Effect Of Salvianolic Acid B On Cardio-cerebrovascular Disease And Its Mechanism

BackgroundDiadetes mellitus(DM)is a global health concern.There are about 300 million people suffering from diabetes in the world,and the number is still increasing.It is estimated that about 450 million people will suffer from diabetes by 2030.As one of the complications of diabetes,diabetic cardiomyopathy(DCM)has been attracted great attention because it can cause serious adverse cardiovascular events.Diabetic cardiomyopathy is a myocardial disease that can’t be explained by hypertension,coronary atherosclerosis,valvular heart disease and other cardiac diseases.It is characterized by asymptomatic diastolic dysfunction in the early stage and eventually develops into symptomatic congestive heart failure.Nowadays,the mechanisms of diabetic cardiomyopathy mainly have been focused on hyperglycemia,lipotoxicity,insulin resistance,myocardial fibrosis,myocardial hypertrophy,apoptosis and necrosis,oxidative stress injury and so on.However,as the development of myocardial fibrosis,myocardial hardness increases and compliance decreases,which lead to dysfunction of cardiac relaxation,contraction and conduction,as well as arterial wall hyperplasia,lumen shrinkage,capillary density decrease,myocardial blood supply decrease and myocardial function damage.Studies showed that coronary microcirculation disturbance can aggravate interstitial fibrosis of the heart in diabetic patients,and endothelial dysfunction may be one of the important reasons for the progress of diabetic cardiomyopathy.Therefore,increasing capillary density,which promotes angiogenesis and increases blood supply and oxygen supply can bring light to the treatment of diabetic cardiomyopathy.So it is of great clinical and practical significance to explore effective drugs to promote angiogenesis and study its mechanism.Salvianolic acid B(Sal B)is one of the main effective components of salvia miltiorrhiza.Studies have found that salvianolic acid B can reduce myocardial injury,cerebral infarction volume and neurological deficit score in cerebral infarction animals induced by ischemia-reperfusion surgery via improving microcirculation disturbance and promoting angiogenesis.In addition,studies have shown that salvianolic acid B can increase the number of endothelial cells,promote the formation of tubular structures,and highten the expression of VEGFA,VEGFR2,ANGPT1,ANGPT2,Tie2 and so on.Therefore,this study is to determine whether salvianolic acid B can improve cardiac function in diabetic cardiomyopathy mice by promoting angiogenesis and further explore its molecular mechanism,so as to provide alternative and safe clinical drugsObjective1.To study the effect and mechanism of salvianolic acid B on ventricular remodeling and myocardial fibrosis in diabetic mice;2.To study the effect and mechanism of salvianolic acid B on angiogenesis in diabetic cardiomyopathy mice.Methods1.Establishment of animal model and administration of neferine in vivoC57BL/6J mice were induced by streptozotocin(STZ)solution,and were randomly divided into 3 groups:model group(DM),low dose of Sal B group(10mg/kg/d)(SalB-L)and high dose of Sal B(120mg/kg/d)(SalB-H).Sal B was administered by intraperitoneal injection once a day for 16 weeks.The control group and DM group were given the same amount of normal saline2.Echocardiographic examination of cardiac functionThe cardiac function of mice in all groups was assessed by doppler echocardiography at the end of exprement.3.Histological stainingThe hearts of mice in each group were collected and fixed with formaldehyde for 24 hours,and were prepared as paraffin sections.H&E were performed to show the general shape of hearts.Masson and Sirius red staining were to observe collagen content,meanwhile collagen I and collagen III immunohistochemical staining were used to test the distribution and expression of collagen,CD31 immunohistochemical staining was to measure capillary density in myocardial tissue.4.Immunofluorescence stainingImmunofluorescence staining of a-SMA and CD31 were performed to show capillary density.5.Western blotHeart tissues and