| With lifestyle changes and population aging,the morbidity and mortality of cardiovascular disease(CVD)are increasing year by year.According to the data of"China Cardiovascular Health and Disease Report 2021",CVD deaths account for the first place in the total causes of death among urban and rural residents.It can be estimated that the current number of cardiovascular diseases in China is about 330 million,of which the number of coronary heart disease patients is about 11 million,and the number of patients with cardiovascular disease will continue to grow rapidly in the next 10 years.The main methods of modern medicine for the treatment of coronary atherosclerotic heart disease are lipid lowering,crown expansion,stabilizing plaque,antiplatelet and other treatments.However,on the basis of standardized treatment,there are still many clinical practical problems,such as more side effects and high risk of recurrent cardiovascular events.The CANTOS study which published in the New England Journal of Medicine in 2017,provides high-level evidence-based medical evidence for the reduction of adverse cardiovascular events in anti-inflammatory therapy for the first time,and the theory of immune inflammation has become a new breakthrough in the prevention and treatment of atherosclerosis(AS).Apoptosis and pyroptosis of macrophages play important roles in the formation,rupture and immune inflammatory response of susceptible plaques in AS.In recent years,many evidences show that apoptosis and pyroptosis of macrophages can promote the formation of necrotic cores and increase the vulnerability of plaques.How to reduce or inhibit macrophage apoptosis and pyroptosis during the development of atherosclerosis has become a hotspot and difficulty in the prevention and treatment of AS in recent years.Atherosclerotic plaque formation and luminal stenosis of coronary arteries due to lipid deposition are the main pathological changes in coronary heart disease,which are similar to "blood stasis" and "blood vessel blockage" in traditional Chinese medicine.However,pathological changes such as plaque rupture,immune inflammatory response,oxidative stress injury and tissue necrosis during the acute event of coronary heart disease are far from being summarized by a single cause of blood stasis,which are similar to the characteristics of toxin,such as sudden onset,cruelty and corrosion.On the basis of fully understanding the modern pathophysiological mechanism of atherosclerotic plaques,the scientific research team led by Academician Chen Keji innovatively put forward the hypothesis that acute cardiovascular events are caused by "toxic blood stasis" through the understanding of"stasis" and "toxicity" in traditional Chinese medicine,and comprehensively using clinical epidemiology,proteomics,data mining and other technical methods.The proposal of the etiological pathogenesis theory of "toxic blood stasis" has further promoted the clinical practice of promoting blood circulation and detoxification methods for the prevention and treatment of atherosclerosis and recurrent cardiovascular events of coronary heart disease.The Huoxue Jiedu optimized prescription is created by Academician Chen Keji’s scientific research team based on clinical practice experience,preliminary research basis and relevant research progress at home and abroad.Previous studies had confirmed that the core drug composition of the Huoxue Jiedu optimized prescription inhibited carotid artery thrombosis in rabbits and Wistar rats and reduced the level of serum inflammatory factors.However,does the Huoxue Jiedu optimized prescription have an effect on the formation and development of macrophage foam cells,which are core components of atherosclerotic plaques?Will the combination of Huoxue Jiedu optimized prescription with caspase 1 inhibitor or ASC small interfering RNA further affect the survival status of macrophage foam cells?What are the effects and potential crosstalk mechanisms of the expression of caspase 1,the key effector of the pyroptosis pathway,and the expression of ASC genes on apoptosis and pyroptosis of macrophages under oxidative low-density lipoprotein-induced conditions?The above problems have not been reported in relevant studies at home and abroad.On the basis of this work,we carried out literature research and in vitro experiments around the theory of "toxic blood stasis",aiming to explore the effect and mechanism of Huoxue Jiedu optimized prescription on the apoptosis and pyroptosis of J774A.1 macrophages induced by oxidized low-density lipoprotein and provides certain evidence support and enlightenment for the treatment of cardiovascular-related diseases from the perspective of immunoinflammation.Literature research The effects of statin plus trimetazidine on inflammatory factor levels and ventricular remodeling in coronary atherosclerotic heart disease:A systematic review and meta-analysis.Objective Systematically evaluate the effect of inflammatory factors and ventricular remodeling based on inflammasomes/immune injury response theory of statin combined with trimetazidine in coronary atherosclerotic heart disease from the perspective of anti-inflammation,to provide certain evidence support and enlightenment for the treatment of coronary