Analysis Of Caffeic Acid Para-nitro Phenylethyl Ester On Organ Injury And Expression Of Galectin-3 Related Proteins In Chronic Myocardial Ischemia Rats

Myocardial ischemia?MI?results from the decrease of myocardial blood flow,which leads to the abnormal regeneration of blood vessels,inflammation,fibrosis infiltration and cardiovascular dysfunction.Clinically,chronic myocardial ischemia?CMI?is common,with mild symptoms,slow formation and high incidence rate.TGF-?1?TGF-?1?and Galectin-3?Gal-3?are reported to be involved in apoptosis,fibrosis,inflammation and angiogenesis,and TGF-?1 can regulate the expression of Gal-3.They are both involved in heart disease related research,and Gal-3 is also used as a biomarker to predict heart failure.Caffeic acid p-nitro phenylethyl ester?CAPE-p NO₂?is a derivative of caffeic acid phenethyl ester?CAPE?designed and synthesized in our laboratory,which has protective effects on the organ function of diabetic cardiomyopathy mice,myocardial ischemia-reperfusion injury rats and other animal models.This research aimed to study the protective effect of CAPE-p NO₂ on the heart and lung of CMI rats,as well as the possible mechanism related to Gal-3 and its relating proteins from cell experiment and animal experiment.CAPE was used as positive control.Animal experiment: the CMI model was established by feeding SD rats with high fat-and-cholesterol diet for 8 weeks and intraperitoneal injection of vitamin D3?2000 U/kg/d?for 3 consecutive days at the third week.The level of blood lipid,ST segment elevation of electrocardiogram?ECG?and the degree of lipid deposition in aortic sections of randomly sampled rats were regarded as the basis for successful modeling.The experiment was divided into five groups: 1)control group,normal rats intraperitoneally injected with equal volume saline;2)CMI model group,model rats intraperitoneally injected with equal volume saline;3)CAPE group,model rats intraperitoneally injected with 1 mg/kg/d CAPE;4)the High-p NO₂ group,model ratsintraperitoneally injected with 1 mg/kg/d CAPE-p NO₂;5)the Low-p NO₂ group,model rats intraperitoneally injected with 0.7 mg/kg/d CAPE-p NO₂.After 4 weeks of treatments,the rats were euthanized after ECG measurement,and the levels of serum lipids?TC,TG,HDL-C and LDL-C?,serum myocardial enzymes?HBDH,LDH,CK and CK-MB?and serum Gal-3 were measured.The heart,lung and other tissues were collected.The results showed that the expression of Gal-3 and TGF-?1 increased in serum and tissues of CMI rats,and decreased after CAPE-p NO₂ treatment.Treatment with CAPE-p NO₂,the body weight,heart index and serum HDL-C level of CMI rats increased,while the lung index decreased;the serum TC,TG,LDL-C,HBDH,LDH,CK and CK-MB levels decreased;the heart rate was slowed down,and the elevation of ST segment of ECG decreased;the morphology of cardiomyocytes were also improved.CAPE-p NO₂ could reduce the collagen deposition in heart tissue and regulate the expression of downstream proteins of TGF-?1/Gal-3 pathway in heart and lung tissue,which was shown as down regulating the expression of MMP-9,Smad2,Col-I,Col-III,Bax and caspase 3,up regulating the expression of Bcl-2,and down regulating the expression of TNF-?,NF-?B and IL-6.These results indicated that CAPE-p NO₂ might improve the fibrosis and inflammation of heart and lung,as well as reduce the apoptosis of cardiac and lung cells by regulating TGF-?1/Gal-3pathway,and its effect was stronger than that of CAPE at the same dose.Cell experiment: The CMI cell model was established by stimulating H9c2 cardiomyocytes with lipopolysaccharide?LPS?.The specific method was to culture the cells with low glucose medium containing 1 ?g/m L LPS for 12 hours.The cells of treatment groups were pre-treated with drug containing medium for 12 hours before modeling.The cells were divided into 5 groups: 1)control group,cells cultured in high glucose medium for 24 h;2)CMI model group,cells cultured in high glucose medium for 12 h,and then cultured in low glucose medium containing LPS for 12hours;3)CAPE group,cells cultured in high glucose medium containing 20 ?M CAPE for 12 h,and then cultured in low glucose medium containing LPS for 12 h;4)in CAPE-p NO₂ group,cells cultured in high glucose medium containing 20 ?M CAPE-p NO₂ for 12 h,and then cultured in low glucose medium containing LPS for12 h;5)inhibitor group?-p NO₂ + MCP group?,cells cultured in high glucose medium containing 20 ?M CAPE-p NO₂ and 5 mg/ml MCP for 12 h,and then cultured in low glucose medium containing LPS for 12 h.The fluorescence intensity of Gal-3,TGF-?1,Col-I,Col-III,TNF-? and IL-6 in the cells were measured byimmunofluorescence test.Then the apoptosis of cardiomyocytes was detected by AO staining.Finally,the expression of proteins in cardiomyocytes was analyzed by WB assay.Similar to results of animal experiments,these results showed that in the model group,the expression of Gal-3 increased and then decreased after CAPE-p NO₂ treatment.After treatment with CAPE-p NO₂,the cardiomyocytes apoptosis decreased,the expression of Bcl-2 increased and the expression of Bax,caspase 3,Col-I,Col-III,TNF-? and IL-6 decreased.The effect of CAPE-p NO₂ was stronger than that of CAPE.The regulating effect of CAPE-p NO₂ on the proteins was influenced by MCP,the Gal-3 inhibitor.The results of cell experiments and animal experiments indicated that the level of endogenous Gal-3 increased with the development of CMI,and CAPE-p NO₂ could decrease the level of Gal-3.CAPE-p NO₂ played a protective role in alleviating the degree of inflammation,apoptosis and fibrosis in the heart and lung of CMI rats,and the effects might be through regulating Gal-3 expression and TGF-?1/Gal-3 pathway.