MTOR Signalling Pathway Involve In VSMC Calcification Induced By High Phosphate

【Objective】 The aim of this paper is to characterize the possible role of mTOR Signalling Pathway in the calcification of VSMC induced by high Phosphate.【Methods】 1. VSMCs were cultured from media layers of aorta.Morphological observation of cells were by inverted phase contrast microscope, and VSMCs were identified by a positive staining of α-SMA. 2. VSMCs at passage 3-8 were used for experiments. The cells were divided into two groups randomly:(1) high Phosphate group(2.6mmol/L);(2) normal Phosphate group(1.3mmol/L). We observe the morphology and visualize the calcium deposition by Alizarin Red S staining at day 7. Real-time PCR was performed to measure Cbfα1, OPN m RNA expression, and Western blot was used to detect p-mTOR(S2448), Cbfα1 and OPN protein expression. 3. When The p-mTOR(s2448) expression of VSMCs was enhanced by high Phosphate, rapamycin, the mTOR inhibitor was added in high Phosphate group.VSMCs of high Phosphate group were divided into 4 groups randomly:(1) high Phosphate group(2.6mmol/L);(2)high Phosphate(2.6mmol/L)+rapamycin(1ng/ml) group;(3) high Phosphate(2.6mmol/L)+rapamycin(10ng/ml) group;(4)high Phosphate(2.6mmol/L) +rapamycin(100ng/ml) group. After 24 h, the m RNA expression of Cbfα1 and OPN were measured, after 48 h,the protein expression of p-mTOR(S2448), Cbfα1, OPN were detected. 4. Effect of rapamycin on calcium deposition: VSMCs which had been treated with high phosphate for 7 days were divided into two groups randomly:(1) high phosphate group(2.6mmol/L)(2)high Phosphate(2.6mmol/L)+rapamycin(100ng/ml) group.Cultured another 7 days,calcium deposition was visualized by Alizarin Red S staining. All experiments were repeated 3 times.【Results】 1. We found the VSMC was characterized by the ribbon spindle shape and showed“hill and valley”morphological appearance under inverted phase contrast microscope. Direct immunohistochemical staining of α-SMA showed positive staining in the VSMC. 2. 7 days after the intervention, Alizarin Red S staining showed that calcium deposition was obvious in high phosphate group,, while the normal phosphate group had no significant calcium deposition; Cbfα1 and OPN m RNA expression were increased in high phosphate group(P<0.05); p-mTOR(S2448), Cbfα1 and OPN protein were upregulated in high phosphate group(P<0.05). 3. After VSMC calcification model induced by high phosphate was established, VSMCs were treated with different concentrations of rapamycin.After 24 hours, Cbfα1 m RNA expression were decreased in high Phosphate(2.6mmol/L)+rapamycin(1ng/ml) group,high Phosphate(2.6mmol/L)+rapamycin(10ng/ml) group and high Phosphate(2.6mmol/L)+rapamycin(100ng/ml) group,compared with high Phosphate group. The OPN m RNA expression also decreased in high Phosphate(2.6mmol/L)+rapamycin(10ng/ml)group and high Phosphate(2.6mmol/L)+rapamycin(100ng/ml) group,while there were no significant difference between high Phosphate(2.6mmol/L)+rapamycin(1ng/ml) group and high Phosphate group(P<0.05). After 48 hours, p-mTOR(S2448),OPN and Cbfα1 protein expression all decreased in high Phosphate(2.6mmol/L)+rapamycin(1ng/ml) group,high Phosphate(2.6mmol/L)+rapamycin(10ng/ml) group,high Phosphate(2.6mmol/L) +rapamycin(100ng/ml) group, Compared with high Phosphate group. 4. Alizarin red S staining showed that calcium deposition was inhibited in high Phosphate(2.6mmol/L)+rapamycin(100ng/ml)group,compared with high Phosphate group.【Conclusion】 1. mTOR signaling pathway is activated In VSMC calcification induced by high Phosphate, and the expression of Cbfαl and OPN are upregulated. 2. When rapamycin inhibits the expression of mTOR signaling molecule, Cbfαl and OPN expression levels decrease, and the VSMC calcification induced by high Phosphate was attenuated. 3. mTOR Signalling Pathway plays an important role in the regulation of VSMC calcification induced by high Phosphate.