| Objective: Acute myocardial infarction(AMI)is the leading cause of death worldwide.In addition to myocardial damage caused by ischemia and hypoxia,post-MI injury is aggravated by excessive local inflammation,which promote cardiomyocyte pyroptosis and accelerates extracellular matrix synthesis,leading to myocardial fibrosis,adverse cardiac remodeling,and heart failure.Studies have shown that anti-inflammatory therapy can reduce myocardial injury after acute myocardial infarction and improve atherosclerosis.In recent years,the protective effect of colchicine on cardiovascular disease has been paid more and more attention.However,the clinical application of high dose of colchicine is limited due to the high incidence of adverse reactions.In order to overcome the adverse effects of colchicine.In this study,the calcium carbonate nano delivery system was used to load colchicine,and then colchicine was transported targetly to the myocardium of myocardial infarction site,so as to increase the efficacy of colchicine and reduce adverse reactions.Methods: In this study,a calcium carbonate drug delivery system was used as a carrier,and colchicine was wrapped to made colchicine nanoparticles.At the same time,this study also prepared a calcium carbonate free nano delivery system for colchicine delivery.Colchicine calcium carbonate nanoparticles were studied in vivo and in vitro.In vitro,umbilical vein endothelial cells and macrophages have been studied in this study.Cell injury models induced by ox LDL stimulation,and divided into five groups according to different drug treatments.The control group(control,no ox LDL and drug stimulation),colchicine group(Col,ox LDL and colchicine stimulation),colchicine calcium carbonate nanoparticles group(CCa NPs group,ox LDL and colchicine calcium carbonate nanoparticles stimulation),nanoparticle group(NPs,ox LDL and calcium carbonate nanoparticles stimulation),Model group(ox LDL stimulation,no drug intervention).MTT was used to evaluate the cell viability of different groups.ELISA was used to detect the contents of inflammatory factors such as TNF-α and IL-1β.The level of ROS in endothelial cells was detected by DCFH-DA,and apoptosis of endothelial cells and macrophages was detected by flow cytometry.In vivo:(1)in this study,colchicine was divided into model group,low dose group,middle dose group and high dose group for appropriate drug concentration screening..The levels of serum TNF-α,IL-1β,CRP in myocardial infarction rats were measured by ELISA,the expression of TLR4,NFκB,NLRP3,caspase-1,TNF-α,IL-1β in myocardial infarction were detected by western-blot,and the pyroptosis of myocardial cells was detected by TUNEL.(2)The appropriate drug concentration was selected according to the preliminary experimental results,and corresponding doses of colchicine calcium carbonate nanoparticles,colchicine non-calcium carbonate nanoparticles and nanoparticles were made.Forty-two rats were divided into six groups according to different drug interventions: model group(MI group),nano-particle group(NPs),colchicine group(Col),and colchicine Calcium carbonate nanoparticle group(CCa NPs),colchicine-free calcium carbonate nanoparticle group(CNPs)and sham operation group(Sham),serum TNF-α,IL-1β,CRP and LDH were detected by ELISA,and expression of inflammatory related proteins such as TLR4,NFκB,NLRP3,Caspase-1,IL-10,TGF-β,TNF-α,and IL-1β in myocardial infarction were detected by western blot,TUNEL method to detect cardiomyocyte pyroptosis,Masson staining to evaluate myocardial fibrosis,CD206 and CD11 C positive macrophages were detected by histochemical method to understand the polarization of macrophages.(3)the safety of colchicine and its nano-delivery system was evaluated by detecting the liver and kidney function and pathological results of liver and kidney function in rats.Results: 1.Characterization of colchicine nanoparticles.The particle size of colchicine calcium carbonate nanoparticle,colchicine calcium carbonate free nanoparticle and calcium carbonate nano carrier were about 243,248 and 273 nm,respectively.The drug loading of colchicine nanoparticles and colchicine calcium carbonate free nanoparticles were 4.25% and 3.5%,respectively.2.The protective effects of colchicine and colchicine nanoparticles on endothelial cells.MTT results showed that colchicine calcium carbonate nanoparticles(CCa NPs),compared with colchicine,nanoparticle(NPs)and model group,can significantly improved endothelial cell viability;Col,CCa NPs significantly reduce the TNF-α,IL-1 β levels of damaged endothelial cells;CCa NPs reduce the ROS level of damaged endothelial cells,and CCa NPs significantly inhibit the apoptosis of damaged endothelial cells.3.Colchicine and colchicine nanoparticles significantly inhibited macrophage apoptosis.The apoptosis of macrophages in colchicine and colchicine nano group was significantly lower than that in ox LDL,NPs group,and colchicine nano group could further reduce the apoptosis of macrophages compared with colchicine group.4.The suitable concentration of colchicine was screened.The middle dose colchicine group(0.2mg/kg caudal vein injection)significantly inhibited the levels of CRP,TNF-α,IL-1 β and other inflammatory factors,reduced the expression of TLR4,NF-κ B,NLRP3,caspase-1,IL-1 β,TNF-α and other inflammatory related proteins in myocardium after myocardial infarction,and the middle dose colchicine group had the least TUNEL positive cells.5.Anti-inflammatory effects of colchicine and colchicine nanoparticles.Compared with MI group,CCa NPs and Col group significantly reduced the levels of serum CRP,TNF-α,IL-1 β and other inflammatory factors,and the anti-inflammatory effect of CCa NPs was better than Col group;CCa NPs group significantly reduced the expression of TLR4,NF-κB,NLRP3,IL-1β and other inflammatory proteins;the expression of NF-κB relative protein reduced significantly in Col,CNPs and sham group;the expression of IL-1 β relative protein reduced significantly in CNPs and sham group;The expression of IL-10,TGF-β in CCa NPs and Sham group were higher than that in MI group,and the expression of TGF-β in CCa NPs group was significantly higher than that in col group.6.Colchicine and colchicine nanoparticles improved myocardial fibrosis after MI by inhibiting myocardial pyroptosis. Compared with MI group,the release of LDH in CCa NPs,CNPs and sham group were significantly reduced;the expression of TUNEL positive cells in myocardium was significantly reduced after myocardial infarction;the expression of caspase-1 in CCa NPs,CNPs,Col and sham group wer significantly lower than MI group.7.Effects of colchicine and colchicine nanoparticles on macrophage polarization.CCa NPs increased the expression of CD206-positive cells and reduced the expression of CD11c-positive cells,which means regulate the polarization of macrophages polarization;8.Safety evaluation of colchicine and colchicine nanoparticles.In the CCa NPs,CNPs,and NPs groups,compared with the sham operation group and MI group,liver and kidney function were not significantly difference,and pathological results were normal.Conclusions 1.The calcium carbonate nano delivery system based on colchicine was successfully prepared.2.Colchicine calcium carbonate nanoparticles can protect endothelial cells by inhibiting the release of inflammatory factors,improving the oxidative stress of injured endothelial cells and reducing the apoptosis of endothelial cells.3.0.2mg/kg colchicine can inhibit inflammatory factors with few adverse reactions,and has the strongest effect on inhibiting inflammatory protein and improving cardiomyocyte pyroptosis after myocardial infarction;4.Colchicine calcium carbonate nanoparticles may inhibit cardiomyocyte pyroptosis and inflammatory response by regulating TLR4 / NF κ B / NLRP3 signal pathway;5.Colchicine calcium carbonate nanoparticles improved myocardial fibrosis by inhibiting cardiomyocyte pyroptosis and regulating macrophage polarization;6.The nanoparticles loaded with colchicine have good safety. |
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