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Objective To investigate the mechanism that receptor activator of NF-κB ligand (RANKL) promotes arterial calcification.Methods Firstly,RANKL was added into the culture media,in which the monocyte precursor cells alone were cultured.Morphological observation and tartrate resistant acid phosphatase(TRAP)stain were used to assess whether RANKL could induce the monocyte precursor cells to differentiate into osteoclast-like cells.During arterial calcification,both in vivo and in vitro expressions of RANKL and osteoprotegerin (OPG,as RANKL inhibitor)were measured via real-time PCR.The extent of osteoclast-like cell differentiation was also assessed.Results It was found that RANKL could induce osteoclast-like cell differentiation.There were no both in vivo and in vitro expressions of osteoclast-like cells in the early stage of calcification.At that time,the ratio of RANKL to OPG was very low.In the late stage of calcification,a small amount of osteoclast-like cell expression coincided with a relatively high ratio of RANKL to OPG.According to the results,the ratio of RANKL to OPG was very low during most of the arterial calcification period.This made it possible for OPG to completely inhibit RANKL-induced osteoclast-like cell differentiation.The ratio of RANKL to OPG was (0.36 ± 0.08) (F =36) and (1.68 ± 0.08) (F =36) respectively in the early and late subgroup of calcification group in the animal model,but was zero in the control group(both P＜0.05).The ratio of RANKL to OPG was(0.42±0.09) (F=16)and(1.50 ± 0.10)(F=16)respectively in the early and late subgroup of calcification group in the cell model,but was zero in the control group(both P＜0.05).Conclusions Our result likely explains why RANKL has the ability to induce osteoclast-like cell differentiation,but acts as a promoter of calcification.
RESUMO
Objective To study the change and significance of osteoprotegerin(OPG)/receptor activator of NF κB ligand(RANKL)during the process of rat arterial smooth muscle cells (SMC) to differentiate subgroups:7 d (early) subgroup and 14 d (late) subgroup.into osteoblast-like cells.Methods The rat arterial SMCs were divided randomly into the SMC group,atorvastatin group,and osteoblast-like group.Each group was also divided into 2.The osteoblast-like group was given β-glycerophosphate and vitamin C in culture medium,the atorvastatin group was given both β-glycerophosphate,vitamin C and atorvastatin.Von Kossa staining,Ca2+ content assay,ALP activity assay and osteocalcin assayed with Western blot were used to check the level of calcification.Real-time PCR was used to check the mRNA expressions of OPG and RANKL.Results There was high expression of OPG (early 2.71 ±0.08,late 2.69 ±0.02) but no RANKL in the SMC group all the time.The expressions of OPG were increased in the early subgroup and decreased in late subgroup in the atorvastatin group (early 3.52±0.05,late 2.50±0.03) and osteoblast-like group (early 4.18±0.10,late 2.30 ± 0.11).And the expressions of RANKL were both increased in the atorvastatin group and osteoblast-like group dnring the calcification process (from 1.01 ± 0.19 to 2.40 ± 0.10,and from 1.70±0.07 to 3.22±0.11,respectively).Among the three groups,the ratio of OPG/RANKL was decreased with the increasing calcification level (F =52.93,2.33,both P＜0.05).And compared with the early subgroups,the ratio of OPG/RANKL in the late subgroups was also decreased along with the increase of calcification level in each group(F=38.71,1.74,both P＜0.05).Conclusions The ratio of OPG/RANKL has a negative correlation with the level of the osteoblast-like cells differentiation,and atorvastatin could inhibit the calcification.
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Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly increased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.
RESUMO
Recent studies showed that activation of Wnt/β-catenin pathway promoted the differentiation of osteoblast-like cells in the arterial calcification, but its mechanism remains unknown. In this study, the hypothesis that Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by upregulating the expression of receptor activator of NF-κB ligand (RANKL) was examined. LiCl was used to activate the Wnt/β-catenin pathway. The differentiation of osteoblast-like cells was observed by Von Kossa staining, calcium content assay, alkaline phosphatase (ALP) activity assay, and detection of osteocalcin expression. Real-time PCR was performed to detect the expression of RANKL and osteoprotegerin (OPG, the decoy receptor of RANKL) during the osteoblast-like cell differentiation. Different concentrations of OPG were added to the culture media respectively to inhibit the function of RANKL, and the change in the differentiation of osteoblast-like cells was evaluated. The results showed that when the Wnt/β-catenin pathway was activated by LiCl, the expression of RANKL was significantly increased, which coincided with the differentiation of osteoblast-like cells (P<0.05), and the OPG treatment could partly attenuate the promoting effect of Wnt/β-catenin pathway on the differentiation of osteoblast-like cells (P<0.05), but it failed to completely abolish such effect. It was concluded that activation of Wnt/β-catenin pathway promotes the differentiation of osteoblast-like cells by both RANKL-dependent and RANKL-independent mechanisms.