| Myocardial infarction(MI),a disease in which acute and persistent coronary artery occlusion results in ischemic anoxic necrosis of myocardial tissue,shows an increasing incidence and death rate over the years.Inflammation is the key pathophysiological process after myocardial infarction.In pathological conditions,immune cells migrate and chemotaxis to the heart and blood vessel walls through the blood circulation and lymphatic circulation,releasing inflammatory factors and forming an inflammatory microenvironment,participating in local inflammation,immune effects and tissue damage,and mediating heart and blood vessel remodeling.It is particularly important to study the injury mechanism of the upstream ring of inflammatory response.Myocardial injury and inflammatory microenvironment after MI repair based on mitochondrial pathway has become a new target for drug research,which is of great significance.Mitochondrial injury is one of the cores of myocardial injury and the regulatory center of apoptosis.In recent years,recent studies have shown that mitochondria is not only the main organelles of cellular energy supply and apoptotic pathway,but also the transmitters and regulators of biological signals.Repairing myocardial injury and inflammatory microenvironment based on mitochondrial pathway has become a new target for drug research.Experiments were carried out from animal,cell and pharmaceutical chemistry aspects to study the biological targets of regulating the mitochondrial function by Huoxuewentong formula and improving the inflammatory microenvironment of ischemic myocardial muscle.This study aims to explore the targeted regulation of inflammation by mitochondria,and elucidate the molecular mechanism of promoting blood circulation and warming through traditional Chinese medicine to improve long-term clinical prognosis of myocardial ischemia.Part Ⅰ.The study on the regulation of Huoxuewentong Formula on cardiac function and inflammatory microenvironment in myocardial infarction ratsObjective:To observe the effects of Huoxuewentong formula onleft ventricular ejection fraction,pathological changes and changes of inflammatory factors in SD rats after MI,and to explore the mechanism of Huoxuewentong formula on inhibiting myocardial apoptosis and reducing myocardial inflammation and improving cardiac function.Methods:The left anterior descending branch of the rat coronary artery was treated with ligation and ischemia.The rats were randomly divided into 4 groups with 9 rats in each group:In Sham group(Model group),Model group(Model group),Huoxuewentong formula(HXWTF group)and positive control group,cardiac function and radial changes were detected by high-resolution echocardiography,infarct size,fibrosis,angiogenesis and inflammatory cell aggregation were analyzed by cardiac histology.Enzyme-linked immunosorbent assay and western blotting were used to detect the changes of pro-inflammatory and anti-inflammatory factors in peripheral blood and myocardial tissue of rats.Results:(1)Compared with Sham group,EF,FS,SV and CO in Model group were significantly decreased(P<0.05),while LVESV was significantly increased(P<0.05).Compared with Model group,EF and FS in HXWTF group were significantly increased(P<0.05),SV and CO showed an increasing trend,but there was no statistically significant difference between groups.(2)Compared with Sham group,GRS Seg2 in Model group was significantly decreased(P<0.05),GRS Segl and GRS Seg3 had a downward trend,but there was no statistically significant difference between groups,and MOWD was significantly increased(P<0.05).Compared with Model group,GRS Seg2 in HXWTF group was significantly increased(P<0.05),GRS Segl and GRS Seg3 had an increasing trend,but no significant difference was found among groups;MOWD had a decreasing trend,but no significant difference was found among groups.(3)Compared with Model group,collagen fiber deposition was significantly reduced in HXWTF group,and CD31 molecular expression was significantly increased in HXWTF group compared with Model group.(4)Compared with Model,the infarct size of HXWTF group was reduced,myocardial fibers were arranged in an orderly manner,and inflammatory cell infiltration was inhibited.(5)Compared with Sham group,the levels of TNF-α,IL-6 and IL-10 in Model group were significantly increased(P<0.05);Compared with Model,the TNF-α,IL-6 and IL-10 in HXWTF group were significantly decreased(P<0.05)and significantly decreased(P<0.05).