heart disease from antiimmunoinflammation method.Methods A total of 9 databases were searched,including EMbase,The Cochrane Library,Web of Science,Medline,Pubmed,WanFang Data,CNKI,China Biomedical Literature Service System(CBM),and Weipu Database(VIP),and the search period was from the date of database creation to April 12,2021.Reference lists of all included studies were handsearched,according to the Cochrane systematic review methodology,we searched for randomised controlled trials(RCTs)in Chinese and English comparing atorvastatin plus trimetazidine with usual care(antiplatelet,blood pressure control,diuretic,coronary dilation,etc.)in patients with coronary atherosclerotic heart disease,and data extraction and quality evaluation of literature materials,RevMan 5.4 software was used for meta-analysis.Outcomes included inflammatory factor indicators:C-reactive protein(CRP),IL-6(interleukin-6),tumor necrosis factor α(TNF-α),and ventricular remodeling-related outcomes:left ventricular end-diastolic diameter(LVEDD),left ventricular end-systolic diameter(LVESD).Results 12 randomized controlled trials with a total of 1120 patients with coronary heart disease were included.Meta-analysis results:(1)In terms of inflammatory factors,statin plus trimetazidine group significantly reduced serum concentrations of CRP(n=770,SMD=-2.70,95%CI[-2.55,-1.40],TNF-α(n=678,SMD=-2.25,95%CI[-3.39 to-1.12],P<-0.0001,IL-6(n=770,SMD=-2.10,95%CI[-3.10,-1.10],P<0.00001)in patients with coronary heart disease compared with the control group(statin alone or trimetazidine).(2)In terms of ventricular remodeling:Compared with the control group(statin alone or trimetazidine),statin plus trimetazidine significantly reduced the left ventricular end-diastolic diameter(n=626,SMD=-4.99,95%CI[-6.83 to-3.15],P<0.00001)and left ventricular end-systolic diameter(n=626,SMD=-4.74,95%CI[-5.91 to-3.58],P<0.00001)in patients with coronary heart disease before treatment.Conclusion As a first-line treatment for cardiovascular system-related diseases,statin combined with trimetazidine significantly reduced serum concentrations of IL-6,TNFα,and CRP in patients with coronary heart disease compared with statin or trimetazidine alone,further reduces the left ventricular end-diastolic diameter and left ventricular end-systolic diameter,and improves ventricular remodeling in patients with coronary heart disease.It suggests that in addition to the known mechanisms such as lipids lowering and cardiomyocyte energy metabolism improvement,inhibition of the immune inflammatory response may be a potential mechanism for statins and trimetazidine to exert a protective effect on the cardiovascular system.Experimental Study Effect and mechanism of Huoxue Jiedu optimized prescription on apoptosis/pyroptosis of J774A.1 macrophages induced by oxidative low-density lipoprotein based on inflammasomes/caspase pathwayObjective Explore the mechanism of Huoxue Jiedu optimized prescription(hereinafter referred to as HXJDII)inhibit J774A.1 macrophages apoptosis/pyroptosis induced by ox-LDL from the perspective of regulating the key effectors of inflammasomes/caspases signaling pathway which affecting the apoptosis/pyroptosis pathwayMethods Foam cell models were established by inducing J774A.1 macrophages with ox-LDL,and randomly divided into blank control group,model control group(ox-LDL 100μg/ml,24h),AC inhibitor group(AC-YVAD-CMK 50μmol/L,24h),Huoxue Jiedu group(HXJDII drug-containing serum 25%,24h),Huoxue Jiedu+AC inhibitor group(HXJDII drug-containing serum 25%+AC-YVAD-CMK).50μmol/L),ASC gene silencing group(ASC siRNA),ASC gene silencing+ox-LDL group(ASC siRNA+ox-LDL 100μg/ml,24h),ASC gene silencing+ Huoxue Jiedu group(ASC siRNA+HXJDII containing serum,25%,24h).Each group was given corresponding interventions according to the established experimental protocol,the morphological changes of macrophages were observed by transmission electron microscopy;TUNEL assay was used to detect the positive rate of TUNEL in cells.The expression levels of key effector genes of apoptosis:caspase 3,caspase 6,caspase 7 and key effector genes of pyroptosis:NLRP3,ASC,IL-1β,IL-18 and caspase 1 in J774A.1 macrophages were detected by qRT PCR;The expression levels of key effector proteins of apoptosis:caspase 3,caspase 6,caspase 7 and key effector proteins of pyroptosis:NLRP3,ASC,IL-1β,IL-18,caspase 1 and Gasdermin D in J774A.1 macrophages were detected by western blot;The expression levels of immunoinflammation factors TNF-α,hs-CRP,IL-6 and NF-κB in the supernatant of each group were detected by ELISA assay;MTT assay was used to detect the relative viability of cells in each experimental group as the intervention time was 24h arid 48h.Results1.The results of transmission electron microscopy showed that compared with the blank control group,a large number of lipid vacuoles could be seen in J774A.1 macrophages in the model control group,cell swelling and rupture,dead cells and cell debris could be observed obviously,cell membrane microvilli and nucleoli disappeared,chromatin was wrinkled and distributed along the nuclear membrane;The cell membrane structure of the Huoxue Jiedu group,AC inhibitor group,AC inhibitor+Huoxue Jiedu group was complete,no cell swelling and rupture were seen,and the