cells of each group proteins were collected to measure protein expression in all groups after protein extraction,electrophoresis and membrane transfer.6.Transcriptome sequencingTranscriptome sequencing of myocardial tissue in each group were conducted to explore its molecular mechanism according to standard procedures.7.Cell cultureHUVECs was purchased from ATCC and cultured in ECM medium containing 5%fetal bovine serum and 1%growth factor.Cells were changed fluid and passaged by conventional methods8.Real-time quantitative RT-PCRTissue and cellular in all groups were collected to measure changes of mRNA after RNA extracted,reverse transcription and PCR amplification.9.CCK-8The cell viability was measured by CCK-8 according to standard procedures.10.Matrigel HUVEC tube formation assayHUVECs was divided into control group,hypoxia group and Sal B group.HUVECs were grown on Matrigel Basement Membrane Matrix in 24-well plates.The plate was coated with 70μL/well of cold Matrigel and incubated for 30 min at 37℃to allow for gelation.HUVECs were resuspended in culture medium and seeded in each Matrigel-coated 24-well plate at 4×104/well.The number of tubes were observed and calculated after 4 hours.11.Immunocytochemistry stainingHUVECs were fixed in 4%paraformaldehyde,washed with PBS and then incubated primary antibody overnight at 4℃.And then cells were incubated with fluorescent secondary antibodies for 1h at 37℃ on next day.Nuclei were counterstained with DAPI.12.Methylation detectionMethylation sequencing of HUVECs in each group were conducted to explore the level of methylation of IGFBP3 promoter.13.Intervention of gene expressionIn vitro experiments,the expression of IGFBP3 was inhibited by transient transfection of siRNA-IGFBP3.14.Cell migration experimentsThe cell migration was observed by cell scratch test and transwell chamber.15.Cell cycle distributionFlow cytometry was performed to measure the proportion of G1,S or G2 of cell cycle.16.Statistical analysisSPSS 18.0 software was used to analyze the experimental data.The results were expressed as the mean ± standard deviation.Variables between groups were examined using one-way ANOVA.P less than 0.05 was considered statistically significant.Results1.The general characteristics of miceCompared with normal control mice,diabetes brought about more higher random blood glucose in mice.However,low-dose or high-dose Sal B treatment had no significant effect on blood glucose.In adition,Sal B administration attenuated polydipsia,polyphagia,urorrhagia and emaciation in diabetic mice.2.Sal B reduces diabetes-induced cardiac remodeling and cardiac dysfunctionAfter 16 weeks of administration of high and low doses of Sal B,the left ventricular remodeling of mice hearts in each group was observed.Compared with normal mice,diabetic mice showed enlarged heart and blunt apex.Above changes in SalB-L and SalB-H group were reversed.In addition,we found Sal B treatment reduced heart weight ratio(HW/BW)and myocardial cell diameter in diabetic mice.Compared with natural control mice,diabetes contributed to a decrease in cardiac function in mice,and Sal B treatment can improve the cardiac function parameters.3.Sal B attenuates diabetes-induced cardiac fibrosisHistological staining and western blot analysis showed that diabetes resulted in myocardial extensive fibrosis.However,Sal B administration downregulated collagen content in myocardial tissue.4.Sal B increases myocardial angiogenesisCD31 immunohistochemical staining results showed that compared with normal mice,the expression of CD31 in diabetic mice was significantly reduced,and high and low doses of Sal B intervention promoted CD31 expression.CD31 and α-SMA co-localization fluorescence staining showed same results,diabetes caused less mature blood vessels in myocardium of diabetic mice which were reversed by high and low doses of Sal B treatment.Western blot results further showed that the expression of VEGFA and VEGFR2 in myocardial tissue of diabetic mice was decreased,while Sal B treatment could increase their expression.5.Sal B promotes angiogenesis by reducing the expression of insulin-like growth