AMPK/PGC-1α/PPARα/UCP2 Pathway Of Hibernating Myocardium And The Effect Of Regulating Qi And Activating Blood Therapy

Cardiovascular diseases are most life-threatening disease all over the world.Once the myocardial necrosis occurs,the necrotic myocardium will remain as scar tissue and lose the contractile capability permanently,and ultimately develop into heart failure.seriously affect the long-term outcome and quality of life of patients with cardiovascular disease.How to protect the myocardium before the ischemia had not fully leads to myocardial necrosis is an important key to preserve the function of the ischemic heart.Hibernating myocardium(HM)refers to the myocardial dysfunction due to the reduced perfusion of the coronary artery.This is a protective phenomenon that HM compromised between myocardial oxygen consumption and myocardial function.The myocardial dysfunction of HM can probably recover by restoration of coronary blood flow and optimized energy metabolism.HM widely exists in many kinds of cardiovascular diseases,such as stable angina pectoris,unstable angina pectoris,myocardial infarction,heart failure etc.HM can exist in the ischemic area,either it can exist in the border area of infarcted myocardium.How to guarantee the viability and recover the contractile function of HM is the key problem of improving the cardiac function of patients who suffer from cardiovascular diseases.So far,the clinical researches on HM are rare,and most of them resulted in a negative conclusion.HM belongs to "Xiong bi" in traditional Chinese medicine(TCM),based on its theory of Qi and Blood,TCM showed positive results on "Xiong bi".Regulating Qi and activating Blood therapy is a commonly used and effective therapy,it can relieve the symptoms of angina,improving the cardiac function,increase the quality of life,improve the long-term prognosis.The mechanism includes anti-inflammatory effect,anti-platelet effect,activating the angiogenesis,regulating the function of endothelial,optimize the cell energy metabolism etc.Among these,the mechanism that how TCM improve the energy metabolism is not well established so far.This research will target on the energy metabolism of HM,observe the changes of HM and the effect of regulating Qi and activating Blood TCM on HM cardiac function and mitochondrial function,especially on uncoupling protein 2(UCP2)and its up-stream proteins such as AMPK/PGC-1α/PPARα.The complex of Panax notoginseng,Salvia miltiorrhiza,Dalbergia odorifera oil which play the role of regulating Qi and activating Blood were chosen as the intervention method.This research includes following parts:1 Literature reviews.Respectively review the advances of HM on TCM and western medicine,including current understanding of HM,therapeutic strategies,and the relationship between regulating Qi and activation Blood and HM.Also review the change of energy metabolism and mitochondria function of HM,and pharmacological agents targeting myocardial metabolism.2 Guanxin Danshen dropping pills on coronary heart disease:a systematic review and meta-analysisObjective:Systematically review the effect and safety of Guanxin Danshen dropping pills(GXDS)which composed of Panax notoginseng,Salvia miltiorrhiza,Dalbergia odorifera oil on coronary heart disease.Method:Computerized searching in PubMed,Cochrane Library,Embase,MEDLINE,Chinese national knowledge infrastructure(CNKI),VIP information database,wanfang data,Chinese Biomedical Literature Database(SinoMed)was done.Select the randomized controlled tials(RCTs)according to the inclusion and exclusion criteria.The main outcome is major adverse cardiovascular events(MACE),the second outcomes include symptom of angina pectoris,improvement of electrocardiogram,frequency of angina attack,duration of angina pectoris,reduction of nitroglycerin,adverse reactions.Data extraction were done by two researchers separately.The data were synthase using Revman 5.3.Results:After the selection,eleven RCTs were retrieved in this study,involving 1253 patients(intervention group:control group = 643:610).The overall methodological quality of RCTs were low according to Cochrane handbook risk of bias tool.Reduction of MACE(RR:0.42;95%CI 0.19 to 0.90 P =0.02),alleviation of angina pectoris symptoms(RR:1.18;95%CI 1.11 to 1.25 P<0.00001),improvement of ECG(RR:1.18;95%CI 1.08 to 1.30 P =0.0005)were observed in this study.The rate of non-inferiority