硫代硫酸钠抑制高磷诱导的血管平滑肌细胞钙化的机制
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摘要
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.
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.
引文
[1] Liu S, Zhang DL, Guo W, et al. Left ventricular mass index and aortic arch calcification score are independent mortality predictors of maintenance hemodialysis patients[J].Hemodial Int,2012,16(4):504-511.
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[16] Shuto E, Taketani Y, Tanaka R, et al. Dietary phosphorus acutely impairs endothelial function[J].J Am Soc Nephrol,2009,20(7):1504-1512.
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[20] Bijarnia RK, Bachtler M, Chandak PG, et al. Sodium thiosulfate ameliorates oxidative stress and preserves renal function in hyperoxaluric rats[J].PLoS One,2015,10(4):e0124881
[21] Adirekkiat S, Sumethkul V, Ingsathit A, et al. Sodium thiosulfate delays the progression of coronary artery calcification in haemodialysis patients[J].Nephrol Dial Transplant,2010,25(6):1923-1929.
[22] 余毅,霍苗苗,王琰,等. 硫代硫酸钠早期干预对高磷诱导的残肾大鼠血管钙化的影响[J].中华高血压杂志,2016,24(4):348-353.
[23] 余毅,毕智敏,王琰,等. 硫代硫酸钠对维持性血液透析患者冠状动脉钙化干预作用[J].中华医学杂志,2016,96(46):3724-3728.
[24] Pasch A, Schaffner T, Huynh-Do U, et al. Sodium thiosulfate prevents vascular calcifications in uremic rats[J].Kidney Int,2008,74(11):1444-1453.
[25] Mendoza FJ, Lopez I, Montesde O, et al. Metabolic acidosis inhibits soft tissue calcification in uremic rats[J].Kidney Int,2008,73(4):407-414.
[26] Krishnaraj P, Ravindran S, Kurian GA. The renal mitochondrial dysfunction in patients with vascular calcification is prevented by sodium thiosulfate[J].Int Urol Nephrol,2016,48(11):1927-1935.
[27] O'Neill WC, Hardcastle KI. The chemistry of thiosulfate and vascular calcification[J].Nephrol Dial Transplant,2012,27(2):521-526.
[28] Liu Y, Shanahan CM. Signalling pathways and vascular calcification[J].Front Biosci(Landmark Ed),2011,16:1302-1314.
[29] Mukherjee A, Rotwein P. Selective signaling by Akt1 controls osteoblast differentiation and osteoblast-mediated osteoclast development[J].Mol Cell Biol,2012,32(2):490-500.
[30] Zhan JK, Wang YJ, Wang Y, et al. The mammalian target of rapamycin signalling pathway is involved in osteoblastic differentiation of vascular smooth muscle cells[J].Can J Cardiol,2014,30(5):568-575.
[31] Sun H, Kim JK, Mortensen R, et al. Osteoblast-targeted suppression of PPARγ increases osteogenesis through activation of mTOR signaling[J].Stem Cells,2013,31(10):2183-2192.
[2] Hill KM, Martin BR, Wastney ME, et al. Oral calcium carbonate affects calcium but not phosphorus balance in stage 3-4 chronic kidney disease[J].Kidney Int,2012,83(5):959-966.
[3] Vo TM, Disthabanchong S. Are there ways to attenuate arterial calcification and improve cardiovascular outcomes in chronic kidney disease?[J].World J Cardiol,2014,6(5):216-226.
[4] Mathews SJ, de Las Fuentes L, Podaralla P, et al. Effects of sodium thiosulfate on vascular calcification in end-stage renal disease: a pilot study of feasibility, safety and efficacy[J].Am J Nephrol,2010,33(2):131-138.
[5] Mukherjee A, Rotwein P. Selective signaling by Akt1 controls osteoblast differentiation and osteoblast-mediated osteoclast development[J].Mol Cell Biol,2012,32(2):490-500.
[6] Duan XY, Xie PL, Ma YL, et al. Omentin inhibits osteoblastic differentiation of calcifying vascular smooth muscle cells through the PI3K/Akt pathway[J].Amino Acids,2011,41(5):1223-1231.
[7] Li H, Tao HR, Hu T, et al. Atorvastatin reduces calcification in rat arteries and vascular smooth muscle cells[J].Basic Clin Pharmacol Toxicol,2010,107(4):798-802.
[8] Sarnak MJ. Cardiovascular complications in chronic kidney disease[J].Am J Kidney Dis,2003,41(5 Suppl):S11-17.
