硫代硫酸钠抑制高磷诱导的血管平滑肌细胞钙化的机制

Sodium thiosulfate attenuates vascular smooth muscle cell calcification induced by high phosphate

目的观察硫代硫酸钠(STS)对高磷干预的血管平滑肌细胞(VSMC)蛋白激酶B(Akt)/雷帕霉素靶蛋白(mTOR)分子的影响,探讨STS抑制高磷诱导的VSMC钙化的可能机制。方法体外原代培养SD大鼠VSMC,3～6代细胞用于后续实验。①将细胞随机给予正常磷(1.3 mmol/L)或高磷(2.6 mmol/L)干预,7 d后茜素红染色检测细胞钙盐沉积,邻甲苯酚络合酮比色法测定细胞钙含量;Western blot法检测p-Akt(ser473)、p-mTOR(ser2448)表达。②细胞随机给予高磷、高磷+不同浓度(0.5、1.0、2.0 mmol/L)STS干预。24 h后检测细胞钙盐沉积和钙含量。③细胞随机给予高磷、高磷+Akt特异抑制剂渥曼青霉素(100 nmol/L)或mTOR抑制剂雷帕霉素(100 ng/mL)、高磷+STS(2.0 mmol/L)干预,48 h后检测各组p-Akt或p-mTOR表达。所有实验重复5次,每次实验设置3个复孔。结果干预7 d后,与正常磷相比,高磷干预细胞钙含量增高[(77.33±12.86)比(20.33±7.09)μg/mg pro,P<0.05];与正常磷相比,高磷干预细胞p-Akt(0.57±0.06比0.26±0.08)、p-mTOR(0.68±0.08比0.42±0.07)表达增高(均P<0.05)。与高磷相比,0.5 mmol/L STS及1.0 mmol/L STS干预24 h后钙盐沉积无明显减轻,而2.0 mmol/L STS干预后钙盐沉积明显减轻,细胞钙含量减少。与高磷相比,渥曼青霉素干预后细胞p-Akt蛋白表达减少(0.36±0.15比0.85±0.22,P<0.05),雷帕霉素干预后细胞p-mTOR蛋白表达减少(0.38±0.18比0.86±0.22,P<0.05),而2.0 mmol/L STS干预后细胞p-Akt和p-mTOR蛋白表达无明显变化(P>0.05)。结论高磷体外诱导大鼠VSMC钙化,激活Akt/mTOR信号分子。渥曼青霉素和雷帕霉素抑制VSMC Akt/mTOR信号分子,减轻高磷诱导的VSMC钙化。STS可减轻高磷诱导的VSMC钙化,但非通过Akt/mTOR信号分子起作用。

Objective To observe the effect of sodium thiosulfate(STS)on protein kinase B(PKB,Akt)and rapamycin target protein(mTOR)in high-phosphorus treated vascular smooth muscle cells(VSMC),and to explore the possible mechanism of STS inhibiting high-phosphorus induced VSMC calcification.Methods VSMC of SD rats were cultured in vitro.Passage 3 to 6 were used for experiments.①VSMC were randomly treated by normal(1.3 mmol/L)or large dose of phosphorus(2.6 mmol/L).Seven days later,protein expression of p-Akt and p-mTOR were examined by Western blot.②VSMC were treated by large dose of phosphate with or without different concentrations(0.5,1,2 mmol/L)of STS.After 24 hours,calcium deposition and calcium content in VSMC were detected.③VSMC were treated by large dose of phosphorus,phosphorus+Akt inhibitor(wortmannin),phosphorus+mTOR inhibitor(rapamycin)and phosphorus+STS,respectively.After 24 hours,calcium deposition and calcium content in VSMC were detected.The protein expression of p-Akt and p-mTOR were detected.All experiments were repeated 5 times at least.Results Compared with normal dosage of phosphate,the calcium deposition and calcium content were significantly increased in high-phosphate treated VSMC after 7 days((77.33±12.86)vs(20.33±7.09)μg/mg pro,P<0.05)The protein expression of p-Akt(0.26±0.08 vs 0.57±0.06)and p-mTOR(0.42±0.07 vs 0.68±0.08)were also significantly increased after high-phosphate treatment(P<0.05).Compared with high-phosphorus treatment,calcium deposition and calcium content were decreased in VSMC treated by 2.0 mmol/L STS,but not in VSMC treated by 0.5 and 1.0 mmol/L STS.After treated by wortmanin or rapamycin for 24-48 hours,the protein expression of p-Akt(0.36±0.15 vs 0.85±0.22,P<0.05)and p-mTOR(0.38±0.18 vs 0.86±0.22,P<0.05)were obviously decreased,while after treated by 2.0 mmol/L STS,the protein expression of p-Akt and p-mTOR were not decreased compared with high-phosphate treated VSMC.Conclusion High-phosphate induces VSMC calcification in vitro.Signal molecules Akt and mTOR are activated in high-phosphate induced VSMC calcification.Wortmannin and rapamycin can inhibit high-phosphate induced VSMC calcification by inhibiting the activation of Akt and mTOR,respectively.Sodium thiosulfate may attenuate high-phosphate induced VSMC calcification.However,it has no effects on Akt and mTOR molecules.