| BackgroundMyocardial ischemia-reperfusion injury?MIRI?refers to the pathological process when myocardial ischemia is caused by partial or complete obstruction of the coronary arteries,then the ischemic myocardium returns to normal blood flow perfusion for a certain period of time but myocardial injury becomes worse than before.Its manifestations mainly include myocardial diastolic dysfunction and irreversible damage to myocardial tissue.In recent years,with the continuous improvement of medical standards,cardiopulmonary bypass?CPB?technology has been widely used in the field of cardiac surgery,and CPB cardiac arrest surgery has become the norm for surgical treatment of cardiovascular diseases.However,the MIRI caused by CPB technology has always been a problem that plagues clinicians.How to reduce the myocardial cell damage caused by myocardial ischemia reperfusion?IR?,so as to effectively protect the myocardium and improve the prognosis of patients has become a hot spot of current research.Hydrogen sulfide?H2S?is the third biologically active endogenous gas signal molecule found after nitric oxide?NO?and carbon monoxide?CO?.The distribution of endogenous H2S in the body is organ-specific.Mainly regulated and synthesized by the highly expressed cystathionine-?-lyase?CSE?in myocardial tissues,H2S plays a pathological and physiological role.Previous research has confirmed that endogenous H2S has a variety of biological activities and plays a very important roles in pathology and physiology in the cardiovascular system,such as anti-oxidation,anti-apoptosis,anti-inflammatory,etc.In the process of MIRI,H2S exerts myocardial protection mainly through S-sulfhydrated modification,regulation of mi RNA expression,anti-apoptosis,anti-oxidative stress,and regulation of ion channel activity.Although the positive role of H2S in IR has been confirmed,its physical and chemical properties,such as its highly toxic,unstable water-soluble properties,and explosiveness when mixed with air,have hindered its clinical application as a drug.At present,research on H2S donor drugs mainly focuses on exogenous and endogenous H2S.The former is represented by a new water-soluble H2S sustained-release agent GYY4137,which was first synthesized in 2008.Although exogenous H2S donors are easy to prepare,their shortcomings such as lack of targeting effect and poor stability restrict their further clinical application;the latter is mainly focused on targeted drug delivery technology.Among many targeted drug delivery technologies,nanocarriers have the advantages of low toxicity and easy preparation compared with traditional delivery systems?such as adenovirus,lipid microspheres,etc.?,and can be used as both a water-soluble drug carrier and a fat-soluble drug carrier Drug carrier.In addition,nanocarriers have a variety of administration routes to choose from,in addition to improving the stability of the drug,it helps to achieve the targeted effect of the drug.The development of nanometer drug-loaded microparticles is a new field of cross-penetration between nanotechnology and medicine.Using nanocarriers to load specific antibodies and other technical modifications,a CSE ischemic myocardial tissue targeted drug delivery system was constructed to specifically overexpress ischemia myocardial tissue for up-regulation of endogenous H2S expression.It's expected to obtain new H2S donor drugs with precise curative effect and good safety.Objectives1.To construct a CSE myocardial targeting nanocarrier and evaluate whether the nanocarrier can specifically affect ischemic myocardial tissue and increase the level of endogenous H2S in the myocardium at IR.2.To elucidate whether endogenous H2S can reduce mitochondrial autophagy by regulating mitochondrial autophagy in ischemia-reperfusion cardiomyocytes.Method1.Construction of CSE Myocardium Targeting Nanocarrier:The target peptide-PEG quantum dots?PPQD?and CSE gene plasmid vectors were electrostatically assembled with polyethyleneimine?PEI?.It made target the quantum dots and CSE gene plasmid vectors assembled into nanoclusters,where positively-charged PEI has cell transfection effect,and targeting peptides on the surface of quantum dots have the effect of targeting and localizing myocardial ischemic tissue.2.Validation of CSE nanocarrier targeted targeting:IR myocardium of rats?The left anterior descending coronary artery?LAD?of the coronary artery was ligated for 30minutes,and the myocardial tissue of the left ventricular wall was taken after 120 minutes of release?,non-IR myocardium,lung,spleen,pancreas and aortic tissues were compared for CSE protein expression in each tissue using Western-blot method.3.Subjects and groups:Based on the verification of the targeted localization of CSE nanocarriers,healthy male Sprague-Dawley?SD?rats were selected and divided into the following 4 groups:?1?normal control group:SD rats,suture after thoracotomy passed under LAD without ligation,suture was drawn and chest closed after 30min,and normal saline was injected intravenously after 120min;?2?IR group:SD rats,after the thoracotomy,the suture passed under the LAD,the LAD was blocked after 30 minutes,and the blood flow was restored for 120 minutes after intravenous injection of saline.;?3?CSE up-regulation group:CSE overexpression nano-target plasmid was injected intravenously immediately after IR to up-regulate the expression of CSE in ischemic myocardium.?4?CSE down-regulation group:Construction of si RNA,liposome as a carrier,and local myocardial injection reduced the CSE expression of ischemic myocardium.4.The left ventricular function changes?