过表达TNIP3对脓毒症小鼠心房颤动易感性的影响及其机制研究
The effect of TNIP3 overexpression on susceptibility to atrial fibrillation in septic mice and the potential mechanisms
杨宏杰 1帅维1
作者信息
- 1. 武汉大学人民医院心内科武汉大学心血管病研究所心血管湖北省重点实验室(湖北武汉 430060)
- 折叠
摘要
目的 研究肿瘤坏死因子α诱导相互作用蛋白3(TNIP3)对脓毒症小鼠心房颤动(简称房颤)的影响及其潜在的机制.方法 40只雄性C57BL/6小鼠随机分为4组:对照+9型腺相关病毒(AAV9)-绿色荧光蛋白(GFP)组(CTL+AAV9-GFP 组)、对照+AAV9-TNIP3 组(CTL+AAV9-TNIP3 组)、脂多糖(LPS)+AAV9-GFP 组(LPS+AAV9-GFP组)、脂多糖+AAV9-TNIP3组(LPS+AAV9-TNIP3组),每组10只小鼠.按分组于尾静脉注射携带TNIP3或仅空载GFP的AAV9,4周后按分组于腹腔注射LPS 10 mg/kg或等量生理盐水连续3天.以心脏超声检测左室射血分数(LVEF)、左室短轴缩短率(LVFS).以右房Burst刺激检测房颤诱发率.对心房肌组织切片行Masson染色进行心房肌纤维化检测.以免疫组化法检测TNIP3在心房肌的表达,免疫荧光法检测巨噬细胞标志物CD68在心房肌的表达,及Western-blot法检测TNIP3、Toll样受体4(TLR4)、P65总蛋白(T-P65)及其磷酸化蛋白(P-P65)的表达.结果 与 CTL+AAV9-GFP 组相比,CTL+AAV9-TNIP3 组心房组织 TNIP3 增加显著(3.02±0.39 vs 0.46±0.04,P＜0.05).LPS+AAV9-GFP组与CTL+AAV9-GFP组比较,心房肌组织中TNIP3蛋白表达亦升高(1.44±0.09 vs 0.54±0.08,P＜0.05),免疫组化 TNIP3 阳性细胞占比增加[(17.43±1.68)％vs(5.67±0.60)％,P＜0.05].LPS+AAV9-TNIP3 组与 LPS+AAV9-GFP 组相比房颤诱发率低(10％vs 80％,P＜0.05),LVEF 和 LVSF 均增加(0.72±0.046 vs 0.40±0.02,0.37±0.015 vs 0.28±0.021,P＜0.05);心房肌纤维化减轻,纤维化面积占比减小[(9.97±1.57)％vs(18.43±0.71)％,P＜0.05].LPS+AAV9-TNIP3 组与 LPS+AAV9-GFP 组相比,炎症浸润明显减轻,CD68 阳性细胞占比显著减小[(7.77±0.61)％vs(17.03±1.22)％,P＜0.05];TLR4 蛋白、P-P65 蛋白以及 P-P65/T-P65 蛋白表达下调.结论 过表达TNIP3通过抑制脓毒症相关的心脏纤维化和炎症细胞浸润,降低房颤易感性,其机制与TLR4/NF-κB信号通路相关,TNIP3可能是防治脓毒症相关房颤的新靶点.
Abstract
Objective To investigate the effects and potential mechanisms of tumor necrosis factor alpha induced interacting protein 3(TNIP3)on atrial fibrillation(AF)in septic mice.Methods Forty male C57BL/6 mice were randomly divided into four groups:control+AAV9-GFP group(CTL+AAV9-GFP group),control+AAV9-TNIP3 group(CTL+AAV9-TNIP3 group),lipopolysaccharide+AAV9-GFP group(LPS+AAV9-GFP group),and lipopolysaccharide+AAV9-TNIP3 group(LPS+AAV9-TNIP3 group).According to the group(n=10 each),type 9 adeno-associated virus(AAV9)carrying TNIP3 or only green fluorescent protein(GFP)were injected by the tail vein.Four weeks after AAV9 injection,LPS 10 mg/kg or an equal volunme of saline were intraperitoneal injec-ted for 3 days.Cardiac ultrasound examination were performed to measure left ventricular ejection fraction(LVEF)and left ventricular short axis fractional shortening(LVFS).Electrical burst stimulation on right atrium were used to detect AF induction rate.Masson staining was used to detect atrial fibrosis.Immunohistochemistry was used to detect the expression of TNIP3 in atrium,and immuno-fluorescence was used to detect the expression of mac-rophage marker CD68.Western blot was used to detect the expression of TNIP3,toll like receptor 4(TLR4),P65 total protein(T-P65),and its phosphorylated protein(P-P65).Results Atrial TNIP3 expression was signif-icantly increased in CTL+AAV9-TNIP3 group compared to the CTL+AAV9-GFP group(3.02±0.39 vs 0.46±0.04,P＜0.05).Compared with the CTL+AAV9-GFP group,both atrial TNIP3 expression(1.44+0.09 vs 0.54+0.08,P＜0.05).and the percentage of TNIP3 positive cells[(17.43±1.68)％vs(5.67±0.60)％,P＜0.05]signifi-cantly increased in the LPS+AAV9-TNIP3 group.The susceptibility to AF was significantly reduced in the LPS+AAV9-TNIP3 group compared to the LPS+AAV9-GFP group,with lower AF induction rates(10％vs 80％,P＜0.05).Compared with the LPS+AAV9-GFP group,both of the LVEF and LVSF increased(0.72±0.046 vs 0.40±0.02,0.37±0.015 vs 0.28±0.021,P＜0.05)the fibrosis of atrium was reduced and the proportion of fibrosis area was decreased[(9.97±1.57)％vs(18.43±0.71)％,P＜0.05]in the LPS+AAV9-TNIP3 group.Compared with the LPS+AAV9-GFP group,the LPS+AAV9-TNIP3 group showed a significant reduction in inflammatory infil-tration with significant decrease in the proportion of CD68 positive cells[(7.77±0.61)％vs(17.03±1.22)％,P＜0.05].In addition,the expression of TLR4,P-P65 protein and P-P65/T-P65 ratio were decreased.Conclusion Overexpressed TNIP3 led to the inhibition of atrial fibrosis,inflammatory remodeling,and AF susceptibility in sepsis through the pathway of TLR4/NF-κB.TNIP3 may be a new target for the prevention and treatment of sepsis related AF.[Chinese Journal of Cardiac Pacing and Electrophysiology,2024,38(5):342-348]
关键词
心血管病学/肿瘤坏死因子α诱导相互作用蛋白3/心房炎性重构/心房颤动/脓毒症Key words
Cardiology/Tumor necrosis factor alpha induced interacting protein 3/Atrial inflammatory remode-ling/Atrial fibrillation/Sepsis引用本文复制引用
基金项目
湖北省自然科学基金项目(2023AFB140)
出版年
2024
NETL
NSTL
万方数据