Mechanism Research Of Tongxinluo Inhibiting Apoptosis And Lipid Accumulation In Macrophages Of Advanced Atherosclerotic Plaque

BackgroudCardiovascular disease is one of the three leading causes of chronic noncommunicable diseases that cause the most deaths,and goes far beyond the cancer’s death rate.From 2006 to 2016,the number of deaths caused by global cardiovascular and cerebrovascular diseases increased by 14.5%Atherosclerosis is a type of circulatory system diseases which was closely associated with cardiovascular and cerebrovascular events(ischemic heart disease and brain disease).Its etiology is unclear,which mechanism is complex with many factors involved.Macrophages play a crucial role in the development and progression of atherosclerosis.Due to the injury of the vascular endothelium,a large amount of monocyte chemotactic factor is released,monocytes in the blood are recruited,migrated under the endothelium,and induced to macrophages.Macrophages entering the vessel wall constantly phagocytose lipoproteins until they become foamy cells.In advanced plaques,apoptosis of macrophages can lead to plaque necrosis and aggravation of inflammation,resulting in the production of acute intraluminal thrombus caused by vulnerable plaque,leading to unstable angina,myocardial infarction and even heart sudden death or stroke.Therefore,inhibiting such apoptosis may be a very important approach to delay the progression of atherosclerosis,improve plaque stability and thus reduce the incidence of acute cardiovascular events in the future treatment.Autophagy is a lysosomal-dependent cell self-protection mechanism which is evolved in the response to external environmental stimuli during eukaryotic cell evolution and is also an important cell death-related mechanism as well as apoptosis.Autophagy and apoptosis are not mutually exclusive pathways of the two mechanisms,the relationship between them is complicated and varies with the external environment.Experiments by knocking out Atg7 or Beclin-1,or utilizing the autophagy inhibitor 3-MA have shown that inhibiting autophagy will inhibit caspase activation and reduces apoptosis.And in contrast,by inhibiting apoptosis,the induction of autophagy markers is accelerated and the death of some cells is reduced,which means autophagy is an upstream functions of apoptosis.Tongxinluo is a traditional Chinese medicine made of 12 kinds of ingredients.Since its approval by the State Drug Administration of China in 1996,it has been widely used in the treatment of atherosclerosis,coronary heart disease,angina pectoris,myocardial infarction and stroke etc.Previous experimental study found that TXL has a variety of roles in the aspect of cardiovascular system protection,such as anti-oxidation,anti-inflammatory,anti-angiogenesis and protection of vascular endothelial function.Previous studies showed that TXL could stabilize atherosclerotic rabbit vulnerable plaque;inhibit plaque inflammatory angiogenesis;promote myocardial ischemia in mice after myocardial angiogenesis and so on.However,whether TXL regulates the apoptosis of macrophages in advanced atherosclerotic plaques,thus affects plaques stability still remains unknown.In this study,we examined the therapeutic effects of TXL on macrophages apoptosis and plaque stability both in vitro and in vivo,and explored the underlying mechanismsObjective1)To investigate the effect of TXL on the apoptosis of macrophages in advanced plaques of atherosclerosis;2)To investigate if there is cell autophagy involved in the mechanism of TXL in inhibiting apoptosis of macrophages in advanced plaques of atherosclerosis;3)To investigate the possible molecular mechanism of TXL in altering macrophage autophagy and apoptosis in plaque.Methods1.Preparation of Tongxinluo(TXL)superfine powder solutionIn vitro experiments,the superfine powder was dissolved in serum-free medium with an ultrasound machine to aid the dissolution,and centrifugation at 2500g for 15 minutes.The supernatant was collected in a clean bench filter