| Background and objective:Doxorubicin is one of the commonly used tumor chemotherapeutics.It is widely used in the clinical treatment of breast cancer,malignant lymphoma,lung cancer and other malignant tumors.It is an anthracycline antibiotic produced by streptomyces bacteria,which is a non-specific anti-tumor drug,but the dose-dependent cardiotoxicity caused by it is an important factor restricting its therapeutic effect during clinical use,and it cannot be ignored and serious side effects.Therefore,how to reduce the toxic and side effects of these drugs without affecting the chemotherapy effect is a problem that researchers currently need to solve.Current research has not fully elucidated the mechanism of adriamycin-induced cardiotoxicity,but existing studies have suggested that possible factors include oxidative stress,calcium overload,cardiac cell apoptosis,mitochondrial damage,autophagy disorders,and inflammation.Dox metabolism in the body can generate oxygen free radicals.Excessive accumulation of oxygen free radicals causes calcium ions in the cell to be overloaded,which in turn causes mitochondrial damage and myocardial cell apoptosis.In turn,the increase of intracellular calcium ions will further increase the accumulation of oxygen free radicals.Therefore,these factors interact with each other to form the result of cardiotoxicity.Extracellular vesicles(EVs)are ultra-micromembrane vesicles secreted by cells ranging in diameter from 30 to 4000 nm.They are found in serum,urine,saliva,amniotic fluid,and milk.Among various body fluids such as synovial fluid and joint fluid,it is a new type of intercellular signal transmission method,which is a hot spot of current research.According to the formation process and particle size,extracellular vesicles are mainly divided into three subtypes: microvesicles,exosomes,and apoptotic bodies.Microvesicles are small vesicles with a diameter of 200nm-1μm that are directly detached from the cell membrane;exosomes are vesicles that are generated through the endosome pathway and then fused with the plasma membrane.The diameter is between 30-100 nm,which is the smallest subtype of extracellular vesicle;apoptotic bodies are vesicles with diameters between 1 μm and 4 μm produced during the apoptotic process.In view of the fact that the current extraction technology is not mature enough,it is difficult for researchers to extract exosomes with higher purity,usually vesicles except for small apoptosis,so in this study,these vesicle structures are collectively referred to as extracellular vesicles.Therefore,these vesicle structures are collectively called extracellular vesicles in this study.Existing research results suggest that many types of cells secrete extracellular vesicles have a good application prospect in reducing tissue and organ damage.In addition,in the cardiov-ascular system,EVs also act on cells in the heart through adjacent secretion,paracrine secretion and long-distance secretion,and participate in processes such as cardiac muscle cell apoptosis,ventricular remodeling,and angiogenesis.Fibroblasts are the main component of non-cardiomyocytes in the heart.They are widely present in cardiac tissues and surround cardiomyocytes.They can produce a variety of substances that affect the function and growth of cardiomyocytes,so they are unique in the protection of cardiomyocytes.The advantages.Therefore,this project aims to study the protective effects of human cardiomyocyte fibroblasts(Human Cardiac Fibroblasts,HCF)secreted EVs on adriamycin-induced apoptosis of human cardiomyocytes and related mechanisms from a paracrine perspective.Methods:1.Construct a cell model of adriamycin-induced human cardiomyocyte apoptosis,and observe the cell morphology and Western blotting to detect the expression of apoptosis-related protein PARP.2.To confirm the protective effect of EVs secreted by HCF on myocardial cell apoptosis induced by doxorubicin:(1)Extracellular vesicles were extracted from the culture supernatant of human primary myocardial fibroblasts,and immunized by particle size,electron microscopy and protein The marker protein was detected by blotting to identify the extracted extracellular vesicles.(2)The extracted EVs were added to the cell culture supernatant,and the changes of cardiomyocyte apoptosis were detected by microscopic observation and Western blot.3.Exploring the protective mechanism of EVs secreted by HCF: Based on the existing research basis,we speculate that EVs secreted by HCF play a protective role by mediating the MAPK signaling pathway.Therefore,we detected the expression of key proteins in the pathway by western blotting..Result:1.With the increase of Dox concentration,the viability of cardiomyocytes decreases,and the more severe the damage,the greater the shear of the apoptosisrelated protein PARP.2.The particle size test results show that the particle size of EVs is mainly distributed around 150 nm,and the size distribution range is 80-400 nm.The morphology of EVs is observed by transmission electron microscopy,and the vesicle-like structure can be seen.The membrane structure of the protein;the results of the detection of the marker protein showed that the expression of TSG101,CD63,CD9 was positive;in addition,the marker protein vimentin of the fibroblasts was also tested,which also showed that EVs were different from the source cells in the expression of certain proteins be consistent.3.EVs secreted by HCF can significantly reduce the cleavage of apoptosis-related protein PARP and reduce the rate of cardiomyocyte apoptosis induced by Dox.4.Compared with the Dox-treated group,HCF-secreted EVs significantly increased the expression of p-ERK1 / 2,a key protein in the MAPK pathway.Conclusion:Extracellular vesicles secreted by human fibroblasts can inhibit the apoptosis of cardiomyocytes induced by adriamycin and promote the survival of cardiomyocytes,and this protective effect is mediated through the ERK pathway. |
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