| The skeleton as an important organ play the indispensable role in the human body.At the same time, bone disease also becomes one of the serious diseases for human.Theincidence of inflammation bone disease that caused by bacterial infection grew graduallyrecent year. The chronic parodontitis and periapical periodontitis were commoninflammation bone disease, whose main reseaon was bacteria infection.TheGram-negative bacteria holds a very major position, it can produce theLipopolysaccharides, which influenced the bone metabolism of bone alveolar, could createinflammation and absorption of bone alveolar, eventually caused loss of the tooth. Thebone reconstruction main dependented on mutual replacement of osteoblast and osteoclastnormally, the intercoordination of osteoblast and osteoclast in this process will be harassed when inflammation, thus caused to the abnormal of bone metabolic.The present study reported TLR4may participate in inflammatory reaction induced byLPS, and eventually induced the expression of inflammatory genes including cytokinesand chemokines, led to the occurrence of inflammation. Classic Wnt signal pathway couldnot only regulate the proliferation, differentiation and apoptosis of the osteoblast and theosteoclast, but also played an important role in inflammation bone disease such asperiodontitis and rheumatoid arthritis.According to the latest research reports, TLR4coulddown-regulate the Wnt signal pathway in neonatal mouse ileal epithelium; however theinteraction mechanism between TLR4and the classic Wnt signals in osteoclast had notbeen reported. Classic TLR4and Wnt signaling pathway in bone disease played such animportant role, we conjectured that if these two in bone metabolic process will interact toaffect bone resorption and formation? In this experiment, we used LPS as the simulationinflammatory microenvironment medium of osteoblasts and osteoclasts, then used the LPSas the activator of TLR4in the osteoclasts. The expression and function of TLR4wasdetected by immunofluorescence, real-time fluorescence quantitative PCR, Western blot,cell migration technology in osteoclasts, at the same time we also studied the role ofclasssic Wnt signaling pathways in osteoclasts, the aim was to explore further theinteraction mechanism of TLR4and classic Wnt signal pathway in osteoclasts.The study content is divided into three parts:1. LPS imitate the inflammation micro-environment of rat osteoblasts and osteoclastsin vitroObjective: LPS as a inflammation micro-environment simulation of osteoblastsand osteoclasts, detect the biological characteristics of osteoblasts and osteoclasts, wheninflammation.Method: configure with different concentrations (0,10ng/ml, and100ng/ml,and1ug/ml, and10ug/ml) in DMEM medium of LPS respectively, stimulate theosteoblasts, osteoclasts respectively at different times, using MTT, ALP, TRAP, ALP cintentfor detecting its principal biological characteristics. Results:(1) Different concentrations of LPS can inhibit the proliferation and differentiationability of osteoblast and there is concentration-dependent.10ug/ml LPS act on osteoblastsafter24h, its main ALP, Runx2mRNA expression level of osteogenic marker enzymesignificantly reduced than control groups（P<0.05）.(2) Different concentrations of LPS can promote the proliferation and differentiationability of osteoclasts and increase osteoclastic related marker enzyme TRAP, MMP-9andCathinK mRNA expression level（P<0.05）.Conclusion: As a simulation of inflammation micro-environment in osteoblastsand osteoclasts, LPS may result in the reduction of bone forming, increasing boneresorption.2. The Expression of TLR4in normal osteoclasts and its biological effect.Objective: To detecte the expression changes of TLR4, the cell migration rate andosteoclastic related marker enzyme mRNA expression level of osteoclasts wheninflammation.Method: The experiment is divided into2group:(1) control group: normal DMEMmedium (2) DMEM medium with10ug/ml LPS, respectively stimulute cell for24h, usingPCR, Western, real-time fluorescent quantitative PCR, and Cell immuno-fluorescencetechnology, cell migration, and other methods to detect changes of TLR4expression inosteoclasts, the migration rate and the osteoclastic related marker enzymes wheninflammation.Results:(1) TLR4can be expressed in the normal osteoclast, LPS can activate its expression ofmRNA and protein and the fluorescence intensity in osteoclast（P<0.05）.(2) TLR4is activated by LPS can act on osteoclast and increased the cell migrationrate and the osteoclastic related marker enzymes（P<0.05）.Conclusion: TLR4is activated by LPS can increase the activity of osteoclast. 3. The main role of classic Wnt signaling pathway in osteoclasts and the interactivemechanism between TLR4and classic Wnt signaling pathwayObjective: To study the major role of Wnt signal pathway on osteoclastic relatedmarker enzyme and the interaction with TLR4in osteoclasts.Method: The experiment is divided into6Group randomly: blank group (normalmedium), LPS group (10ug/ml), Wnt3a group (100ng/ml), DKK1group (200ng/ml), LPSand DKK1group, LPS and Wnt3a group, respectively coculture with osteoclast for24h;using RT-PCR to detect the mRNA expression levels of osteoclastic related enzymetartrate acid phosphate enzyme (TRAP), matrix metal protease9(MMP9), organizationprotease k (Cathin k), TLR4, and the main signaling molecule β-catenin and LRP-6ofclassic Wnt signaling pathway.Results:(1) The exogenous Wnt3a, DKK1acts on osteoclast, Wnt3a can reducethe osteoclastic related maker enzyme mRNA expression level of osteocalst, DKK1had the opposite effect(P<0.05); But had no obvious effect on TLR4mRNA expressionlevels(P>0.05).(2) LPS can upregulate the TLR4mRNA expression and osteoclastic related makerenzyme mRNA expression level and downregulate the mRNA expression levels of β-catenin and LRP-6in Wnt signaling(P<0.05).(3) Wnt3a, DKK1, LPS+Wnt3a, LPS+DKK1respectively act on osteoclasts for24h,compared with LPS stimulation alone, the downregulation of LPS combined Wnt3a on theosteocalstic related enzymes mRNA expression of osteoclasts reduce(P<0.05); Conversely,the upregulation of LPS combined DKK1on the osteocalstic related enzymes mRNAexpression of osteoclasts enhance further enhanced(P<0.05).Conclusion:TLR4can promote osteoclastic related maker enzyme activity by inhibiting Wntsignal; similarly, the canonical Wnt signaling can also inhibit the inflammation activity inwhich TLR4participate by LPS to decrease the osteoclastic activity. |
