| BackgroundAcute myocardial infarction(AMI)is an ischemic heart disease with arrhythmia,pain,heart failure,shock and other symptoms.AMI is one of the main causes of death of human diseases,which seriously endangers human health.In recent years,the incidence rate of our country is also increasing.The difficulties in treatment and high medical costs increase the sufferings of patients,but also bring heavy economic pressure to the family and society.The effective treatment of AMI is to reconstruct the blood circulation of the ischemic site.Thrombolysis,coronary intervention and other methods are the effective methods to treat AMI.However,reperfusion after myocardial ischemia can not improve and recover the patients,but aggravate the myocardial injury.In the stage of myocardial ischemia-reperfusion,there are many causes of myocardial stress injury,including arrhythmia,reactive oxygen species injury,calcium overload,inflammatory cell infiltration,etc.The damage caused by the restoration of blood supply on the basis of myocardial ischemia is called myocardial ischemia-reperfusion injury.The prevention and treatment of MIRI is of great significance in the treatment of acute myocardial infarction.Myocardial cells need high energy metabolism,so they have more mitochondria.After myocardial ischemia,many reasons cause mitochondrial damage.Mitochondrial autophagy removes damaged mitochondria.On the one hand,it avoids the release of apoptosis promoting factors such as reactive oxygen species and cytochrome c into the blood,on the other hand,it provides material basis for energy metabolism of cardiomyocytes.However,in the stage of reperfusion after myocardial ischemia,with the recovery of blood flow,the damage of reactive oxygen species,calcium overload,inflammatory cell infiltration and so on can aggravate the mitochondrial damage.Mitochondrial autophagy is over activated,and the over activated mitochondrial autophagy increases the death of myocardial cells.Mitochondrial autophagy is a double-edged sword for MIRI.Its timely and moderate occurrence is closely related to the survival and death of cardiomyocytes.FUNDC1 is a mitochondrial membrane receptor protein,which plays an important role in hypoxia induced mitochondrial autophagy.Through the binding of LIR domain and LC3,it can promote the recognition of damaged mitochondria by autophagy precursor and mediate the occurrence of mitochondrial autophagy.Shuangshen Ningxin capsule(SSNX)has the effect of Invigorating Qi,promoting blood circulation,removing blood stasis and relieving pain.It is clinically used to treat "chest obstruction" and "heartache" of qi deficiency and blood stasis syndrome.SSNX can prevent MIRI by inhibiting the decrease of mitochondrial membrane potential,the opening of mitochondrial membrane permeability transition pore and the release of cytochrome C,promoting the activity of mitochondrial complex I,reducing the swelling rate of mitochondria induced by Ca2+,reducing the apoptosis of mitochondrial pathway and protecting the functional structure of mitochondria.It is suggested that SSNX may play a regulatory role in mitochondrial autophagy.It was found that there are 29 kinds of active components in SSNX,and many of them can regulate autophagy,such as ginsenoside Rg1(Rg1)and salvianolic acid B(Sal B).In this paper,Rgl,Sal B and tetrahydropalmatine(THP)were selected to study the material basis.Purpose and SignificanceTo explore the role of SSNX in the regulation of mitochondrial autophagymediated by FUNDC1 in MIRI.And to determine whether Rg1,THP and Sal B arepart of the material basis of SSNX regulates of FUNDC1 mediated mitochondrialautophagy to protect MIRI in rats.Although a large number of literatures have reported a lot of drugs against myocardial ischemia-reperfusion injury,at present,there is no corresponding drug for clinical treatment of this link,and there is no way to completely resist myocardial ischemia-reperfusion injury,so it is still necessary to further explore the treatment target and drugs of myocardial ischemia-reperfusion injury.Part I The mechanism of Shuangshen Ningxin capsule protecting myocardial ischemia/reperfusion injury in rats1.1 Protective action of Shuangshen Ningxin capsule on myocardial ischemia/reperfusion injury in ratsObjectiveTo explore the protective action of SSNX on myocardial ischemia-reperfusion injury in ratsMethodsThe rats were divided into three groups:45mg□kg-1,90mg□kg-1 and 180mg□kg-1 of SSNX,normal group and model group.After 7 days of