Effect And Mechanism Of Calcitriol On Osteoblastic Differentiation In Porcine Aortic Valve Interstitial Cells Induced By Lipopolysaccharide

Objective: To investigate the effect and mechanism of calcitriol on osteoblastic differentiation in porcine aortic valve interstitial cells(PVICs)induced by lipopolysaccharide(LPS),and to provide theoretical and experimental support for intervention and treatment of calcified aortic valvular disease.Methods: The normal group valvular tissues were taken from patients with surgical treatment of aortic dissection and the CAVD group valvular tissues were taken from patients undergoing aortic valve replacement because of calcified aortic valve stenosis.Runx2,OPN and TGF-β1 protein levels by western blot and immunohistochemistry.The porcine aortic valve interstitial cells(AVICs)were isolated by continuous collagenase digestion from healthy pigs.Its phenotypes were identified by immunofluorescence staining.LPS(4ug/ml)was employed to induce osteogenic differentiation.In PVICs,early and late osteogenic differentiation abilities were determined by Alkaline phosphatase(ALP)staining and Alizarin red Sstaining.Runx2 and OPN mRNA levels by RT-qPCR and Runx2,OPN,TGF-β1,TGF-βR,p-Smad2/3 protein levels by western blot.LPS(4μg/ml)and calcitriol(0.1μmol/L)was employed to treat PVICs and osteogenic differentiation abilities were determined by Alkaline phosphatase(ALP)staining and Alizarin red S staining.Runx2 and OPN mRNA levels by RT-qPCR and Runx2,OPN,TGF-β1,p-Smad2/3 protein levels by western blot.Result: In human valve tissue,the expression of Runx2,OPN and TGF-β1 in CAVD group was significantly higher than that in non-CAVD group(P < 0.01).PVICs were successfully isolated and the staining ofα-smooth muscle actin(α-SMA)and Vimentin were positive,the staining of von Willebrand factor(vWF)was negative.In PVICs,LPS(4μg/ml)treatment increased the alkaline phosphatase activity,the deposition of calcium salts,and the Runx2 and OPN mRNA levels(P<0.05).In addition to an increase in osteoblastic differentiation markers,LPS-treated PVICs exhibit significantly increased protein levels expression of TGF-β1,TGF-βR,Smad2/3(P <0.05).Finally,LPS(4μg/ml)and calcitriol(0.1μmol/L)treatment reduced the alkaline phosphatase activity,the deposition of calcium salts,and the Runx2 and OPN mRNA levels(P<0.01).In addition to an reduced in osteoblastic differentiation markers,Calcitriol-treated PVICs exhibit significantly reduced protein levels expression of TGF-β1 and Smad2/3(P<0.001).Conclusion: These results indicate LPS may be promote osteoblastic differentiation in PVICs,and calcitriol may be inhibited of osteoblastic differentiation in PVICs induced by LPS,and TGFβ1-Smads2/3 signaling pathway may play an important role.