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| 基于Mekk2-/-小鼠研究复方贞术调脂胶囊调控β-catenin泛素化的成骨机制 |
| Research on the osteogenesis of β-catenin ubiquitination by FTZ in Mekk2-/- mice |
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| DOI：10.3969/j.issn.1006-7108.2020.12.002 |
| 中文关键词: 中医中药 复方贞术调脂胶囊 促分裂原活化蛋白激酶激酶激酶2 β-catenin泛素化 敲除小鼠 |
| 英文关键词:traditional Chinese medicine FTZ MEKK2 β-catenin ubiquitination knockout mice |
| 基金项目:国家自然科学基金项目青年科学基金（81603641） |
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| 中文摘要: |
| 目的 观察促分裂原活化蛋白激酶2敲除（Mekk2-/-）小鼠的表型变化以及复方贞术调脂胶囊（FTZ）含药血清对MEKK2表达影响，探讨FTZ对MEKK2-β-catenin通路以及β-catenin去泛素化的作用机制。方法 建立Mekk2-/-小鼠，通过MicroCT检测其与野生型的表型差别；通过FTZ干预Mekk2-/-小鼠和野生型的成骨细胞，运用Western blot观察对β-catenin磷酸化水平的作用；通过FTZ与FGF2干预野生型小鼠原代细胞，观察对MEKK2磷酸化水平的作用；通过C3H10T1/2细胞转染后进行FTZ和Wnt3a干预，观察β-catenin活性变化。结果 Mekk2-/-小鼠与野生型小鼠相比，BV/TV、Tb.N、Tb.Th值均较后者降低（P<0.05），Tb.sp无差异变化（P>0.05）。FTZ含药血清增强了野生型小鼠细胞β-catenin的磷酸化水平（P<0.05），但是在Mekk2-/-小鼠细胞中则明显降低（P<0.05）。FTZ含药血清和FGF2干预后的原代细胞中，p-MEKK2/3表达水平均比不干预时细胞增强（P<0.05），且二者提高水平不存在明显差异（P>0.05）。FTZ含药血清和Wnt3a均促进β-catenin活化，而FTZ含药血清和Wnt3a共同干预细胞后，可见β-catenin活化程度更高，效果呈累加效应（P<0.05）。结论 FTZ通过激活MEKK2通路，独立于Wnt经典通路促进β-catenin的去泛素化进程。 |
| 英文摘要: |
| Objective To observe the phenotypic changes of mitogen-activated protein kinase 2 knockout (Mekk2-/-) mice and the effect of FTZ containing serum on the expression of MEKK2;To investigate the mechanism of FTZ on the MEKK2-β-catenin pathway and the deubiquitination of β-catenin. Methods Mekk2-/- mice were established by CRISPR/Cas9. The phenotypic differences between Mekk2-/-mouse and wildtype mouse were detected by MicroCT. The osteoblasts of Mekk2-/- mouse and wild-type mouseare treated by FTZ and the phosphorylation of β-catenin was detected by Western blot. The phosphorylation level of MEKK2 was observed in osteoblasts wildtype mousetreated by FTZ and FGF2; β-catenin activity was evaluated in transfection of C3H10T1/2 cells treated by FTZ and Wnt3a. Results The BV/TV, Tb.N, and Tb.Th values of Mekk2-/- mice were lower than those of the wildtype mouse (P<0.05), and there was no difference in Tb.sp (P>0.05). FTZ containing serum enhanced the phosphorylation of β-catenin in osteoblasts of wild type mice (P<0.05), but significantly decreased in Mekk2-/- mouse (P<0.05). In the primary osteoblasts after FTZ containing serum and FGF2 treatments, the expression of p-MEKK2/3 was higher than that of non-treatment group (P<0.05), and there was no significant difference between the two levels (P>0.05). Both FTZcontaining serum and Wnt3a promoted the activation of β-catenin activation. After FTZ-containing serum and Wnt3a treatment in the cells, the activation of β-catenin was higher and the co-treatment effect was additive (P<0.05). Conclusion FTZ promotes the deubiquitination of β-catenin by activating the MEKK2 pathway, independent of the Wnt classical pathway. |
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