intragastric administration(sham operation group and control group were given the same amount of normal saline),ligating the left anterior descending coronary artery to establish the model of myocardial ischemia-reperfusion injury.The ligation time was 45 minutes and the reperfusion time was 3 hours.The indexes of left ventricular end diastolic pressure,left ventricular peak pressure,the maximum rate of left ventricular pressure decrease and the maximum rate of left ventricular pressure rise were measured by hemodynamics.Serum CK,CK-MB and LDH were detected by serology.Double staining of TTC and Evan’s blue in myocardial tissue to observe the changes of myocardial infarction area in rats.Myocardial tissue HE staining observes the morphological structure and inflammation-of myocardial cells in rats.ResultsThe results showed that the contents of CK,LDH and CK-MB in the model group were higher than those in the normal group.Compared with the model group,SSNX significantly reduced the serum CK-MB content of rats,the content of serum CK,LDH in middle dose group and LDH in low dose group was reduced.The results of hemodynamics showed that HR、DBP、MAP、LV+dp/dtmax、LV-dp/dtmax in the model group were significantly lower than those in the normal group.Compared with the model group HR,SBP、DBP、MAP、LVSP、LVEDP、LV+dp/dtmax、LV-dp/dtmax were all increased.HE staining showed that normal group myocardial tissue structure was complete,most of myocardial fibers were normal and arranged in bundles,myocardial cells were arranged in order,nuclear morphology was mild.In the model group,there were swelling and rupture of some myocardial fibers,necrosis of focal cardiomyocytes,infiltration of neutrophils under epicardium,congestion of capillaries between muscle fibers and infiltration of inflammatory cells.Myocardial eosinophilic,wavy fiber and contraction band decreased in SSNX group.In SSNX group myocardial eosinophilic,wavy fiber and contraction band decreased.The large and medium dose group of SSNX has a better effect on myocardial pathological changes.TTC and Evan’s blue double staining showed that the ratio of ischemic area to total myocardial area was stable at about 50%.No obvious white staining area was found in the myocardium of normal rats.Compared with the noramal group,the white staining area in the model group was larger.The area of white staining area in the middle dose group was significantly smaller than that in the model groupSummaryThe establishment of myocardial ischemia-reperfusion injury model was stable.SSNX has protective action on myocardial ischemia-reperfusion injury model in rats.The protective action of SSNX at 90mg□kg-1 and 180mg□kg-1 was better than that at 45 mg□kg-1.The follow-up mechanism was explored by selecting the dose of 90 mg□kg-1.1.2 Shuangshen Ningxin capsule mediates FUNDC1 pathway to regulate mitochondrial autophagy and protect myocardial ischemia/reperfusion injury in ratsObjectiveSSNX mediates FUNDC1 pathway to regulate mitochondrial autophagy to protect rat myocardial MIRIinjury.MethodsThe rats were divided into three groups:the normal group and the model group.After 7 days of intragastric administration(sham operation group and control group were given the same amount of normal saline),the left anterior descending of ligated coronary artery was observed and the model of myocardial ischemia-reperfusion injury was established.The ligation time was 45 minutes and the reperfusion time was 3 hours.Macroscopical characterization was used to detect the tongue coating and pulse of rats,to observe the effect of SSNX on the tongue and pulse of rats;Serum cTnT content of rats was detected by serology;various indexes of heart function of rats were detected by small animals by ultrasound to evaluate the improvement of heart function from the perspective of imaging;the microstructure of rat heart tissue was observed by hematoxylin eosin staining;LC3-Ⅰ,LC3-Ⅱ and cleaved were detected by wersing blot Cleavrd caspase-3,BNIP3,NIX,FUNDC1 expression.ResultsThe changes of the pulse in the normal group showed that the pulse was smooth and powerful with moderate amplitude and even rhythm.Compared with the normal group,the pulse amplitude of the model group decreased significantly,which was not fluent enough,the communication was difficult,the amplitude became smaller and the rhythm became faster.Compared with the model group,SSNX group can increase the pulse amplitude and relieve the pulse condition of rats.In the normal group,the tongue was reddish and