甲基奈醌-4对血管平滑肌细胞钙化的影响
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摘要
目的探讨甲基奈醌-4对血管平滑肌细胞钙化的影响。方法分别采用生长培养基(磷1. 4 mmol/L)和钙化培养基(磷3. 0 mmol/L),培养人脐静脉血管平滑肌细胞,使用甲基酚酞络合铜法检测细胞基质钙、比色法检测碱性磷酸酶活性、液相色谱法检测细胞内甲基奈醌-4水平,茜素红S染色观察细胞基质钙盐沉积。在钙化培养基下,改变甲基奈醌-4水平,观察细胞基质钙、碱性磷酸酶活性的变化。结果钙化培养基能增加人脐静脉血管平滑肌细胞基质钙含量、碱性磷酸酶活性,减少细胞内甲基奈醌-4水平,细胞外基质出现明显的钙盐沉积。减少甲基奈醌-4的水平能增加基质钙含量、碱性磷酸酶活性。添加甲基奈醌-4能减少基质钙含量、碱性磷酸酶活性。结论甲基奈醌-4能抑制磷诱导的血管平滑肌细胞钙化。
Objective To investigate the effect of menaquinone-4 on vascular smooth muscle cell calcification. Methods With either growth medium( 1. 4 mmol/L,Pi) or calcification medium( 3. 0 mmol/L,Pi),primary human umbilical vein smooth muscle cells( HUVSMCs)were incubated. Cell matrix calcium was quantitated using o-cresolphthalein complexone method. Alkaline phosphatase activity was assessed colorimetrically. Menaquinone-4 was measured by ultra-performance liquid chromatography-tandem mass spectrometry. Matrix calcification morphology was by Alizarin Red S staining. Matrix calcium morphology and alkaline phosphatase activity were observed under different levels of Menaquinone-4. Results Calcification medium increased matrix calcium,alkaline phosphatase activity and reduced menaquinone-4 level compared with growth medium( P < 0. 05). Abundant mineral deposits could be detected in the extracellular matrix upon calcification medium. Decreased menaquinone-4 in HUVSMCs increased matrix calcification and alkaline phosphatase activity,and vice-versa. Conclusion Menaquinone-4 might inhibit phosphate-induced vascular smooth cells calcification.
Objective To investigate the effect of menaquinone-4 on vascular smooth muscle cell calcification. Methods With either growth medium( 1. 4 mmol/L,Pi) or calcification medium( 3. 0 mmol/L,Pi),primary human umbilical vein smooth muscle cells( HUVSMCs)were incubated. Cell matrix calcium was quantitated using o-cresolphthalein complexone method. Alkaline phosphatase activity was assessed colorimetrically. Menaquinone-4 was measured by ultra-performance liquid chromatography-tandem mass spectrometry. Matrix calcification morphology was by Alizarin Red S staining. Matrix calcium morphology and alkaline phosphatase activity were observed under different levels of Menaquinone-4. Results Calcification medium increased matrix calcium,alkaline phosphatase activity and reduced menaquinone-4 level compared with growth medium( P < 0. 05). Abundant mineral deposits could be detected in the extracellular matrix upon calcification medium. Decreased menaquinone-4 in HUVSMCs increased matrix calcification and alkaline phosphatase activity,and vice-versa. Conclusion Menaquinone-4 might inhibit phosphate-induced vascular smooth cells calcification.
引文
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[22]Cheung CL,Sahni S,Cheung BM,et al.Vitamin K intake and mortality in people with chronic kidney disease from NHANES III[J].Clin Nutr,2015,34(2):235-240.
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[2]Tatami Y,Yasuda Y,Suzuki S,et al.Impact of abdominal aortic calcification on long-term cardiovascular outcomes in patients with chronic kidney disease[J].Atherosclerosis,2015,243(2):349-355.
[3]Benz K,Varga I,Neureiter D,et al.Vascular inflammation and media calcification are already present in early stages of chronic kidney disease[J].Cardiovasc Pathol,2017,27:57-67.