factor binding protein 3(IGFBP3)Transcriptome sequencing results of myocardial tissue in mice showed that IGFBP3 was highly expressed in diabetic mice,while high and low doses of Sal B treatment reduced the expression.Real-time quantitative RT-PCR results of myocardial tissue further showed that high and low doses of Sal B adiministration can rescue the high expression of IGFBP3 mRNA level in myocardium of diabetic mice.6.Sal B promotes angiogenesis by activating p-AKT and p-ERK signaling pathwaysThe western blot analysis of myocardial tissue of mice in each group showed that the level of phosphorylated AKT and ERK signaling pathway were activated in myocardial tissue of mice in high and low dose Sal B treatment group.7.Sal B enhances the activity of HUVECsCCK-8 experiment showed that 50ug/ml Sal B has the strongest promoting effect on endothelial cell activity,so 50ug/ml Sal B is used in subsequent experiments.8.Sal B promotes HUVECs angiogenesisWestern blot analysis showed that Sal B can promote much more expression of VEGFA and VEGFR2 of HUVECs in hypoxia environment for 24h than for 12h.In vitro cell tube-forming experiments showed that hypoxia environment significantly reduces the tube-forming ability of endothelial cells,while Sal B treatment can improve the tube-forming ability of cells.9.Sal B promotes IGFBP3 translocation to cytoplasmWestern blot and cellular immunofluorescence analysis showed that IGFBP3 was mainly expressed in the nucleus of endothelial cells under hypoxia,but less in the cytoplasm.After Sal B treatment,IGFBP 3 expression were decreased in the nucleus of endothelial cells and increased in the cytoplasm.10.Sal B enhances methylation of IGFBP3 promoter regionThe reduction of IGFBP3 expression by Sal B is achieved by enhancing methylation of IGFBP3 promoter region.11.Sal B enhances cell tube forming ability by inhibiting IGFBP3.After inhibiting IGFBP3 expression with siRNA,the tubulation ability of cells was enhanced.12.Sal B promotes cell migration by inhibiting IGFBP3Scratch and transwell chember analysis showed that the cell migration ability increased after inhibiting IGFBP3.13.Sal B promotes cell proliferation by inhibiting IGFBP3Flow cytometry showed that inhibition of IGFBP 3 increased the proportion of S and G2 phases of cells.14.Sal B regulates IGFBP3 expression through p-AKT and p-ERK signaling pathwaysWestern blot experiment showed that Sal B treatment or si-IGFBP3 could increase the phosphorylation level of AKT and ERK.Conclusion1.Sal B attenuated diabetes-induced myocardial remodeling,dysfunction and fibrosis in diabetic mice;2.Sal B promoted myocardial angiogenesis in diabetic cardiomyopathy mice by reducing IGFBP3 activity;3.Sal B promoted cytoplasmic translocation of IGFBP3;4.Sal B reduced the expression of IGFBP3 by promoting methylation of IGFBP3 promoter region and cytoplasmic translocation of IGFBP3;5.Sal B regulated IGFBP3 by enhancing p-AKT and p-ERK signaling pathway to improve angiogenesis in diabetic cardiomyopathy.BackgroundStroke has been extremely widespread with serious mortality and long-term disability worldwide.Ischemic stroke accounts for about 87%of the stroke cases.Few medications are effective for acute ischemic stroke management in conjunction to vascular recanalization and supportive care measures.Therefore,exploring a drug that is able to repair ischemic cerebral apoplexy is an significant task worldwide.Restoring cerebral blood flow and saving dying neurons are considered effective therapies for ischemic brain injury.Angiogenesis has been long in high interest to explore promising approaches,as emerging evidence suggests that angiogenesis promotes longer periods of survivor after ischemic stroke,reduce rodent cerebral infarction volumes and improve neurological function.Salvianolic acid B(Sal B)is one of the main active components of Salvia miltiorrhiza,which has been widely used for treating cardio-cerebral vascular diseases.It has been reported that salvianolic acid can promote angiogenesis in acute myocardial infarction and protect endothelial cells in vitro.Meanwhile,our previous study found Sal B can alleviate cardiac