the fur was thin and white.Compared with the normal group,the tongue in the model group was darker red,and the coating was darker white,and the R,B and G values of the tongue surface were reduced.Compared with the model group,the rats in SSNX group showed improvement in dark red tongue and dark white fur,and the values of R,G and B on the tongue surface were increased.The level of cTnT in serum of model group was significantly higher than that of normal group.The content of cTnT in serum of SSNX group was significantly lower than that of model group.Compared with the normal group ejection fraction and short axis shortening fraction decreased significantly,and the end systolic diameter,end systolic volume,end diastolic diameter and end diastolic volume increased in the model group.Compared with the model group,the ejection fraction and short axis shortening fraction increased,and the end diastolic volume decreased in the SSNX group.Heidenhain staining showed that a small amount of punctate dark gray staining was seen in normal myocardial cells.In the model group,dark gray color could be seen that involved the whole layer of myocardium,and vasodilation and inflammatory cell infiltration can be seen.In the SSNX group,there were multiple dark gray staining in cardiomyocytes,some of which involved the whole myocardial layer,vasodilation and inflammatory cell infiltration got better.Compared with the normal group,the expression of LC3-Ⅱ/LC3-Ⅰ,cleared caspase-3 and FUNDC1 in the model group were increased.Compared with the model group,the expression of LC3-Ⅱ/LC3-Ⅰ,cleared caspase-3 and FUNDC1 in the SSNX group were decreased.However,there were no significant changes in mitochondria1 autophagy receptor protein NIX and BNIP3 in the model group as well as the SSNX group.SummaryThe increased level of mitochondrial autophagy during MIRI may be related to the activation of FUNDC1 pathway.BNIP3 and NIX mediated mitochondrial autophagy did not show any activation.SSNX has inhibitory effect on mitochondrial autophagy in myocardial cells during MIRI in rats.The protective action of SSNX on MIRI may be related to this inhibitory effect.Further study showed that the inhibition may be related to the inhibition of the activation of FUNDC1 pathway of mitochondrial autophagy,but no regulatory effect was found on BNIP3 and NIX mediated mitochondrial autophagy.Part Ⅱ Study on the mechanism of the effective components of Shuangshen Ningxin capsule Rgl,THP and Sal B protecting H9c2 cardiomyocytes from hypoxia/reoxygenation injury2.1 Study on the optimal protective dose of Rg1,THP and Sal B on the hypoxia/reoxygenation injury of H9c2 cardiomyocytesObjectiveTo determine the appropriate dose of Rg1,THP and Sal B for the subsequent protective effect and mechanism research.MethodsIn the experiment of the effect on the activity of H9c2 cardiomyocytes,we take H9c2 cardiomyocytes as the research object.The experiment was divided into normal group and drug dosage group.Normal group was culture with complete DMEM medium.Each drug group was cultured with DMEM medium containing drugs.The cells were seeded in 96 well cell plates at a density of 1×105·ml-1 for 36 hours,and then discard the old culture medium,wash the holes twice with PBS,then change the corresponding culture medium for each group,and conduct 24 hours normal culture in the constant temperature cell incubator with 37℃ and 5%CO2.After 24 hours of drug action,CCK-8 method was used to detect cell activity.In the experiment of the effect on the activity of H9c2 cardiomyocytes injured by hypoxia/reoxygenation,we take the model of H9c2 cardiomyocytes injured by hypoxia/reoxygenation as the research object.The experiment was divided into normal group,model group and drug dosage group.Normal group was cultured with complete DMEM medium.Model group was cultured with DMEM medium without sugar and oxygen,and no drugs were added in the process of anoxia and reoxygenation.All drug groups were added with test drug at the same time of hypoxia,and the rest process was the same as model group.The cells were inoculated with 1×105·ml-1 density in 96 well cell plate for 36 hours.Then discard the old medium and wash the holes twice with PBS,change the corresponding medium.The cells were cultured in an anoxic chamber filled with nitrogen for 4 hours.At the end of hypoxia,to make the final concentration of glucose 4.5g·L-1wwe add 49.5g·L-1 glucose 10μLto each hole.2 hours reoxygenation and glucose culture in a 37℃ 5%CO2 incubator.Then CCK-8 