AIM: To study the interaction between Toll-like receptor 4 (TLR4) and canonical Wnt signa-rnling pathway in osteoclasts-like cells. METHODS: Primary rat osteoclast-like cells were cultured and identified by tartrate resistant acid phosphatase(TRAP) staining and morphological observation. The cells were then stimulated with LPS (10 g/mL), Wnt3a (100 ng/mL), DKK1 (200 ng/mL), LPS plus Wnt3a and LPS plus DKK1 respectively. Cells without stimulation served as the controls. 24 hours after stimulation, the mRNA expression levels of TRAP, MMP-9, cathin K, TLR4 and LRP6, β-catenin were examined by RT-PCR. RESULTS; Wnt3a stimulation decreased MMP-9, TRAP and Cathin K mRNA expression (P<0.05). DKK1 stimulation increased MMP-9, TRAP and CathinK mRNA expression ( P < 0.05). However, neither Wnt3a nor DKK1 showed significant influence on TLR4 expression (P>0.05). LPS increased TLR4, TRAP, MMP-9 and cathin K mRNA expression (P<0. 05), decreased p-catenin and LRP6 mRNA expression (P <0.05). Co-stimulation of LPS plus Wnt3a attenuated the effectsrnof LPS (P < 0.05). Co-stimulation of LPS plus DKK1 significantly augmented the effects of LPS (P < 0.05). CONCLUSION : TLR 4 can promote osteoclastic activity of osteoclast-like cells via inhibition of Wnt signaling pathway.%目的:观察Toll样受体4(TLR4)与经典Wnt信号在破骨样细胞中的相互作用.方法:体外分离培养大鼠破骨样细胞,经抗酒石酸酸性磷酸酶染色、形态学观察鉴定后,随机分为6组:空白对照组(正常培养液)、LPS组、Wnt3a组、DKK1组、LPS+ DKK1组、LPS+ Wnt3a组,分别与破骨样细胞共同培养24h后；利用RT-PCR分别检测破骨样细胞中TLR4、破骨相关酶酒石酸酸性磷酸酶(TRAP)、基质金属蛋白酶9(MMP9)、组织蛋白酶K(cathin K)以及经典Wnt信号通路中主要信号分子β-catenin、LRP-6 mRNA表达水平.结果:体外分离培养的大鼠破骨样细胞数目多,状态良好.Wnt3a能下调细胞内破骨相关标志酶mRNA的表达水平、DKK1则上调其mRNA的表达水平(P＜0.05)；但两者对TLR4 mRNA的表达水平均无明显作用(P＞0.05).LPS可激活破骨样细胞中TLR4的表达,且可上调破骨样细胞内各破骨相关酶mRNA的表达水平；而下调Wnt信号分子β-catenin、LRP-6 mRNA的表达水平(P＜0.05).将Wnt3a、DKK1、LPS+ Wnt3a、LPS+ DKK1分别作用破骨样细胞24h后,与LPS单独刺激相比,LPS联合Wnt3a作用后对相关酶表达的下调作用明显降低(P＜0.05)；相反,LPS联合DKK1作用后对相关酶表达的上调作用进一步增强(P＜0.05).结论:TLR4可通过抑制经典Wnt信号而促进破骨细胞内破骨相关酶的活性；同样,经典Wnt信号受到TLR4的调节后也能反向抑制TLR4的激活而降低破骨细胞内酶的活性.
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