| BackgroundAcute Myocardial Infarction(AMI)has become one of the leading causes of death as the prevalence and mortality rates are increasing worldwide.Currently,the most effective methods to reconstruct myocardial blood flow are thrombolysis and Percutaneous Coronary Intervention(PCI).Even though reperfusion therapy has resulted in revascularization and restoration of blood flow,but the ischemic area does not receive adequate perfusion patients with myocardial ischemia,which a phenomenon known as no-reflow(NR),which can cause a prolonged and superimposed ischemia in myocardial tissue damage.The prevention,management,and treatment of no-reflow have become a common focus of cardiovascular physicians and researchers.The main reasons for the occurrence of myocardial no-reflow are closely related to swelling of microvascular endothelial cells,increased intertissue pressure caused by exudate in the extra-microvascular interstitium,and microvascular occlusion caused by platelet aggregation or leukocyte embedding.Mitochondria play a major role in signaling in endothelial cells.Some properties of mitochondria,such as mitochondrial oxidative stress,mitochondrial division,mitochondrial fusion and mitochondrial autophagy,play an important role in endothelial cell function.The mitochondria may play a more complex role in the development of endothelial cell dysfunction,it is that to analyze mitochondrial function and mitochondrial dynamics may provide new ideas for the treatment of ischemia-reperfusion in the cardiac microvasculature.NR is a pathological state after PCI and belongs to the same category as AMI.According to TCM,the main pathogenesis of AMI is "interconnection of toxins and stasis,paralysis of veins and ligaments".Based on this theory,combined with modern medical understanding of the pathogenesis of NR(inflammation),"activating blood circulation and removing blood stasis" and "clearing heat and detoxifying toxins Based on this theory,combined with modern medical understanding of the pathogenesis of NR(inflammation),"invigorating blood stasis" and "clearing heat and detoxification" should be the main treatment for NR.SL extract consists of different extracted parts of Salvia miltiorrhiza and Andrographis paniculate,which have the effect of activating blood circulation and removing blood stasis,clearing heat and detoxifying toxins.According to our previous study,SL extract has been shown to have a significant effect on atherosclerosis.In rat,rabbit and ApoE knockout mouse models,it inhibited atherosclerotic plaque formation by reducing inflammatory cytokines in serum,regulating lipid metabolism,inhibiting vascular endothelial damage,improving vascular endothelial function,and inhibiting platelet aggregation.In recent years,we have also found that SL extract has a significant protective effect in anesthetized canine models of ligated coronary arteries and ameliorates myocardial infarction.Purpose of the study1.To investigate whether the ameliorative effect of SL extracts on post-ischemia reperfusion-induced no-reflow and its mechanism are related to protection against microvascular endothelial injury.2.To investigate whether the protection of microvascular endothelial injury by SL extract is related to its modulation of PINK/Parkin pathway to improve mitochondrial dysfunction.Research Methods and Results1.Pharmacodynamic study of SL extract on rats without recurrent flow modelMethods:①Experimental animal grouping and administration:SD rats were randomly grouped into sham-operated group,model group,SL extract each dose group(58.015 mg/kg,116.03 mg/kg,232.06 mg/kg for low,medium and high doses,respectively),and atorvastatin group(ACT,0.9 mg/kg),administered by gavage once a day for 7 days,and administered Modeling was done after the end of administration.②Model establishment and evaluation:the rat model without recurrent flow was prepared by ligating the left anterior descending branch of the coronary artery for 2h and then reperfusing it for 24h.The successful surgical ligation was indicated by cyanosis or whitening of the myocardium in the area dominated by LAD after ligation,weakening of the heart beat and elevation of the ST segment on the ECG,and the successful model without recurrent flow was indicated by obvious fluorescent dark areas in the myocardium visible after thioflavin S staining.③SOPTOP NIRII-MS imaging and LS-18 micro-optical imaging were used to observe the perfusion in the left inferior coronary micro-artery of rats.④Rats in each group were injected intravenously with 0.6%thioflavin S staining to distinguish the no-reflow area from the reflow area,and the area percentage of the no-reflow area was analyzed to evaluate the effect of SL extract on the occurrence of no-reflow.⑤ Small animal ultrasound system was used to detect cardiac function,and three consecutive cardiac cycles were measured,and left ventricular ejection fraction(EF)and left ventricular short-axis shortening(FS)were calculated.⑥ The levels of serum markers(CK,CK-MB,cTnT and cTnI)of myocardial injury were measured.