Effects And Mechanisms Of Novel Nano-oxygen-carrier On Improving Myocardial Injury In Septic Rats

ObjectiveSepsis is one of the leading causes of death in the intensive care unit(ICU).Studies have shown that septic patients with myocardial dysfunction have a significant increase in mortality.The pathophysiology of septic myocardial injury is complex,among which hypoxia and oxidative stress injury play a crucial role in the process of septic myocardial injury.The purpose of this study was to establish a polydopamine(PDA)-coated hemoglobin(Hb)nanosystem,evaluate its antioxidant effect and biocompatibility in vitro,test its effects on improving myocardial injury in septic rats,and further explore the possible mechanisms of the system to improve septic myocardial injury.MethodsThe polydopamine-coated hemoglobin(Hb-PDA)nanosystem was prepared and characterized by transmission electron microscopy(TEM).The particle size was measured by dynamic light scattering(DLS)particle size analyzer.The UV-visible spectrum and Fourier infrared spectrum(FT-IR)were analyzed to characterize the feasibility of PDA for surface modification of Hb.The antioxidant capacity of the nanoparticles was evaluated by detecting the total antioxidant capacity.The nanoparticles were co-cultured with rat H9c2 cell to evaluate the cytocompatibility of the system.Forty healthy female SD rats were divided into Sham operation group(Sham+ NS group),sepsis group(CLP+ NS group),hemoglobin group(CLP+ Hb group)and polydopamine-coated hemoglobin group(CLP+Hb-PDA group)according to random number table method,10 rats in each group.30 min before modeling,1 m L 0.9% sodium chloride solution,100 ug/m L Hb solution and 100ug/m L Hb-PDA solution were injected into Sham group and CLP group,Hb group and HbPDA group through internal jugular vein,respectively.Sham group was performed laparotomy,while Cecal Ligation and Puncture(CLP)was used to establish sepsis model in other groups.The vital signs and behavioral changes of rats in each group were observed at 6 h,12 h and 24 h after surgery,and the mortality rate was calculated 24 h after modeling.0.5 m L blood was collected through internal jugular vein for blood gas analysis after anesthesia.The intestinal microcirculation was observed after laparotomy with SDF.1 m L blood was taken from the inferior vena cava for subsequent biochemical detection.The sternum was cut promptly at the level of the xiphoid.The heart was fully exposed and heart perfusion was performed with physiological saline.Then all rats were euthanized.Left ventricular myocardial tissue was fixed in 4% formaldehyde,which subsequently used to observe the myocardial pathology change via hematoxylin eosin staining(HE).The apex cordis tissue was cut at about 1mm thick and fixed in glutaraldehyde for observing ultrastructural changes of myocardial fibers and mitochondria under the TEM.The levels of tumor necrosis factor(TNF-α),interleukin-1β(IL-1β)and interleukin-6(IL-6)inflammatory factors in serum of rats were detected by ELISA,and the levels of creatine kinase isoenzyme(CK-MB)and cardiac troponin(c Tn-I)in peripheral blood of rats were detected by ELISA.The level of superoxide dismutase(SOD)in myocardial tissue of rats in each group was detected via WST-1 method.Western blotting(WB)was used to detect the expression levels of caspase-3,Bcl-2 and Bax apoptosis proteins in myocardium of rats.Results1.Compared with Hb,the particle size of Hb-PDA nanoparticles increased under TEM,and the particle size measured by DLS was about 50 nm.UV-visible spectrum and FT-IR of HB-PDA showed all characteristic peaks of Hb and PDA,and Hb almost had no T-AOC antioxidant ability in ABTS+ scavenging experiment.Compared with Hb,Hb-PDA exhibited obvious antioxidant capacity(P<0.0001).Co-culturing Hb-PDA with H9c2 cells for 24 h,no marked cytotoxicity was observed at the concentration below 100 ug/m L.2.24 h after modeling,the survival rates of Sham group,CLP group,Hb group and Hb-PDA group were 100%,76.9%,76.9%,and 92.3% respectively,of which Hb-PDA group was higher than CLP group.Compared with Sham group,blood lactic acid level of rats in CLP group was significantly increased(P<0.0001),SDF showed decreased capillary density.Blood flow heterogeneity was observed at microcirculation level.And intermittent flow and even blood stasis were also observed.HE staining showed loose and disordered myocardial tissue,and obvious red blood cell exudation in blood vessels.At the same time,inflammatory cell infiltration and severe damage were observed.The cardiac muscle fibers were disordered under TEM,and some mitochondria were slightly swollen,cristae was broken,and ultrastructure was destroyed.There was no significant difference in serum TNF-α level,while il-1 β and IL-6 levels were significantly increased(P<0.001).The levels of CK-MB and c Tn-I were significantly increased(P<0.0001),the activity of SOD in myocardium was significantly decreased(P<0.05),caspase-3 and Bax were significantly increased,and Bcl-2 was significantly decreased(P<0.05).3.Compared with CLP group,the death rate and blood lactic acid level in Hb-PDA group decreased significantly(P<0.01),capillaries in microcirculation did not decrease significantly,blood flow was relatively smooth,HE myocardium pathological inflammatory changes were relatively light,cardiac muscle fiber arrangement was relatively regular under TEM microscopy,and no obvious mitochondrial swelling and structural damage were observed.There was no significant difference in serum TNF-α level,the expression levels of IL-1β(P<0.01)and IL-6(P<0.0001)were significantly decreased,the levels of myocardial injury markers CK-MB and c Tn-I were significantly decreased(P<0.0001),and SOD activity in myocardial tissue was significantly increased(P<0.0001).The contents of Caspase-3 and Bax were significantly decreased(P<0.05),while the content of Bcl-2 was significantly increased(P<0.05).4.Compared with Hb group,the death rate and blood lactic acid level in Hb-PDA group decreased significantly(P<0.01),and the microcirculation was relatively improved.The pathological inflammatory changes of HE myocardium were lighter,mitochondrial accumulation and swelling were obvious in Hb group under TEM,while mitochondrial damage was lighter in Hb-PDA group.The expression levels of IL-1β(P<0.01)and IL-6(P<0.001)were significantly decreased,SOD activity was significantly increased in myocardial tissue(P<0.001),and caspase-3,Bax and Bcl-2 expression levels were not significantly different.Conclusion1.In this study,PDA was successfully modified on the surface of Hb to obtain HbPDA,which exhibited great antioxidant performance and biocompatibility at a certain concentration;2.Hb-PDA can improve the myocardial injury of septic rats,and it is superior to Hb alone.3.The ameliorative effect of Hb-PDA on septic myocardial injury may be related to the improvement of microcirculation,reduction of myocardial oxidative stress level and inhibition of myocardial cell apoptosis.