method for cell activity detection.ResultsThe results showed that Rg1,THP and Sal B at the concentrations of 800μmol□L-1,180μmol□L-1,180μmol□L-1 and above inhibited the activity of normal cultured cardiomyocytes.The results of the study on the effects of hypoxia/reoxygenation injury on the activity of H9c2 cardiomyocytes showed that compared with the normal group,the activity of cardiomyocytes in the model group was significantly decreased,compared with the model group,the three dose groups of Rg1 could significantly increase the activity of cardiomyocytes,when the concentration was 100 μmol□L-1,the cell survival rate was the highest.When the concentration of THP was 45μmol□L-1,the cell activity was significantly increased.Sal B can significantly increase cell activity in three dose groups,and the cell survival rate is the highest at 45μmol□L-1.SummaryWhen the concentration of Rg1 is 100μmol□L-1,THP 45 μmol□L-1,Sal B 45μmol□L-1,the protective effect of Rg1 on the hypoxia/reoxygenation injury of H9c2 cardiomyocytes is better.2.2 Protective actions of Rgl,THP and Sal B on hypoxia/reoxygenation injury of H9c2 cardiomyocytesObjectiveTo study the protective actions of Rg1,THP and Sal B on the H/R injury of H9c2 cardiomyocytes.MethodsH9c2 cardiomyocytes were divided into normal group,model group,Rg1 group,THP group and Sal B group.The methods of cell culture and the intervention measures as same as 2.2.After 2 hours of reoxygenation,to carry out index tests.The content of LDH in the supernatant of cells cultured in 96-well cell plate was detected by micro enzyme labeling method.The ROS level of cells cultured in 6-well cell plate was measured by chemical fluorescence method,and the fluorescence intensity was observed under the living cell workstation.ResultsIn this study,H9c2 cardiomyocyte H/R injury model was used to simulate myocardial ischemia-reperfusion injury.After H/R operation,the cardiomyocyte viability decreased,the LDH leakage and the intracellular ROS level increased.Rg1,THP and Sal B could reduce the LDH leakage and the ROS level of H9c2 cardiomyocytes after H/R injury.Rg1,THP and Sal B had obvious protective action on H9c2 cardiomyocytes after H/R injury.2.3 Rg1,THP,Sal B regulate FUNDC1 mediated mitochondrial autophagy to protect H9c2 cardiomyocytes from hypoxia/reoxygenation injuryObjectiveTo explore whether the protective actions of Rg1,THP and Sal B on H/R injury of H9c2 cardiomyocytes are related to the regulation of FUNDC1 mediated mitochondrial autophagy.MethodsThe establishment,grouping and administration methods of the model as same as those in 2.2.After 2 hours of reoxygenation,the ATP content in the cells inoculated in the 6-well plate was detected by fluorescein enzyme method,and the ΔΨm changes were detected by chemical fluorescence method.Observe the change of mitochondrial membrane potential in the living cell workstation.Western blot was used to detect the expression of cleaved caspase-3,LC3-Ⅰ,LC3-Ⅱ,FUNDC1.ResultsThe results showed that H/R injury decreased the ATP content and ΔΨm.Compared with the model group,the ATP content and ΔΨm in Rg1 group,THP group and sal B group were increased.Compared with the normal group,the ratio of LC3-Ⅱ/LC3-Ⅰ,the expression of cleaved caspase-3 and FUNDC1 were increased in the model group.Compared with the normal group,the ratio of LC3-Ⅱ/LC3-Ⅰ,the expression of cleaved caspase-3 and FUNDC1 were decreased in Rg1 group,THP group and sal B group.SummaryH/R injury caused the over activation of mitochondrial autophagy in H9c2 cardiomyocytes and induced the apoptosis of mitochondrial pathway.The over activation of mitochondrial autophagy was related to the mitochondrial autophagy mediated by FUNDC1.Rg1,THP and Sal B could increase the mitochondrial membrane potential,improve energy metabolism and protect H9c2 cardiomyocytes from H/R injury by inhibiting mitochondrial autophagy mediated by FUNDC1.ConclusionSSNX can protect myocardial ischemia-reperfusion injury by inhibiting mitochondrial autophagy mediated by FUNDC1.Rg1,THP and Sal B protect H9c2 cardiomyocytes from H/R injury by inhibit mitochondrial autophagy mediated by FUNDC1.It is speculated that Rg1,THP and Sal B are part of the material basis of SSNX regulating mitochondrial autophagy mediated by FUNDC1 to protect myocardial ischemia-reperfusion injury.SSNX is expected to be an effective therapeutic drug to regulate mitochondrial autophagy against myocardial ischemia-reperfusion injury. |
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