Sodium Thiosulfate Attenuates Vascular Smooth Muscle Cell Calcification Induced By High Phosphate

?Objective?Our objective is to clarify the possible mechanism of STS attenuating VSMC calcification induced by high phosphate.?Methods?1.SD rat VSMC were cultured in vitro.Cell shape and growth were observed by inverted phase contrast microscope.Immunohistochemical staining of ?-SMA was used to identify VSMC.Passage 3 to 6 were used for experiments.2.VSMC were randomly divided into two groups:(1)normal phosphate(Pi1.3mmol/L)group,(2)high phosphate(Pi 2.6mmol/L)group.After 7 days,calcium deposition and calcium content in VSMC were detected by Alizarin red staining and o-cresolphthalein complexone method,respectively.The m RNA expression of Cbf?1 and OPN were examined by Real-time PCR,and the protein expression of p-Akt,p-m TOR,Cbf?1 and OPN were examined by Western blot analysis.3.VSMC were treated with STS after Akt and m TOR were activated.The cells were divided into four groups:(1)high phosphate(Pi 2.6mmol/L)group,(2)high phosphate +STS(0.5mmol/L)group,(3)high phosphate+STS(1.0mmol/L)group,(4)high phosphate+STS(2.0mmol/L)group.After 24 hours,calcium deposition and calcium content in VSMC were detected.4.VSMC were treated with Akt inhibitor(Wortmannin),m TOR inhibitor(Rapamycin)and STS respectively.Cells were divided into two groups:(1)high phosphate(Pi2.6mmol/L)subgroup,high phosphate+Wortmanin(100nmol/L)subgroup,high phosphate+STS(2.0mmol/L)subgroup;(2)high phosphate(Pi 2.6mmol/L)subgroup,high phosphate +Rapamycin(100ng/ml)subgroup,high phosphate+STS(2.0mmol/L)subgroup.After 24 to 48 hours,calcium deposition and calcium content in VSMC were detected.The m RNA expression of Cbf?1 and OPN were examined,and the protein expression of p-Akt,p-m TOR,Cbf?1 and OPN were detected.All experiments were repeated 3 times at least.?Results?1.Rat VSMC were spindle-shaped and presented with “hill and valley” adherent growth under inverted phase contrast microscope.Positive staining of ?-SMA was observed,with a large amount of brown granules in the cytoplasm.2.After 7 days,compared with normal phosphate group,the calcium deposition and calcium content were significantly increased in high phosphate group(P<0.05).The m RNA expression of Cbf?1 and OPN were significantly increased in high phosphate group(P<0.05).Also,the protein expression of p-Akt,p-m TOR,Cbf?1 and OPN were significantly increased in high phosphate group(P<0.05).3.After 24 hours treatment of STS,compared with high phosphate group,the calcium deposition and calcium content were obviously decreased in high phosphate+STS(2.0mmol/L)group.4.After treated with Wortmanin and STS 24-48 hours,compared with high phosphate group,the calcium deposition and calcium content were obviously decreased in high phosphate+Wortmanin(100nmol/L)group and high phosphate+STS(2.0mmol/L)group(P<0.05).Also,the m RNA and protein expression of Cbf?1 and OPN were significantly decreased in high phosphate+Wortmanin(100nmol/L)group and high phosphate+STS(2.0mmol/L)group(P<0.05).However,compared with high phosphate group,the protein expression of p-Akt was not decreased in high phosphate+STS(2.0mmol/L)group(P>0.05).5.After treated with Rapamycin and STS 24-48 hours,compared with high phosphate group,the calcium deposition and calcium content were both obviously decreased in high phosphate+Rapamycin(100ng/m L)group and high phosphate+ STS(2.0mmol/L)group.Also,the m RNA and protein expression of Cbf?1 and OPN were significantly decreased in high phosphate+Rapamycin(100ng/m L)group and high phosphate+STS(2.0mmol/L)group(P<0.05).However,compared with high phosphate group,the protein expression of p-m TOR was not decreased in high phosphate+STS(2.0mmol/L)group(P>0.05).?Conclusion?1.High phosphate induces VSMC calcification in vitro.Signal molecules Akt and m TOR are activated in high phosphate induced VSMC calcification,also Cbf?1 and OPN expression are significantly up-regulated.2.Wortmannin and Rapamycin could inhibit high phosphate induced VSMC calcification by inhibiting the activation of Akt and m TOR,respectively.Also,Cbf?1and OPN expression are significantly down-regulated.3.Sodium thiosulfate may attenuate high phosphate induced VSMC calcification by inhibiting m RNA and protein expression of Cbf?1 and OPN.however,it has no effects on Akt and m TOR molecules.