sterilized with a sterile filter,made of 2000mg/L drug solution,long-term storage at-20℃ conditions.When doing the experiment,dilute the medium to the working concentration.In vivo experiments,the superfine powder dissolved in saline,ultrasound can be used to help dissolve,prepared into TXL-Physiological-saline suspension,experimental animals were given gavage daily.2.Culture and processing of human monocyte/macrophage cell lines(THP-1)2.1 Macrophage culture and inductionHuman monocyte/macrophage cell lines were purchased from the American Type Culture Collection(ATCC)and cultured in RPMI-1640 medium containing 10%fetal bovine serum and 1%penicillin/streptomycin f for 2-4×105/mL of cell density culture.When the cell density reached 8×105/mL,the cells were collected by centrifugation(centrifugation conditions:1000 r/min,5 min).After centrifugation,discard the supernatant,add new complete medium,blow slowly,resuspend the cells,transfer to a new cell culture flask,and incubate the culture flask in a 5%CO2 cell culture incubator at 37℃.The maximum cell density should not exceed 1×106/mL.Induction of preparation of experimental model macrophages need to culture medium added 50 ng/mL phorbbarin(PMA)differentiation for 48 hours adherent macrophages.2.2 Macrophage interventionMacrophages cultured in vitro were pretreated with TXL solution of different concentration gradient.The control group was added with normal medium.After 24 hours,50 mg/L of human ox-LDL and/or 3-M A was stimulated.Group A(3)100 mg/L TXL+ox-LDL group(4)200 mg/L TXL+ox-LDL group(5)500 mg/L TXL+ox-LDL group;Group B was as follows:(1)control group(2)ox-LDL group(3)TXL+ox-LDL group(4)3-MA+ox-LDL group.3.Establishment of animal modeSixty-eight male apoE-/-mice,8 weeks old,were given a high-fat diet for one week and then underwent carotid artery cannulation.One week later,they were randomly divided into four groups:saline group(24 rats),low dose TXL group(TXL-L,0.38 g/kg/d,24),middle dose TXL group(TXL-M,0.75 g/kg/d,24 mice)and high dose TXL(TXL-H,1.5 g/kg/d,24 mice).Four weeks after gavage,shBeclin-1 lentivirus was injected into the tail vein for transfection,and each subgroup was again divided into NS group(12 mice)and shBeclin-1 group(12 mice).Four weeks after the continuous gavage administration,the experimental animals were euthanized,and the blood and tissues of the animals were collected.4.Western blot analysisDifferent groups of macrophages and experimental mouse tissue proteins were extracted and subjected to SDS-PAGE electrophoresis.Then 0.2%PVDF was used to transfect the membrane and blocked with 5%non-fat dry milk.The primary antibody was incubated overnight at 4℃ and the secondary antibody was incubated overnight at 4℃.After ECL chemiluminescence,use Photoshop to analyze the gray value of the corresponding protein band.5.Transfection of cellsBeclin-1 siRNA and Negative Control Negative control siRNA were synthesized by GenePharma in Shanghai and transfected into cells according to the product manual of Invitrogen’s liposome Lipo-3000.6.Construction of viral vectors containing the gene of interestThe gene silencing Beclin-1 gene adenovirus vector was constructed by Genechem in Shanghai and the GFP carrying green fluorescent protein(GFP)was used as the control group.7.Co-immunoprecipitationDifferent groups of cell non-denatured protein lysis buffer,5%added 5x sample buffer was boiled as a control,the other lysate was added magnetic beads and IP primary antibody mixture,4℃ overnight rotation,the eluent eluted magnetic beads After adding 2x loading buffer.8.Histological stainingThe aortic root of each experimental group was collected and frozen sections were prepared.Immunohistochemical staining was used to observe the co-localization of TUNEL and MOMA-2 in blood vessels and the co-localization of LC3B and MOMA-2.9.Streaming detectionBD’s Annexin V-FITC Apoptosis Detection Kit detects macrophage apoptosis.Each sample was first incubated with Annexin V-FITC for 15 minutes and then with PI for 5 minutes,and cell flow analysis was completed within 1 hour of staining.10.Cell immunofluorescenceMacrophages were pre-treated and stimulated for 30 min with immunostaining fixative.After incubation with PBS,0.05%Triton X-100 was incubated for 8 min at room temperature,blocked with 5%BSA for 30 min,washed with PBS and incubated with LC3B and(/or)LysoTracker Red,overnight at 4℃,the next day incubated with fluorescent secondary antibody for 2 hours,DAPI stained nuclei,observed and photographed under a laser confocal microscope.11.Statistical analysisThe experimental data were expressed as mean±S.E.M。