⑦TTC staining to distinguish myocardial infarcted area from non-infarcted area.Results:① Changes in microarterial blood flow velocity were tracked over 1 min during NIR two-zone imaging,and the results showed that microarterial blood flow velocity under the left coronary artery branches was significantly slower in the model group compared with the sham group;compared with the model group,microarterial blood flow velocity under the left coronary artery branches was significantly faster in the SL extract high-dose group rats(all p<0.01).②During the process of tissue hyalinization,we observed a significant non-perfused area in the apical region of the model group,while no significant non-perfused area was observed in the SL extract group.After the completion of the transparency using LS-18 micro-optical imaging,the blood vessels with blood flow through them could only be captured and imaged,and the results showed that the blood vessels were densely distributed on the heart surface of the rats in the sham-operated group,and almost no clear vessels were observed in the left coronary artery distribution area of the rats in the model group,and some clear vessels could be observed in the left coronary artery distribution area of the SL extract group compared with the model group.③The results of thioflavin S staining showed that,compared with the sham-operated group,rats in the model group showed more obvious areas of no-reflow after reperfusion,and the percentage of area of myocardial no-reflow in rats in the model group was 37.04%±9.67%,compared with the model group,each dose of SL extract and ACT could effectively reduce the occurrence of no-reflow,and the percentage of area of no-reflow in rats in the low,medium and high dose groups of SL extract The percentage of reflux-free area was 18.31%±4.01%,13.79%±4.77%and 12.67%±2.47%in the low,medium and high dose groups of SL extract,respectively,and 13.63%±6.90%in the ACT group.④The ultrasound results of small animals showed that the rats in the shamoperated group had normal pulsation of the anterior and posterior walls of the left ventricle and normal ventricular systolic and diastolic functions,while the rats in the model group had nearly flat pulsation of the anterior wall of the left ventricle and impaired ventricular systolic and diastolic functions,and the rats in the SLadministered group and ACT-administered group had improved pulsation of the anterior wall of the left ventricle compared with the model group,indicating that the ventricular systolic and diastolic functions were improved;data analysis showed that compared with the sham The analysis of the data showed that the EF and FS of rats in the model group were significantly lower than those in the sham-operated group,and the EF and FS of rats in the low,medium and high dose groups of SL extracts and the ACT drug group were significantly higher than those in the model group(all p<0.01).⑤The TTC staining results showed that,compared with the shamoperated group,rats in the model group showed a more obvious infarct area after reperfusion,with the infarct area accounting for 29.13%±10.00%.The percentage of infarct area in the low,medium and high dose groups of SL extract was 16.80%±10.16%,15.64%±6.53%,10.96%± 3.09%,and the percentage of area without recurrent flow in the ACT group was 12.67%± 7.32%,respectively.⑥Compared with the sham-operated group,the serum levels of CK,CK-MB,cTnT,and cTnI were significantly higher in the model group,and the serum levels of CK,CK-MB,cTnT,and cTnI were significantly lower in the low,medium,and high dose groups of SL extracts and ACT group compared with the model group(all p<0.01).2.Pharmacological effect study of SL extract on microvascular endothelial injuryMethods:① Evaluation of microvascular injury:Transmission electron microscopy was used to observe the microvascular injury in each group of rats;CD31 was double-stained with VE-cadherin by immunofluorescence to assess the tight junctions of microvessels.②Evaluation of vascular inflammation:neutrophils were labeled with Gr-1 and myocardial tissues were labeled with troponin T.The migration of neutrophils into myocardial tissues was observed in the heart tissues of each group of rats;HE staining was used to observe the inflammatory infiltration in the heart tissues and the aggregation of erythrocytes in the microvessels of each group of rats.③ Extraction,isolation,purification and identification of primary cardiac microvascular endothelial cells(CMECs):CMECs cells were extracted and isolated by complex enzyme digestion and gradient centrifugation,and purified by passaging.The three aspects were identified.④Cell administration and modeling:The experimental cells were divided into normal control group,model group,SL extract 1.25μg/mL,2.5μg/mL and 5μg/mL groups,and incubated in a 5%CO2 incubator at 37℃ for 24 h.After that,DMEM complete medium was added to the normal control group and model group,and the SL extract dose groups were incubated with the corresponding concentration of drug-containing medium for 12h.After 12 h,the model group and SL extract administration group discarded the original culture medium,added sugar-free serum-free medium,and incubated in anoxic incubator for 2 h.After 2 h,the model group and SL extract administration group discarded the sugar-free serum-free medium,and the normal control group discarded the DMEM base medium.⑤Cell proliferation and apoptosis assays:CCK8 to detect the survival rate of cells after OGD/R injury,visible light photometric assay to detect the content of LDH in cell supernatant;Annexin V-FITC/PI double-staining assay to detect the apoptosis rate,Western Blot to detect the expression of apoptosis-related proteins Bcl2 and Bax.