Mesoporous Silica Nanoparticles Loaded Panax Notoginseng R1 For Targeted Treatment Of Myocardial Infarction And Its Mechanism

ObjectivesCardiovascular disease has a great negative impact on human beings and society.It plays a major role in the morbidity and mortality of human beings all over the world,especially myocardial infarction（MI）.After MI,the damaged cardiomyocytes are gradually replaced by fibroblasts,which leads to ventricular remodeling and thinning,and eventually leads to heart failure and even sudden death.In the prevention and treatment of cardiovascular disease,traditional Chinese medicine has the advantages of multi-target,multi-component,less side effects,low cost and so on.Notoginsenoside R1（NGR1）is the main monomer extracted from the dry roots and rhizomes of Panax notoginseng,which can play the roles of neuroprotection,anti-inflammation,anti-apoptosis and anti-ischemia.However,due to the different distribution,absorption and metabolism of different drugs in the body,it is not necessarily able to play the most effective pharmacological activity after administration.It has been proved that the amount of NGR1 absorbed into the blood circulation by oral administration is very low.Therefore,in this study,we established H₂O₂ injury model of H9C2 cells in vitro to verify the protective effect of NGR1 and its possible mechanisms.On this basis,the mouse model of MI was established by ligating the anterior descending branch of left coronary artery,and NGR1 was loaded by CD11b antibody modified mesoporous silica nanoparticles（MSN）to improve the utilization and myocardial targeting of NGR1,and to explore its therapeutic effects and possible mechanisms on MI.MethodsIn cell experiments,H9C2 cells were cultured in vitro and pretreated with NGR1.H₂O₂ was used to simulate the injury model in vitro.CCK8,TUNEL and annexin V-PI flow cytometry were used to detect the effect of NGR1 on the proliferation activity of H9C2 cells and the effect of NGR1 on the injury induced by H₂O_2.The levels of p-AKT,p-ERK/2,LC3b and Beclin-1 were detected by western blot to explore the protective mechanisms of NGR1 on H9c2 cells in vitro.TUNEL staining and Annexin V-PI flow cytometry were used to compare the protective effects of NGR1 and insulin-like growth factor-1（IGF-1）.In animal experiments,MI models were established in nude mice and C57BL/6 mice.NGR1 loaded mesoporous silica nanoparticles（MSN-NGR1-CD11b antibody nanoparticles）were injected into the tail vein to target the myocardial infarction site.The targeting effect of MSN-NGR1-CD11b antibody nanoparticles was detected by in vivo imaging.The ejection fraction（EF）,shortening fraction（FS）and other cardiac function indexes were detected by small animal echocardiography.TUNEL was used to detect the apoptosis of myocardial cells in the infarcted area.Masson trichrome staining was used to detect the infarcted area and collagen deposition.Immunofluorescence was used to detect the expression of CD31 and CD206 in the infarcted area And the Elisa experiment detects the expression of inflammatory factors in the infarct area.Finally,the expression of p-AKT,p-ERK1/2,LC3b and Beclin-1 was detected by Western blot after 3 days of treatment in each group to explore the possible mechanisms of MSN-NGR1-CD11b antibody improving myocardial function after MI.ResultsThe results showed that NGR1 at 100μM could significantly promote the proliferation of H9C2 cells.350μM H₂O₂ could cause the death rate of H9C2 cells to be about 50%.CCK8 activity assay showed that NGR1 at 100μM had the most significant protective effect on H9C2 cells against injury induced by H₂O₂.Therefore,100μM NGR1 is the best concentration to protect H9C2 cells,which was further verified by Annexin V-PI flow cytometry.Western blot showed that NGR1+H₂O₂ group significantly activated p-AKT and p-ERK1/2 level compared with control group and H₂O₂group.However,NGR1 had no significant effect on LC3b and Beclin-1 protein levels.After H9C2 cells were treated with NGR1 and H₂O₂,CCK8 activity,TUNEL and Annexin V-PI flow cytometry showed that the number of apoptotic cells in NGR1+H₂O₂ group was significantly reduced,and the protective effect of NGR1 was weakened after adding PI3K/AKT signaling pathway and MAPK/ERK signaling pathway inhibitorsThe best concentration of IGF-1 to protect H9C2 from induce of H₂O₂is 10 ng/ml.TUNEL results showed that IGF-1 could alleviate the injury of H9C2 cells induced by H₂O₂.However,compared with IGF-1,the protective effect of NGR1 was more obvious under the same treatment time.Annexin V-PI flow cytometry showed that the number of apoptotic H9C2 cells decreased by 9.71%after IGF-1 treatment,while the number of apoptotic H9C2 cells decreased by 14.80%after NGR1 addition.After MI,the number of CD11b positive cells in the infarct site increased,and most of the CD11b positive cells came from peripheral blood.One hour after MI modeling,MSN-NGR1-CD11b antibody nanoparticles were injected into the tail vein of nude mice.In vivo imaging results of 3-48 hours showed that MSN could target the infarct site and stay at the infarct site for 24 hours.One day after MI,MSN-NGR1-CD11b antibody nanoparticles were injected into tail vein.The cardiac function,infarct size and collagen deposition of MSN-NGR1-CD11b antibody nanoparticles injection group were significantly improved.In addition,the expression of CD206 protein,which on the surface of M2 macrophage,was increased.ELISA results showed that in the MSN-NGR1-CD11b antibody nanoparticle treatment group,the inflammatory factors TNF-αand IL-6 were significantly reduced,indicating that NGR1 reduced the inflammatory response in the infarct site.Compared with the control group and non-CD11b antibody modified nanoparticles group,the angiogenesis of MSN-NGR1-CD11b antibody nanoparticles group was more obvious.Western blot results showed that MSN-NGR1-CD11b antibody nanoparticles activated the levels of p-AKT and p-ERK1/2,but did not affect the expression of LC3b and Beclin-1 protein.ConclusionsNGR1 can protect H9C2 cells against H₂O₂ induced injury,which may be related to the activation of PI3K/AKT signaling pathway and MAPK/ERK signaling pathway.MSN nanoparticles coupled with CD11b antibody can significantly enhance NGR1 targeting myocardial infarction site,activate PI3K/AKT and MAPK/ERK signaling pathway,reduce inflammatory response and inhibit cardiomyocyte apoptosis in myocardial infarction site of MI mice,so as to effectively improve cardiac function after MI.