| BackgroundIschemic heart disease is still the leading cause of mortality and disability worldwide.Although timely reperfusion is the most efficient treatment of this disease clinically,it can also lead to a further cardiomyocyte damage that is referred to myocardial ischemia/reperfusion(I/R)injury.However,there are little effective drugs that can protect myocardial from I/R injury.Matrine is a quinolizidine alkaloid compound that extracted from the traditional Chinese herb Radix Sophorae flavescentis.Studies have found that matrine exhibits various pharmacological activities including anti-tumor,anti-inflammatory,anti-allergic,anti-virus,anti-fibrosis、 antiarrhythmic effects and improving cardiac insufficiency.Studies demonstrated that matrine attenuated hepatic I/R injury as well as focal cerebral ischemic injury.Recently,it has been found that matrine exerts cardiovascular protective effects in isoproterenol-induced acute myocardial injury.However,the role of matrine in myocardial I/R injury remains not fully understood.The Janus kinase/signal transducer and activator of transcription(JAK/STAT)pathway plays an important role in mediating cardio-protection against I/R injury.Numerous data indicate that the JAK2/STAT3 signaling pathway is specificallyassociated with the prevention of myocardial I/R injury.Recent studies showed that matrine suppresses the growth of human chronic myeloid leukemia cells and cholangiocarcinoma cells via suppression of the JAK2/STAT3 signaling.However,the effect of matrine on the activation of the JAK/STAT signaling is still unknown in I/R-injured cardiomyocytes.Heat shock proteins(HSPs)are a family of stress response proteins that are found in all species exposure to stressful conditions.HSPs suppress the phosphorylation of some proteins and elevate the stability and function of these proteins.As a representative member of HSPs,HSP70 is proven to be a cardiac protective molecule and can be induced to inhibit cardiomyocyte apoptosis after myocardial I/R injury.Recent studies showed that HSP70 inhibited cardiomyocyte necroptosis through suppressing autophagy in myocardial I/R injury.Studies have shown that HSP70 can be activated by the JAK/STAT pathway;however,the relationship between HSP70 and the JAK2/STAT3 signaling pathway remains unknown in I/R-injured cardiomyocytes.Meanwhile,whether the function of matrine in myocardial I/R injury is associated with HSP70 expression has rarely been investigated.On the basis of the above data,we speculated that matrine may attenuate myocardial I/R injury.The first part of this study explored the protective effect of matrine on myocardial I/R by establishing the myocardial ischemia reperfusion model in vitro,and further revealed the protective effect of matrine on myocardial I/R by detecting changes in JAK2/STAT3 signaling pathway and HSP70.In the second part of this study,the regulation of matrine to JAK-STAT-HSP70 was observed by culture primary myocardial cells.First,to observe whether matrine has a protective effect on myocardial I/R injury,and secondly to observe whether matrine exerts protective effect on myocardial I/R injury by regulating the expression of HSP70 up-regulated by JAK2/STAT3 pathway,and to understand the molecular mechanism of matrine in myocardial I/R injury.This study is divided into the following two parts:Part Ⅰ The effect of matrine on myocardial I/R injury in SD rats and the regulation of the expression of JAK2-STAT3Methods(1)establishment of animal model: 24 male SD rats were randomly divided into4 groups,6 for each group.(1)Sham group(2)I/R group(3)I/R + matrine(50mg/kg)Group(4)I/R + matrine(100 mg/kg)group.To create a model of myocardial I/R injury in rats,the rats were subjected to ischemia for 30 minutes and then reperfusion for 24 hours.Rats in the sham group underwent the same procedure without LAD occlusion.(2)Treatment: rats in the I/R + matrine group were intraperitoneally injected with 50 mg/kg or 100 mg/kg(1 mL)of matrine every day for 7 days before I/R,and the rest mice were given the same volume of saline.