Lithium chloride inhibits vascular smooth muscle cell calcification by increasing pyrophosphate levels / 中华老年医学杂志

ABSTRACT

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.