ądp/dt max?of the rats were detected at 24hours after operation,and then the rats were sacrificed for specimen collection:?1?the rat myocardium was stained with TTC after sectioning;?2?the ischemic myocardial tissue was homogenized Frozen in slurry or liquid nitrogen;?3?the serum was frozen at-20°C.5.Comparison of related indexes of myocardial injury:?1?The method of TTC staining is used,in which the pale area is the infarcted area?INF?,the red stained area and the pale area are collectively referred to as the dangerous area?AAR?,the blue stained area is the non-infarcted area,The sum of the red and blue stained areas is the left ventricular area?LV?.The ratio of AAR/LV was used to evaluate the size of the dangerous area?%?,and the ratio of INF/AAR was used to evaluate the size of the infarct area?%?.?2?The levels of serum LDH,CK-MB and c Tn T in each group were measured using a fully automatic biochemical analyzer.6.Comparison of mitochondrial autophagy related indexes:?1?the m RNA expression changes of mitochondrial autophagy related genes?Beclin1,Parkin,Nix?in each group were compared by RT-PCR method;?2?the protein expression changes of autophagy marker proteins LC3-?,Beclin1,Parkin and Nix in each group were compared by Western-blot method.7.Statistical method:All the measurement data are expressed by?MeanąSD?,and the counting data are expressed by percentage.One-way ANOVA was used to compare the measurement data among different groups,and chi-square test or Fisher exact probability test was used to compare the counting data.SPSS 21.0 was used for statistical analysis.P<0.05,it was considered that the difference was statistically significant.Results1.Preparation of CSE Nano-Carrier?1?Transmission electron microscopy observations revealed that thiol-containing glutathione molecules transferred oil-soluble quantum dots to the aqueous phase,and the quantum dots modified by the aqueous phase had good uniform dispersion,with an average particle size of about 7-10 nm.?2?Agarose gel electrophoresis revealed that the migration distance of glutathione-modified quantum dots was the longest without obvious tailing,indicating that the glutathione-modified quantum dots had good particle size and charge uniformity.After modification with polyethylene glycol?PEG?,the electrophoretic bands of the quantum dots are slightly diffused and the migration distance is reduced,indicating that PEG has been modified to the surface of the quantum dots,and at the same time it has some effects on the hydrated particle size distribution of the quantum dots.But the impact is small.After further targeted peptide modification,the electrophoretic bands of the quantum dots become wider,indicating that the targeted peptide can be combined with the PEG modified quantum dots,but it will cause the aggregation of some quantum dots.?3?Agarose gel electrophoresis revealed that when the PEI/QD molar ratio?N/P?was0.2,almost all of the quantum dots were blocked in the loading wells of the electrophoresis,indicating that the amount of PEI at this N/P was sufficient to make the quantum dots completely agglomerated completely by electrostatic self-assembly.In the end,we chose to make PEI a DNA carrier cluster with an appropriate excess PEI/QD N/P ratio of 0.3.?4?Agarose gel electrophoresis revealed that when the number of DNA plasmids was fixed,when the PEI-QD/DNA N/P was 0.5,most of the plasmids were already wrapped in quantum dot clusters.2.Verification of targeting and Localization of CSE Nano-CarrierBy Weston-blot method,compared with non-IR myocardium,lung,spleen,pancreas and aortic tissue,the expression of CSE protein in myocardial tissue at IR significantly increased after CSE nanocarrier injection,and the difference was statistically significant?P<0.05?.It is confirmed that CSE nanoparticles can specifically and highly express in the myocardium of IR.3.Changes of cardiac function?1?Before blocking LAD,there was no significant difference in cardiac function indexes?