(3)Identification of results: levels of Creatine Kinase-Myocardial Band(CK-MB)and Cardiac Troponin I(cTnI)were measured using an enzyme-linked immunosorbent assay kit.Infarct area was detected by chlorinated triphenyl tetraazole(TTC)staining.Expressions of HSP70,JAK2,p-JAK2,STAT3,p-STAT3,Bcl2 and Bax were detected by Western blot.Results(1)In rat models,CK MB and cTnI levels in myocardial cells were significantly increased in the I/R group compared with the sham group(P < 0.01),while in the matrine treatment group CK MB and cTnI were significantly reduced in a dose-dependent manner(P<0.01 compared with the I/R group).(2)The area of myocardial infarction in rats was analyzed by TTC staining,and the area of infarction induced by I/R injury was reduced by matrine treatment in a dose-dependent manner(P<0.01 compared with the I/R group).(3)Bax protein imprinting analysis detection,the expression of Bcl-2 and caspase 3 protein,the result shows the I/R injury significantly increased activation of caspase 3 levels and the proportion of Bax/Bcl-2(compared with the sham operationgroup,P < 0.0),and treatment of matrine in dose dependent manner reduce apoptosis related proteins Bax expression(compared with the I/R group,P < 0.01),quantitative Bax/Bcl-2,the ratio of activated caspase 3 levels(compared with the I/R group,P <0.01).(4)The protein levels of HSP70,p-JAK2,JAK2,p-STAT3 and STAT3 were measured after treatment with either 50mg/kg or 100mg/kg of matrine in I/R animal models.The results showed that the expression of HSP70 was significantly increased in I/R group.Myocardial I/R injuries resulted in decreased p-JAK2/JAK2 and p-STAT3/STAT3,(compared with the sham operation group,p <0.01).While the expression of HSP70 was significantly increased in a dose-dependent manner by matrine(p <0.05 or p <0.01 in I/R group).while p-JAK2/JAK2 and p-STAT3/STAT3 were significantly increased after treatment with matrine(p <0.05 or p <0.01 compared with the I/R group).Part Ⅱ the regulation of matrine on primary cultured cardiac myocytes JAK2-STAT3-HSP70Methods:(1)Myocardial cells were isolated and cultured in sprague-dawley(SD)rats within 24 hours and the cells were treated with matrine of different concentrations(0,50,100,200,400,600,800uM).Myocardial cells treated with Hypoxia/Reoxygenation(H/R).The cells were treated with matrine of different concentrations(200,400uM),and after 4 hours of hypoxia,then incubated in a culture box containing normal oxygen for 6 hours for reoxygenation.The experiment was divided into 4 groups and comparison group.(1)Control group;(2)H/R group;(3)H/R+200uM matrine group;(4)H/R+400uM matrine group.The activity of cardiac myocytes was determined by Cell Counting kit-8(CCK8).The activity of LDH and creatine kinase(CK)was assessed by enzyme-linked immunoassay.Western blot was used to detect Bcl2,Bax and Active Caspase 3 protein levels and the expression of Bax/Bcl2 ratio.(2)To observe the effects of JAK2/STAT3 pathway blocker AG490 on the H/R effect of matrine on myocardial cells.The experiment was divided into 3groups.(1)H/R group;(2)H/R+400 uM matrine group;(3)H/R+400 uM matrine+2uM AG490 group.Activity of lactate dehydrogenase(LDH)and creatine kinase(CK)was assessed using an enzyme-linked immunoassay.The level of HSP70 mRNA was detected by PCR.The expression levels of HSP70,Bcl2,Bax and Active Caspase 3and the ratio of Bax/Bcl2 were detected by Western blot.(3)To observe the effect of HSP70 siRNA blocking HSP70 on H/R effect of matrine on myocardial cells.The experiment was divided into 3 groups.