氯化锂通过上调焦磷酸水平抑制血管平滑肌细胞钙化

Lithium chloride inhibits vascular smooth muscle cell calcification by increasing pyrophosphate levels

目的探讨氯化锂对血管平滑肌细胞钙化的影响及其可能的作用机制。方法体外培养人主动脉血管平滑肌细胞,用钙化培养基(无机磷浓度为3 mmol/L)建立平滑肌细胞钙化模型。药物处理组用氯化锂(10 mmol/L)预处理细胞4 h后加入3 mmol/L的无机磷,继续培养细胞数天后用茜素红染色检测细胞钙盐沉积水平,同时应用焦磷酸偶联酶反应底物烟酰胺腺嘌呤二核苷酸(NADH)消耗法,酶标仪(OD340)检测细胞外焦磷酸的分泌;实时荧光定量聚合酶链式反应(real-time PCR)和免疫印迹法(Western blot)检测细胞中ankh基因的表达;采用慢病毒感染人主动脉平滑肌细胞的方法,建立对照细胞组和ankh敲低的实验细胞组,并观察其对细胞钙化的影响。结果高磷细胞组钙化检测值为(65.00±2.11)ng/g,较对照细胞组(12.39±0.38)ng/g钙化水平明显升高(P<0.01)。氯化锂预处理细胞的钙化检测值为(24.92±1.87)ng/g,与高磷对照组(60.94±4.51)ng/g比较能显著抑制高磷诱导的血管平滑肌细胞钙化(P<0.01);氯化锂预处理明显上调细胞外液中焦磷酸的含量,基础条件下预处理组为(51.70±7.26)×10^-3 mmol/g,对照组为(28.71±2.55)×10^-3mmol/g(P<0.01);高磷刺激下预处理组为(34.35±4.27)×10^-3mmol/g,对照组为(20.89±4.93)×10^-3mmol/g(P<0.05),同时氯化锂预处理显著升高细胞ankh表达水平(P<0.01);而敲低ankh使无机磷诱导的血管平滑肌细胞钙化程度显著加重,敲低ankh细胞钙化检测值为(71.73±2.45)ng/g,对照组为(56.19±3.59)ng/g(P<0.01)。结论氯化锂通过上调平滑肌ANKH的表达,升高细胞外液中的焦磷酸水平,抑制高磷诱导下血管平滑肌细胞的钙化。

Objective To investigate the effects of lithium chloride on vascular smooth muscle cell calcification and the potential underlying mechanisms.Methods Human aortic smooth muscle cells were cultured in vitro,and a smooth muscle cell calcification model was established by using a calcification medium(the concentration of inorganic phosphorus was 3 mmol/L).Cells in the drug treatment group were pretreated with lithium chloride(10 mmol/L)for 4 hours and then with inorganic phosphorus at 3 mmol/L.After several days in culture,calcium deposition in cells was measured by alizarin red S staining.The secretion of extracellular pyrophosphate was detected by measuring nicotinamide adenine dinucleotide hydrogen(NADH)consumption of pyrophosphate-coupled enzyme reactions,which were monitored spectrophotometrically at 340 nm.Real-time PCR and Western blotting were used to detect mRNA and protein expression of the human progressive ankylosis(ankh)gene.Human aortic smooth muscle cells were infected with the scramble control lentivirus and the sh-ankh lentivirus,respectively,to establish the control cell group and the ankh knockdown cell group.The effects of ankh knockdown on cell calcification were examined.Results The calcification level in vascular smooth muscle cells increased in the high inorganic phosphorus group,compared with the control group[(65.00±2.11)ng/g vs.(12.39±0.38)ng/g,(P<0.01)].Compared with the high-phosphorus control group,lithium chloride evidently inhibited high phosphate-induced vascular smooth muscle cell calcification[(24.92±1.87)ng/g vs.(60.94±4.51)ng/g,(P<0.01)].Lithium chloride pretreatment clearly increased extracellular pyrophosphate levels under unstimulated conditions[(51.70±7.26)×10^-3mmol/g vs.(28.71±2.55)×10^-3mmol/g(P<0.01)]and under high phosphorus stimulation[(34.35±4.27)×10^-3mmol/g vs.(20.89±4.93)×10^-3mmol/g(P<0.05)],and increased the expression of ankh as well(P<0.01).In addition,ankh knockdown markedly enhanced the extent of inorganic phosphorus-induced vascular smooth muscle cell calcification(71.73±2.45 ng/g vs.56.19±3.59 ng/g,P<0.01).Conclusions Lithium chloride inhibits high phosphorus-induced vascular smooth muscle cell calcification by enhancing the level of extracellular pyrophosphate via increased ankh expression.