⑥Scratch assay and Transwell migration assay were performed to evaluate the migration ability of CMECs cells.⑦Tubulogenesis assay was performed to evaluate the tubulogenic ability of CMECs cells.⑧ NO and ET1,IL-10 and IL-1β in cell supernatants were measured by nitrate reductase assay and enzyme-linked immunosorbent assay,respectively.⑨The cellular barrier function of CMECs was evaluated by Transwell-Evans Blue monolayer permeation assay and transmembrane resistance.Results:① Transmission electron microscopy and immunofluorescence results showed that compared with the sham-operated group,the microvascular endothelium of NR model rats showed swelling,irregularity in the vessel wall,and luminal narrowing,etc.The fluorescence intensity of VE-cadherin was reduced to 74.05%of that of the sham-operated group,while the SL extract high-dose group was able to significantly improve the damaged microvascular structure,and the expression of VE-cadherin in the SL extract low,The expression of VE-cadherin increased to 89.87%,82.23%and 89.69%in the medium and high dose groups of SL extract,respectively.②Compared with the sham-operated group,neutrophils migrated to the myocardial tissue in NR model rats,and a large amount of inflammatory infiltration and erythrocyte aggregation in the microvessels were observed;compared with the model group,the SL extract group could significantly inhibit the migration of neutrophils to the myocardial tissue and reduce the inflammatory infiltration and erythrocyte aggregation in the myocardial tissue.③ The extracted CMECs cells showed pavement-like growth and cyclic growth characteristics,were able to express both CD31 and VWF,and had good tube-forming ability and migration ability.④Compared with the normal group,the model group showed a significant decrease in cell survival,a significant increase in LDH content in cell supernatant,a significant increase in apoptosis,a significant increase in Bax expression and a significant decrease in Bcl2 expression after OGD/R injury;compared with the model group,all dose groups of SL extract significantly increased the survival rate of model cells and the Bcl2 expression,and decreased the apoptosis rate and Bax expression.⑤Compared with the normal group,the wound healing ability and the number of tranwell permeable cells in the model group were significantly decreased,and the migration ability of cells was significantly improved in each dose group of SL extract(p<0.05 or p<0.01).⑥Compared with the normal group,the vascular length of CMECs cells after OGD/R injury was significantly shortened,and the number of nodes and branches were significantly reduced,and SL extracts significantly increased the vascular length,number of nodes and branches of CMECs cells compared with the model group(p<0.05 orp<0.01).⑦Compared with the normal group,the ET-1 secreted by cells in the model group was significantly increased and NO was significantly decreased.Compared with the model group,SL extract was able to significantly decrease the content of ET-1 and increase the content of NO in all dose groups(p<0.05 or p<0.01).⑧ Compared with the normal group,the permeation of Evans Blue was significantly increased and the transmembrane resistance was significantly decreased in the model group;compared with the model group,the permeation of Evans Blue was significantly decreased and the transmembrane resistance was significantly increased in each dose group of SL extract(p<0.05 orp<0.01).3.Study on the effect of SL extract on mitochondria of microvascular endothelial cells after OGD/R injuryMethods:① The kits were used to detect the intracellular ATP,calcium ion content and the degree of mPTP opening in each group.②Flow cytometry was used to detect the mitochondrial membrane potential and the level of ROS in the cells.③Laser confocal observation of cell mitochondrial morphology and Western Blot to detect the expression of mitochondrial fusion-related proteins DRP1,MFF,OPA1 and MFN2.④ Representative ultrastructural morphology of autophagosomes and autolysosomes and bilayer membrane structure with closed mitochondria(mitochondrial autophagy)were observed under transmission electron microscopy,and the expression of mitochondrial autophagy-associated proteins p62,LC3Ⅱ/Ⅰ was detected by laser confocal and western blot.Results:① Compared with the normal group,the model group showed a significant decrease in ATP production and mitochondrial membrane potential,a significant increase in intracellular calcium ion content and ROS accumulation,and a significant increase in the openness of mPTP;compared with the model group,the SL extract doses of 1.25,2.5,and 5 μg/mL were able to significantly increase ATP production,increase mitochondrial membrane potential,decrease intracellular calcium ion,decreased the accumulation of ROS in the cells and reduced the opening of mPTP(all p<0.05 or p<0.01).