Effect Of All-Trans Retinoic Acid On Myocardial Cells In Atherosclerotic Rabbits And Its Mechanism

Objective To investigate the effects of all-trans-retinoic acid(ATRA)on cardiomyocyte in atherosclerosis(AS)and explore its possible mechanisms.Methods(1)Establishment of animal model:24 normal purebred white rabbits,normal rabbits were given normal rabbit feed;model group was given high fat diet,high fat diet consisted of normal rabbit feed,1% cholesterol,5% lard;the rabbits of the ATRA group was given high fat diet,at the same time,feed with the drug of ATRA 5 mg/(kg.d)every day.Collected serum,artery,heart tissue of each group after 12 weeks,serum was used for blood lipid testing;oil red staining was used to observe atherosclerotic plaques;heart tissue was embedded in paraffin sections,morphological changes of myocardial tissue was detected by HE staining and Masson staining;Td T-mediated d UTP-biotin nick end labeling(TUNEL)was used to detect apoptosis of myocardial tissue in each group;mmunohistochemical staining was used to detect the expression of Caspase-12,CHOP,Caspase-3 in each group.Western blot analysed the expression of MLCK and ERK protein phosphorylation levels,and also detected apoptosis-related proteins(Caspase-12,Bax,Bcl-2,JNK,p-JNK),endoplasmic reticulum stress-related proteins(ATF-6?,PERK,ATF)-4,CHOP)expression levels in the hearts of rabbits in each group.(2)Induction of cell model: Used the palmitic acid(PA)to stimulate H9c2 cells.MTT was used to detect the inhibition rate of different concentrations of palmitic acid on H9c2,and determined the optimal concentration of palmitic acid.After the stimulation of PA,MTT was used to detect the survival rate of ATRA,and determined the optimal concentration of ATRA.Cultured the H9c2 in vitro and divided into normal group,model group,ATRA group and ERK/MAPK selective inhibitor(PD98059,PD) group.The expression of MLCK protein in each group was detected by Western blot.Cultured the H9c2 in vitro and divided into normal group,solvent control group,model group and ATRA group.Hoechst33258 staining method was used to detect the degree of apoptosis in cardiomyocytes;expression levels of related apoptosis-related proteins(Caspase-12,JNK,p-JNK)and endoplasmic reticulum stress-related protein(CHOP)in H9c2 cells were detected by Western blot.Results(1)Establishment of animal model: A large number of oil red plaques appeared in the rabbit arteries in the model group,and the serum total cholesterol and total triglyceride levels increased significantly.The above results suggest that the model is successful..After the intragastric administration of ATRA,the atherosclerosis was significantly relieved.HE staining showed that the myocardial morphology and nuclear arrangement of the ATRA group were significantly less disordered than the model group.Masson staining showed that the ATRA treatment could effectively improve the accumulation of collagen fibers in myocardial tissue;TUNEL showed that the number of apoptotic cells in the model group was higher than that in the ATRA group;the immunohistochemical results showed that the expression of Caspase-12,CHOP,Caspase-3 in the ATRA group was much lower than that in the model group;The results of Western blot showed that the expression of MLCK protein and phosphorylation levels of ERK in heart tissue of rabbits were promoted after fed with high fat diet,and the expression of Caspase-12,JNK,p-JNK and Bax were significantly higher than those in normal group.MLCK,p-ERK,Caspase-12,JNK,p-JNK,Bax was down-regulated after the treatment of ATRA.,and the expression of anti-apoptotic protein Bcl-2 was increased.(2)Induction of cell model:MTT results showed that the ability of PA to inhibit cell proliferation increased with increasing concentration within a certain concentration range.After ATRA culture,it effectively slowed the inhibition of cell proliferation by PA,and finally chose the concentration of 0.1m M PA which inhibition rate was close to 50%.and chose the concentration of 10?M ATRA which the survival rate was highest;the expression of MLCK decreased after ATRA and PD administration.the results of Hoechst33258 staining showed that the cells showed more apoptotic bodies after apoptosis by PA,and apoptotic bodies were effective reduction after the culture of ATRA.Western blot results showed that the expression of endoplasmic reticulum stress-related protein(CHOP)and apoptosis-related proteins(Caspase-12,JNK,p-JNK)was significantly increased in cells stimulated with palmitic acid.Related proteins are all reduced.Conclusions The disorder of myocardial tissue structure and the increase of cardiomyocyte apoptosis in atherosclerotic rabbits may be due to the activation of apoptosis-related proteins by the increased expression of MLCK in the heart which triggers the activation of ERK/MAPK pathway and endoplasmic reticulum stress pathway.ATRA may protect atherosclerotic cardiomyocytes by regulating the expression of ERK/MAPK pathway and endoplasmic reticulum stress-related proteins in atherosclerotic heart.