in adverse effects were not statistically significant between two groups(RR:0.87;95%CI 0.06 to 13.38 P =0.92).Conclusion:Dropping pills composed of Panax notoginseng,Salvia miltiorrhiza,Dalbergia odorifera oil are beneficial to CHD patients as adjunct therapy,however,more rigorously designed,and high-quality RCTs are still needed due to the generally low methodological quality of the included studies.3 Experimental researches:Part.l Regulating Qi and activating Blood therapy on HM and its effect on cardiac function and high energy phosphate compoundObjective:To establish the bama mini pig HM model,observe the functional changes and the high energy phosphate compound level of HM,evaluate the effect of regulating Qi and activating Blood therapy on HM.Method:Hibernating myocardium of pig model(18-23kg)were established by a thoracotomy approach,after anesthetized,the hearts were exposed,the hibernating myocardium group receive Ameriod constrictor(2.5mm)on the left anterior descending coronary artery and the sham group only underwent the thoracotomy approach without placement of a Ameriod constrictor.Four weeks later,regular echocardiography and dobutamine stress echocardiography were performed to evaluate the cardiac function(LVEDV,LVESV,LVIDd,LVIDs,EF%,FS%,WT%)and detect the hibernating myocardium(through wall motion score index,WMSI)for further analysis.At last sixteen pigs were survived and judged as HM model.Randomly divided into four groups,they are Sham,Model,TMZ and GXDS group.Sham and Model group were fed by normal fodder.The TMZ and GXDS add Trimetazidine and Guanxin Danshen dropping pill to the normal fodder.After six weeks,regular echocardiography and dobutamine stress echocardiography were performed again.Animals were sacrificed and mitochondria were extracted immediately.Transmission electron microscopy,HE staining,PAS staining were done to observe the histopathology of HM.High performance liquid chromatography were done to quantify the high energy phosphate compound.Results:(1)The EF%and WT%of HM were significantly decreased compared with Sham group(P<0.05).Six weeks after intervention,the EF%and WT%of Model group decreased further.While the EF%and WT%of TMZ and GXDS groups were increased,and the difference was statistically significant(P<0.05).(2)Before intervention Model,TMZ,GXDS groups respectively has 16(25%),14(21.85%)and 13(20.31%)section with abnormal ventricular wall movement,mainly distributed in the left anterior descending branch suppling area,during DSE 12,14,12 sections which showd biphasic response were judged as HM.After intervention,the Model,TMZ,GXDS groups respectively have 20(31.25%),11(17.19%)and 10(15.62%)section with abnormal ventricular wall movement,during DSE 7,9,8 sections showd a biphasic response and judged as HM.(3)Histopathology of HM:Transmission electron microscopy observation showed the myocardium of sham group presented the integral and regularly distributed sarcomeres,normal size and shape of mitochondria.The myocytes of HM showed disorganization and loss of contractile material and mitochondria,inhomogeneity on the shape and size of mitochondria,an occurrence of slight nonspecified cytoplasm,destruction of mitochondrial cristae.HE staining showed the normal myocardium showed regularly arranged and integral myocardium.However,the HM had dark small nuclear,disordered myocardium observed in intercellular substance,with inflammatory cells infiltration.PAS staining showed loosely arranged and enlarged hypertrophied myocytes in HM as compared with the normal myocardium,increased glycogen in HM compare with the normal myocardium.The intervention of TMZ and GXDS partially suppressed the above-mentioned histopathologic changes.(4)The result of high performance liquid chromatography showed that in HM ATP,ADP,TAN,EC were significantly decreased compared with Sham group,the difference was statistically significant(P<0.05).The level of AMP was virtually unchanged(P>0.05).In GXDS group and TMZ group,the level of ATP and ADP were higher than Model group,leading to the increase of EC,and the difference was statistically significant(P<0.05).TAN did not show a statistically significant increase(P>0.05).Conclusion:The cardiac function of HM significantly decreased along with abnormal ventricular wall movement.The high energy phosphate compound and energy reservation of HM were poor.Intervention with GXDS can improve