| BackgroundAfter Acute myocardial infarction(AMI),myocardial damage is often further aggravated by thrombolysis,percutaneous coronary intervention and percutaneous coronary intervention to re-establish the blood supply to the heart.Schemia reperfusion(I/R)has been found to cause coronary microcirculation disorder(CMD)in some patients.Previous studies have mostly focused on reperfusion injury in cardiomyocytes,and there is a lack of research on the mechanism of microcirculation injury.Vascular endothelial dysfunction is an important cause of CMD due to I/R.Mitochondria play an important role in endothelial cells,maintaining their homeostasis through continuous division and fusion,transmitting intracellular and extracellular signals,and regulating apoptosis.The mitochondrial dynamin-related protein 1(Drp1)is a key molecule in the regulation of mitochondrial division.It has been shown that inhibition of mitochondrial division can reduce myocardial ischemia-reperfusion injury.Shuangshen Ningxin(SSNX),a component herbal medicine developed by our team over the years for the treatment of coronary heart disease with Qi deficiency and blood stasis,is composed of total ginsenoside,total phenolic acid of Salvia miltiorrhiza,and total alkaloids of Yanhu,which have the efficacy of benefiting Qi and activating blood circulation,dispelling blood stasis and relieving pain.Previous studies have shown that SSNX protects MIRI in rats by inhibiting Ca2+overload in cardiomyocytes and FUNDC1-mediated mitochondrial autophagy,and protects MIRI in miniature pigs by inhibiting PINK 1/Parkin pathway-mediated mitochondrial autophagy,while SSNX was found to have some ameliorating effects on coronary microcirculation disorders established by microsphere embolization in rats.The current animal models of CMD are mainly coronary microembolism and myocardial ischemia-reperfusion without recurrent flow.In this study,we will use in vivo ligation of the left anterior descending branch of rat coronary artery to simulate myocardial ischemia-reperfusion coronary microcirculation disorder model and Hypoxia Reoxygenation(H/R)model of rat cardiac microvascular endothelial cells(CMECs)to investigate the effects of mitochondrial.The study investigated the protective mechanism of myocardial ischemia-reperfusion coronary microcirculation disorder by SSNX and its active ingredients from the perspective of fragmentation.This study was divided into three main parts.Part I Protective effect of SSNX on myocardial ischemia-reperfusion coronary microcirculation disorder in ratsObjectiveExploring the protective effect of SSNX on myocardial ischemia-reperfusion coronary microcirculation disorder in rats.MethodsMyocardial ischemia-reperfusion coronary microcirculation disorder model was prepared by ligating the left anterior descending branch of coronary artery in SD rats,randomly divided into sham-operated group,model group,Nicorandil group,SSNX 180 mg/kg group,SSNX 90 mg/kg group and SSNX 45 mg/kg group,administered by gavage for 7 days,and observed the pulse and tongue of rats after the last administration,and echocardiography to observe the cardiac function of rats.The myocardial perfusion of rats was detected by acoustic imaging,the serum CK,CK-MB and LDH were detected by biochemical analysis,the plasma APTT and FIB were detected by platelet agglutination factor analyzer,the area of myocardial infarction of rats was determined by Evans blue staining,and the area without recurrent flow was determined by thioflavin staining.Results1.Compared with the sham-operated group,the cardiac function indexes in the model group were significantly lowered,the stroke volume(SV),cardiac output(CO),left ventricular ejection fraction(EF)and Fractional Shortening(FS)were significantly lowered.Left ventricular anterior wall end systolic diameter(LVAWs),left ventricular posterior wall end diastolic diameter(LVAWd)and left ventricular posterior wall end systolic diameter(LVPWs)were lowered,the peak myocardial perfusion time was significantly increased,the tongue of rats was purplish-dark,the R,G and B values were lowered,the pulse amplitude of rats was smaller,the pulse count was increased,the serum