Peng Zefang, Zhang Ming, Ouyang Minzhi, Ouyang Xiangnan, Xu Ganqiong, Zhou Jiawei, Zhang Meixiang
Department of Ultrasound Diagnosis, the Second Xiangya Hospital, Central South University, Changsha 410011, Hunan, China. Corresponding author: Zhang Ming, Email:
Zhonghua Wei Zhong Bing Ji Jiu Yi Xue. 2020 Aug;32(8):953-959. doi: 10.3760/cma.j.cn121430-20200217-00168.
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.
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).
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): 169.22±8.95 vs. 189.79±6.79, IL-6 mRNA (2): 19.90±1.60 vs. 23.74±1.45, TNF-α mRNA (2): 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.
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.
制备以L-精氨酸（LA）为核心的原代心肌细胞（PCM）特异性肽偶联介孔硅纳米颗粒（MSN）（PCM-MSN@LA），并评估其对脓毒症心肌的特异性保护作用。
通过缩合反应制备PCM-MSN@LA，检测PCM-MSN@LA的表征、LA修饰量及释放情况，观察PCM-MSN@LA的吞噬作用及其与心肌组织的亲和力。（1）实验一：将SD新生大鼠心肌细胞分为对照组（Con组）、脂多糖（LPS）组、MSN@LA/LPS组和PCM-MSN@LA/LPS组。LPS组用5 mg/L LPS刺激16小时，而MSN@LA/LPS组和PCM-MSN@LA/LPS组用5 mg/L LPS和含25 mg/L LA的纳米颗粒（MSN@LA和PCM-MSN@LA）处理16小时。检测细胞活力和活性氧（ROS）产生水平。通过末端脱氧核苷酸转移酶介导的dUTP缺口末端标记法（TUNEL）观察细胞凋亡。采用蛋白质免疫印迹法检测内皮型一氧化氮合酶（eNOS）和诱导型一氧化氮合酶（iNOS）蛋白的变化。（2）实验二：将64只健康雄性C57BL/6小鼠按随机数字表法分为假手术组（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组在腹腔注射LPS后立即经尾静脉注射0.5 mg/kg MSN@LA和PCM-MSN@LA。每组8只动物用于观察24小时生存率，另外8只小鼠在术后12小时通过超声心动图检测心功能；采用实时荧光定量聚合酶链反应（RT-qPCR）检测白细胞介素（IL-1、IL-6）和肿瘤坏死因子-α（TNF-α）的mRNA表达。
PCM-MSN@LA呈球形，粒径约180 nm，Zeta电位约-21 mV，负载有LA。LA修饰量和释放率分别为12.3%和24.3%。细胞吞噬实验表明，PCM-MSN@LA具有心肌细胞和心肌组织靶向能力。实验一：心肌细胞经LPS刺激后，细胞活力下降，而ROS生成、细胞凋亡、eNOS和iNOS蛋白表达增加。与LPS组相比，MSN@LA/LPS组和PCM-MSN@LA/LPS组细胞活力更高，ROS水平和细胞凋亡减少，eNOS和iNOS表达增加。PCM-MSN@LA/LPS组比MSN@LA/LPS组更能进一步改变上述效应[细胞活力（A值）：0.51±0.08 vs. 0.41±0.03，ROS（相对荧光强度）：28 450±1 941 vs. 35 628±2 551，TUNEL阳性细胞/总细胞：0.27±0.03 vs. 0.35±0.04，eNOS/β-微管蛋白：1.467±0.046 vs. 1.201±0.131，iNOS/β-微管蛋白：1.700±0.033 vs. 1.577±0.068，均P < 0.05]。实验二：MSN@LA/LPS组和PCM-MSN@LA/LPS组24小时存活数高于LPS组（数量：2、4 vs. 1，P值分别为0.36和0.03）。与Sham组相比，LPS组心功能明显受抑制，炎症因子mRNA表达增加。PCM-MSN@LA/LPS组左心室射血分数（LVEF）和左心室短轴缩短率（LVFS）高于LPS组，IL-1、IL-6和TNF-α mRNA表达低于LPS组[LVEF：0.456±0.019 vs. 0.337±0.017，LVFS：（21.97±1.78）% vs. （15.53±1.67）%，IL-1 mRNA（2）：169.22±8.95 vs. 189.79±6.79，IL-6 mRNA（2）：19.90±1.60 vs. 23.74±1.45，TNF-α mRNA（2）：8.21±0.81 vs. 11.00±1.48，均P < 0.05]。MSN@LA/LPS组与LPS组各指标无明显差异。
具有心肌靶向特性的PCM-MSN@LA显著提高心肌细胞活性，下调炎症因子表达和ROS产生，减轻脓毒症时的心功能不全，实现脓毒症心肌损伤的靶向治疗。