the cardiac function and energy level of the HM.Part.2 Regulating Qi and activating Blood therapy on HM mitochondrial function and AMPK-UCP2 pathwayObjective:To establish the bama mini pig model of HM,observe the HM mitochondrial function and AMPK/PGC-la/PPARa/UCP2 pathway,and investigate the mechanism of the results gained from part 1.Method:Hibernating myocardium of pig model(18-23kg)were established by a thoracotomy approach,after anesthetized,the hearts were exposed,the hibernating myocardium group ’receive Ameriod constrictor(2.5mm)on the left anterior descending coronary artery and the sham group only underwent the thoracotomy approach without placement of a Ameriod constrictor.Four weeks later,dobutamine stress echocardiography were performed to detect the hibernating myocardium(through wall motion score index,WMSI)for further analysis.At last sixteen pigs were survived and were judged as HM model and randomly divided into four groups,they are Sham,Model,TMZ,and GXDS group.Sham and Model group were fed by normal fodder.The TMZ and GXDS add Trimetazidine and Guanxin Danshen dropping pill to the normal fodder.After six weeks dobutamine stress echocardiography were performed again.After sacrifice,mitochondria were extracted immediately.The activity of mitochondria respiratory chain complexes Ⅰ,Ⅱ,Ⅲ and ATP synthase were tested.The mitochondrial membrane potential(MMP)were tested via the JC-1 method.Western blotting technique were processed to quantify the protein level of AMPK,pAMPK,PGC-1α,PPARα,UCP2.Real Time PCR were done to quantify the gene level of AMPK,PGC-1α,PPARα,UCP2.The Statistical analysis was performed using SPSS.Results:(1)The activity of complex Ⅰ,complex Ⅱ,complex Ⅲ in Model group were reduced compared with the normal myocardium(Sham),the intervention with GXDS and TMZ could improve the activity of complex Ⅰ and complex Ⅲ compared with Model group,the difference was statistically significant(P<0.05).The activity of complex Ⅱwere increased but did not show the statistical difference(P>0.05).(2)The activity of ATP synthase were reduced in Model group compared with the Sham group,the difference was statistically significant(P<0.05).TMZ could enhance the activity of HM,but there was no statistical difference(P>0.05)between Model gourp and TMZ group.GXDS group showed increased activity of ATP synthase,the difference was statistically significant(P<0.05)compared with Model group.(3)The MMP of each group showed the following result.The MMP of Model group were decreased significantly compared with Sham group(P<0.05),the difference was statistically significant.The intervention of TMZ and GXDS suppress the loss of MMP,the changes were statistically significant(P<0.05).(4)The protein expression of AMPK,pAMPK,PGC-1α,PPARα,UCP2 were detected by western blotting technique,the result showed that the protein level of AMPK and pAMPK were increased in HM,and the down-stream protein including PGC-1α,PPARa,UCP2 were up-regulated,the difference was statistically significant compared with Sham group(P<0.05).The ratio of pAMPK/AMPK was increased in HM(Sham vs Model=78.57 ± 9.25%vs 91.93 ± 1.61%),the difference was statistically significant(P<0.05).In TMZ group and GXDS group,the ratios of phosphorylated AMPK were down-regulated(81.0 9 ± 1.75%and 80.51 ± 2.12%),there were statistical difference compared with Model group(P<0.05).The UCP2 and the up-stream proteins such as PGC-1α,PPARα,UCP2 were down-regulated in TMZ group and GXDS group,there were statistical differences compared with Model group(P<0.05).(5)The Real Time PCR was performed to observe the mRNA level of AMPK,PGC-1α,PPARα,UCP2 pathway,HM showed up-regulated mRNA of and upstream regulators,including AMPK,PGC-1α and PPAR compare with Sham group,the difference was statistically significant(P<0.05).TMZ and GXDS could decrease the level of AMPK,PGC-1α,PPARα,UCP2 gene level compare with no treatment,the difference was statistically significant(P<0.05).Conclusion:The activity of complex Ⅰ,complex Ⅱ,complex Ⅲ were decreased in HM,AMPK/PGC-1α/PPARα/UCP2 pathway were activated,lead to the loss of MMP along with the decreased activity of ATP synthase.GXDS could increase the activity of complex Ⅰ and complex Ⅲ,suppress the activation of AMPK/PGC-1α/PPARα/UCP2 pathway,up-regulate the MMP,increase the electrochemical gradient of ischemic myocardium,as the result,increase the activity of ATP synthase.