Effect of Bushen Huoxue decoction on inhibiting osteogenic differentiation of vascular smooth cells by regulating OPG/RANK/RANKL system in vascular calcification
Effect of Bushen Huoxue decoction on inhibiting osteogenic differentiation of vascular smooth cells by regulating OPG/RANK/RANKL system in vascular calcification
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DOI：
10.21037/atm.2019.02.33
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发表时间：
2019-03-01
影响因子：
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通讯作者：
Zhang, Ning
中科院分区：
文献类型：
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作者：
Liu, Shi-Yi;Meng, Xiang-Fei;Zhang, Ning
背景：探讨补肾活血汤（BSHXD）通过调节骨保护素（OPG）和核因子κ B配体受体激活因子（RANKL） mRNA表达抑制血管平滑肌细胞（VSMCs）血管钙化成骨分化的作用及其分子机制。方法：体外培养大鼠主动脉VSMCs。高水平的无机磷酸盐培养基（2.4 mM）诱导VSMCs成骨分化。采用血清药理学方法制备含bshxd的血清。采用6孔板，以约4 × 10(4)个细胞/mL的密度对VSMCs进行接种，培养10天。然后应用不同浓度的含bshxd的血清。高、中、低剂量组含bshxd的血清浓度百分比分别为20%、10%和5%。茜素红S染色评价钙结节，并于第3、6、10天按试剂盒说明检测碱性磷酸酶（ALP）活性和钙沉积。Western blot检测大鼠第3、6、10天ALP和α -平滑肌肌动蛋白（a-SMA）的表达水平。在第3、6、10天采用实时荧光定量PCR检测OPG和RANKL mRNA的表达。结果：与对照组相比，BSHXD能显著减弱茜素红- s染色观察到的钙结节。BSHXD组a-SMA蛋白表达水平上调，ALP表达水平下调（P< 0.05）。BSHXD对VSMCs的ALP活性和钙沉积也有抑制作用（P< 0.05）。这些变化与BSHXD在VSMCs成骨分化过程中上调OPG mRNA表达、下调RANKL mRNA表达有关。结论：BSHXD对抑制高水平磷酸盐诱导的VSMCs成骨分化具有有益作用。其潜在机制似乎与VSMCs中OPG mRNA和RANKL mRNA的表达调节有关，从而阻止VSMCs向成骨表型的表型变化。
Background: To investigate the effects of Bushen Huoxue Decoction (BSHXD) and its underlying molecular mechanisms on inhibiting osteogenic differentiation of vascular smooth muscle cells (VSMCs) in vascular calcification via regulating the mRNA expression of osteoprotegerin (OPG) and the receptor activator of the nuclear factor-kappa B ligand (RANKL).Methods: VSMCs from the aortas of rats were cultured in vitro. Osteogenic differentiation of VSMCs was induced by high levels of an inorganic phosphate medium (2.4 mM). BSHXD-containing serum was prepared using the serum-pharmacological method. VSMCs were plated using 6-well plates at an approximate density of 4.0x10(4) cells/mL and cultured for 10 days. This was followed by the application of different concentrations of BSHXD-containing serum. The percentage of concentrations of BSHXD-containing serum in high, middle and low dosage group was 20%, 10% and 5%, respectively. Calcium nodules were evaluated by alizarin red S staining, and alkaline phosphatase (ALP) activity and calcium deposition were both examined as per the instruction of the test kits on the 3rd, 6th, and 10th days. Protein expression level of ALP and alpha-smooth muscle actin (a-SMA) were detected by Western blot on the 3rd, 6th, and 10th days. The mRNA expression of the OPG and RANKL were also detected by real-time PCR on the 3rd, 6th, and 10th days.Results: Compared with the control group, BSHXD significantly attenuated the calcium nodules that were examined by alizarin red-S staining. Protein expression levels of a-SMA were up-regulated and ALP were down-regulated on the BSHXD group (P< 0.05). BSHXD also attenuated the ALP activity and calcium deposition of the VSMCs (P< 0.05). These changes were associated with the effect of BSHXD on up-regulating the expression of OPG mRNA and down-regulating the expression of RANKL mRNA in the process of osteogenic differentiation of VSMCs.Conclusions: BSHXD has a beneficial effect on inhibiting osteogenic differentiation of VSMCs induced by high levels of phosphate. The underlying mechanism appears to be related to the modulation of expressions of OPG mRNA and RANKL mRNA in the VSMCs, thereby preventing the phenotypic changes of VSMCs to an osteogenic phenotype.