Studies On The Effects And Mechanism Of Melatonin On Myocardial Apoptosis In Atherosclerosis Rabbits

Objective To explore whether melatonin protect myocardial tissue of experimental atherosclerosis rabbits and the possible mechanism.Methods(1)Rabbit Model of Atherosclerosis: Common purebred New Zealand white rabbits were randomly divided into 3 groups: normal group: normal feed;model group: high fat diet(normal feed + 1% cholesterol + 5% Lard);melatonin group: fed high-fat diet and treated with MLT 10 mg/(kg·d)intragastrically.One week after purebred big-eared white rabbits were adapted for feeding,they were induced by high-fat diet and treated with melatonin.After 12 weeks,rabbits were sacrificed to get blood,detection in serum lipid indexes.Artery oflipid plaque was observed by oil red staining.Heart tissue was taken paraffin-embedded HE staining and Masson staining were used to observe the cytoplasmic nuclear staining and distribution of collagen fibers in myocardial tissue;TUNEL was used to observe the apoptosis of myocardial tissue;immunohistochemistry(IHC)was used to detect CHOP.Caspase-12 the endoplasmic reticulum stress-related protein expression levels.In addition,in order to investigate its possible molecular mechanism,the expression of apoptosis-related proteins(Caspase-9,Caspase-3,bcl-2,bax)and endoplasmic reticulum stress-related proteins(GRP78,CHOP,PERK,ATF6?,ATF4,Caspase-12)expression were detected by Western blot.(2)Cultured rat cardiomyocytes H9c2: H9c2 cells were cultured with palmitate as a model.The effects of palmitic acid on the proliferation of H9c2 cells were detected by MTT assay,and the effects of palmitic acid and melatonin on cell apoptosis were detected by Hoechst 33258 staining.Western blot was used to detect the related protein levels of ERS pathway related proteins(CHOP,PERK and ATF4).Results AS rabbit models were copied successfully.The results showed that the model group with a large number of lipid plaques that the model was successful.The results of lipid profile showed that total triglycerides(TG)was(6.947 ± 2.382)mmol /L and total cholesterol(TCH)was(55.279±14.913)mmol / L in model group,both were significantly higher than normal group,whereas the melatonin group decreased compared with the model group.The results of HE staining showed that the cytoplasm of myocardial cells in the red part of the rabbits in the model group was unevenly stained,and there was a breakage of myocardial fibers,which could be improved after treatment with MLT.Masson staining showed that the rabbits in the model group exhibited a large number of abnormally accumulating blue collagen fibers also showed uneven dyeing.By TUNEL staining under fluorescence microscopy,the number of apoptotic cells in the model group was red,and the number decreased after treatment with MLT.IHC find that MLT could down-regulate the expression of CHOP,Caspase-12 protein in rabbits with AS.The normal and treatment groups were light brown or colorless.Western blot showed that the expression of these protein PERK,ATF6?,ATF4,GRP78,CHOP,and Caspase-12 were significantly increased,and the apoptosis-related protein Csepase-3,Casepase-9,bax expression increased,the same phenomenon also appears in the phosphorylation level of JNK.After treatment with MLT in AS rabbits,the expression of related protein were down-regulated.(2)H9c2 cell model of palmitate culture: high concentrations of palmitate stimulation can inhibit the proliferation of H9c2 cells,and increase with increasing concentrations in a certain range;Hoechst 33258 staining showed that high concentration of free fatty acids could induce apoptosis of H9c2 cells,while cells with MLT treated had no obvious apoptosis.Western blot results showed that the expression of ERS-related proteins CHOP,PERK and ATF4 were significantly increased in H9c2 cells afterr palmitate stimulation for 24 h compared with the normal group.However,in the cells treated with MLT at the same time,the expression of these proteins was significantly decreased compared with PA treatment group.Conclusions The atherosclerotic rabbit myocardial tissue is structurally altered and has an increase in apoptosis.The mechanism may be the imbalance of oxidative stress caused by elevated blood lipids,resulting in endoplasmic reticulum stress response via CHOP,Casepase-12,JNK pathway activation is regulated by apoptosis-related proteins;MLT protects atherosclerotic rabbit heart by lowering blood lipids and reducing myocardial cell apoptosis.