Protective effects of myocardium-targeted nanoparticles loaded L-arginineon on sepsis-induced myocardial injury / 中华危重病急救医学

ABSTRACT

Objective:To prepare primary cardiomyocyte (PCM) specific peptide-conjugated mesoporous silicon nanoparticles (MSN) with L-arginine (LA) as a core (PCM-MSN@LA), and evaluate its specific protective effect on septic myocardium.Methods:PCM-MSN@LA was prepared by condensation reaction, the characterization of PCM-MSN@LA, the amount of LA modification and release was detected, and the phagocytosis of PCM-MSN@LA and its affinity to myocardial tissue was observed. ① Experiment one: SD neonatal rat cardiomyocytes were divided into control group (Con group), lipopolysaccharide (LPS) group, MSN@LA/LPS group and PCM-MSN@LA/LPS group. The LPS group was stimulated with 5 mg/L LPS for 16 hours, while the MSN@LA/LPS group and PCM-MSN@LA/LPS group were treated with 5 mg/L LPS and 25 mg/L LA-containing nanoparticles (MSN@LA and PCM-MSN@LA) for 16 hours. Cell viability and reactive oxygen species (ROS) production levels were detected. Apoptosis was observed via terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method (TUNEL). Western Blot was used to detect the changes in endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS) proteins. ② Experiment two: 64 healthy male C57BL/6 mice were divided into Sham group, LPS group, MSN@LA/LPS group and PCM-MSN@LA/LPS group by random number table method, 16 mice in each group. LPS group were injected 50 mg/kg LPS intraperitoneally. MSN@LA/LPS group and PCM-MSN@LA/LPS group were injected with 0.5 mg/kg MSN@LA and PCM-MSN@LA via tail vein immediately after intraperitoneal injection of LPS. Eight animals in each group were used to observe the 24-hour survival rate, and the other 8 mice were used to detect cardiac function by echocardiography at 12 hours after operation; mRNA expressions of interleukin (IL-1, IL-6) and tumor necrosis factor-α (TNF-α) were measured by real-time fluorescent quantitative polymerase chain reaction (RT-qPCR).Results:PCM-MSN@LA was spherical, with particle size of about 180 nm, Zeta potential of about -21 mV, with LA loaded. The amount of LA modification and release rate were 12.3% and 24.3%, respectively. Cell phagocytosis experiments showed that PCM-MSN@LA had the targeting ability of cardiomyocytes and myocardial tissue. Experiment one: after LPS stimulation of myocardial cells, cell viability decreased, while ROS generation, apoptosis, eNOS and iNOS protein expressions increased. Compared with LPS group, MSN@LA/LPS group and PCM-MSN@LA/LPS group had higher cell viability, reduced ROS levels and apoptosis, increased expressions of eNOS and iNOS. PCM-MSN@LA/LPS group changed the above effect further than MSN@LA/LPS group [cell viability ( A value): 0.51±0.08 vs. 0.41±0.03, ROS (relative fluorescence intensity): 28 450±1 941 vs. 35 628±2 551, TUNEL positive cells/total cells: 0.27±0.03 vs. 0.35±0.04, eNOS/β-Tubulin: 1.467±0.046 vs. 1.201±0.131, iNOS/β-Tubulin: 1.700±0.033 vs. 1.577±0.068, all P < 0.05]. Experiment two: the number of 24-hour survive in MSN@LA/LPS group and PCM-MSN@ LA/LPS group were higher than LPS group (number: 2, 4 vs. 1, P values were 0.36 and 0.03 respectively). Compared with Sham group, the cardiac function of LPS group was significantly inhibited and the mRNA expression of inflammatory factors increased. The PCM-MSN@LA/LPS group had higher left ventricular ejection fraction (LVEF) and left ventricular short-axis shortening rate (LVFS) than LPS group, and lower mRNA expressions of IL-1, IL-6, and TNF-α mRNA [LVEF: 0.456±0.019 vs. 0.337±0.017, LVFS: (21.97±1.78)% vs. (15.53±1.67)%, IL-1 mRNA (2 -ΔΔCT): 169.22±8.95 vs. 189.79±6.79, IL-6 mRNA (2 -ΔΔCT): 19.90±1.60 vs. 23.74±1.45, TNF-α mRNA (2 -ΔΔCT): 8.21±0.81 vs. 11.00±1.48, all P < 0.05]. There was no significant difference in each index between the MSN@LA/LPS group and LPS group. Conclusion:PCM-MSN@LA with myocardial targeting characteristic significantly increased the activity of myocardial cells, down-regulated the expression of inflammatory factors and the production of ROS, alleviated cardiac insufficiency in sepsis, and achieved the targeted treatment of myocardial injury in sepsis.