骨关节炎软骨下骨细胞中Wnt 5a/Smad 2/3激活及Sclerostin的抑制促进骨小梁硬化

doi:10.3870/j.issn.1672-8009.2025.06.003

摘要/Abstract

摘要： 目的探讨骨关节炎(osteoarthritis,OA)软骨下骨细胞通过激活非经典Wnt信号通路抑制硬化蛋白(sclerostin)促进骨小梁硬化的机制。方法建立软骨细胞-成骨细胞共培养体系,细胞实验分组:Control组、OE-vector组、LIF OE组、shRNA-NC组、shRNA-LIF组;通过RNA免疫共沉淀(RIP)检测LIFR与Sclerostin编码基因SOST mRNA的直接相互作用;qRT-PCR检测SOST mRNA的表达水平;软骨细胞过表达/敲低LIF,蛋白质印迹检测软骨细胞中LIF和LIFR及成骨细胞中Sclerostin的表达。40只7~8周龄雄性C57BL 6J小鼠随机分为诱导4周和8周的对照组、假手术组、模型组,及诱导8周的模型+LIFR拮抗剂处理组;Micro-CT检测小鼠关节骨体积分数(BV/TV)、小梁骨厚度(Tb.Th)和小梁骨数量(Tb.N);HE染色和番红O-固绿染色观察小鼠关节组织并进行组织病理学分析;蛋白质印迹检测Wnt 5a、Smad 2/3、Sclerostin、白血病抑制因子(leukemia inhibitory factor,LIF)/LIF受体(LIF receptor,LIFR)、抗酒石酸酸性磷酸酶(tartrate-resistant acid phosphatase,TRAP)、基质金属蛋白酶-13(matrix metalloproteinases 13,MMP-13)蛋白的相对表达水平。结果 RIP实验结果显示LIFR与SOST mRNA有直接相互作用。LIF过表达后LIFR表达水平升高(P<0.05),Sclerostin表达水平降低(P<0.05);LIF敲低后以上结果相反。模型诱导8周后,模型组小鼠后肢骨关节部位相比于假手术组BV/TV和Tb.Th升高(P<0.05),Tb.N降低(P>0.05);模型+LIFR拮抗剂处理组小鼠后肢骨关节部位相比于模型组BV/TV和Tb.Th降低(P<0.05),Tb.N升高(P>0.05)。HE染色和番红O-固绿染色结果显示,模型组软骨被破坏,细胞间基质降解,细胞排列紊乱,软骨层解体,软骨下骨受累;模型+LIFR拮抗剂处理组软骨表面区和增殖区被破坏,但软骨肥大带相对完整,提示LIFR拮抗剂处理后软骨区域破坏相对缓解。4周或8周后,模型组Wnt 5a、p-Smad 2/3表达水平相比于假手术组升高(P<0.05),Sclerostin表达水平降低(P<0.05),LIF、LIFR、TRAP、MMP-13表达水平升高(P<0.05)。结论软骨下骨细胞可能通过激活Wnt 5a/Smad 2/3上调LIF抑制Sclerostin,促进骨小梁硬化。

关键词: 骨关节炎, 软骨下骨, 骨小梁硬化, 硬化蛋白, 白血病抑制因子

Abstract: Objective To investigate the effect of Wnt 5a/Smad 2/3 activation and sclerostin inhibition in subchondral bone cells on trabecular sclerosis in osteoarthritis. Methods The co-cultured system of chondrocytes and osteoblasts was established.Cells were divided into 5 groups:Control group,OE-vector group,LIF OE group,shRNA-NC group and shRNA-LIF group.RNA immunoprecipitation(RIP)was used to detect the direct interaction between LIFR and sclerostin encoding gene SOST mRNA.The relative expression levels of SOST mRNA was detected by qRT-PCR.LIF expression in chondrocytes was overexpressed or knocked-down,and the relative expression levels of LIF and LIFR was detected by Western blotting.fourty male C57BL 6J mice aged 7-8 weeks were randomly divided into 7 groups:Control group,Model group,Sham group with 4-week or 8-week induction,and Model+LIFR antagonist group with 8-week induction.Micro-CT was used to measure the bone volume fraction(BV/TV),trabecular thickness(Tb.Th)and trabecular number(Tb.N).HE staining and safranin O-fast green staining were used to analyze the histopathological changes in mice joint tissues.The relative expression levels of Wnt 5a,Smad 2/3,Sclerostin,leukemia inhibitory factor(LIF)/LIF receptor(LIFR),tartrate-resistant acid phosphatase(TRAP)and matrix metalloproteinases 13(MMP-13)were detected by Western blotting. Results RIP results showed that LIFR and SOST mRNA had direct interaction.Overexpression of LIF increased LIFR expression levels(P<0.05)and decreased Sclerostin expression levels(P<0.05);knocking down LIF produced the opposite results.After 8 weeks of induction,the BV/TV and Tb.Th were increased(P<0.05),and the Tb.N was decreased(P>0.05)in the Model group when compared with those in the Sham group.Compared with those in the Model group,the BV/TV and Tb.Th were decreased(P<0.05)in the Model+LIFR antagonist group,and the Tb.N was increased(P>0.05).HE staining and Safranin O-fast green cartilage staining results showed that in the Model group,cartilages was destroyed,the intercellular matrix degraded,cell arrangement was disrupted,the cartilage layer disintegrated,and the subchondral bone was affected.In the Model+LIFR antagonist group,the cartilage surfaces area and proliferation area were destroyed,but the hypertrophy zone remained relatively intact,suggesting that cartilage zone destruction was relatively alleviated following LIFR antagonist treatment.After 4 weeks or 8 weeks of induction,the expression levels of Wnt 5a,p-Smad 2/3 in the Model group were increased(P<0.05),the expression levels of sclerostin was decreased(P<0.05),and the expression levels of LIF,LIFR,TRAP and MMP-13 were up-regulated(P<0.05)when compared with those in the Sham group. Conclusion Wnt 5a/Smad 2/3 activation and sclerostin inhibition in subchondral bone cells promote trabecular sclerosis.

Key words: osteoarthritis, subchondral bone, trabecular sclerosis, sclerostin, leukemia inhibitory factor

中图分类号:

R684.3
R332

艾克热木江·阿尔肯, 韩小平, 崔泳, 黄涛. 骨关节炎软骨下骨细胞中Wnt 5a/Smad 2/3激活及Sclerostin的抑制促进骨小梁硬化[J]. 医学分子生物学杂志, 2025, 22(6): 548-556.

Ekermujiang Arken, HAN Xiaoping, CUI Yong, HUANG Tao. Wnt 5a/Smad 2/3 Activation and Sclerostin Inhibition in Subchondral Bone Cells Promote Trabecular Sclerosis in Osteoarthritis[J]. Journal of Medical Molecular Biology, 2025, 22(6): 548-556.

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