(1)H/R group;(2)H/R+400 uM matrine group;(3)H/R+400 uM matrine+HSP70 siRNA group.Patients were in the HSP70 siRNA group.HSP70 siRNA was transfected into myocardial cells for 24 H,followed by H/R stimulation.The cells were cultured in culture medium with or without 400 uM matrine,and after 4 hours of hypoxia.They are incubated in an aerobic culture box for 6 hours and then reoxygenation.Lactate dehydrogenase(LDH)release and creatine kinase(CK)activity were assessed using an enzyme-linked immunoassay.Detection of HSP70 mRNA level by real-time fluorescent quantitative PCR;Expression of HSP70 protein was detected by Western blot.Results:(1)Matrine protects cardiomyocytes from injury induced by H/R in vitro.After treatment with different concentrations of matrine,the myocardial cells of the original generation of rats were measured with CCK8 analysis.There is no effect on the viability of primary rat cardiomyocytes treated with matrine at a concentration of50-400 μM.Myocardial cells were treated with 200 uM or 400 uM matrine,and after 4hours of hypoxia,then incubated in an oxygen culture box for 6 hours before reoxygenation.Cell viability was measured with CCK8 and it was found that the viability of myocardial cells treated with 200 uM or 400 uM matrine was significantly increased(P<0.05 or P<0.01 compared with the hypoxia/reoxygenation group).LDH release levels(C)and CK activity(D)were measured(P<0.05 or P<0.01 comparedwith the hypoxia/reoxygenation group or control group).(2)Protein imprinting was used to analyze the protein level of Bax,bcl-2 and active cysteine protease 3,the ratio of Bax/bcl-2 and the quantitative level of active cysteine protease 3(P<0.01 compared with the hypoxia/reoxygenation group or the control group).(3)Matrine inhibits H/R induced myocardial cell damage by activating JAK2/STAT3 signaling.The expression levels of p-JAK2,JAK2,p-STAT3 and STAT3 were detected by western blot analysis,and the proportion of p-JAK2/JAK2 and p-STAT3/STAT3 was analyzed(p <0.05 or p <0.01 compared with control group or H/R group).(4)The myocardial cells were pretreated with the JAK2 inhibitor AG490(2u M)30 minutes before H/R treatment,and the whole H/ R process was incubated,and the LDH content was determined and the activity of CK was analyzed.While in the matrine treatment group LDH and CK were significantly reduced in a dose-dependent manner(P<0.01 compared with the H/R group).AG490 can reverse the effect of matrine(P<0.05 or P<0.01 compared with matrine group or H/R group).(5)Protein imprinting analysis of Bax,bcl-2 and active cysteine protease 3levels;The quantitative value of Bax/bcl-2 ratio in different treatment groups and the level of active cysteine protease 3 in different treatment groups.AG490 can reverse the effect of matrine(P<0.05 or P<0.01 compared with matrine group or H/R group)(6)HSP70 mediates the effect of matrine on H/R induced myocardial cell injury.HSP70 mRNA expression and HSP70 protein level in myocardial cells were measured(P<0.05,or P<0.01).Myocardial cells were transfected with the control siRNA or HSP70 siRNA and the protein level of HSP70 was measured(P<0.01 compared with the control group).Myocardial cells were transfected with HSP70 siRNA and then stimulated by H/R,and the cells were cultured in culture medium containing 400 uM matrine,and the LDH level and CK activity of myocardial cells were detected(with H/R group,P<0.01).HSP70 siRNA can reverse the above effect of matrine(P<0.01)(7)the expression of HSP70 was increased by activation of JAK2/STAT3 pathway by matrine.The myocardial cells were pretreated with the JAK2 inhibitorAG490 2uM 30 minutes before H/R treatment,and the whole H/R process was incubated.HSP70 mRNA level and HSP70 protein expression were measured(P<0.01 compared with H/R group).AG490 can reverse the effect of matrine(P<0.01 compared with matrine group)Conclusions(1)matrine has the protective effect of myocardial I/R injury;The molecular mechanism of matrine in myocardial I/R injury mainly focuses on the activation of JAK2/STAT3 pathway and the expression of HSP70.(2)the protective effect of myocardial I/R injury of matrine is blocked by JAK2 inhibitors AG490 and HSP70 siRNA,and AG490 can reduce the expression of increased HSP70 of matrine.(3)matrine can alleviate myocardial cell I/R injury by activating the JAK2/STAT3 pathway and up-regulating the expression of HSP70. |
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