PFKFB3抑制剂减轻高磷诱导的大鼠血管平滑肌细胞钙化

牛津; 张敏; 王巧娟; 刘振华; 顾晓明; 冯娜; 张淑苗; 贾敏; 樊荣; 李娟; 裴建明

doi:10.12125/j.chj.202104010

PFKFB3抑制剂减轻高磷诱导的大鼠血管平滑肌细胞钙化

Effect of PFKFB3 inhibitor on calcification in rat vascular smooth muscle cells treated with β-GP

摘要

摘要:
目的探究果糖-2, 6-二磷酸酶3(6-bisphosphatase 3, PFKFB3）抑制剂3-PO对高磷诱导大鼠血管平滑肌细胞（vascular smooth muscle cell, VSMC）钙化的作用及其潜在机制。
方法将原代培养的VSMC随机分为：对照(Con)组、高磷诱导（β-GP）组、高磷诱导+ 3-PO（β-GP +3-PO）组、对照+乳酸钠（Con +Lactate）组、对照+乳酸钠+ 3-PO（Con +Lactate+3-PO）组。实验前后用微板法检测细胞内钙和碱性磷酸酶（ALP）含量；茜素红染色检测VSMC钙化程度；CCK 8试剂盒检测细胞活力；蛋白免疫印迹法（Western blot）检测表型转化相关蛋白(RUNX2, BMP-2, SM22a)及糖酵解相关蛋白(PFKFB3, HKⅡ, GLUT1与GLUT4)表达水平，比色法检测乳酸含量及乳酸脱氢酶（LDH）活力。
结果与Con组比较，β-GP组VSMC钙化程度及成骨蛋白表达显著增加（P<0.01），细胞活力显著降低（P<0.01），而给予3-PO可显著逆转β-GP组的VSMC表型转化蛋白的表达变化（P<0.05），并使细胞活力增加（P<0.01）。进一步的研究证实，β-GP组中PFKFB3表达水平，乳酸含量及LDH活力显著增加（P<0.01），而在β-GP +3-PO组中显著下调（P<0.01），其余糖酵解蛋白表达水平未明显改变。另外，乳酸钠也可引起VSMC钙化程度及表型转化蛋白表达的增加（P<0.01），给予3-PO可逆转乳酸钠引起的VSMC钙化和表型转化（P<0.05）。
结论 PFKFB3抑制剂3-PO可能通过降低PFKFB3表达抑制糖酵解水平，从而抑制了高磷诱导的VSMC的钙化，研究结果为临床治疗血管钙化提供了潜在的药物靶点。

Abstract:
AIM To explore the role of 6-bisphosphatase 3 (PFKFB3) in phosphorus-induced calcification of primary vascular smooth muscle cells (VSMC) in rats and its underlying mechanism.
METHODS VSMC were randomly divided into 5 groups: control (Con) group, hyperphosphatemia induction (β-GP) group, β-GP+3-PO (a selective PFKFB3 inhibitor) group, Con+Lactate group, and Con+Lactate+3-PO group. Before and after the experiment, the contents of intracellular calcium, alkaline phosphatase (ALP), lactate dehydrogenase (LDH) and lactate were detected. Alizarin red staining was used to detect the calcification degree of VSMC and CCK8 kit was used to detect cell viability. The expression levels of RUNX2, BMP-2, SM22a (as the key molecules of calcification-related proteins) and PFKFB3, HKⅡ, GLUT1, GLUT4 (as the key enzyme of glycolysis) were detected by Western bolt.
RESULTS Compared with the control group, the β-GP group showed a lower cell viability (P<0.01) and SM22a expression (P<0.01) and significantly higher RUNX2 and BMP-2 expressions (P<0.01). However, treatment with 3-PO, the above effects were abrogated (P<0.05). Further studies confirmed that glycolysis and PFKFB3 expressions were significantly increased in the β-GP group compared with those in the control group (P<0.01), while significantly lower expressions were found in the β-GP+3-PO group (P<0.01). The HKⅡ, GLUT1 and GLUT4 expressions were unaltered. Lactate treatment significantly increased VSMC calcification and the phenotypic transforming protein expression (P<0.01), while this phenomenon could be reversed by 3-PO administration. (P<0.05).
CONCLUSION Inhibition of PFKFB3 by 3-PO decreases VSMC calcification and increases cell viability by reducing glycolysis and PFKFB3 expression, which provides a potential drug target and strategy for clinical treatment of vascular calcification.

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