nucleoli morphology was relatively normal,and microvilli could be seen on the surface of some cells;Compared with ASC gene silencing+ox-LDL group,the volume of intracellular vesicles in the ASC gene silencing+Huoxue Jiedu group was significantly reduced,and microvilli were visible on the surface of some cells.The results of TUNEL staining showed that the positive rate of TUNEL in J774A.1 macrophages in the model control group was significantly higher than that in the blank control group(P<0.05);The positive rate of TUNEL in Huoxue Jiedu group,AC inhibitor group,AC inhibitor+Huoxue Jiedu group,and ASC gene silencing+Huoxue Jiedu group was significantly lower than that in the model control group(P<0.05).qRT PCR results showed that compared with the model control group,Huoxue Jiedu optimized prescription could reduce the mRNA expression of key pyroptosis effectors such as caspase 1,NLRP3,ASC,IL-1β,IL-18 and the key effector of apoptosis caspase 3 in macrophages,but could not effectively reduce the mRNA expression of caspase 6 and caspase 7,which were key effector genes for apoptosis.The results of Western blot showed that compared with the model control group,the expression of key pyropyrosis effector proteins NLRP3,ASC,IL-1β,IL-18 and Gasdermin D proteins in Huoxue Jiedu group,AC inhibitor group,AC inhibitor+Huoxue Jiedu group was significantly downregulated(P<0.05),but could not effectively reduce the protein expression of key apoptosis effector caspase 3,caspase 6 and caspase 7(P>0.05).The ELISA results showed that compared with the model control group,the expression of TNF-α,hs-CRP,IL-6 and NF-κB inflammatory factors in the supernatant of macrophages mediated by ox-LDL was significantly downregulated(P<0.05)in the Huoxue Jiedu group,AC inhibitor group,and AC inhibitor+Huoxue Jiedu group.2.The results of transmission electron microscopy showed that compared with the blank control group,the cells in the ASC gene silencing group were swollen,the volume increased,and obvious vesicle formation was visible in the cells.In the ASC gene silencing+ox-LDL group,nucleoli disappeared,cell volume increased,microvilli disappeared on the surface of cell membrane,and obvious vesicle formation was visible in cells.The results of TUNEL assay showed that the positive rate of TUNEL in J774A.1 macrophages in the ASC gene silencing group was significantly higher than that in the blank control group,and the positive rate of TUNEL in the ASC gene silencing+ox-LDL group was significantly lower than that in the model control group(P<0.05).The results of MTT assay showed that the relative survival rate of J774A.1 macrophages in the ASC gene silencing+ox-LDL group was significantly higher than that in the model control group,and the relative survival rate of J774A.1 macrophages in the ASC gene silencing group was significantly lower than that in the blank control group(P<0.05).The results of qRT PCR and Western blot showed that the downregulation of the expression levels of ASC mRNA and protein in the ASC gene silencing group significantly increased the expression levels of IL-1β,IL-18 and Gasdermin D(P<0.05)compared with the blank control group.Compared with the model control group,the expression levels of NLRP3,ASC,IL-1β,IL-18 and Gasdermin D in the ASC gene silencing+ox-LDL group were significantly downregulated(P<0.05).3.The results of qRT PCR and Western blot showed that there was no significant difference in the expression levels of caspase 1 mRNA and protein molecules in the ASC gene silencing group compared with the blank control group(P>0.05).Compared with the model control group,the expression level of the other party was decreased after the inhibition of caspase 1 or ASC in the AC inhibitor group,ASC gene silencing+ox-LDL group respectively.In addition,the expression levels of caspase 3,6,7,NLRP3,IL-1β,IL-18 and GSDMD in the AC inhibitor group and ASC gene silencing+ox-LDL group were inhibited compared with the model control group(P<0.05).Conclusion1.Huoxue Jiedu optimized prescription can inhibit ox-LDL-mediated J774A.1 macrophage pyroptosis and inflammatory factor expression levels,reduce the positive rate of TUNEL,enhance the relative activity of cells,and improve the survival status of macrophages mediated by ox-LDL,and the inhibitory effect of blood detoxification optimization on macrophage pyroptosis is significantly better than that of macrophage apoptosis.2.In the absence of ox-LDL-induced injury,ASC gene silencing can downregulate ASC gene and protein expression levels,and promote J774A.1 macrophage pyroptosis.In the case of ox-LDL-induced macrophage damage,ASC gene silencing can inhibit macrophage pyroptosis.It can be seen that under different physiological and pathological conditions,ASC has the dual effect of promoting and inhibiting the pyroptosis signaling pathway.3.Caspase 1 and ASC are not only involved in cell pyroptosis,but also in the protein cascade that regulates the apoptosis signaling pathway.In the protein molecular cascade of the pyroptosis pathway,caspase 1 and ASC interact with each other,and there is a positive feedback regulation mechanism between the expression levels of ASC and caspase 1 under ox-LDL induction conditions. |
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