[9] Zitt E, Rosenkranz AR. Vascular calcification and increased mortality in dialysis patients:is the baroreflex sensitivity the answer?[J].Nephrol Dial Transplant,2005,20(11):2580.
[10] Adragao T, Pires A, Lucas C, et al. A simple vascular calcification score predicts cardiovascular risk in haemodialysis patients[J].Nephrol Dial Transplant,2004,19(6):1480-1488.
[11] Sage AP, Tintut Y, Demer LL. Regulatory mechanisms in vascular calcification[J].Nat Rev Cardiol,2010,7(9):528-536.
[12] Covic A, Kanbay M, Voroneanu L, et al. Vascular calcification in chronic kidney disease[J].Clin Sci,2010,119(3):111-121.
[13] Peres LA, P rcio PP. Mineral and bone disorder and vascular calcification in patients with chronic kidney disease[J].J Bras Nefrol,2014,36(2):201-207.
[14] Ciceri P, Elli F, Brenna I, et al. Lanthanum prevents high phosphate-induced vascular calcification by preserving vascular smooth muscle lineage markers[J].Calcif Tissue Int,2013,92(6):521-530.
[15] Burger D, Levin A. Shedding light on mechanisms of hyperphosphatemic vascular dysfunction[J].Kidney Int,2013,83(2):187-189.
[16] Shuto E, Taketani Y, Tanaka R, et al. Dietary phosphorus acutely impairs endothelial function[J].J Am Soc Nephrol,2009,20(7):1504-1512.
[17] 余毅,曹翠云. 核心结合因子和Ⅱ型胶原在高磷诱导的残肾大鼠血管钙化中的表达[J].中华高血压杂志,2012,20(9):847-851.
[18] 余毅,颜开平,王琰,等. 核心结合因子和Ⅱ型胶原在慢性肾脏病5期患者桡动脉中的表达[J].中华肾脏病杂志,2012,28(11):868-872.
[19] Nigwekar SU, Brunelli SM, Meade D, et al. Sodium thiosulfate therapy for calcific uremic arteriolopathy[J].Clin J Am Soc Nephrol,2013,8(7):1162-1170.
[20] Bijarnia RK, Bachtler M, Chandak PG, et al. Sodium thiosulfate ameliorates oxidative stress and preserves renal function in hyperoxaluric rats[J].PLoS One,2015,10(4):e0124881
[21] Adirekkiat S, Sumethkul V, Ingsathit A, et al. Sodium thiosulfate delays the progression of coronary artery calcification in haemodialysis patients[J].Nephrol Dial Transplant,2010,25(6):1923-1929.
[22] 余毅,霍苗苗,王琰,等. 硫代硫酸钠早期干预对高磷诱导的残肾大鼠血管钙化的影响[J].中华高血压杂志,2016,24(4):348-353.
[23] 余毅,毕智敏,王琰,等. 硫代硫酸钠对维持性血液透析患者冠状动脉钙化干预作用[J].中华医学杂志,2016,96(46):3724-3728.
[24] Pasch A, Schaffner T, Huynh-Do U, et al. Sodium thiosulfate prevents vascular calcifications in uremic rats[J].Kidney Int,2008,74(11):1444-1453.
[25] Mendoza FJ, Lopez I, Montesde O, et al. Metabolic acidosis inhibits soft tissue calcification in uremic rats[J].Kidney Int,2008,73(4):407-414.
[26] Krishnaraj P, Ravindran S, Kurian GA. The renal mitochondrial dysfunction in patients with vascular calcification is prevented by sodium thiosulfate[J].Int Urol Nephrol,2016,48(11):1927-1935.
[27] O'Neill WC, Hardcastle KI. The chemistry of thiosulfate and vascular calcification[J].Nephrol Dial Transplant,2012,27(2):521-526.
[28] Liu Y, Shanahan CM. Signalling pathways and vascular calcification[J].Front Biosci(Landmark Ed),2011,16:1302-1314.
[29] Mukherjee A, Rotwein P. Selective signaling by Akt1 controls osteoblast differentiation and osteoblast-mediated osteoclast development[J].Mol Cell Biol,2012,32(2):490-500.
[30] Zhan JK, Wang YJ, Wang Y, et al. The mammalian target of rapamycin signalling pathway is involved in osteoblastic differentiation of vascular smooth muscle cells[J].Can J Cardiol,2014,30(5):568-575.
[31] Sun H, Kim JK, Mortensen R, et al. Osteoblast-targeted suppression of PPARγ increases osteogenesis through activation of mTOR signaling[J].Stem Cells,2013,31(10):2183-2192.