Cardioprotective Effect And Mechanism Of Allicin On Myocardial Ischemia In Rat

Myocardial ischemia is one of the primary cause of harm to human health,and the higher incidence of myocardial infarction result from it is an important factor to cause rising mortality.Myocardial ischemia can induce the production of oxygen radicals in myocardium,the oxidative stress injury from which can further aggravate the injury of myocardial ischemia.Meanwhile,the ventricular remodeling after myocardial infarction is the main pathological basis of the cardiac function.Myocardial hypertrophy,inflammation and fibrosis were considered to be the main pathological features of compensatory repair tissue after myocardial infarction,and these features can lead to a further decline in the heart function.At present,the therapeutic method for myocardial ischemia is finite and effectiveness.Therefore,it is of great significance to find a safe and effective therapeutic drug.Allicin is generally regarded as the main active substance of garlic.But the research about it is rare in China for the reason that we regard garlicin as the main active substance of garlic.Allicin has been studied extensively for its beneficial properties to the inflammation,neoplasms and oxidative stress.The aim of this study was to determine whether allicin produced cardioprotective effect to myocardial ischemia and exhibit beneficial effects to myocardial fibrosis and cardiac fibroblasts.Objective1.Identify the cardioprotective and anti-oxidant effect of allicin administration after myocardial ischemia.2.Investigate effect of long term allicin application on cardiac remodeling and function after myocardial ischemia,and clarify the effect of allicin on myocardial fibrosis and TGFβ/Smads signaling pathway after myocardial infarction.3.Clarify the effect of allicin on cardiac fibroblast proliferation,differentiation and collagen secretion,and further research the effect of allicin on cardiac fibroblast electrophysiology.Methods1.Cardioprotective effect of allicin on acute myocardial infarction250 Wistar rats were randomly divided into seven groups.Then the rat model of myocardial ischemia was replicated using coronary artery ligation method.The allicin was administrated by intravenous injection immediately after the operation.Rats were sacrificed after 24h and the hearts and serum were collected to examine the following indexes:serum troponin(cTnⅠ),myoglobin(Mb),muscle acid kinase isoenzyme(CK-MB)and oxidative stress index,including SOD,MDA,CAT and GSH-Px.The hearts were used to examine myocardial infarct size by NBT staining.2.Effect of allicin on cardiac remodeling and myocardial fibrosis after myocardial infarction60 Wistar rats were randomly divided into six groups.Then the rat model of myocardial ischemia was replicated using coronary artery ligation method.The drugs was administrated by intraperitoneal injection from the second day after the operation for 21 days.Then ultrasonic test was performed to examine the structure and function of the heart.After that,the rats were sacrificed and the hearts were excised to observe the extent of cardiac fibrosis by masson staining and examine the type Ⅰ,Ⅲ collagen,TGFβ1,Smad3 and Smad7 expression by immunohistochemistry.3.Effect of allicin on rat cardiac fibroblast.Neonatal rat cardiac fibroblasts was isolated and cultured in vitro,then the fibroblast induced by Ang Ⅱ,while giving the allicin three dose intervention.After 24h,the cell proliferation was detected by MTS,immunofluorescence staining was used to detect the expression of intracellular alpha-SMA,Western blot and qRT-PCR were used to detect collagen expression,patch clamp was used to record current density of intermediate-conductance Ca2+-activated potassium channel(Kca3.1),and Western blot and qRT-PCR were applied to determine the effect of allicin on the expression of Kca3.1 protein and mRNA.Results1.The cardioprotective effect of allicin on acute myocardial infarction in rats.(1)Myocardial infarction size and serum indexes in myocardial infarction.Compared with the myocardial infarction group,allicin treatment(1.2,1.8,3.6mg/kg)significantly decreased myocardial infarction area(P<0.01).Meanwhile,allicin(3.6mg/kg)significantly decreased serum cTnⅠ and Mb level and allicin(1.8mg/kg)decreased serum cTnⅠ level(P<0.05).Compared with myocardial infarction group,the serum CK-MB and Mb have the tendency of decreasing,but the discrepancy of CK-MB was not significant.The serum Mb of allicin high dose group was significantly decreased compared with model group(P<0.05).(2)Oxidative stress indexes of acute myocardial infarction.The SOD and GSH-Px level of serum in allicin treatment groups had increasing inclination,but there was no significant difference between groups(P>0.05).Compared with myocardial infarction group,allicin treatment(1.2,1.8,3.6mg/kg)significantly increased serum CAT level,and allicin(1.8mg/kg)decreased serum MDA level significantly(P<0.05).2.Effect of long term application of allicin on cardiac remodeling and myocardial fibrosis in rats(1)Echocardiogram results of ratsCompared with myocardial infarction group,allicin(1.2mg/kg,1.8mg/kg,3.6mg/kg)significantly increased LVAWd/s,EF,FS and SV(P<0.05)and decreased LVIDd/s(P<0.05).