The Effect And Mechanism Of Autophagy On LPS- Induced Osteoblast-like Phenotype Transformation Of Porcine Aortic Valvular Interstitial Cells

Objective: To investigate the effect and mechanism of autophagy on osteoblast-like phenotype transformation of Valvular Interstitial Cells induced by LPS(lipopolysaccharide).Methods: To investigate the relationship between inflammation,autophagy and osteoblast-like phenotype transformation at the level of human aortic valve,the expression level of osteogenesis index(RUNX2,OPN),inflammation index(p-p65)and autophagy index(LC3,p62)in calcific(3 cases)and normal(3 cases)aortic valve samples were detected by immunohistochemistry.Then,porcine aortic valvular interstitial cells(pAVICs)(2~6 generations)were treated by LPS with concentration gradients and length of time gradients and expression of osteogenesis,inflammation and autophagy index were measured by Western blot.For estimating the formation of autophagosome,changes of LC3Ⅱ were determined by Western blot after Bafilomycin A1(Baf A1)treatment,an inhibitor of autophagolysosomes.To study effect of autophagy on osteoblastlike phenotype transformation in pAVICs,GFP-LC3 virus were transfected to observe green fluorescence distribution after autophagy agonists and inhibitors treatment,and Western blot was used to measure autophagic and osteogenic alterations.Changes of inflammation and osteogenesis were detected by Western blot after combined treatment of NF-κB inhibitors and LPS,which reminders whether NF-κB involved in LPS-induced osteoblastlike phenotype transformation.Western blot and immunofluorescence were used to detect p-p65 activation and nuclear translocation after autophagy agonists and inhibitors combined treatment with LPS,which indicates regulation of autophagy on inflammatory pathway.Results: The results suggested that the expression of the osteogenic,inflammatory and autophagy index in the lesion aortic valve specimens were higher than those in the normal control group.Western Blot results showed an up-regulation of osteogenesis index,autophagosome formation and activation of inflammatory pathway in primary pAVICs treated by LPS.Autophagy agonists and inhibitors could inhibit and promote LPS-induced osteoblast-like phenotype transformation of pAVICs,respectively.NF-κB inhibitors could alleviate LPS-induced phenotype transformation.Autophagy agonists and inhibitors could inhibit and promote the activation of NF-κB induced by LPS,respectively.Conclusions: It is possible that autophagy negatively regulates the osteogenic phenotype transformation of VICs induced by LPS,which may play a regulatory role by inhibiting the inflammatory pathway,NF-κB.