天麻素通过Nrf2/Keapl信号通路促进成骨细胞分化和抑制骨质疏松作用

Roles of Gastrodin on promoting osteoblast differentiation and inhibiting osteoporosis by Nrf2/Keapl signaling pathway

目的研究天麻素调控核因子E2相关因子2(Nrf2)/Kelch样ECH相关蛋白1(Keap1)信号通路促进成骨细胞分化和抑制骨质疏松的作用机制。方法将新提取的新生(出生24 h内)SD大鼠颅盖骨制成的原代成骨细胞分为4组:对照组、模型组、低、高剂量实验组,低、高剂量实验组细胞预先给予1,5μmol·L-1天麻素预处理,之后和模型组一起,加入100μmol·L-1地塞米松处理;用细胞增殖/毒性检测(CCK-8)法、Hoechst 33258染色法检测各组成骨细胞生物学行为;JC-1线粒体膜电位检测及ATP生成实验证实Nrf2/Keapl信号通路的作用机制;茜素红染色观察细胞成骨分化的功能性保护作用;蛋白质印迹法(Western blot)检测线粒体凋亡途径关键蛋白、Nrf2/Keapl信号通路关键因子的表达水平。结果对照组、模型组、低、高剂量实验组原代成骨细胞的增殖率分别为(513.35±28.67)%,(231.67±15.32)%,(337.62±21.37)%和(418.14±20.96)%;凋亡率分别为(11.53±3.71)%,(77.26±10.52)%,(52.37±6.31)%和(24.91±4.69)%;原代成骨细胞的中红绿光信号比率分别为0.23±0.03,1.93±0.52,1.01±0.26和0.77±0.22;成骨结节相对形成率分别为(100.00±0.05)%,(25.00±0.05)%,(51.00±1.24)%和(65.00±3.22)%;Nrf2的表达量分别为1.07±0.08,0.51±0.03,0.78±0.04和0.92±0.08;Keap1的表达量分别为0.19±0.05,0.96±0.09,0.69±0.05和0.57±0.05;NQO1的表达量分别为0.81±0.07,0.36±0.02,0.53±0.04和0.75±0.05;HO-1的表达量分别为1.02±0.06,0.46±0.02,0.58±0.06和0.78±0.06。以上指标,低、高剂量实验组分别与模型组比较,差异均有统计学意义(均P<0.05)。结论天麻素可能通过提高原代成骨细胞增殖活性,激活Nrf2/Keapl信号通路降低线粒体氧化应激损伤,维持线粒体膜电位稳态及ATP正常生成来抑制细胞凋亡,同时促进成骨钙结节形成来促进成骨分化从而改善骨质疏松。

Objective To study the action mechanism of Gastrodin promoting osteoblast differentiation and inhibiting osteoporosis(OP) by regulating nuclear factor erythroid 2-related factor 2(Nrf2)/Kelch-like ECH-associated protein 1(Keap1) signaling pathway. Methods The primary osteoblasts made by newly extracted craniums of neonatal SD rat(within 24 h after birth) were enrolled and divided into control group, model group, test-L group and test-H group, respectively. Cells in test-L group and test-H group were pretreated with 1 and 5 μmol·L-1 Gastrodin beforehand. Then, cells in the two groups and model group were treated with 100 μmol·L-1 dexamethasone. The biological behaviors of osteoblasts in each group were detected by Cell Couting Kit-8(CCK-8) and Hoechst 33258 staining methods. The action mechanism of Nrf2/Keapl signaling pathway was confirmed by JC-1 mitochondrial membrane potential(MMP) detection and ATP generation experiment. The functional protective roles of osteoblasts differentiation were observed by Alizarin Red staining. Expression levels of mitochondrial apoptosis pathway key proteins and Nrf2/Keapl signaling pathway key factors were detected by Western blot. Results The proliferation rates of primary osteoblasts in control group,model group,test-L group and test-H group were(513. 35 ± 28. 67) %,(231. 67 ± 15. 32) %,(337. 62 ± 21. 37) %,(418. 14 ± 20. 96) %. The apoptosis rates were(11. 53 ± 3. 71) %,(77. 26 ± 10. 52) %,(52. 37 ± 6. 31) %,(24. 91 ± 4. 69) %,respectively. The ratios of red to green light signals in primary osteoblasts of control group,model group,test-L group and test-H group were 0. 23 ± 0. 03,1. 93 ± 0. 52,1. 01 ± 0. 26 and 0. 77 ± 0. 22,respectively.The relative formation rates of osteoblasts nodules in primary osteoblasts were(100. 00 ± 0. 05) %,(25. 00 ± 0. 05) %,(51. 00 ± 1. 24) % and(65. 00 ± 3. 22) %,respectively. The relative expression of Nrf-2 were1. 07 ± 0. 08,0. 51 ± 0. 03,0. 78 ± 0. 04,0. 92 ± 0. 08;Keap1 were 0. 19 ± 0. 05,0. 96 ± 0. 09,0. 69 ± 0. 05,0. 57 ± 0. 05;NQO1 were 0. 81 ± 0. 07,0. 36 ± 0. 02,0. 53 ± 0. 04,0. 75 ± 0. 05;HO-1 were 1. 02 ± 0. 06,0. 46 ± 0. 02,0. 58 ± 0. 06,0. 78 ± 0. 06),respectively. The differences between model group and test-L and-H group in the above indices were statistically significant(P < 0. 05). Conclusion Gastrodin may inhibit apoptosis and promote formation of osteogenesis calcium nodules to promote osteogenesis differentiation by increasing proliferation activity of primary osteoblasts, activating Nrf2/Keapl signaling pathway, reducing mitochondrial oxidative stress damage,and maintaining homeostasis of MMP and normal ATP production,thus improving OP.