CK,CK-MB and LDH levels of rats were increased,the plasma APTT,PT and TT time were shortened,the FIB level was increased,the area of myocardial infarction and the area without recurrent flow in rats were significantly increased.2.Compared with the model group,cardiac function in the Nicorandil group was significantly improved,with significantly higher SV,CO,FS,EF.significantly shorter myocardial perfusion peak time,dark red tongue,moderate pulse amplitude,reduced serum CK,CK-MB and LDH levels,prolonged plasma APTT times,reduced FIB levels,and significantly reduced myocardial infarct area and no-reflow area.3.Compared with the model group,the cardiac function of the rats in each group of SSNX was significantly improved,with increased SV,FS,EF.significantly shorter myocardial perfusion peak time,dark red tongue,moderate pulse amplitude and reduced serum CK,CK-MB and LDH levels.In the group of SSNX 180 and 90 mg/kg,LVAWs and LVAWd were increased,plasma APTT time were prolonged,FIB content was reduced,and myocardial infarct area and area without recurrent flow were significantly reduced.ConclusionsThis part of the study better simulated the myocardial ischemia-reperfusion coronary microcirculation disorder model in rats by ligating the left anterior descending branch of coronary artery.The experimental results showed that SSNX effectively improved cardiac function and microcirculatory disorders,reduced serum CK,LDH and CK-MB contents,prolonged ATPP time,reduced FIB content,improved coagulation function,reduced myocardial infarct area and no-reflow area,improved myocardial blood perfusion,and effectively improved myocardial ischemia-reperfusion coronary microcirculatory disorders.Comparing all the indexes,it was found that SSNX 180 and 90 mg/kg had better protective effect on myocardial ischemiareperfusion coronary microcirculation disorder in rats,and combined with the laboratory pre-experimental basis,SSNX 90 mg/kg was selected for the subsequent mechanism study to explore.Part Ⅱ Mechanism of SSNX regulating NR4A1 inhibiting mitochondrial mitotic perfusion and protecting myocardial ischemia-reperfusion coronary microcirculation disorderObjectiveTo explore whether SSNX protects myocardial ischemia-reperfusion coronary microcirculation disorders by regulating NR4A1 and inhibiting mitochondrial division.MethodsSD rats were randomly divided into sham-operated group,model group,Nicorandil group and SSNX group.In the model group and the drug administration group,the rats were administered the drug by gavage after ligating the left anterior descending branch of the coronary artery for 7 days,and the serum ET-1 and eNOS levels were measured after the last administration.The expression of NR4A1,Mff,Drp1,VDAC1,HK2,Cyt-c and Caspase 9 was detected by Western Blot,and NR4A1,Mff and Drpl gene expression was detected by Q-PCR and Immunohistochemistry NR4A1,Caspase 9 protein expression levels were detected.Results1.Compared with the sham-operated group,the serum ET-1 level increased and eNOS level decreased in the model group;HE staining results showed that the arrangement of myocardial fibers in rats was disordered,ill-defined,cytoplasmic wrinkled,the vessel wall was degenerated and swollen,inflammatory cells were infiltrated,and cell necrosis was visible;The frozen section results showed that gelatin ink was significantly less filled in the vessels and the density of ink was reduced;Western Blot results showed NR4A1,Mff,Drp1,VDAC1,HK2,Cyt-c,Caspase 9 protein expression increased;qRT-PCR results found NR4A1,Mff,Drp1 gene expression increased;Immunohistochemistry results showed NR4A1,Caspase 9 protein expression increased;ELISA results showed mPTP openness increased.Transmission electron microscopy revealed that myocardial fibers were disrupted and disorganized mitochondrial cristae were structurally disturbed,swollen or even broken,local expansion between outer and inner membranes formed vacuoles,vascular vessel walls were depressed and fractured,and a few inflammatory cells were visible.2.Compared with the model group,the Nicorandil group decreased serum ET-1 level and increased eNOS level;HE staining results showed that the structural damage