Statistical analysis was performed using SPSS 16.0 software.The unpaired t-test was used to compare the two samples.Comparison of multiple samples was performed using one-way ANOVA.P<0.05 was considered statistically significant.Results1.Effect of TXL on the Apoptosis of Macrophages Induced by ox-LDLFlow cytometry and TUNEL assay showed that ox-LDL-induced apoptosis was inhibited in the concentration of 200mg/l and 500mg/l TXL solution(P<0.05).This phenomenon was related to the results of Western Blot,cleaved-Caspase3,bax and bcl-2 protein expression changes are consistent.2.Effect of TXL on autophagy of macrophages stimulated by ox-LDLImmunofluorescence staining showed that the concentration of autophagy bodies and autophagy lysosomes in the concentration of 200 mg/L and 500mg/L TXL increased compared with the control group,and the level of autophagy increased(P<0.05)Phenomenon and Western Blot results,LC3B and Beclin-1 protein expression changes are consistent.3.TXL attenuates ox-LDL-impaired apoptosis in THP-1 Macrophages by enhancing autophagyFlow cytometry and TUNEL assay showed that TXL can inhibit the increase of apoptosis induced by 3-MA+ox-LDL(P<0.05),which is related to the results of Western Blot,cleaved-caspase3,bax and bcl-2 protein expression changes are consistent.4.TXL Effect of Beclin-1,Beclin-1 and Bcl-2 complex on apoptosis of macrophagesCompared with the control group,the effect of TXL on macrophage apoptosis was inhibited in the Beclin-1 interference group(P<0.05).Immunoprecipitation results showed that TXL group reduced the increase of Beclin-1 and Bcl-2 complex binding induced by ox-LDL.5.TXL Decreased Apoptosis of Macrophages in advanced atherosclerotic plaque in VivoThe results of immunohistochemistry showed that there was a dose-dependent decrease in the apoptosis of macrophages in the plaque compared with the saline group(P<0.05).In addition,macrophage autophagy in the plaque increased Trend,and most of the cell autophagy occurred in macrophages(P<0.05).However,Beclin-1 intervention after the disappearance of this phenomenon.Conclusions1)TXL inhibited the apoptosis of macrophages in advanced atherosclerotic plaques2)TXL inhibited apoptosis by enhancing macrophage autophagy in advanced atherosclerotic plaques;3)TXL can inhibit macrophage apoptosis by changing the interaction between Beclin-1 and Beclin-1 and Bcl-2.BackgroudAtherosclerosis is a chronic inflammatory disease derived from lipid dysfunction that occurs in the walls of the large and middle arterial blood vessels.In the initial stages of atherosclerosis,endothelial function is disturbed and apolipoprotein B lipoproteins,such as low-density lipoprotein(LDL),are retained in the subendothelium and the endothelium is activated to secrete chemokines and monocyte adhesion.As soon as monocytes enter the vascular endothelium,they differentiate into macrophages that can take up the subcutaneous lipoproteins.With the accumulation of lipids in macrophages,large amounts of lipid droplets(LDs)accumulate in the cytoplasm Macrophages eventually become foam cells.As a major component of atherosclerotic lesions,foam cells play a very important role in the development of atherosclerosis.The formation of foam cells can promote the development of atherosclerosis.Therefore,the reduction of macrophages Cell foaming will be an effective therapeutic strategy to reverse plaque lipid accumulation.Cholesteryl esters(CEs),which