载精氨酸心肌靶向纳米颗粒对脓毒症心肌的保护作用研究

Protective effects of myocardium-targeted nanoparticles loaded L-arginineon on sepsis-induced myocardial injury

目的制备心肌特异性多肽(PCM)修饰介孔硅纳米粒(MSN)包载精氨酸(LA)的纳米颗粒(PCM-MSN@LA),评价其对脓毒症心肌的特异性保护作用.方法通过缩合反应制备PCM-MSN@LA,检测PCM-MSN@LA表征、LA修饰量和释放量,观察PCM-MSN@LA的细胞吞噬行为及其对组织的亲和力.①实验一:将SD乳鼠原代心肌细胞分为正常对照组(Con组)、脂多糖(LPS)组、精氨酸纳米粒组(MSN@LA/LPS组)、心肌靶向精氨酸纳米粒组(PCM-MSN@LA/LPS组).LPS组用5 mg/L LPS刺激细胞16 h;MSN@LA/LPS组和PCM-MSN@LA/LPS组分别用含25 mg/L LA的MSN@LA及PCM-MSN@LA与LPS共同刺激细胞16 h.测定细胞活性和活性氧(ROS)产生水平;用原位末端缺刻标记法(TUNEL)检测细胞凋亡情况;用蛋白质免疫印迹试验(Western Blot)检测内皮型一氧化氮合酶(eNOS)、诱导型一氧化氮合酶(iNOS)蛋白表达.②实验二:按照随机数字表法将64只健康雄性C57BL/6小鼠分为假手术组(Sham组)、LPS组、MSN@LA/LPS组和PCM-MSN@LA/LPS组,每组16只.LPS组腹腔注射50 mg/kg LPS;MSN@LA/LPS组和PCM-MSN@LA/LPS组腹腔注射LPS后立即经尾静脉分别注射0.5 mg/kg的MSN@LA及PCM-MSN@LA.每组8只小鼠用于观察24 h存活情况;另外8只于术后12 h用超声心动图评价心功能,用实时荧光定量聚合酶链反应(RT-qPCR)测定心肌组织白细胞介素(IL-1、IL-6)、肿瘤坏死因子-α(TNF-α)的mRNA表达.结果PCM-MSN@LA呈球形,粒径约180 nm,Zeta电位约-21 mV,且可负载LA,LA修饰量及释放率分别为12.3%、24.3%,细胞吞噬实验显示PCM-MSN@LA具有心肌细胞和心肌组织靶向性.实验一:LPS刺激后心肌细胞活性下降,ROS生成增多,凋亡增多,eNOS及iNOS蛋白表达增多.与LPS组比较,MSN@LA/LPS组和PCM-MSN@LA/LPS组细胞活性增高,ROS及凋亡减少,eNOS、iNOS表达增多;且PCM-MSN@LA/LPS组较MSN@LA/LPS组可进一步提高细胞活性,减少ROS产生和细胞凋亡,增加eNOS、iNOS蛋白表达〔细胞活性(A值):0.51±0.08比0.41±0.03,ROS水平(相对荧光强度):28450±1941比35628±2551,凋亡细胞/总细胞数:0.27±0.03比0.35±0.04,eNOS/β-Tubulin:1.467±0.046比1.201±0.131,iNOS/β-Tubulin:1.700±0.033比1.577±0.068,均P<0.05〕.实验二:MSN@LA/LPS组和PCM-MSN@LA/LPS组术后24 h存活动物数多于LPS组(只:2、4比1,P值分别为0.36、0.03).与Sham组比较,LPS组小鼠心功能明显下降,心肌组织炎性因子mRNA表达增多.与LPS组比较,PCM-MSN@LA/LPS组左室射血分数(LVEF)、左室短轴缩短率(LVFS)明显升高,IL-1、IL-6、TNF-α的mRNA表达明显下降〔LVEF:0.456±0.019比0.337±0.017,LVFS:(21.97±1.78)%比(15.53±1.67)%,IL-1 mRNA(2-ΔΔCT):169.22±8.95比189.79±6.79,IL-6 mRNA(2-ΔΔCT):19.90±1.60比23.74±1.45,TNF-αmRNA(2-ΔΔCT):8.21±0.81比11.00±1.48,均P<0.05〕;而MSN@LA/LPS组与LPS组各指标比较差异无统计学意义.结论PCM-MSN@LA具有心肌靶向性,可显著提高心肌细胞活性,下调炎性因子表达,减少ROS产生,减轻脓毒症心功能不全,从而实现脓毒症心肌损伤的靶向治疗.

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.①Experiment 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.②Experiment 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):28450±1941 vs.35628±2551,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-αmRNA[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-αmRNA(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.