of myocardial tissue was reduced,myocardial cells were more neatly arranged and relatively well-defined,and the swelling of vessel walls was significantly better;Gelatin ink-filled vessels increased in density and the average length of vessels increased;Western Blot results showed that,Mff,Cyt-c,Caspase 9 protein expression decreased;qRT-PCR results showed that NR4A1,Mff,Drpl gene expression was significantly reduced;Immunohistochemistry results showed that NR4A1,Caspase 9 protein expression was significantly reduced;ELISA results showed that mPTP opening was reduced;Transmission electron microscopy observation revealed that myocardial fibers were arranged The results of ELISA showed that the openness of mPTP was reduced.3.Compared with the model group,serum ET-1 level decreased and eNOS level increased in the SSNX group;myocardial tissue structure damage was reduced in rats,myocardial cells were arranged in an orderly manner with clear boundaries,swelling of the vessel wall was significantly better,and myocardial cell necrosis was less common;Gelatin ink-filled vessel density increased and the average length of vessels increased;Western Blot results showed that NR4A1,Mff,Drpl,VDAC1,HK2,Cyt-c,Caspase 9 protein expression was reduced.Drp1,VDAC1,HK2,Cyt-c,Caspase 9 protein expression was decreased;qRT-PCR results showed that NR4A1,Mff,Drp1 gene expression was significantly decreased;Immunohistochemistry results showed that NR4A1,Caspase 9 protein expression was significantly decreased;ELISA results showed that mPTP opening was significantly decreased;Transmission electron microscopy observation Myocardial fibers were found to be in orderly arrangement,the number of mitochondrial fragmentation was reduced,the edema of the vessel wall was reduced,the inflammatory cell infiltration was reduced,and the vessel wall was intact.ConclusionsThis part of the study further investigated the mechanism of the effect of SSNX in protecting myocardial ischemia-reperfusion coronary microcirculation disorders based on the previous study.The experiments showed that SSNX could reduce ET-1 and eNOS content,downregulate NR4A1 protein and gene expression,reduce mitochondrial division-related Mff and Drpl protein and gene expression,regulate VDAC1/HK2/mPTP signaling pathway,reduce Cyt-c release,inhibit Caspase 9 expression level,and reduce apoptosis to protect myocardial I/Rcoronary artery microcirculatory disorders.Part Ⅲ Protective mechanism of SSNX and active component THP on H/R injury of rat microvascular endothelial cellsSection Ⅰ Examination of the toxicity of SSNX and the active ingredient THP to CMECsObjectiveTo investigate whether SSNX and the active ingredient THP are toxic to CMECs.MethodsCMECs were divided into normal group,SSNX group,THP group and NR4A1 antagonist DIM-C-pPhOH group,inoculated in 96-well plates at a density of 2×105 units/mL,placed in CO2 incubator,and after the growth density reached above 80%,the complete medium in each well was discarded,washed twice with PBS,100 μL complete medium was added to each well of the normal group,and each drug administration group was given Each well of the normal group was added with 100 μL of complete medium and each well of the drug administration group was given 100 μL of drug-containing complete medium and placed in a CO2 incubator.After 24 h of drug intervention,the 96-well plate was taken out of the CO2 incubator,10 μL CCK8 was added to each well and the 96-well plate was placed back into the CO2 incubator for 2 h.After incubation,the absorbance(OD)was measured at 450 nm by an enzyme marker.Results1.SSNX was not significantly toxic to CMECs in the concentration range of 3.125200 μg/mL,and enhanced the viability of CMECs in the concentration range of 6.2525 μg/mL.2.THP was not significantly toxic to CMECs in the concentration range of 1.56-200 μM,and significantly enhanced the viability of CMECs in the concentration range of 3.125-50μM.3.The antagonist DIM-C-pPhOH was not significantly toxic to CMECs in the concentration range of 0.9375-15μM,and significantly enhanced the viability of CMECs in the concentration range of 3.75-15μM.ConclusionsThe viability of CMECs was significantly enhanced