Tnfaip3基因通过调控线粒体影响结肠炎小鼠的机制研究

Mechanism study of Tnfaip3 gene affecting colitis in mice via regulating mitochondria

目的探讨肿瘤坏死因子α诱导蛋白3(Tnfaip3)基因通过调控线粒体影响葡聚糖硫酸钠(DSS)诱导的结肠炎小鼠肠道炎症的机制。方法取雌性Tnfaip3基因杂合缺陷(Tnfaip3^(+/-))小鼠及野生型(WT)小鼠各14只,分为野生型(WT)组、肠炎模型(WT-DSS)组、Tnfaip3杂合缺陷(Tnfaip3^(+/-))组、Tnfaip3杂合缺陷肠炎模型(Tnfaip3^(+/-)-DSS)组,每组7只。WT-DSS组及Tnfaip3^(+/-)-DSS组持续7 d饮用含有3%DSS的水诱导结肠炎,WT组及Tnfaip3^(+/-)组小鼠饮用自来水。每天观察各组小鼠体质量,评估疾病活动指数(DAI)变化。造模第8天处死小鼠,测量小鼠结肠长度,显微镜下观察小鼠结肠组织损伤情况并进行结肠组织病理学评分。实时荧光定量PCR检测小鼠结肠组织炎性因子即肿瘤坏死因子α(TNF-α)、白细胞介素1β(IL-1β)、核因子κB(NF-κB)的mRNA表达,检测结肠组织丙二醛(MDA)、总抗氧化能力(T-AOC)、谷胱甘肽过氧化物酶(GSH-PX)和超氧化物歧化酶(SOD)等氧化应激指标。透射电镜观察线粒体、溶酶体及自噬线粒体结构并计数。检测线粒体膜电位JC-1及三磷酸腺苷(ATP)水平。Western blot检测线粒体及细胞质中线粒体自噬相关蛋白PINK1、P62、LC3BⅡ/LC3BⅠ比值等的表达。结果各组小鼠存活率均为100%。与WT小鼠相比,Tnfaip3^(+/-)小鼠的体质量更低,结肠长度更短,组织病理学评分更高,差异均具有统计学意义(均P<0.05)。与WT-DSS组相比,Tnfaip3^(+/-)-DSS组小鼠体质量更低,DAI评分更高,结肠更短,组织病理学评分更高,结肠组织TNF-α、IL-1β、NF-κB的mRNA表达更高,差异均具有统计学意义(均P<0.05)。Tnfaip3^(+/-)小鼠结肠组织的MDA水平高于WT小鼠,Tnfaip3^(+/-)-DSS组MDA水平显著高于WT-DSS组,抗氧化指标T-AOC、GSH-PX和SOD均低于WT-DSS组,差异均具有统计学意义(均P<0.05)。相较于WT-DSS组,Tnfaip3^(+/-)-DSS组小鼠肠道组织线粒体结构紊乱程度更重,线粒体数量更少,线粒体膜电位JC-1更高,线粒体ATP含量更低,溶酶体和自噬线粒体数量更多,线粒体和细胞质中自噬相关蛋白PINK1、P62和LC3BⅡ/LC3BⅠ比值表达均更高,差异均具有统计学意义(均P<0.05)。结论Tnfaip3基因可能通过调控氧化应激、线粒体损伤及线粒体自噬在结肠炎小鼠的肠道炎症中起保护作用。

Objective To investigate the mechanism of tumor necrosis factor alpha-induced protein 3(Tnfaip3)gene affecting the intestinal inflammation in mice with dextran sulfate sodium(DSS)-induced colitis by regulating mitochondria.Methods Female Tnfaip3 heterozygous deficiency(Tnfaip3^(+/-))mice(n=14)and wild-type(WT)mice(n=14)were collected and divided into 4 groups including wild type(WT)group,colitis model(WT-DSS)group,Tnfaip3 heterozygous deficiency(Tnfaip3^(+/-))group,Tnfaip3 heterozygous deficiency colitis model(Tnfaip3^(+/-)-DSS)group,with 7 mice in each group.Mice colitis in WT-DSS and Tnfaip3^(+/-)-DSS groups were induced by drinking 3%DSS water for 7 days,while mice in WT and Tnfaip3^(+/-)groups were treated with tap water.Body weight and disease activity index(DAI)were evaluated daily.The mice were sacrificed after 7 days of modeling,the colon length was measured,the colon tissue damage was observed under the microscope,and the colon histopathological score was evaluated.The mRNA expressions of inflammatory factors as tumor necrosis factorα(TNF-α),interleukin 1β(IL-1β),and nuclear factorκB(NF-κB)in colon tissue were detected by quantitative real-time PCR.Oxidative stress indicators such as malonic dialdehyde(MDA),total antioxidant capacity(T-AOC),glutathione peroxidase(GSH-PX)and superoxide dismutase(SOD)in colon tissue were detected.Transmission electron microscopy was used to observe the structures of mitochondria,lysosomes,and autophagic mitochondria,and calculate the numbers.Mitochondrial membrane potential JC-1 and adenosine-triphosphate(ATP)levels were detected,and Western blot was used to detect the expressions of mitophagy-related proteins PINK1,P62,and LC3BⅡ/LC3BⅠin mitochondria and cytoplasm.Results The survival rate of mice in all groups was 100%.Compared with WT group,the mice in Tnfaip3^(+/-)group had lower body weight,shorter colon and higher histopathological scores(all P<0.05).Compared with WT-DSS group,the mice in Tnfaip3^(+/-)-DSS group had lower body weight,higher DAI score,shorter colon,higher histopathological score,and higher mRNA expressions of inflammatory factors TNF-α,IL-1βand NF-κB in colon tissues(all P<0.05).MDA level in colon tissues of Tnfaip3^(+/-)mice was higher than that of WT mice(P<0.05).Compared with WT-DSS group,the MDA level in Tnfaip3^(+/-)-DSS group was higher,the levels of T-AOC,GSH-PX and SOD were lower(all P<0.05).Compared with WT-DSS group,Tnfaip3^(+/-)-DSS mice had more severe intestinal disordered mitochondrial structure,less mitochondrial number,higher mitochondrial membrane potential JC-1 level and lower mitochondrial ATP content,more number of lysosomes and autophagic mitochondria and higher expressions of proteins such as PINK1,P62 and ratio of LC3BⅡ/LC3BⅠin mitochondria and cytoplasm(all P<0.05).Conclusion Tnfaip3 gene may play a protective role in intestinal inflammation of colitis mice by regulating oxidative stress,mitochondria damage and mitophagy.