Protective Effects Of Astragalus Polysaccharide Nanoparticles On Myocardial Injury In Sepsis

Background:Myocardial injury caused by sepsis is a complication of common clinical critical illness and an important cause of high mortality in ICU patients.It was found that multiple factors may participate in its pathophysiological process,in which excessive release of inflammatory factors may be the initiating factor of this disease.The NF-κB signaling pathway was the classic way to activate inflammatory factors.Toll-like receptors(TLRs)was shown to trigger the NF-κB signaling pathway and resulted the excessive release of inflammatory mediators.Astragalus polysaccharides(APS),as a common traditional Chinese medicine,have been applied to improve immune system in antioxidant,antiviral,anti-inflammatory clinic therapy.It has been demonstrated that TLR4 could mediate immune regulation of APS after binding to APS.Unfortunately,such bulky biomacromolecules were usually difficult for absorption through the small intestinal mucosa and only absorbed via the paracellular route to a minor extent.The low absorption resulted in an inadequate availability of this drug.In order to solve the problem of clinical application of APS,in this thesis,the astragalus polysaccharide nanoparticles(APS-CS/TPP)were synthesized by the nanotechnology to intervene in sepsis myocardial injury,and the mechanism of the APS-CS/TPP nanoparticles was explored.New therapeutic regimen targeting the treatment of sepsis myocardial injury for the application of traditional chinese medicine could have important clinical strategies.Purpose:The effects of this prepared APS-CS/TPP nanoparticles on the myocardial injury induced by sepsis were evaluated in vivo and in vitro.Furthermore,the possible therapeutic mechanism for APS-CS/TPP nanoparticles was investigated.Methods:1.Preparation of the APS-CS/TPP nanoparticles and analysis its characteristicsAPS-CS/TPP nanoparticles were synthesized using the ionic gelation technique.Then the Fourier Transform Infrared Spectra of APS,CS/TPP and APS-CS/TPP nanoparticles were analyzed to evaluate the cross-linking of APS with CS/TPP.The morphological characteristics of the APS-CS/TPP nanoparticles were observed by scanning electron microscope.The physical characteristics of the APS-CS/TPP nanoparticles,such as its size,distribution and stability,were characterized by testing the particle size,Zeta potential and dispersion index.Finally,the APS-CS/TPP nanoparticles was co-cultured with cardiomyocytes(H9c2 cells),and the cell compatibility of this drug was evaluated by detecting changes in cell viability.2.The effect of APS-CS/TPP nanoparticles on lipopolysaccharide(LPS)-induced H9c2 cellsH9c2 cells were divided into 5 groups,named Control group,LPS+PBS group,LPS+APS group,LPS+CS/TPP group and LPS+APS-CS/TPP group.The 5×10~4 per well of H9c2 cells were inoculated in the culture plate and cultured for 4 h.Then APS,CS/TPP and APS-CS/TPP nanoparticles at a concentration of 50μg/ml were given respectively.After incubating for 24 h,the LPS at a concentration of 10μg/ml were added for 24 h.For the Control group,only PBS buffer was added.MTT method was used to detect changes in cell viability,cell morphology by Wright staining.DAPI and Mito tracker staining were used to observe apoptotic nuclei and mitochondrial morphology.Flow cytometry was used to detect the amount of apoptosis and DCFH-DA needle method was used to detect the level of reactive oxygen species(ROS)in H9c2 cells.3.Therapeutic effect of APS-CS/TPP nanoparticles on mice with sepsis myocardial injuryNinety C57BL/6 male mice were randomly divided into five groups:sham operation control group(Control group),model control group(CLP+NS group),astragalus polysaccharide group(CLP+APS group),chitosan nanoparticle group(CLP+CS/TPP group)and astragalus polysaccharide nanomedicine group(CLP+APS-CS/TPP group).Mice in the last three groups were received intragastric administration for APS,CS/TPP,APS-CS/TPP nanoparticles at 200 mg/kg once a day for 3 days.Meanwhile,the NS groups received isovolumic saline.After that,mice in all groups were treated with CLP to induce polymicrobial sepsis.Following CLP,mice were respectively treated with saline or APS,CS/TPP,APS-CS/TPP nanoparticles(200 mg/kg,i.p.)at 6 h,12 h and 24 h.The levels of bacterial load,WBC,C-reactive protein and cardiac troponin I in peripheral blood of each group were compared at 6 h,12 h and 24 h after CLP,and the changes of myocardial histopathology in each group were analyzed too.4.Exploring the mechanism of APS-CS/TPP nanoparticles in treating sepsis myocardial injuryThe effects of the APS-CS/TPP nanoparticles on the TLR-4/NF-κB signaling pathway in mouse myocardium was explored after treating APS-CS/TPP nanoparticles in mice with sepsis myocardial injury.The effects of inflammatory mediators on myocardial tissues were discussed applying multiplex cytokine