are taken up into macrophage lipoproteins,are hydrolyzed to free cholesterol(FC)and fatty acids.It has been found that the ABC transporters ABCA1 and ABCG1 play an important role in the transfer of FCs to the extracellular.In addition,FC re-esterifies cholesterol fatty acid esters by endoplasmic reticulum A:cholesterol ester transferase(ACAT).Knockout of ABCA1 and ABCG1 in macrophages promotes atherosclerosis in mice.Autophagy is a conserved cellular catabolism process in which the cytoplasmic components are encapsulated by autophagosomes that fuse with lysosomes to form autophagic lysosomes where they undergo substance degradation and then cycle.By increasing autophagy,cells can enhance the absorption and re-decomposition of oxidized low-density lipoprotein and acetylated lipoprotein,thereby enhancing plaque stability.So,in addition to the cholesterol transport pathway,autophagy-lysosome system is another important way to regulate intracellular cholesterol metabolism.A study found that specific knockout atg5,will inhibit mouse macrophage autophagy and promote the occurrence of oxidative stress and plaque necrosis.Objective1)To investigate the effect of TXL on the lipid metabolism of macrophages in advanced plaques of atherosclerosis;2)To clarify whether TXL can inhibit autophagy in the mechanism of lipid metabolism in macrophages of atherosclerotic plaque;3)To investigate the possible molecular mechanism of TXL in altering the lipid metabolism of plaque macrophages.Methods1.Serological metabolic testsSerum triglyceride(TG),total cholesterol(TC),low density lipoprotein cholesterol ester(LDL-C)and high density lipoprotein cholesterol ester(HDL-C)were detected by zymography.2.Histopathology Immunohistochemical stainingUsing microscopic tweezers to strip off connective tissue and adipose tissue around the aorta of mice.The entire blood vessel was dissected longitudinally along the curved side,and then the aorta was roughly stained with oil red O.OCT was embedded in the root of aorta,and the frozen sections were prepared by serial sectioning.The sections were stained with hematoxylin-eosin,oil red O,Sirius red,immunohistochemistry and immunofluorescence.Measurement and calculation of aortic plaque load,aortic root cross-sectional plaque area percentage,plaque lipid content,plaque collagen content,plaque macrophage content,plaque smooth muscle cell content,Bodipy and LC3B Binding percentage3.Lipid Bodipy and autophagy LC3B dual immunofluorescent stainingThe autophagosomes were labeled with LC3B and the lipids were labeled with BODIPY(a neutral-lipid fluorescent dye)for co-localization of lipid droplets and autophagosomes in the mouse aortic root plaque.It was examined whether TXL reduces lipid accumulation in atherosclerotic plaques by promoting macrophage autophagy.4.Cell culture and cell slide preparationThe THP-1 cell line was purchased from American ATCC and cultured in RPMI 1640 medium containing 10%fetal bovine serum.The cell suspension was added to an eight-chamber culture plate,and after successful induction with 160 nM PM A as macrophages,the corresponding culture medium and stimulus were added.5.Intracellular lipid staining5.1 Cell oil red O stainingThe different groups of macrophages slide was placed in 4%formaldehyde solution to fix,after PBS washed three times,placed in oil red O working solution and incubated for 10 minutes,wash away the floating color with PBS,The nuclei were stained by hematoxylin solution,and the cells were sealed with glycerol gelatin after staining.Images were taken as soon as possible.5.2 Lipid Bodipy stainingThe macrophages from different groups were fixed in 4%formaldehyde solution,washed three times with PBS,and placed in pre-made Bodipy staining solution for 30 minutes.After washing with PBS,the nucleus was stained with DAPI,and the antibody was used after staining.Quenching the immunofluorescence film to seal and take photos as soon as possible for data analysis6.Dual immunofluorescence staining of cellsAfter washing with