fibrosis and remodel in diabetic cardiomyopathy by promoting angiogenesis.What’s more,Sal B can allievate celebral injury of stroke animals.However,whether Sal B enhances vascular densities in ischemic stroke and decreases neurological injury by promoting angiogenesis is still unclear.Therefore,in this study,the ischemic stroke rats are used as experimental models to explore the molecular mechanism of Sal B in improving neurological function of ischemic stroke rats,so as to provide alternative and safe clinical drugs.Objective1.To study the effect of Sal B on nerve injury in ischemic stroke rats;2.To study the effect and mechanism of Sal B on angiogenesis in ischemic stroke rats;3.To study the effect of Sal B on STC1 expression and its mechanism.Methods1.Establishment of animal model and administration of Sal B in vivo230g male SD rats were randomly divided into operation group and sham operation group.The rats in the operation group were induced by Longa suture method.Rats in sham operation group were given the same operation method without inserting thread plugs.These pMCAO rats were randomly divided into 3 groups:the operation group(OG),low dose of Sal B group(15mg/kg/d)(SalB-L)and high dose of Sal B(30mg/kg/d)(SalB-H).Sal B was administered by intraperitoneal injection once a day for 2 weeks or 3 weeks.The sham group was given the same amount of normal saline.2.Neurological functional assessmentsBefore the rats were sacrified,the Longa 5 scoring system was used to evaluate the neurological functional deficits.3.Western blotThe infarcted brain tissues of rats in each group were collected to measure protein expression according to standard procedures.4.Assessment of infarct volumeThe rats were sacrificed under deeply anesthesia.After transcardially perfused with ice cold PBS,their brains were sliced into 2.0 mm sections and immersed in 2%2,3,5-triphenyltetrazolium chloride(TTC)in dark for 30 min.Normal brain tissue appeared red,and infarcted tissue appeared in pale gray.5.Histological stainingBrain tissues of rats in each group were collected,fixed with 4%formaldehyde for 24 hours,and embedded in paraffin to prepare paraffin sections.H&E and Nissl staining were performed to observe the pathological conditions of brain tissues.6.Tissue fluorescence immunohistochemistryBrain tissues of rats in each group were collected and fixed with 4%formaldehyde for 24 hours.After precipitated with sugar,celebral tissues were embedded with OCT Immunofluorescence staining was used to observe neuronal damage,capillary density and STC1 expression in brain tissue.7.TUNEL stainingBrain tissues of rats in each group were collected and TUNEL staining was performed to observe neuronal apoptosis.8.Extraction of primary cortical neuronsPrimary neurons were extracted from newborn rats within 24 hours,and neuronal apoptosis was detected after identification.9.Aortic ring sprouting experimentThe thoracic aortic rings of rats were cultured with Matrigel matrix glue,and the effects of drug-containing serum on the sprouting of aortic rings were observed.10.Cell cultureHUVECs was purchased from American ATCC Company and cultured in ECM medium containing 5%fetal bovine serum and 1%growth factor.The fluid was changed and passaged by conventional methods.11.Intervention of gene expressionIn vitro experiments,the expression of STC1 in cells was inhibited by transient transfection of siRNA-STC112.In vitro tube formation analysisHUVECs were divided into control group,hypoxia group,hypoxia plus Sal B group,hypoxia plus inhibition of STC1 expression group,hypoxia plus Sal B and inhibition of STC1 expression group.HUVECs were grown on Matrigel Basement Membrane Matrix in 24-well plates.The plate was coated with 70 μL/well of cold Matrigel and incubated for 30 min at 37℃ to allow for gelation.HUVECs were resuspended in culture medium and seeded in each Matrigel-coated 24-well plate at 4×104/well.13.Cell migration experimentThe cell migration was observed by scratch test and transwell-chamber.14.Cell proliferation experimentEdu proliferation experiment was used to observe the proliferation of cells.15.Statistical analysisSPSS 18.0 