| Coronary Heart Disease(CHD)is an acronym for coronary atherosclerotic heart disease,also known as ischemic heart disease,one of the cardiovascular diseases that seriously threaten human health.Coronary heart disease is a common and frequently-occurring disease in the world,and the morbidity and mortality increase year by year,and the age of onset becomes younger in the average age.Therefore,prevention and treatment of coronary heart disease has become a major issue facing the medical community.Myocardial ischemia and hypoxia is an important pathological basis of coronary heart disease.Coronary heart disease belongs to the category of "heartache" and "chest" in traditional Chinese medicine.Traditional Chinese medicine believes that heart dystrophy and blood stasis is the most important pathogenesis of coronary heart disease.Coronary heart disease qi deficiency and blood stasis syndrome become the most common clinical syndromes.Qi deficiency is the symptom while blood stasis is the root cause.The onset process ismostly virtual and real,mixing with each other.Yiqi Huoxue Fang(YQHXF)is a formula granule developed by this group on the basis of ancient Fangyang Huanwu Decoction.It consists of Yiqi Recipe Huangqi,Huoxue Recipe Taoren,Honghua,Chishao,Chuanxiong,Danggui and Dilong.Compound traditional Chinese medicine has many advantages of multi-links,multi-targets,and multi-pathways in treating diseases.By deeply illustrating the function mechanism of traditional Chinese medicine compound and clarifying the function target of traditional Chinese medicine,a powerful basis and reference for clinical practice will be provided.In recent years,the role played by mitochondria in the development of cardiovascular diseases has become more and more eye-catching.Mitochondria are the center of cellular energy metabolism.Most of the energy is provided by mitochondria through oxidative phosphorylation in the form of ATP,which is the main source of ATP in cardiomyocytes with high energy consumption.Mitochondria are not only the site for energy supply,but also the center of cell signal transduction.They participate in a large number of signaling cascades and regulate important biological functions such as intracellular calcium homeostasis,ROS production,cell apoptosis,and autophagy,thereby regulating the cell's survival and death.Under ischemic and hypoxic conditions,there will be obvious oxidative stress response,mitochondria will produce ATP through the electron transfer body and oxidative phosphorylation process,and meanwhile produce a large amount of reactive oxygen species(ROS).Excessive ROS will in turn attack cells and sub-areas,triggering lipid peroxidation and destroying the structural integrity of the mitochondrial membrane.In the course of respiratory oxidation,mitochondria store the energy in the form of electrochemical potential energy in the mitochondrial inner membrane,causing an asymmetric distribution of protons and other ions on both sides of the inner membrane to form a mitochondrial membrane potential ??m.Normal membrane potential is a prerequisite for the maintenance of oxidative phosphorylation and ATP production by mitochondria.The stability of membrane potential is conducive to the maintenance of normal physiological functions of cells.Therefore,the destruction of mitochondrial membrane potential is an important indicator of mitochondrial structure and function damage,and is also the earliest event in the process of apoptosis.Therefore,reducing mitochondrial damage and protecting the integrity of mitochondrial structure and function are crucial for the protection of myocardial cell injury.Studies have shown that damaged mitochondria can be specifically degraded through autophagy-lysosome pathway thereby alleviating oxidative stress damage caused by mitochondrial dysfunction.The above-mentioned way that specifically degrades mitochondria through autophagy is called mitochondrial autophagy.Under normal circumstances,through normal mitochondrial autophagy,cells can degrade and eliminate impaired or dysfunctional mitochondria to maintain the balance of intracellular mitochondrial mass and quantity as well asto protect cell function.However,studies have also shown that excessive autophagy also promotes myocardial cell loss.Mitochondrial autophagy,apart from the removal of damaged mitochondria,also degrades normal mitochondria,moreover,excessive degradation of key proteins and organelles is not conducive to the maintenance of cell homeostasis.The function of mitochondrial autophagy has been studied more frequently in cerebrovascular and neurodegenerative diseases,and less frequently in cardiovascular diseases.The main mechanism of its protective effect on cardiac function also remains unclear.Based on the above information,the present study was designed to investigate the protective effect of Yiqi Huoxue Fang and its active ingredients on cardiomyocytes protection via a rat model of coronary heart disease caused by qi deficiency and blood stasis and a hypoxia/reoxygenation injury model of H9C2 cardiomyocyte cell line.Moreover,from the perspective of mitochondrial autophagy,the protective effect of Yiqi Huoxue Fang on mitochondrial structure and function and its mechanism will be discussed in this paper.The main research contents and methods of animal experiments are as follows:1.Establish a disease-syndrome combined model of coronary heart diseasein rats with qi deficiency and blood stasis syndrome,and preliminarily evaluate the protective effect of YQHXF on model myocardium.The main experimental methods is to observe the macroscopic signs