②Mitochondrial network fragmentation increased after OGD/R injury,and the aspect ratio and shape factor coefficients of mitochondrial network were significantly decreased,and the expression of mitochondrial division-related proteins Drp1 and MFF was significantly increased,and the expression of fusion-related proteins MFN2 and OPA1 was significantly decreased.Compared with the model group,SL extracts significantly increased the aspect ratio and shape factor coefficients of mitochondrial network,decreased the expression of Drpl and MFF,and increased the expression of MFN2 and OPA1(all p<0.05 or p<0.01).③A small number of double-membrane vacuoles containing autophagic components were seen in the sham group,while a large number of autophagic vesicles and lysosomes containing autophagic vesicles were observed in the model group,fewer mitochondrial autophagy-related structures in the SL extract group.④Compared with the normal group,mitochondria and lysosomes fusion was significantly increased in the model group after OGD/R injury,Pearson correlation coefficient was significantly increased,and the expression of mitochondrial autophagy-related proteins p62 and LC3Ⅱ/Ⅰ was significantly increased;compared with the model group,SL extracts significantly decreased the Pearson correlation coefficient of mitochondria and lysosomes,and significantly decreased the expression of p62 and LC3Ⅱ/Ⅰ(all p<0.05).4.To investigate the molecular mechanism of SL extract protection of microvascular endothelial cells based on mitochondrial dysfunctionMethods:①The experimental cells were divided into normal control group,model group,SL extract administration group,CCCP group and CCCP+SL group.The final administration concentration of SL extract administration group was 2.5μg/mL and CCCP administration concentration was 5 mM.The model group,SL extract administration group,CCCP group and CCCP+SL group were subjected to OGD/R injury.After the end of modeling,cellular proteins of each group were collected,mitochondrial proteins and cytoplasmic proteins were separated,and the expression of PINK and Parkin proteins in cells,cytoplasm and mitochondria were detected by Western Blot.②Detection of ATP,calcium ion content,openness of mPTP,mitochondrial membrane potential,and the level of ROS in the cells of each group.③ Laser confocal observation of cell mitochondrial morphology,fusion of mitochondria and lysosomes,Western Blot to detect the expression of mitochondrial division and fusion related proteins DRP1,MFF,OPA1,MFN2 and mitochondrial autophagy related proteins p62,LC3Ⅱ/Ⅰ.④The migration ability and tube-forming ability of cells were evaluated by scratch assay and tube-forming assay,and the cell barrier function was evaluated by Transwell-Evans Blue monolayer permeation test and transmembrane resistance,respectively.Results:①After OGD/R injury,the PINK/Parkin pathway was significantly activated compared with the normal group,and the expression of total PINK and Parkin proteins in cells was significantly increased;further detection of PINK and Parkin protein expression in mitochondria and cytoplasm showed that the expression of PINK and Parkin proteins in mitochondria was significantly increased and in cytoplasm Compared with the model group,SL extracts at all doses significantly reduced the expression of PINK and Parkin protein,and significantly reduced the accumulation of PINK and Parkin protein in mitochondria(all p<0.05 or p<0.01).②CCCP is an activator of PINK,and the findings revealed that after OGD/R injury,CCCP was able to significantly activate the PINK/Parkin pathway and increase the expression of total PINK and Parkin proteins in cells significantly compared with the normal group,while the expression of PINK,Parkin proteins in mitochondria was significantly increased and decreased in cytoplasm significantly.Compared with SL group,CCCP+SL group could significantly reverse the inhibitory effect of SL on PINK/Parkin pathway(bothp<0.05 or p<0.01).③Compared with the SL group,the CCCP+SL group showed increased fragmentation of cellular mitochondrial network,significantly decreased aspect ratio and shape factor coefficient of mitochondrial network,significantly increased expression of DRP1 and MFF,and significantly decreased expression of MFN2 and OPA1(both p<0.05 or p<0.01).Compared with the SL group,the Pearson correlation coefficient of mitochondria and lysosome co-labeling in the CCCP+SL group was significantly increased,and the expressions of mitophagy-related proteins p62 and LC3II/I were significantly increased(allp<0.05 or p<0.01).④ Compared with the SL group,the CCCP+SL group was able to significantly decrease the content of ATP,mitochondrial membrane potential,increase the openness of mPTP,and significantly increase the intracellular calcium ion and ROS content(all p<0.05 or p<0.01).⑤Compared with the SL group,the CCCP+SL group showed a significant decrease in cell migration rate,a significant shortening of cell tubule length,a significant decrease in the number of nodes and branches,a significant increase in Evans blue permeation in cells,and a significant decrease in transmembrane resistance(all p<0.05 orp<0.01).Conclusion(1)SL extract significantly improved no-reflow induced by ischemia-reperfusion,and its pharmacological mechanism of action was closely related to the reduction of microvascular endothelial injury by SL extract.(2)The protection of microvascular endothelial injury by SL extract was closely related to its inhibition of PINK/Parkin pathway to improve mitochondrial dysfunction. |
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