software was applied to analyze and process the experimental data.The continuous variables in the diagram are expressed as mean standard deviation One-way ANOVA was used to test the variables among multiple groups.P<0.05 is considered statistically significant.Results1.Salvianolic acid B promotes cerebral angiogenesis in ischemic stroke ratsAfter ischemic stroke rats were induced successfully,Sal B was injected intraperitoneally for 2 weeks or 3 weeks.Western blot of brain tissue in each group showed that Sal B could further promote the expression of VEGFR2 and VEGFA proteins after 3 weeks rather than these after 2 weeks.So Sal B administration for 3 weeks was used for the subsequent experiments.2.Salvianolic acid B can reduce neurological damage in ischemic stroke ratsNeurological deficit scores were examined.We found sham operation group didn’t show any abnormal symptom,and the rats of operation group subjected to MCAO showed higher neurological deficit scores.Sal B administration could dose-dependently improve motor behavioral deficits compared with operation rats.3.Salvianolic acid B can reduce infarct volume of ischemic stroke ratsTTC staining indicated that infarct volumes were significantly extensive in operation group while sham operation group showed no infarctions,and Sal B reversed this effect with low and high treatment doses.4.Salvianolic acid B can reduce brain tissue damage in ischemic stroke ratsH&E staining in the cerebral cortex region and Nissl staining in the hippocampus region showed Sal B administration reversed the damage in a dose-dependent manner induced by pMCAO operation.Cleaved-caspase3 immunohistochemistry staining and a TUNEL detection kit showed increased apoptotic neurons in in the cerebral cortex region of operation rats compared with that of sham operation rats,and Sal B treatment markedly reduced apoptotic neurons.Western blot further confirmed an increase of apoptosis in the MACO rats that was reversed by Sal B administration.5.Salvianolic acid B can promote cerebral angiogenesis in ischemic stroke ratsThe results of CD31 immunoistochemistry showed that ischemia stroke substantially diminished vessels density at 3 weeks after surgery.However,Sal B administration reversed the downregulation induced by the surgery.The aortic ring sprouting experiment showed that the aortic ring sprouting decreased under hypoxia condition.After cultured with salvianolic acid B-containing serum,the aortic ring sprouting increased,which further indicates that salvianolic acid B promotes angiogenesis.6.Salvianolic acid B promotes angiogenesis by promoting STC1 expressionThe results of STC1 immunohistochemical in rat brain tissue showed that STC1 was almost not expressed in the brain tissue of rats in sham-operation group,STC1 expression increased in ischemic stroke rats,while high and low doses of Sal B treatment further increased STC1 expression.Results of western blot in vivo and in vitro further showed that Sal B treatment could promote the expression of hypoxia-dependent STC1.7.Salvianolic acid B promotes migration of human umbilical vein endothelial cells by increasing STC1Sal B can promote the migration of human umbilical vein endothelial cells under hypoxia.After transfection of siRNA to inhibit the expression of STC1,the promotion of salvianolic acid B on cell migration is reduced.8.Salvianolic acid B promotes angiogenesis by activating mTOR/AKT signaling pathwayThe results of immunoblotting experiments in brain tissues of rats in each group showed that the phosphorylation levels of AKT and ERK signal pathways in brain tissues of rats were further increased in high and low dose Sal B treatment group.Conclusion1.Salvianolic acid B can improve neurological deficit score and cerebral infarction volume of ischemic stroke rats;2.Salvianolic acid B can reduce neuronal apoptosis in ischemic stroke rats by promoting angiogenesis in brain tissue;3.Salvianolic acid B promotes cerebral angiogenesis in ischemic stroke rats by increasing STC1 activity.4.The mechanism of Salvianolic acid B alleviating neurological injury may be related to the activition of mTOR/AKT phosphorylation.