PBS for three times,the macrophages of different groups were stained with lyso-Tracker Red or fixed in 4%formaldehyde solution and incubated with the LC3B antibody,and the corresponding secondary antibody and Bodipy dye at a concentration of 20 mg/L were added on the next day,DAPI stained nuclei.7.Western-Blot testThe macrophages were scraped off the culture flask wall with a cell scraper,the cell pellet was collected by centrifugation,the total protein was extracted,separated by SDS-polyacrylamide gel electrophoresis,transferred to membrane,incubated with antibodies,chemiluminescent colorimetric and other steps to ’detect the corresponding protein expression.8.Statistical analysisThe experimental data were expressed as mean±S.E.M.Statistical analysis was performed using SPSS 16.0 software.The unpaired t-test was used to compare the two samples.Comparison of multiple samples was performed using one-way ANOVA.P<0.05 was considered statistically significant.Results1.TXL reduces lipid deposition in advanced plaques of atherosclerosisOil red O en face staining,H&E staining and colocalization of lipid droplets with autophagosomes in plaques showed that TXL was able to reduce plaque load and plaque cross-sectional area effectively in a dose-dependent manner compared with the saline group.2.TXL reduce atherosclerotic plaque vulnerabilityCompared with the saline group,the content of lipid and macrophage in plaque of TXL group was significantly decreased,while the contents of collagen and smooth muscle cells were significantly increased,while the vulnerability index was significantly decreased.However,in Beclin-1 silencing group,above effects are weakened3.Effect of TXL on Lipid Metabolism of Macrophages Induced by ox-LDLCompared with ox-LDL group,the lipid deposition of macrophages in 200mg/L and 500mg/L TXL concentration groups decreased significantly(P<0.05),while in 500mg/L group,Close to the physiological level of the control group,indicating that TXL suppresses macrophage lipid deposition in a dose-dependent manner.In addition,TXL regulates plaque lipid deposition through Beclin-1-mediated macrophage autophagy.4.Effect of Tongxinluo on lipid deposition in macrophages stimulated by ox-LDLThe results of cell oil red O staining and lipid droplet Bodipy staining showed thatcompared with the ox-LDL group,macrophage lipid deposition was significantly reduced in the 200 mg/L and 500 mg/L Tongxinluo concentration groups(P<0.05).The 500 mg/L TXL+ox-LDL group has been able to fall close to the physiological level of the control group,which suggested that Tongxinluo inhibits macrophage lipid deposition and is dose-dependent.5.TXL affect macrophage ox-LDL-impaired lipid metabolism by enhancing autophagyLipid deposition in 3-MA group was significantly higher than that in TXL group,but decreased in 3-M A+TXL group(P<0.05).Breakdown of lipid in LD in TXL group was higher than that in ox-LDL group(P<0.05).The above results indicated that TXL can regulate the lipid metabolism by increasing the level of autophagy.In order to study the role of autophagy key gene Beclin-1 in TXL inhibiting ox-LDL-induced macrophage lipid deposition,Beclin-1 small interfering RNA was used.Compared with TXL+ox-LDL group,the expression of ABCA1 and ABCG1 in TXL+Beclin-1 siRNA+ox-LDL group was decreased(P<0.05),and LOX-1 protein expression was increased(P<0.05).6.Effects of Tongxinluo pharmacology on histone deacetylaseTXL reversed the high expression of class Ⅰ histone deacetylases induced by ox-LDL(P<0.05).Compared with TXL+ox-LDL group,after addition of histone deacetylase agonist,the ratio of LC3Ⅱ/LC3Ⅰ decreased,the expression of Beclin-1 protein decreased,and the expression of ABCA1 and ABCG1 decreased.Conclusions1)TXL inhibited lipid deposition in macrophages in advanced atherosclerotic plaques;2)TXL inhibited macrophage lipid deposition by enhancing autophagy in advanced atherosclerotic plaques;3)TXL affects the decomposition of autophagic intracellular lipid droplets by changing intracellular HD AC.