| Objectives:1.To compare the effects of different current intensity of electroacupuncture preconditioning(EAP)against myocardial ischemia reperfusion injury(MIRI),and explore the relative suitable intensity of EAP against MIRI.2.To explore the relationship between mTORC1 and mitophagy in the protective effect of EAP against MIRI.3.To explore the mechanism of EAP based on mTORCl-ULK1-FUNDC1 signaling pathway to reduce mitophagy in MIRI rats.Methods:1.120 specific pathogen free(SPF)grade adult male sprague-dawley(SD)rats were randomly divided into 8 groups of 15 rats and were categorized as:a)model group(I/R group)b)model+non-EAP control group(I/R+SEA group)c)model+EAP with 0.2 mA current intensity for 4d(I/R+0.2 mA group)d)model+EAP with 1 mA current intensity for 4d(I/R+1 mA group)e)model+EAP with 2 mA current intensity for 4d(I/R+2 mA group)f)model+EAP with 4 mA current intensity for 4d(I/R+4 mA group)g)model+EAP with 6 mA current intensity for 4d(I/R+6 mA group)h)model+EAP with 8 mA current intensity for 4d(I/R+8 mA group).Each EAP group was intervened 4 days(d)before MIRI operation and PC6 rats were selected for 20 minutes of EAP(2/100Hz)once a day.The I/R+SEA group was anesthetized with isoflurane in the same manner and simultaneously with the other groups of rats,with the exception of undergoing EAP.The left anterior descending branch(LAD)of the coronary artery was ligated on the 5th day for 30 minutes,and reperfusion was performed for 4 hours in each group to establish a model of myocardial ischemia reperfusion injury.Simultaneously,the electrocardiography(ECG)monitoring was conducted.The body weight of all the rats were monitored daily and the ventricular arrhythmia scores(VAS)were calculated after 4 hours of reperfusion.Survival curves of the rats in each group were also analyzed.The area ratio of myocardial infarction(Infarct size,IS%)and the risk area(Risk size)was calculated by Evans blue-TTC double staining.The concentrations of serum creatine kinase(CK),creatine kinase isoenzyme(CK-MB),lactate dehydrogenase(LDH)and troponin T(cTnT)were measured by enzyme-linked immunosorbent assay(ELISA).2.On the basis of determining the optimum current intensity of electro-acupuncture pretreatment for MIRI,Experiment 2 and experiment 3 were carried out.140 SPF adult male SD rats were randomly divided into 5 groups:sham operation group(Group S),with the same operation as model group except for the LAD ligation;sham operation+electro-acupuncture pretreatment group(Group S+EA),the operation was same as that of the model+electro-acupuncture pretreatment group with the exception of LAD ligation;the model group(I/R group),the operation was the same as the modeling method in method 1;the model+non electro-acupuncture pretreatment group(I/R+SEA group),on the skin surface of PC6 on both sides of rats,stuck self-made bamboo signet with the same size as electro-acupuncture for 20 min/time/d,for 4d,and the model was made on the 5th day;the model+electro-acupuncture pretreatment group(I/R+EA group),electro-acupuncture was performed once a day(2mA,2/100Hz,20min),for 4d,the model was made on the 5th day.28 rats/group.experiment 3:196 SPF adult male SD rats were randomly divided into 7 groups:I/R group;I/R+EA group;blank control group(group B),without intervention;model+negative control pretreatment group(I/R+C Group),with intraperitoneal injection of the same amount of cosolvent as that in the inhibitor group,once a day for 4 days,and the model was made on the 5th day;model+negative control+electro-acupuncture pretreatment group(I/R+C+EA group),electro-acupuncture was performed once a day,with intraperitoneal injection of the same amount of cosolvent as that in the inhibitor group,for 4 days,and the model was made on the 5th day;model+mTORC1 inhibitor(rapamycin)pretreatment group(I/R+R group),with intraperitoneal injection of Rapamycin,once a day,3.0 mg/kg,for 3 days,and the model was constructed 24 hours after the last injection;the model+mTORC1 inhibitor+electro-acupuncture pretreatment group(I/R+R+EA group),electro-acupuncture was performed once a day for 4 days;On the second day of electro-acupuncture,rapamycin was given