The Effect And Mechanism Of Dihydrotanshinone Ⅰ On Osteogenic Phenotypic Transition Of Aortic Valve Interstitial Cells Induced By Osteogenic Medium

Objective:To explore the effect and mechanism of dihydrotanshinone I(DHI)on the osteogenic phenotypic transition of primary porcine valve interstitial cells(PVICs)induced by osteogenic medium(OM).Provide theoretical and scientific basis for clinical intervention in calcific aortic valve disease(CAVD).Methods:3 cases of calcified aortic valves and 2 cases of noncalcified aortic valves were selected,and the expression of osteogenic indicators Runx2,OPN and inflammation indicators IL-1β and p-NF-κB were detected by immunohistochemical staining(IHC).Healthy porcine aortic valve interstitial cells(PVICs)were isolated in vitro and phenotype identification was performed by immunofluorescence.Through OM induced osteogenic phenotype transition of PVICs,CCK8 method determines the optimal concentration and time of dihydrotanshinone I(DHI)intervention.Early calcification is detected by alkaline phosphatase staining(ALP staining),and late osteogenesis changes are indicated by Alizarin Red S staining.q PCR and Western blot were used to detect the changes in nucleic acid and protein levels of the osteogenic indicators Runx2 and OPN.Western blot was used to detect the changes of NF-κB,ERK1/2 and Smad1/5/8 pathways.Results :In calcified aortic valves,the expression of osteogenic indicators(Runx2,OPN)and inflammation-related indicators(IL-1β,p-NF-κB)were significantly up-regulated compared with the normal control group.The porcine aortic valve interstitial cells(PVICs)were successfully isolated,v WF was negative,α-SMA was positive and vimentin was positive.The experiment was divided into Blank,OM,OM+DMSO and OM+DHI.On the basis of the osteogenic phenotypic transformation of PVICs induced by OM,the expression of osteogenic indicators Runx2 and OPN was increased in the OM group and OM+DMSO group compared with the blank group.ALP staining and Alizarin Red S staining showed calcium deposition increase.After DHI(10μM)treatment,compared with OM group and OM+DMSO group,the expression levels of calcification indicators Runx2 and OPN were down-regulated,ALP staining and Alizarin Red S staining showed that calcium salt deposition decreased,and the phosphorylation levels of NF-κB,ERK1/2 and Smad1/5/8 were decreased.Conclusions : DHI(10μM)treatment can reverse the osteogenic phenotypic transition of PVICs induced by osteogenic medium,and the mechanism may be related to NF-κB、ERK 1/2 and Smad1/5/8 pathways.