(6)Compared with Sham group,the levels of NF-κB,FOXO1 and p53 in Model group were significantly increased(P<0.05);Compared with Model,NF-κB and FOXO1 in HXWTF group was significantly decreased(P<0.05),and FOXO1 had a decreasing trend,but there was no significant difference between groups.Conclusions:(1)Huoxuewentong formula can improve myocardial ischemia injury in rats,mainly reflected in improving the ejection fraction and other cardiac function status,myocardial fibrosis level and angiogenesis.(2)Huoxuewentong formula can improve the inflammatory microenvironment of ischemic myocarde.it is mainly reflected in improving the inflammatory cell aggregation in myocardial tissue,the levels of proinflammatory and anti-inflammatory factors in serum,and the expression of inflammator-related pathway proteins in myocardial tissue.Part Ⅱ.The study of Huoxuewentong Formula regulating mitochondrial damage in H9c2 oxygen-deprived model to improve the inflammatory microenvironment Objective:To observe the effects of Huoxuewentong formula on cell survival rate,lactate dehydrogenase release and mitochondrial function of H9c2 cell model deprived by blood oxygen,and to explore the possible mechanism of the mitochondrial regulation of immune response.Methods:H9c2 cells were cultured,and H9c2 oxygen deprivation model was established by blood oxygen deprivation methods(1%O2,5%CO2 and 94%N2).The medicated serum and blank serum of Huoxuewentong formula were prepared in SD rats.H9c2 cells were randomly divided into Control group(Control group),Model group(Model group),Huoxuewentong prescription(HXWTF group)and blank serum control group.Cell survival rate was measured by CCK-8 assay,cell damage was assessed by lactate dehydrogenase release,and cell apoptosis was assessed by Hoechst 33342 staining.Enzyme-linked immunosorbent assay(ELISA)and western blotting was used to detect the changes in cell supernatant and inflammation-related factors.The mitochondrial function of H9c2 model and Huoxuewentong formula were evaluated by reactive oxygen species(ROS)levels,mitochondrial membrane potential changes,mitochondrial ATP content and mitochondrial number.Results:(1)24 hours of the blood oxygen deprivation was selected as the best time for injury intervention to establish the model.(2)1%,2%and 4%were selected as the optimal dosage concentration for follow-up experiments.(3)Blood oxygen deprivation can induce the apoptosis of H9c2 cells,and Huoxuewentong formula can alleviate the apoptosis caused by ischemia and hypoxia,promote the proliferation of cardiomyocytes and protect cardiomyocytes.(4)Compared with the Control group,the level of IL-6 in the Model group was significantly increased(P<0.05),and the TNF-α and IL-10 showed an increasing trend,but the statistical difference was not obvious among the groups.Compared with Model,IL-6 in HXWTF group was significantly decreased(P<0.05),IL-10 was significantly increased(P<0.05),and TNF-α had a decreasing trend,but the statistical difference between groups was not obvious.(5)Compared with Control group,the levels of NF-κB,FOXO1 and p53 in Model group were significantly increased(P<0.05);Compared with Model,the expression of NF-kB,FOXO1 and p53 in HXWTF group was significantly decreased(P<0.05).(6)Compared with Control group,ROS activity in Model group was significantly decreased(P<0.05);Compared with Model,ROS activity in low,middle and high dose groups of Huoxuewentong formula was significantly increased(P<0.05).(7)Compared with the Control group,the activity of mitochondrial membrane potential in the Model group was significantly decreased(P<0.05).Compared with the Model group,the fluorescence intensity of mitochondrial membrane potential in the HXWTF group was enhanced,and the damage was significantly decreased(P<0.05).Among the three concentrations of low,medium and high concentrations,the fluorescence expression of medium concentration(HXWTF2,2%)was the highest.