by intraperitoneal injection once a day for 3 days,and the model was made 24 hours after the last injection.There were 28 rats in each group.The body weight of the rats was monitored every day,and the survival curves of the rats in each group were analyzed after the end of the experiment;ECG was used to detect changes in the ST segment of the heart,and VAS statistics were performed in the first 20 minutes of the ischemia period and the first 20 minutes and 220-240 minutes of the reperfusion period;the myocardial infarction area was observed by Evans blue-TTC double staining method,and the IS%and Risk size were calculated;The serum CK-MB,LDH and cTnT levels were measured by ELISA;DUTP notched terminal marker(TUNEL)mediated by terminal deoxynucleotidyl transferase was used to detect the apoptosis of cardiomyocytes,evaluate the survival of cardiomyocytes,and fully evaluate the cardiac function.On the basis of fully understanding the protective effect of EAP on MIRI,the level of autophagic vesicles was observed by transmission electron microscopy;the mitochondria,lysosomes and their combination were detected by immunofluorescence double-label staining to evaluate the correlation between the anti-MIRI effects of EAP and mitophagy.The distribution and expression level of LC3-Ⅰ(mito-LC3-Ⅰ)and LC3-Ⅱ(mito-LC3-Ⅱ),mTORC1,ATG13,ULK1,p-ULK1,FUNDC1,p-FUNDC1 and other substances in myocardial mitochondria were detected by Western blotting(WB)and Immunohistochemistry(IHC)respectively;the ATP production of mitochondria in left ventricular myocardium was detected by rat energy(ATP)ELISA Kit,and the potential mechanism of EAP regulating mitophagy through mTORCl-ULK1-FUNDC1 signaling pathway to reduce the MIRI was analyzed.Results for Objective 1:1.Effects of EAP with different current intensity on body weight and post-operative survival curve of MIRI ratsThe EAP rats showed a normal growth trend in body weight when the applied current intensity was 0.2 mA,1 mA and 2 mA.In contrast,EAP rats treated with 4 mA or more showed a decrease in body weight over the same period.In addition,analysis of the survival curve obtained indicated that EAP rats treated with 0.2 mA,1 mA and 2 mA showed a higher postoperative survival rate whereas the overall survival rate of groups treated with 4 mA,6 mA and 8 mA were noticeably lower,indicating the possibility of a protective effect by EAP on MIRI when the current intensity lies 0.2 mA,1mA,2 mA.Conversely,EAP conducted with≥ 4mA current intensity might have more harmful effects on rats with MIRI.2.Anti-MIRI effects of EAP at different current intensityThe results of VAS,Evans blue-TTC double staining and serum myocardial zymogram showed that EAP could effectively reduce MIRI at different current intensity.VAS results showed that EAP(I/R+ 2mA group)could effectively reduce the the VAS score of ventricular arrhythmias(P<0.05).Evans-blue TTC double staining showed that EAP(I/R+0.2 mA group,I/R+1 mA group,I/R+2 mA group,I/R+4 mA group,I/R+6 mA group,I/R+8 mA group)significantly reduced IS%and risk size(P<0.05),and the IS%and Risk size in I/R+2 mA group were the smallest(P<0.05).Compared with I/R+2 mA group,the IS%in I/R+4 mA group,I/R+ 6 mA group and I/R+8 mA group increased(P<0.05),and the IS%in I/R+6 mA group and I/R+8 mA group increased significantly(P<0.01),Compared with I/R+2 mA group,the risk size in I/R+ SEA group,I/R+ 0.2 mA group,I/R+1 mA group,I/R+6 mA group and I/R+ 8 mA group increased(P<0.05).The results of serum myocardial zymogram showed that EAP with different current intensity could effectively reduce the levels of serum CK(P<0.05),CK-MB(P<0.05),LDH(P<0.05),cTnT(P<0.05).The myocardial zymogram level in the I/R+2 mA group was the lowest and the effect was the best.Based on the results obtained,it can be concluded that the applied current intensity at 2 mA has the best protective effect against MIRI and is optimum for EAP to combat MIRI.Therefore,2 mA was selected for further studies.Results for Objective 2:Study on the effect of EAP on mitophagy in MIRI rats1.Effect of EAP on body weight and survival curve after MIRI operationResults of body weight and postoperative survival curves