The Mechanism Of Endoplasmic Reticulum Stress Pathway Induce Rabbit Valvular Interstitial Cells To Transdifferentiate Into Osteoblast-like Cells

Background:In recent years,with the improvement of social living standards,the aging population is also increasing,which is also particularly important for the study of elderly diseases.Aortic valve calcification disease(Calcific Aortic Valve Disease,CAVD)is the most common senile degenerative valve disease,with aortic valve stenosis,closure insufficiency,and heart failure in its progression to the final stage,which all seriously endanger the patient’s life.At present,surgery is an important treatment to valve calcification.The replacement valves are mainly biological valves and mechanical valves,but because the operation itself also has a load on elderly patients,how to seek the conventional treatment of valve calcification is of great significance.As awareness of CAVD grew,consensus from previous age degenerative lesions was a multifactor active regulatory process.In recent years,endoplasmic mesh stress has been found involved in the lesion process of CAVD,but its specific mechanism is unknown.Objective: The role of endoplasmic reticulum stress in valvular osteogenic transformation was observed by using OX-LDL to stimulate aortic valve interstitial cells to establish the phenotype of valve osteogenesis and endoplasmic reticulum stress inhibitor TUDCA was used to observe the role of endoplasmic reticulum stress in valvular osteoid transformation.Method :1.male,2.5kg,New Zealand rabbits were anesthetized with 25% uratan and their hearts were taken,the heart valves were separated,heart valve stromal cells were cultured,cellular immunofluorescence were used to identified;2.when the VICs reached to 70-80%of the plate,we subcultured it until 3-8 generations which was regarded as stable,gave100μg/ml OX-LDL and cultured 0,1,3,7days,RT-q PCR detected the m RNA changes of bone morphogenetic protein BMP2;3.VICs were harvested after OX-LDL stimulation for 7days,western-blot were used to mesure the changes of BMP2 protein levels;4.VICs were harvested after OX-LDL stimulation for 7 days,we observed the protein changes of endoplasmic reticulum stress-related proteins PERK,downstream effector proteins CHOP,transcription regulators ATF4;5.Applying endoplasmic reticulum stress inhibitors(TUDCA)and grouped by CTL,OX-LDL,OX-LDL+TUDCA,TUDCA,VICs were collected to verify endoplasmic reticulum stress related protein PERK and osteogenic protein BMP2 m RNA and ERS related protein level(perk/atf4/chop)and BMP2 changes.Results :1.The isolated valvular interstitial cells showed α-SMA(+)/ Vimentin(+)/ v WF(-)in immunofluorescence staining;2.OX-LDL stimulated VICs different time(0,1,3,7 days),the expression levels of osteoblastic protein BMP2 m RNA increased on Day 1,3 and 7 in contrast with CTL;3.7 days after OX-LDL stimulated VICs,the protein expression of osteogenic protein BMP2 was elevated;4.7 days after OX-LDL stimulated VICs,raised the expression of endoplasmic reticulum stress related proteins PERK 、 downstream effector proteins CHOP 、 transcription regulators ATF4;5.OX-LDL TUDCA group could reduce the increase of VICs BMP2 and PERK m RNA and protein levels to a certain extent compared with CTL group,Ox-LDL can also alleviate the increase of VICS PERK,ATF4,CHOP,and BMP2 protein levels(P<0.05).Conclusion: Endoplasmic reticulum stress signaling pathway is involved in OX-LDL-induced rabbit valve interstitial cells calcification.Using TUDCA to inhibit endoplasmic reticulum stress can improve the progression of aortic valve calcification in rabbits.