[4]Chen NX,Moe SM.Pathophysiology of Vascular Calcification[J].Curr Osteoporos Rep,2015,13(6):372-380.
[5]Willems BA,Vermeer C,Reutelingsperger CP,et al.The realm of vitamin K dependent proteins:shifting from coagulation toward calcification[J].Mol Nutr Food Res,2014,58(8):1620-1635.
[6]Chatrou ML,Winckers K,Hackeng TM,et al.Vascular calcification:the price to pay for antieoagulation therapy with vitamin K-antagonists[J].Blood Rev,2012,26(4):155-166.
[7]朱杰,王中群,王昭军,等.维生素K2抑制血管钙化作用与机制研究进展[J].中华老年心脑血管病杂志,2016,18(1):94-96.
[8]Nakagawa K,Hirota Y,Sawada N,et al.Identification of UBIAD1 as a novel human menaquinone-4 biosynthetic enzyme[J].Nature,2010,468(7320):117-121.
[9]林赘,冯韵霖.血维生素K水平与慢性肾脏病血管钙化相关性研究[J].成都医学院学报,2017,12(4):461-465.
[10]Aoun M,Makki M,Azar H,et al.High Dephosphorylated-Uncarboxylated MGP in Hemodialysis patients:risk factors and response to vitamin K2,A pre-post intervention clinical trial[J].BMC Nephrol,2017,18(1):191.
[11]Riphagen IJ,Keyzer CA,Drummen NEA,et al.Prevalence and Effects of Functional Vitamin K Insufficiency:The PREVEND Study[J].Nutrients,2017.
[12]Yao L,Sun YT,Sun W,et al.High phosphorus level leads to aortic calcification viaβ-catenin in chronic kidney disease[J].Am J Nephrol,2015,41(1):28-36.
[13]Deng D,Diao Z,Han X,et al.Secreted Frizzled-Related Protein 5Attenuates High Phosphate-Induced Calcification in Vascular Smooth Muscle Cells by Inhibiting the Wnt/β-Catenin Pathway[J].Calcif Tissue Int,2016,99(1):66-75.
[14]Razzaque MS.Phosphate toxicity and vascular mineralization[J].Contrib Nephrol,2013,180:74-85.
[15]Han X,Wang LY,Diao ZL,et al.Apelin:A novel inhibitor of vascular calcification in chronic kidney disease[J].Atherosclerosis,2016,244:1-8.
[16]Neradova A,Schumacher SP,Hubeek I,et al.Phosphate binders affect vitamin K concentration by undesired binding,an in vitro study[J].BMC Nephrol,2017,18(1):149.
[17]van den Heuvel EG,van Schoor NM,Lips P,et al.Circulating uncarboxylated matrix Gla protein,a marker of vitamin K status,as a risk factor of cardiovascular disease[J].Maturitas,2014,77(2):137-141.
[18]Kaesler N,Magdeleyns E,Herfs M,et al.Impaired vitamin K recycling in uremia is rescued by vitamin K supplementation[J].Kidney Int,2014,86(2):286-293.
[19]郭连莹,杜先超,王玲,等.血管钙化对血脂的影响及维生素K干预研究[J].中国食物与营养,2017,23(9):65-68.
[20]朱杰,王中群,王昭军,等.维生素K2对ApoE-/-小鼠动脉粥样硬化内膜钙化和Toll样受体2及4表达的影响[J].中国动脉硬化杂志,2015,23(3):217-223.
[21]CaluwéR,Vandecasteele S,Van Vlem B,et al.Vitamin K2 supplementation in haemodialysis patients:a randomized dose-finding study[J].Nephrol Dial Transplant,2014,29(7):1385-1390.
[22]Cheung CL,Sahni S,Cheung BM,et al.Vitamin K intake and mortality in people with chronic kidney disease from NHANES III[J].Clin Nutr,2015,34(2):235-240.
[23]邱翠婷,吕安林,李寰,等.维生素K依赖蛋白抑制血管钙化研究进展[J/CD].中华临床医师杂志(电子版),2014,8(13):2499-2501.