement under microscope. And it shows negative and positive expression of CD68 and vimentin respectively. The expression of IL-1β and TNF-α mRNA and protein increased significantly under LPS stimulation. The results showed a dose-dependent increase of IL-1β and TNF-α protein and mRNA expression in SFs after stimulating by LPS (0.1,1,10,100 ng/mL) for 12 h, with the peak at 100ng/mL under the stimulation. The proteins expression of IL-1β and TNF-α showed a time-course dependent increasing, with the peak at 12 h. Consistent with proteins changes, IL-1β and TNF-α mRNA expression is also enhanced, with the peak expression at 6 h after the stimulation in a dose-dependent manner. LPS can significantly enhance TLR4, MyD88 mRNA and protein expression, the pretreatment with TAK-242 can significantly inhibit the expression of IL-1β and TNF-a in synovial fibroblasts at mRNA and protein levels.Conclusions:TLR4 play an important role in the expression IL-1β and TNF-α in synovial fibroblasts induced by LPS. The pretreatment with can significantly inhibit the expression of IL-1β and TNF-α in synovial fibroblasts at mRNA and protein levels. TLR4 may participate in the process of the temporomandibular joint disorders. Consistent with proteins changes, IL-1β and TNF-α mRNA expression is also enhanced, with the peak expression at 6 h after the stimulation in a dose-dependent manner. LPS can significantly enhance TLR4, MyD88 mRNA and protein expression, the pretreatment with TAK-242 can significantly inhibit the expression of IL-1β and TNF-a in synovial fibroblasts at mRNA and protein levels.Part two:Effect of TLR4 on Expression of IL-1β and TNF-α in Synovial Fibroblasts of Temporomandibular Joint Exposed to Lipopolysaccharide medited by PI3K/Akt pathwayObjectives:To investigate the immune molecule mechanism of expression of IL-1β and TNF-α in Synovial Fibroblasts of Temporomandibular Joint in rats mediated by TLR4 on expression of IL-1β and TNF-α in Synovial Fibroblasts of Temporomandibular Joint in rats exposed to lipopolysaccharide, and to explore the role of PI3K/Akt signaling pathway in the expression of IL-1β and TNF-β in Synovial Fibroblasts mediated by TLR4.Methods:Stimulated cells using LPS, and detect the expression of PI3K, Akt、 P-PI3K、P-Akt by Western blot. To treat the cells with specific inhibitors of PI3K inhibitor (LY294002) before the LPS stimulation, detect the expression IL-1βand TNF-α mRNA using Real time PCR, and detect the expression IL-1β and TNF-α using enzyme-linked immunoassay (ELISA).Results:LPS can significantly enhance the expression of P-PI3K, P-Akt at both mRNA and protein levels. The LY294002 can significantly inhibit the expression of IL-1β and TNF-α in synovial fibroblasts at mRNA and protein levels.Conclusions:PI3K/Akt plays an important role in the expression IL-1β and TNF-a in synovial fibroblasts induced by LPS. The LY294002 can significantly inhibit the expression of IL-1β and TNF-α in synovial fibroblasts at mRNA and protein levels. PI3K/Akt may participate in the development process of the temporomandibular joint disorders.Part three:Effect of TLR4 on inflammatory response in synovium of temporomandibular joint in ratObjectives:To investigatethe effect of TLR4 on inflammatory response in synovium of temporomandibular joint in rat.Methods:Establish temporomandibular joint synovitis animal model by four methods, in masseter resection group the rats’bilateral masseter muscles were cut off, in occlusal interference group an cast metal crown were bonded on the mandibular right first molar of each rat, in occlusal dimension increase group occlusal pad were bonded on maxillary molars of each rat, in masseter resection and occlusal dimension increase group rats’bilateral masseter muscles were resected and occlusal pads were bonded on their maxillary molars. Pathological changes were observed using hematoxylin and eosin (HE) stains, and pathology scores were evaluated. The expression of TLR4 and MyD88 was detected by immunohistochemical stains, and the expression of TLR4 and MyD88 mRNA was measured by real time PCR. The rats were injected TLR4 inhibitor in bilateral temporomandibular joint, pathological changes of synovium were observed using HE stains, and the expression of IL-1β and TNF-a in the synovium were detected by immunohistochemical stains, and the expression of IL-1β and TNF-α mRNA was detected by real time PCR.Results:The results showed that the masseter resection and occlusal dimension increase group could significantly cause inflammation in the synovium. The expression of TLR4 and MyD88 were increased in the masseter resection and occlusal dimension increase group both at protein and mRNA levels. The treatment with TAK-242 relieved inflammation response in the synovium. Also, it can reduce the expression of IL-1β and TNF-α in the synovium both at protein and mRNA levels.Conclusions:TLR4 may participate in the development process of the temporomandibular joint disorders.Part four:Effect of TLR4 on inflammatory response in synovium of temporomandibular joint in rat medited by PI3K/Akt pathwayObjectives:To investigatethe immune molecule mechanism of the inflammatory response in synovium mediated by TLR4, and to explore the role of PI3K/Akt signaling pathway in the inflammatory response in synovium mediated by TLR4.Methods:Establish temporomandibular joint synovitis rats model by resecting rats’bilateral masseter muscles and bonding occlusal pads on their maxillary molars, the expression of P-PI3K and P-Akt are detected by immunohistochemical stains. The rats were injected PI3K inhibitor in bilateral temporomandibular joint, pathological changes of synovium were observed using HE stains, and the expression of IL-1β and TNF-a in the synovium were detected immunohistochemical stains, and the expression of IL-1β and TNF-a mRNA was detected by real time PCR.Results:The expression of P-PI3K and P-Akt are increased in the masseter resection and occlusal dimension increase group both at protein levels. The treatment of LY294002 relieves inflammation response in the synovium. Also, it can reduce the expression of IL-1β and TNF-α in the synovium both at protein and mRNA levels.Conclusions:PI3K/Akt may participate in the development process of the temporomandibular joint disorders.