(2)Masson stain and expression of collagen Ⅰ and ⅢMasson stain showed increased fibrous tissue in model group,and after allicin treatments the fibrous tissue decreased.Meanwhile,compared model group,allicin treatment can significantly decrease collagen Ⅰ,Ⅲ expression(P<0.05).(3)The changes of TGFβ/Smads signaling proteinsCompared with control,the expression of TGFβ1 and Smad3 increased significantly(P<0.05),while the expression of Smad7 decreased(P<0.05).After treatment of allicin,the expression of TGFPi and Smad3 was inhibited,and the expression of Smad7 was increased(P<0.05).3.Effect and mechanism of allicin on cardiac fibroblast(1)Effect of allicin on cardiac fibroblast proliferation and differentiationAt the level of cell morphology,the cells were adherent and grew into stellate cells with no spontaneous pulsation.After 24h,control group were relatively rare.After stimulated by Ang Ⅱ,the cell number increased.The cell number in allicin groups significantly decrease compared with Ang Ⅱ groups.The MTS results showed that compared with the normal myocardial fibroblast,cell viability was significantly increased in Ang Ⅱ group,and after allicin intervention cell activity of Ang Ⅱgroup decreased significantly(P<0.01).Immunofluorescence staining showed that in Ang II group,a-SMA expression in cardiac fibroblasts significantly increased,and given allicin intervention,the a-SMA expression was significantly decreased.(2)Effect of allicin on expression of collagen Ⅰ and collagen Ⅲ in cardiac fibroblasts.After Ang Ⅱ stimulation,the expression of collagen type Ⅰ and Ⅲ mRNA and protein was significantly higher than that of control group(P<0.05).Compared with the Ang Ⅱ group,allicin(5μM and 1μM)significantly down regulate the expression of collagen Ⅰ and Ⅲ mRNA and protein(P<0.01).(3)Effect of allicin on Kca3.1 of cardiac fibroblasts.① Firstly,the patch clamp was used to record potassium current of whole cell.The results showed that the current density of whole cell potassium current in Ang Ⅱgroup significantly increased,the current density of allicin in high dose group could significantly inhibit the whole cell potassium current.There is no significant difference between the low and middle dose group group and Ang group Ⅱ.Activation curve of potassium channel current density displayed allicin had no effect on activation of potassium channels of allicin.② Kca3.1 is of significant role in proliferation and differentiation of cardiac fibroblasts.In order to prove if the Kca3.1 was included in the current that allicin affected,Kca3.1 blocker TRAM-34 was applied after the whole cell potassium current was recorded.So TRAM-34 insensitive current was recorded,and the results of whole potassium current minus TRAM-34 insensitive current were TRAM-34 sensitive current which is the Kca3.1 current.Compared with the normal group,Ang Ⅱsignificantly increased potassium ion current density of Kca3.1 in rat cardiac fibroblasts,application of allicin in high dose(5μM)decreased the current density of Kca3.1 significantly(P<0.05).The activation curve showed no significant effect of allicin on Kca3.1 activation properties of Kca3.1.(4)Effect of allicin on expression of Kca3.1 mRNA and protein in cardiac fibroblasts.For clarify the mechanism of effect of allicin on Kca3.1,expression of mRNA and protein of Kca3.1 was tested using qRT-PCR and Western blot.The results showed that AngⅡ unregulated expression of Kca3.1 mRNA and protein in cardiac fibroblasts(P<0.01),and after the application of allicin,the expression of Kca3.1 decreased significantly(P<0.05).(5)The relationship of down regulate of Kca3.1 and H2S signaling moleculesH2S is a gas signaling molecules that exhibit cardioprotective and anti-fibrosis effect.Allicin can generate H2S and protect myocardia.Through the application of H2S synthase inhibitor PAG,we found that PAG can inhibit effect of allicin on the expression of KCa3.1 protein and mRNA.After the application of PAG,down regulation of KCa3.1 induced by allicin was canceled,and there was.significant difference compared with the allicin group(P<0.05),indicating that the regulation of allicin on KCa3.1 is related with H2S pathway.Conclusion1.Allicin has cardioprotective effect in myocardial infarction models when administrated immediately after the myocardial ischemia.Allicin can significantly reduced infarct size,decrease serum myocardial infarction sign level and improve the indicators of oxidative stress.2.Allicin administration after myocardial infarction can improve cardiac function,inhibit the development of myocardial fibrosis,the mechanism of which may be related to the regulation of TGFβ1/Smad signaling pathway.3.Allicin can inhibit myocardial fibroblast proliferation and differentiation at the cellular level and down regulate expression of collagen Ⅰ and Ⅲ mRNA and protein expression.4.Allicin also inhibit KCa3.1 cell current density,up regulate the expression of KCa3.1 channel,but does not affect the activation properties of KCa3.1.The regulation of allicin on KCa3.1 channels may be achieved by H2S signaling.