ądp/dt max?among normal control group,IR group,CSE up regulation group and CSE down regulation group?P>0.05?.?2?At 24h after operation,compared with the normal control group,ądp/dt max in IR group,CSE up-regulation group and CSE down-regulation group were significantly decreased,the difference was statistically significant?P<0.05?.It is suggested that myocardial IR has an inhibitory effect on myocardial systolic and diastolic function.At24h after operation,compared with IR group,ądp/dt max increased significantly in CSE up-regulation group,the difference was statistically significant?P<0.05?.It was confirmed that the high expression of H2S in myocardial tissue at IR could alleviate the inhibitory effect of IR on myocardial contractile and diastolic function after injection of CSE nanoparticles into rats.4.Changes of Myocardial Necrosis and Myocardial Morphology?1?Compared with the normal control group,the ratio of INF/AAR in IR group was significantly higher than that in normal control group,the difference was statistically significant?P<0.05?.It is suggested that myocardial injury can be caused by the occurrence of IR in myocardium.?2?Compared with IR group,there was no significant difference in the risk area?%?between the CSE up-regulated group and the CSE down-regulation group?P>0.05?;the ratio of INF/AAR in CSE up-regulation group decreased significantly,while that in CSE down-regulated group increased significantly,the difference was statistically significant?P<0.05?.It was confirmed that the high expression of CSE in myocardial tissue had a protective effect on myocardial injury induced by IR.CSE nano-carrier could make CSE overexpressed in ischemic myocardium caused by IR,reduce the area of myocardial necrosis and myocardial injury.5.Changes of myocardial metabolic function?1?Compared with the normal control group,the contents of serum LDH,CK-MB and c Tn T in IR group were significantly higher than those in normal control group,the difference was statistically significant?P<0.05?.It suggests that myocardial IR can cause obvious damage to cardiomyocytes.?2?At 24h after operation,compared with IR group,the contents of serum LDH,CK-MB and c Tn T in CSE down-regulation group were significantly higher,and the serum LDH,CK-MB and c Tn T contents in CSE up-regulation group were significantly lower than those in IR group,the difference was statistically significant?P<0.05?.It was confirmed that the high expression of H2S in IR could reduce the injury of cardiomyocytes induced by IR after injection of CSE nanoparticles into SD rats.6.Study on the level of mitochondrial autophagy related gene m RNA?1?Compared with the normal control group,the m RNA expression of autophagy-related genes Parkin,Beclin1 and Nix in IR group were significantly up-regulated by RT-PCR method,the difference was statistically significant?P<0.05?.It suggests that myocardial IR can affect the m RNA expression of autophagy-related genes in cardiomyocytes.?2?At 24h after operation,compared with IR group,the m RNA expression of autophagy-related genes Parkin,Beclin1 and Nix in CSE down-regulation group was significantly up-regulated,while the m RNA expression of Parkin,Beclin1 and Nix in CSE up-regulation group was significantly down-regulated,the difference was statistically significant?P<0.05?.It was confirmed that after injection of CSE nanoparticles into rats,the expression of autophagy-related genes Parkin,Beclin1 and Nix in cardiomyocytes was significantly down-regulated.The high expression of H2S in myocardium at IR can reduce the m RNA expression of autophagy-related genes in cardiomyocytes by IR.7.Study on the protein level of mitochondrial autophagy related genes?1?Compared with the normal control group,the expression of autophagy marker proteins LC3-?,Beclin1,Parkin and Nix in IR group were significantly up-regulated by Western-blot method,and LC3-?/LC3-?ratio increased,the difference was statistically significant?P<0.05?.It suggests that myocardial IR can affect the expression of autophagy marker proteins in cardiomyocytes.?2?At 24h after operation,compared with IR group,the protein expression of LC3-?,Beclin1,Parkin and Nix in CSE down-regulation group was significantly up-regulated,LC3-?/LC3-?ratio increased.While the expression of LC3-?,Beclin1,Parkin and Nix protein in CSE up-regulation group was significantly down-regulated,LC3-?/LC3-?ratio decreased,the difference was statistically significant?P<0.05?.It was confirmed that after injection of CSE nano-carrier,the expression of cardiomyocyte autophagy marker protein was significantly down-regulated.The high expression of H2S in myocardium at IR can reduce the expression of autophagy marker protein in cardiomyocytes by IR.Conclusion1.CSE myocardial targeting nanoparticles can specifically act on the myocardial tissue at IR and promote the level of endogenous H2S in ischemic myocardium.2.H2S can reduce the inhibitory effect of IR on myocardial diastolic function and reduce myocardial necrosis and cardiomyocyte damage caused by IR.3.H2S can reduce myocardial IR injury by regulating m RNA and protein of mitochondrial autophagy-related genes in ischemia-reperfusion cardiomyocytes. |
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