Liu Shi-Yi, Meng Xiang-Fei, Liu Shi-Wei, Hao Cong-Li, Li Lan-Fang, Zhang Ning
Department of Nephropathy Diseases, Wang Jing Hospital, China Academy of Chinese Medical Sciences, Beijing 100102, China.
Institute of Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, China.
Ann Transl Med. 2019 Mar;7(6):125. doi: 10.21037/atm.2019.02.33.
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).
VSMCs from the aortas of rats were cultured . 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 4.0×10 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 α-smooth muscle actin (α-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.
Compared with the control group, BSHXD significantly attenuated the calcium nodules that were examined by alizarin red-S staining. Protein expression levels of α-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.
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.
探讨补肾活血汤（BSHXD）对血管钙化中血管平滑肌细胞（VSMCs）成骨分化的影响及其潜在分子机制，通过调节骨保护素（OPG）和核因子κB受体活化因子配体（RANKL）的mRNA表达来实现。
培养大鼠主动脉的VSMCs。用高浓度无机磷酸盐培养基（2.4 mM）诱导VSMCs成骨分化。采用血清药理学方法制备含BSHXD血清。将VSMCs以约4.0×10个细胞/mL的密度接种于6孔板中，培养10天。随后施加不同浓度的含BSHXD血清。高、中、低剂量组含BSHXD血清的浓度百分比分别为20%、10%和5%。通过茜素红S染色评估钙结节，并在第3、6和10天按照试剂盒说明书检测碱性磷酸酶（ALP）活性和钙沉积。在第3、6和10天通过蛋白质印迹法检测ALP和α平滑肌肌动蛋白（α-SMA）的蛋白表达水平。在第3、6和10天通过实时PCR检测OPG和RANKL的mRNA表达。
与对照组相比，BSHXD显著减少了茜素红S染色检测到的钙结节。在BSHXD组中，α-SMA的蛋白表达水平上调，而ALP下调（P<0.05）。BSHXD还减弱了VSMCs的ALP活性和钙沉积（P<0.05）。这些变化与BSHXD在VSMCs成骨分化过程中上调OPG mRNA表达和下调RANKL mRNA表达的作用相关。
BSHXD对抑制高磷诱导的VSMCs成骨分化具有有益作用。其潜在机制似乎与调节VSMCs中OPG mRNA和RANKL mRNA的表达有关，从而防止VSMCs向成骨表型的表型变化。
