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目的:探讨c-Jun氨端激酶(JNK)信号通路在左归丸含药血清调控成骨前体细胞(MC3T3-E1)增殖和成骨特异转录因子核心结合因子(Runx2)mRNA表达中的作用。方法:以MC3T3-E1为研究对象,制备左归丸含药血清,选用JNK特异抑制剂SP 600125,实验分为空白对照组、SP 600125组、左归丸组、左归丸加SP 600125组、倍美力组、倍美力加SP 600125组。孵育48 h后,采用噻唑蓝(MTT)法检测SP600125对左归丸含药血清干预MC3T3-E1成骨前体细胞增殖作用的影响,采用Western blot法分析JNK蛋白磷酸化水平,采用Real Time RT-PCR法检测成骨细胞特异转录因子Runx2 mRNA表达情况。结果:与空白对照组比较,左归丸含药血清组显著促进细胞增殖,明显上调p-JNK蛋白和Runx2 mRNA表达(P<0.01);SP600125显著抑制左归丸含药血清诱导的增殖和p-JNK蛋白表达(P<0.01),对Runx2 mRNA表达的影响不显著。结论:JNK信号通路的激活可能参与了左归丸含药血清诱导的MC3T3-E1成骨前体细胞增殖,但左归丸含药血清诱导的Runx2mRNA高表达对JNK信号通路依赖不显著。
AIM: To investigate the role of JNK signaling pathway in the proliferation of osteoblast precursor cells (MC3T3-E1) and the expression of osteoblast-specific transcription factor Runx2 mRNA in Zuoguiwan drug-containing serum . Methods: MC3T3-E1 was used as the research object to prepare Zuogui Pill serum containing JNK specific inhibitor SP 600125. The experiment was divided into blank control group, SP 600125 group, Zuoguiwan group, Zuoguiwan plus SP 600125 group, Beverly group, Beverly plus SP 600125 group. After incubation for 48 h, the effect of SP600125 on the proliferation of MC3T3-E1 osteoblast cells was detected by MTT assay. The protein level of JNK was analyzed by Western blot. Real Time RT The expression of Runx2 mRNA of osteoblast-specific transcription factor was detected by PCR. Results: Compared with the blank control group, the Zuogui Pill group could significantly promote cell proliferation and up-regulate the expression of p-JNK protein and Runx2 mRNA (P <0.01); SP600125 significantly inhibited the proliferation induced by Zuogui Pill -JNK protein expression (P <0.01), and had no significant effect on Runx2 mRNA expression. CONCLUSION: The activation of JNK signaling pathway may be involved in the proliferation of MC3T3-E1 osteoblast-derived cells induced by Zuogui pill drug-containing serum. However, the high expression of Runx2 mRNA induced by Zuogui pill serum has no obvious dependence on JNK signaling pathway.