of rats in each group indicated that EAP treatment could promote steady weight gain and effectively improved the survival rates after MIRI operation.2.Effects on VAS,Evans blue-TTC stainingThe results showed that EAP could effectively reduce VAS(P<0.05),IS%(P<0.05),risk size ratio(P<0.05).3.EAP reduced MIRI-induced myocardial injury markers and apoptosisThe results showed that,the levels of serum CK-MB(P<0.05),LDH(P<0.05)and cTnT(P<0.05),reduce cardiomyocytes apoptosis(P<0.05),thus inhibiting MIRI and protecting the heart.4.EAP attenuated MIRI-induced mitophagyThe results showed that EAP could effectively reduce the level of autophagic vesicles(P<0.05)and reduce the binding of mitochondria and lysosomes(P<0.05),thus reducing the level of mitophagy and exerting a protective effect on MIRI.5.EAP regulated the expression of mTORC1,ULK1 and FUNDC1 proteinWB and IHC were used to detect the proteins mTORC1,ULK1,FUNDC1 in myocardial tissue.The results showed that EAP could promote the expression of mTORC1 protein and inhibit the expression of the proteins related to mitophagy,such as ULK1,FUNDC1(P<0.05).Results for Objective 3:The role of EAP in regulating mitophagy to resist MIRI via the mTORCl-ULK1-FUNDC1 signaling pathway1.Rapamycin blocked the cardioprotective effect of EAPThe protective effect of EAP on MIRI was negated when the mTORC1 protein was inhibited by the addition of rapamycin.This suggested that mTORC1 may play a key role in the anti-MIRI effects of EAP.When rapamycin was used to inhibit mTORC1 protein,these protective effects of EAP on MIRI were eliminated.The results showed that I/R+R+EA group could induce VAS(P<0.05),IS%(P<0.05),risk size ratio(P<0.05),the levels of serum CK-MB(P<0.05),LDH(P<0.05)and cTnT(P<0.05),induce cardiomyocytes apoptosis(P<0.05),thus deteriorating MIRI.2.Rapamycin attenuated EAP-inhibited mitophagyHowever,the inhibitory effect of EAP on MIRI mitophagy was eliminated after the inhibition of mTORC1 protein by rapamycin.The results showed that I/R+R+EA group could increase the level of autophagic vesicles(P<0.05)and increase the binding of mitochondria and lysosomes(P<0.05),thus increasing the level of mitophagy and exerting a anti-protective effect on MIRI.3.EAP inhibited mitophagy through the mTORC1-ULK1-FUNDC1 signaling pathway.WB and IHC were used to detect the proteins related to mTORC1-ULK1-FUNDC1 signaling pathway:mTORC1,ATG13,p-ULK1,ULK1,p-FUNDC1,FUNDC1 in myocardial tissue and mito-LC3Ⅰ,mito-LC3Ⅱ in mitochondria.The results showed that EAP could promote the expression of mTORC1 protein and inhibit the expression of the proteins related to mitophagy,such as ATG13,p-ULK1,ULK1,p-FUNDC1,FUNDC1,mito-LC3Ⅱ/Ⅰ(all,P<0.05).However,these regulatory effects of EAP were inhibited after the inhibition of mTORC1 protein by rapamycin.It was observed that mitophagy induced by FUNDC1 protein eliminated mitochondria excessively,hindering the production of ATP that serves as an energy source for the cell,which could potentially lead to an ATP/energy production disorder.This absolute deficiency of mitochondria in the cardiomyocytes would then increase the apoptosis rate of the cardiomyocytes(P<0.05).Conversely,the usage of EAP treatment on PC6 could reverse the situation whereby the EAP applied could promote mitochondrial production of ATP(P<0.05),further preventing MIRI.Nevertheless,the usage of rapamycin would inhibit the promotional effect of EAP.Conclusion:1.EAP treatment with different current intensity can effectively prevent MIRI,whereby EAP with 2 mA current intensity showed the best protection effect.2.The effect of EAP on reducing MIRI may be related to the promotion of mTORCl protein expression in myocardial tissue and the down-regulation of ULK1 and FUNDC1 protein expression,thereby inhibiting the occurrence of mitophagy.It shows that mTORC1 may play a key role in EAP anti-MIRI.3.Rapamycin can eliminate the myocardial protective effect of EAP on MIRI rats,and the protective effect of EAP may be achieved through the mTORC1-ULK1-FUNDC1 signaling pathway that regulates mitophagy,thereby increasing mitochondrial-derived ATP. |
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