毛蕊花苷对小鼠异位心脏移植冷缺血再灌注损伤的影响:与NF-κB/NLRP3信号通路的关系

Effect of verbascoside on cold ischemia-reperfusion injury following heterotopic heart transplantation in mice: relationship with NF-κB/NLRP3 signaling pathway

目的评价毛蕊花苷对小鼠异位心脏移植冷缺血再灌注损伤的影响及其与NF-κB/NOD样受体蛋白3(NLRP3)信号通路的关系。方法动物实验:SPF级健康雄性C57BL/6小鼠18只,6~8周龄,体质量24~28 g,采用随机数字表法分为3组(n=6):对照组(C组)、心脏移植冷缺血再灌注组(I/R组)和心脏移植冷缺血再灌注+毛蕊花苷组(I/R+VB组)。采用cuff套管法颈部异位心脏移植的方法制备小鼠心脏移植冷缺血再灌注损伤模型,C组供体心脏取下后立即移植至受体,I/R组供体心脏于4℃保存液保存8 h后移植至受体,I/R+VB组供体与受体小鼠分别于术前3 d时腹腔注射毛蕊花苷20 mg/kg。于再灌注24 h时进行移植物跳动评分后取移植心脏心肌组织,采用ELISA法检测MDA含量和SOD活性;取部分供体心肌组织,观察病理学结果;采用Western blot法检测NF-κB、p-NF-κB、NLRP3、ACS和caspase-1的表达;RT-PCR法检测IL-1β、TNF-α及IL-6 mRNA的表达。细胞实验:建立大鼠心肌细胞H9c2冷缺氧复氧模型,采用随机数字表法将细胞分为3组(n=24):对照组(C组)、冷缺氧复氧组(H/R组)、冷缺氧复氧+毛蕊花苷组(H/R+VB组)。采用10℃缺氧18 h,37℃复氧24 h的方法建立冷缺氧复氧模型,检测细胞活力和LDH活性;Western blot法检测NF-κB和NLRP3的表达;免疫荧光染色法检测p-NF-κB的表达。结果动物实验:与C组比较,I/R组MDA含量升高,移植物跳动评分和SOD活性降低,p-NF-κB、NLRP3、ACS和caspase-1表达、IL-1β、TNF-α及IL-6 mRNA表达上调(P<0.05),心肌组织病理学损伤加重;与I/R组比较,I/R+VB组MDA含量降低,移植物跳动评分和SOD活性升高,p-NF-κB、NLRP3、ACS和caspase-1表达、IL-1β、TNF-α及IL-6 mRNA表达下调(P<0.05),心肌组织病理学损伤减轻。细胞实验:与C组比较,H/R组细胞活力降低,LDH活性升高,p-NF-κB、NLRP3、ACS、caspase-1和p-NF-κB表达上调(P<0.05);与H/R组比较,H/R+VB组细胞活力升高,LDH活性降低,p-NF-κB、NLRP3、ACS、caspase-1和p-NF-κB表达下调(P<0.05)。结论毛蕊花苷可减轻小鼠异位心脏移植冷缺血再灌注损伤,其机制与抑制NF-κB/NLRP3信号通路激活有关。

Objective To evaluate the effect of verbascoside on cold ischemia-reperfusion injury following heterotopic heart transplantation in mice and the relationship with nuclear factor kappa B(NF-κB)/nucleotide-binding oligomerization domain-like receptor containing pyrin domain 3(NLRP3)signaling pathway.Methods This experiment was performed in two parts.PartⅠanimal experiment Eighteen SPF healthy male C57BL/6 mice,aged 6-8 weeks,weighing 24-28 g,were divided into 3 groups(n=6 each)by the random number table method:control group(C group),cold ischemia-reperfusion(I/R)group(I/R group)and cold I/R+verbascoside group(I/R+VB group).Cold I/R injury model of mouse heart transplantation was prepared by neck heterotopic heart transplantation using the modified non-suture cuff technique.The donor heart in group C was immediately transplanted to the recipient after removal,while the donor heart in group I/R was stored in the 4℃University of Wisconsin solution for 8 h before transplantation to the recipient,and verbascoside 20 mg/kg was intraperitoneally injected at 3 days before surgery in donor and recipient mice in I/R+VB group.At the end of reperfusion,the myocardial tissue of the transplanted heart was obtained after assessing the beating score for determination of malondialdehyde(MDA)content and superoxide dismutase(SOD)activity by enzyme-linked immunosorbent assay.Part of the donor myocardium was taken for examination of the pathological results.The expression of NF-κB,p-NF-κB,NOD-like receptor protein 3(NLRP3),ACS and caspase-1 was detected by Western blot.The expression of IL-1β,TNF-αand IL-6 mRNA was detected by real-time polymerase chain reaction.PartⅡcell experiment Rat cardiomyocyte H9c2 cold hypoxia-reoxygenation model was developed,and the cells were divided into 3 groups(n=24 each)by the random number table method:control group(C group),cold hypoxia-reoxygenation group(H/R group),and cold hypoxia-reoxygenation+verbascoside group(H/R+VB group).The cells were exposed to hypoxia for 18 h at 10℃followed by restoration of reoxygenation for 24 h at 37℃to develop the cold hypoxia-reoxygenation model.The cell viability and LDH activity were determined.The expression of NF-κB and NLRP3 was detected by Western blot,and the expression of phosphorylated NF-κB(p-NF-κB)was detected by immunofluorescence staining.Results PartⅠanimal experiment Compared with C group,the MDA content was significantly increased,the beating score of grafts and SOD activity were decreased,and the expression of p-NF-κB,NLRP3,ACS and caspase-1 and IL-1β,TNF-αand IL-6 mRNA was up-regulated(P<0.05),and myocardial histopathological injury was aggravated in I/R group.Compared with I/R group,the content of MDA was significantly decreased,the beating score of grafts and SOD activity were increased,the expression of p-NF-κB,NLRP3,ACS and caspase-1 and IL-1β,TNF-αand IL-6 mRNA was down-regulated(P<0.05),and the myocardial histopathological injury was alleviated in I/R+VB group.PartⅡcell experiment Compared with C group,the cell viability was significantly decreased,the activity of LDH was increased,and the expression of p-NF-κB,NLRP3,ACS,caspase-1 and p-NF-κB was up-regulated in H/R group(P<0.05).Compared with H/R group,the cell viability was significantly increased,the activity of LDH was decreased,and the expression of p-NF-κB,NLRP3,ACS,caspase-1 and p-NF-κB was down-regulated in H/R+VB group(P<0.05).Conclusions Verbascoside can alleviate cold I/R injury following heterotopic heart transplantation in mice,and the mechanism may be related to inhibition of activation of NF-κB/NLRP3 signaling pathway.