immunoassay.The real-time quantitative PCR method was used to detect mRNA levels of tlr4,p38 and Nf-κB in mouse myocardial tissue,and western blot method to detect the expression of TLR4,p38 and NF-κB protein in mouse myocardium.Then the regulation effect of APS-CS/TPP nanoparticles on the immune level of mice to the sepsis myocardial injury were clarfied through the flow cytometry to detect the proportion of T cells in spleen,the multiplex cytokine immunoassay to determine the changes of cytokines in serum,ELISA method to detect the levels of MASP-2,C4a,C3a and C5a in serum.Result:1.APS-CS/TPP nanoparticles with stable properties was successfully constructedAccording to the smallest carrier particle size and the highest encapsulation efficiency,the optimized APS-CS/TPP nanoparticles was prepared under a solution of2 mg/ml CS,1 mg/ml TPP and 1 mg/ml APS respectivly.To investigate APS-CS/TPP nanoparticle formation,FTIR spectra of APS and CS/TPP nanoparticles were recorded.The main IR bands of APS and APS-CS/TPP nanoparticle showed the presence of band shifts at the frequency of 618.3 cm-1,clearly indicating the interaction between APS and CS/TPP nanoparticles.The APS-CS/TPP nanoparticles was spherical and the average particle size was 110±5.09 nm with uniform distribution.The zeta potential was 43.6±0.21 mV,showing a positive charge and stable properties.There was no significant change in cell viability after co-cultivation of the nanomedicine with H9c2 cells(P=0.748),indicating that the APS-CS/TPP nanoparticles has good cell compatibility and can be used for subsequent in vitro and in vivo studies.2.APS-CS/TPP nanoparticles had effect on resisting LPS-induced H9c2 cell damageFor the LPS-induced H9c2 cells given APS-CS/TPP nanoparticles,the cell viability increased by more than 50%compared with the control group,and was about 2 times higher than that of the LPS+PBS group,with a significant difference(P=0.003).In the LPS+APS-CS/TPP group,H9c2 cells grew well,and the cell morphology did not change significantly.The early apoptosis rate of the LPS+APS-CS/TPP group was 47.33%±10.33,which was about 30%lower than that of the LPS+PBS group,with a significant difference(P=0.000).Although the ROS level in the LPS+APS-CS/TPP group was slightly higher than that in the control group,it was about 2 times lower than that in the LPS+PBS group(P=0.013).3.APS-CS/TPP nanoparticles can protect myocardial injury in mice after CLPThe sepsis myocardial injury model of mouse was established by CLP method.After treatment with APS-CS/TPP nanoparticles,it was found that the total number of bacteria in the CLP+APS-CS/TPP group after 12 h was significantly lower than that in the CLP+NS group,and reduced about two times after 24 h,although the bacterial load in peripheral blood increased in a time-dependent manner after CLP(P=0.000).The levers of WBC and CRP in peripheral blood were significantly lower than those in the CLP+NS group.The levers of cTnI was significantly decreased in the CLP+APS-CS/TPP groupcompared with the CLP+NS group(P=0.000).Following treatment with APS-CS/TPP nanoparticles,the myocardial fibers were more neatly arranged and myocardial cells were only partially swollen compared to CLP+NS group.Furthermore,less infiltration of leucocyte cells were observed in APS-CS/TPP nanoparticles receiving group.The score of pathological injury at 24 h after CLP was1.78±0.48,which was significantly lower than that of CLP+NS group(P=0.000).4.APS-CS/TPP nanoparticles played a protective role in sepsis myocardial injury through TLR-4/NF-κB signaling pathway and immunomodulationIt was found that the levels of the proinflammatory cytokines,such as TNF-α,IL-1β,IL-6 in the myocardial tissue of the CLP+APS-CS/TPP group decreased by more than 2 times and the levels of anti-inflammatory cytokines(IL-2,IL-4,IL-5 and IL-10)were significantly increased,compared with the CLP+NS group.The mRNA levels of tlr4,p38 and Nf-κB were significantly reduced,and the expression of TLR4,p38 and NF-κB protein was also reduced.In addition,after applying APS-CS/TPP nanoparticles,the percentage of CD4~+T and CD8~+T cells in the spleen of mice increased significantly,but the proportion of CD4~+CD25~+cells decreased significantly compared with CLP+NS group.However,APS-CS/TPP nanoparticles had no effects on cytokines in peripheral blood.It could significantly reduce the levels of C3a and C5a in serum which had significantly different from CLP+NS group(P=0.000,P=0.011).Conclusion1.APS-CS/TPP nanoparticles with uniform particle size,regular morphology and good biocompatibility were successfully prepared.2.APS-CS/TPP nanoparticles could protect the H9c2 cardiomyocytes exposed LPS.3.APS-CS/TPP nanoparticles had protective effects on mice with CLP-induced myocardial injury.4.APS-CS/TPP nanoparticles could effectively protect myocardial damage against sepsis through TLR4-mediated NF-κB signaling pathway and immunomodulatory.