荜茇明碱抑制BMP2/pSmad1/5信号减轻高钙高磷诱导的主动脉瓣膜间质细胞钙化

Piperlongumine attenuates high calcium/phosphate-induced calcification of aortic valve interstitial cells by inhibiting BMP2/pSmad1/5 signaling

目的:探究荜茇明碱对高钙高磷诱导的主动脉瓣膜间质细胞(aortic valve interstitial cell,AVIC)钙化的保护作用及其机制。方法:体外培养原代猪AVIC,通过高钙高磷诱导钙化并给予不同浓度荜茇明碱处理16 h后,流式细胞仪分析检测凋亡水平;AVIC在高钙高磷条件下,以2.5 mmol/L荜茇明碱或100 ng/mL骨形态发生蛋白2(bone morphogenetic protein 2,BMP2)处理,通过检测钙浓度、碱性磷酸酶(alkaline phosphatase,ALP)活性、茜素红染色反映钙化程度;Western blot、real-time PCR检测成骨相关蛋白和m RNA的表达。结果:①流式细胞分析发现,高钙高磷条件下,荜茇明碱低于5 mmol/L无明显促凋亡作用;②钙浓度、ALP活性、茜素红染色显示,2.5 mmol/L荜茇明碱显著抑制高钙高磷诱导的AVIC钙化;③Western blot、real-time PCR显示,2.5 mmol/L荜茇明碱抑制BMP2、p Smad1/5、RUNX2蛋白和mRNA的表达;④外源性重组BMP2可逆转荜茇明碱抗钙化的作用。结论:荜茇明碱通过抑制BMP2/pSmad1/5/RUNX2信号,减轻高钙高磷诱导的AVIC钙化。

Objective:To explore the role and mechanism of piperlongumine in high calcium/phosphate-induced calcification of aortic valve interstitial cells(AVICs). Methods:Porcine AVICs were isolated and cultured in vitro. AVICs were treated inductively with high calcium/phosphate medium and piperlongumine of different concentrations for 16 hours. Flow cytometry was then performed to measure cell apoptosis. AVICs were treated with 2.5 mmol/L piperlongumine or 100 ng/mL bone morphogenetic protein 2(BMP2)under the condition of high calcium/phosphate. The effect of piperlongumine on calcification was measured using calcium quantification,alkaline phosphatase activity and alizarin red staining. The protein and mRNA levels of osteogenic markers were determined by Western blot and real-time PCR. Results:① Flow cytometry showed that piperlongumine under 5 mmol/L had no obvious effect on AVICs apoptosis treated with high calcium/phosphate medium. ② Calcium quantification,alkaline phosphatase activity and alizarin red staining showed that 2.5 mmol/L piperlongumine attenuated the calcification of AVICs induced by high calcium/phosphate medium. ③Western blot,real-time PCR showed that 2.5 mmol/L piperlongumine inhibited the protein and mRNA expression of osteogenic markers including BMP2,phosphorylated Smad1/5 and RUNX2. ④ Exogenous recombinant BMP2 may reverse the effect of piperlongumine in vitro. Conclusion:Piperlongumine attenuates high calcium/phosphate-induced AVICs calcification by inhibiting BMP2/pSmad1/5 signaling.