骨碎补总黄酮基于Notch信号通路改善骨质疏松的作用及机制研究

Study on davallia mariesil flavones improving condition of osteoporosis via Notch signaling pathway

目的:探讨骨碎补总黄酮(DMF)基于Notch信号通路改善骨质疏松的作用及机制研究。方法:(1)选取2016年1月-2017年1月间我院收治的120例骨质疏松患者作为研究对象,把入选病例随机分为试验组和对照组,每组60例,试验组用强骨胶囊(含骨碎补总黄酮180 mg/粒)联合钙尔奇D治疗,对照组单纯用钙尔奇D治疗。比较两组临床疗效、血钙、和血磷、TNF-α、MCP-1、IL-6水平。(2)分离和培养大鼠骨髓基质细胞,实验分组与给药:NC组:DMEM高糖完全培养液(含10%FBS);RA组:RA(0.4 mmol/L);DMF+RA组:DMEM高糖完全培养液(含DMF的血清)+RA(0.4 mmol/L);Jaggedl+RA组:Jaggedl[1 000μg/L+RA(0.4 mmol/L)];Jaggedl+DMF+RA组:Jaggedl(1 000μg/L)+DMEM高糖完全培养液(含DMF的血清)+RA(0.4 mmol/L);DAPT+RA组:DAPT(16μmol/L)+RA(0.4 mmol/L);DAPT+DMF+RA组:DAPT(16μmol/L)+DMEM高糖完全培养液(含DMF的血清)+RA(0.4 mmol/L)。检测各组Notch-1、Hes-1 mRNA和蛋白水平。结果:(1)临床研究中试验组总有效率高于对照组总有效率(91.67%>76.67%,P<0.05);治疗后试验组血钙和血磷水平高于对照组(P<0.05)。治疗后,试验组TNF-α、MCP-1、IL-6水平低于对照组(P<0.05)。(2)实验研究结果中RT-PCR和Western blot检测结果表明,与RA组相比Jaggedl+RA组中Notch-1、Hes-1 mRNA和蛋白水平均上调,DMF+RA组、DAPT+RA组中Notch-1、Hes-1 mRNA和蛋白均下调(P<0.05)。与Jaggedl+RA组相比Jaggedl+DMF+RA组中Notch-1、Hes-1 mRNA和蛋白水平均下调(P<0.05)。与DAPT+RA组相比DAPT+DMF+RA组中Notch-1、Hes-1 mRNA和蛋白水平均下调(P<0.05)。结论:骨碎补总黄酮可改善骨质疏松症,且其机制可能与抑制Notch信号通路相关。

Objective： To evaluate the effect and mechanism of davallia mariesil flavones（ DMF） improving osteoporosis via Notch signaling pathway. Methods：（ 1） 120 cases patients with osteoporosis in our hospital from January 2016 to January 2017 were analyzed,and divided into experiment group（ 60 cases） and control group（ 60 cases） using digital random grouping method. Experiment group was treated with DMF combined with D-calfor. Control group was treated with D-calfor only. After treatment,the levels of serum calcium,serum phosphor,TNF-α,MCP-1 and IL-6 in serum were detected to evaluate the clinical efficacy.（ 2） Bone marrow stromal cells were separated and cultivate. NC group： DMEM（ contain 10% FBS）. RA group： RA（ 0. 4 mmol/L）. DMF ＋ RA group： DMEM（ contain DMF） ＋RA（ 0. 4 mmol/L）. Jaggedl＋RA group： Jaggedl（ 1 000 μg/L） ＋RA（ 0. 4 mmol/L）. Jaggedl＋DMF＋RA group： Jaggedl（ 1 000 μg/L） ＋DMEM（ contain DMF） ＋RA（ 0. 4 mmol/L）. DAPT＋RA group： DAPT（ 16 μmol/L） ＋RA（ 0. 4 mmol/L）.DAPT＋DMF＋RA group： DAPT（ 16 μmol/L） ＋DMEM（ contain DMF） ＋RA（ 0. 4 mmol/L）. Western blotting assays and PCR were performed to assess mRNA and protein levels of Notch-1,Hes-1. Results：（ 1） In clinical study,the effective rate in treatment group was obviously higher than control group（ 91. 67% 76. 67%,P〈0. 05）. The levels of serum calcium and serum phosphor in the experiment group was higher than in the control group（ P〈0. 05）. The levels of TNF-α,MCP-1 and IL-6 in the experiment group was lower than in the control group（ P〈0. 05）.（ 2） In experimental study,compared with the RA group,the expressions of Notch-1,Hes-1 mRNA and protein were upregulated in Jaggedl＋RA group,but were downregulated in DAPT ＋ RA group,DMF ＋ RA group（ P〈0. 05）. Compared with the Jaggedl＋RA group,the expressions of Notch-1,Hes-1 mRNA and protein were downregulated in Jaggedl＋DMF＋RA group（ P〈0. 05）. Compared with the DAPT＋RA group,the expressions of Notch-1,Hes-1 mRNA and protein were downregulated in DAPT＋DMF＋RA group（ P〈0. 05）. Conclusion： DMF could improve the condition of osteoporosis. The mechanism may be associated with inhibiting the Notch signaling pathway by DMF.