by SSNX in the concentration range of 6.25-25 μg/mL,THP in the concentration range of 3.125-50 μM,and the antagonist DIM-C-pPhOH in the concentration range of 3.75-15 μM.Section Ⅱ Effects of SSNX and the active ingredient THP on the viability effects of anoxic/reoxygenated conditions in CMECsObjectiveInvestigation of the effects of SSNX and the active ingredient THP on the viability of CMECs under anoxic/reoxygenation conditions.MethodsCMECs were divided into normal group(Control group),model group(H/R group),SSNX group,THP group,and antagonist DIM-C-pPhOH group,inoculated in 96-well plates at a density of 2×105/mL,placed in a CO2 incubator,waiting for the growth density to 80%or more,aspirated the old medium,washed 2 times with PBS,added the corresponding medium to each group,placed the 96-well plate(After 15 min,quickly clamp the air inlet and outlet of the box with a hemostat,stop the ventilation,place the box in the CO2 incubator and incubate for 4 h.After 4 h,take out the box and open the hemostat,add 10μL of glucose per well to make the glucose concentration 4.5 g/L,and place the 96-well plate in the incubator.The 96-well plate was placed in the incubator and continued to incubate for 2 h.After the incubation,the absorbance(OD)was detected at 450 nm by an enzyme marker.Results1.The activity of CMECs was significantly lower in the model group compared to the normal group.2.SSNX significantly enhanced the viability of H/R CMECs in 25 pg/mL compared with the model group;THP significantly enhances the viability of H/R CMECs at 12.5 μM;The antagonist DIM-C-pPhOH significantly enhances the viability of H/R CMECs in 7.5 μM.Section Ⅲ Mechanism of mitochondrial division inhibition by SSNX and the active ingredient THP to protect hypoxic/reoxygenated CMECsObjectiveTo investigate the mechanism of action of SSNX and the active ingredient THP in regulating NR4A1 to inhibit mitochondrial division in H/R CMECs.MethodsThe model was established,grouped and administered in the same way as in Section 2,and the optimal dose was selected from the experimental results in Section 2.NO content,mPTP,transendothelial electrical resitrance(TEER)values were measured in CMECs after H/R,and NR4A1,Mff,Drp1,VDAC 1,HK2,Cyt-c,Caspase 9 protein expression levels were detected by Western Blot.Drpl,VDAC1,HK2,Cytc,Caspase 9 protein expression levels were detected by Western Blot,and the ultrastructure of CMECs was observed by transmission electron microscopy.Results1.Compared with the normal group,the NO content in the model group was reduced.mPTP openness was significantly higher.TEER values were significantly reduced,NR4A1,Mff,Drp1,VDAC1,HK2,Cyt-c,Caspase 9 protein expression levels were increased,and transmission electron microscopy revealed that CMECs had crinkled nuclei,multiple swollen mitochondria,broken and fragmented cristae,more autophagy and multiple vesicles vesicles.2.Compared with the model group,THP increased NO content,DIM-C-pPhOH increased TEER and THP,SSNX,and antagonist DIM-CpPhOH decreased mPTP opening,down-regulated NR4A1,Mff,Drp1,VDAC1,HK2,Cyt-c,Caspase 9 protein expression levels,reduced number of mitochondrial swelling fragmentation,reduced autophagy,and ordered internal mitochondrial cristae arrangement in CMECs.ConclusionsThe protective effects of SSNX,THP,and the antagonist DIM-C-pPhOH on hypoxic reoxygenation injury in CMECs were associated with the regulation of NO content and mPTP opening,downregulation of NR4A1 protein expression,inhibition of mitochondrial division-related protein Mff and Drp1 expression levels,regulation of VDAC1/HK2/mPTP signaling pathway,reduction of Cyt-c release,and reduction of Caspase 9 protein expression levels.Conclutions:SSNX has protective effects on myocardial ischemia-reperfusion coronary microcirculation disorders in rats,and its mechanism of action may be related to regulating vasodilation,improving myocardial perfusion,reducing myocardial infarction,no-reflow area,regulating NR4A1 to inhibit Drp1-mediated mitochondrial division,maintaining mitochondrial function in cardiac microvascular endothelial cells,and reducing apoptosis in cardiac microvascular endothelial cells. |
PDF Full Text Request