(7)Compared with Control group,ATP and mitochondria in Model group were significantly decreased(P<0.05);Compared with Model group,ATP in mitochondria of Huoxuewentong formula low-medium-high dose group was significantly increased,and the number of mitochondria was increased(P<0.05).Conclusions:(1)Huoxuewentong formula can improve the cell viability,lactate dehydrogenase release and apoptosis level of H9c2 cell model of blood oxygen deprivation;(2)Huoxuewentong formula can improve the level of inflammation in H9c2 cell oxygen deprivation model,including the level of exoinflammatory factors and the expression of inflammatory pathway proteins;(3)Huoxuewentong formula can improve the mitochondrial function of H9c2 cell model of oxygen deprivation,which is mainly manifested in oxidative stress level,mitochondrial membrane potential changes,mitochondrial ATP content and mitochondrial number.Part Ⅲ.To explore the biological targets of Huoxuewentong formula to improve ischemic myocardial based on mitochondrial function regulation of inflammatory responseObjective:To discuss the biological targets of Huoxuewentong formula for improving mitochondrial function and regulating myocardial inflammatory microenvironment during ischemia and hypoxia.Methods:Based on non-target proteomic studies,gel electrophoresis and mass spectrometry were used to conduct proteomic studies,and further Western blot test was used to verify and siRNA interference techniques of key pathway genes were used to explore the effective targets of mitochondrial function regulation of inflammatory response.Results:(1)Proteomic studies showed that the regulatory target of Huoxuewentong formula was reflected in the migration and aggregation of leukocytes in the inflammatory response after MI.(2)Reclassification of biological processes after protein interaction network analysis,It mainly includes the Relationship between glutathione and NADPH,Integral component of membrane and Intrinsic component of membrane component of Membrane),Plasma membrane bounded cell projection,Membrane and Acetylation may be the target biological processes.(3)The protein expression levels of Sirtl,PGC-1a and VDAC1 in rat myocardium were detected by Western blot.Compared with sham group,the expressions of Sirtl and PGC-1a in Model group were significantly decreased(P<0.05),and the expression of VDAC1 was also significantly decreased(P<0.05).Compared with Model group,the expressions of Sirt1 and PGC-1a in HXWTF group were significantly increased(P<0.05),and the expression of VDAC1 was increased simultaneously(P<0.05).(4)The protein expression levels of Sirtl,PGC-1a and VDAC1 in H9c2 cells were detected by Western blot.Compared with Control group,the expressions of Sirtl and PGC-1a in the model group were significantly decreased(P<0.05),while the expression of VDAC1 was significantly increased(P<0.05).Compared with the model group,the expressions of Sirtl and PGC-1a in HXWTF group were significantly increased(P<0.05),and the expression of VDAC1 was significantly decreased(P<0.05).(5)Western blot detection of Sirtl in H9c2 interfered by siRNA,compared with Control,Sirt1 protein expression was down-regulated(P<0.05)and VDAC1 protein expression was up-regulated(P<0.05)under H/SD conditions.In contrast,the level of Sirtl in HXWTF group was increased(P<0.05)and the level of VDAC1 was decreased(P<0.05)under H/SD condition.At the same time,Control,H/SD and HXWTF groups performed siRNA interference on Sirt1.Compared with si-Control group and si-H/SD group,si-HXWTF group increased Sirtl(P<0.05)and decreased VDAC1 level(P<0.05).Conclusion:(1)The regulatory target of Huoxuewentong formula in inflammatory response after MI is reflected in the migration and aggregation of white blood cells.(2)Huoxuewentong formula can improve mitochondrial function by regulating the Sirtl/PGC1-α/VDAC1 axis,thus achieving the effect of improving the microenvironment of ischemic myocardial inflammation.(3)Sirt1/PGC-1/VDAC1 axis can regulate mitochondrial function and inflammatory response,in which Sirt1 is the upstream target and VDAC1 is an important organelles protein in mitochondria.Part Ⅳ.Huoxuewentong formula by high performance liquid chromatography combined with mass spectrometryObjective:To analyze the chemical constituents of Huoxuewentong prescription qualitatively and quantitatively,standardize the quality control,and further study the material basis of Huoxuewentong prescription,so as to provide evidence for clinical drug use.Methods:The main components of Huoxuewentong formula and their variation were analyzed by HPLC tandem time mass spectrometry.Results and CONCLUSIONS:According to the total ion flow pattern of Huoxuewentong formula,104 of which were TCM compounds.Ranked by content,the top 15 TCM ingredients were salvianolic acid A,paeoniflorin,rorosemary acid,protocatechualdehyde,citric acid,tanshensu,sucrose,L-proline,Paeoniflorin side,cryptotanshinone,hydrothreose,tanshinone IIA,purple oxalic acid and gallic acid. |
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