of rats,collect the color saturation of the tongue,collect the pulse beat amplitude,detect the rat's grip and other aspects to evaluate the TCM syndromes of rats;evaluate heart function of the rat by adopting small animal ultrasonic instrument to collect the rat's echocardiogram,levels of NT-proBNP,ALD and Ang? in serum measured by ELISA;measure myocardial infarct size by MASSON staining;and observe histopathological changes of myocardium by HE staining and light microscopy.2.Based on the confirmation of YQHXF anti-myocardial injury function,further investigate the effects of YQHXF on myocardial mitochondrial structure and function;observe the ultrastructure and morphological changes of mitochondria via transmission electron microscopy;detect serum ATP levels by ELISA kit and evaluate the effects of mitochondrial dysfunction.3.Examine the effect of YQHXF on mitochondrial autophagy.Detect the expression of PINK1,Parkin and HSPO protein in myocardium by immunohistochemistry;detect expression of PINK1 and Parkin by Western Blot assay.In accordance with the characteristics of the compound prescription and literature research basis,astragaloside(AS)and ferulic acid(FA)were selected to partially represent Yiqi and Huoxue drugs for experimental study at the cell level.The main research contents and methods are as follows:1.Investigate the effects of FA and AS on the viability of normal cultured rat H9C2 cardiomyocyte cell line.On this basis,a hypoxia-reoxygenation injury model was established.Measure cell viability via CCK8,measure cell apoptosis level via TUNEL assay and detect Caspase3 level via Western Blot assay,in this way,the protective effect of FA and AS on myocardial H/R injury was preliminarily confirmed.2.Use flow cytometry to analyze the changes of ROS in myocardial cells,use chemiluminescence to detect the change of mitochondrial membrane potential ??m,use ADP/ATP kit to detect myocardial ATP level,therefore their protective effects on mitochondrial function will be investigated.3.Detect the mitochondrial-lysosome binding level by using fluorescent probe Mito Tracker Green-Lyso Tracker Red colocalization;detect the colocalization levels of PINK1-HSP60 and Parkin-HSP60 by immunofluorescence to observe the number of mitochondria and the degree of autophagy.Use Western Blot to detect changes of protein expression of PINK1,Parkin,LCII and P62,investigate the effect of PINK 1/Parkin pathway on mitochondrial autophagyand elucidate its possible mechanism of action.The results of the study are as follows:1.The YQHXF high-dose(349.2 mg·ml-1)group can increase the body weight and grip level of model rats,increase the RGB value of tongue surface,promote the level of TCM syndromes such as pulse amplitude;significantly increase the model rats' indexes of FS,LVIDd,and LVIDs on the echocardiogram.;decrease serum NT-proBNP,ALD,and Ang?levels.YQHXF low-dose(174.6 mg·ml-1)group can restore the body weight and grip level of model rats,increase the RGB value of tongue surface,increase the ejection fraction of heart EF,and decrease the content of serum NT-proBNP,ALD and Ang II;rat pulse amplitude and heart function FS,LVIDd,LVIDs indicators have improved,but not statistically significant.At the same time,YQHXF high and low dose groups can significantly reduce the myocardial injury area,alleviate myocardial cell degeneration,necrosis and inflammatory cell infiltration.2.Upon observance of the ultrastructure of mitochondria,YQHXF can improve myocardial mitochondrial swelling,alleviate the rupture of internal malleolus and maintain mitochondrial morphology.Meanwhile,by measuring serum ATP levels,YQHXF can improve the body's ATP level in rats.3.Immunohistochemistry and immunoblot analysis of myocardial tissue showed that YQ HXF can inhibit the overexpression of mitochondrial autophagy pathway proteins PINK1 and Parkin,increase the expression of mitochondrial matrix protein HSP60,and reduce the expression of apoptotic protein caspase3.4.Both AS and FA can inhibit cardiomyocyte apoptosis caused by H/R injury and protect cardiomyocyte injury.5.Both AS and FA can reduce ADP/ATP ratio,recovery of mitochondrial membrane potential ??m,and decrease ROS level.6.Both AS and FA can inhibit the mitochondrial-lysosome binding,increase the expression of mitochondrial matrix protein HSP60 protein,inhibit the overexpression of mitophagy pathway proteins PINK1,Parkin,increase P62 protein and inhibit the expression of LC3 ?/LC3 ? protein.In summary,the study found:1.YQHXF can improve the syndromes of qi deficiency and blood stasis in the rat model of coronary heart disease with Qi deficiency and blood stasis,and protect the heart function.By protecting the structure and function of mitochondria,relieving degeneration and necrosis of myocardial tissue and inhibiting the apoptosis of cardiomyocytes,YQHXF plays a role in protecting the ischemic myocardium of coronary heart disease with syndromes of Qi deficiency and blood stasis.2.The protective effect of YQHXF on cardiomyocytes of coronary heart disease rats with qi deficiency and blood stasis syndrome is closely related to the mainteinance of the integrity of mitochondrion structure and function and reduction of mitochondrial damage.3.The effective components of YQHXF,AS and FA,have significant anti-H9C2 cardiomyocyte H/R injury effects.This effect inhibits mitochondrial hyperphagy through the PINK1/Parkin pathway,stabilizes mitochondrial numbers,and protects mitochondrial structure and function,thereby inhibiting cells apoptosis toplay a role in myocardial protection. |
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