对氧磷酶3基因过表达对急性中毒小鼠肝损伤的保护作用及机制

doi:10.16098/j.issn.0529-1356.2019.02.007

摘要/Abstract

摘要：

目的探讨对氧磷酶3（PON3）对急性中毒肝损伤的保护作用及作用机制。方法将40只Balb/c小鼠随机分为4组，正常对照（NC）组、敌敌畏（DDVT）对照组、绿色荧光蛋白慢病毒（Lv-GFP）组和重组PON3慢病毒（Lv-PON3）组，每组10只。LV-GFP组和Lv-PON3组小鼠经尾静脉分别注射2×10⁷ TU的Lv-GFP、Lv-PON3慢病毒，3 d后腹腔注射敌敌畏（DDVT）溶液9 mg/kg，DDVT组只需腹腔注射同等剂量DDVT，NC组注射同等剂量生理盐水。各组于DDVT染毒后12 h麻醉处死10只小鼠取肝组织，ELISA法检测各组血清乙酰胆碱酯酶（AChE）、肝功能指标谷丙转氨酶（ALT）和谷草转氨酶（AST）和肝组织氧化应激指标丙二醛（MDA）、过氧化氢酶（CAT）、超氧化物歧化酶（SOD）和谷胱甘肽（GSH），HE染色法观察肝组织形态学改变，Real-time PCR检测肝组织Kelch样环氧氯丙烷相关蛋白-1（Keap1）、血红蛋白加氧酶（HO-1）和转录因子NF-E2相关因子2（Nrf2） mRNA的表达。结果与NC组比较，DDVT组和Lv-GFP组肝组织MDA、ALT、AST水平，HO-1和Nrf2 mRNA表达量明显升高，AChE、CAT、SOD、GSH水平和Keap1 mRNA表达量均明显降低（P<0.05）。与DDVT组比较，Lv-PON3组肝组织MDA、ALT、AST水平，Keap1 mRNA表达量降低，AChE、CAT、SOD和GSH水平均明显著升高，HO-1和Nrf2 mRNA表达量显著上升（P<0.05）。正常对照组肝细胞结构清晰，未出现肝细胞坏死和脂肪病变的情况，DDVT组和Lv-GFP组出现肝细胞坏死和脂肪变性等病理改变，Lv-PON3组肝脏病变组织肝细胞坏死和脂肪变性细胞数有一定的减少。结论 PON3可通过Keap1-Nrf2/HO-1途径缓解氧化应激反应，改善肝功能，对敌敌畏急性中毒小鼠肝损伤有一定的保护作用。

关键词: 对氧磷酶3, 急性中毒, 肝损伤, 氧化应激, 转染, 实时定量聚合酶链反应, 小鼠

Abstract:

Objective To investigate the protective effect and mechanism of oxidative sulfasalase 3 on acute poisoned liver injury. Methods Forty Balb/c mice were randomly divided into 4 groups: normal control (NC) group, dichlorvos (DDVT) control group, green fluorescent protein lentivirus (Lv-GFP) group, and recombinant paraoxonase 3(PON3) lentivirus (Lv-PON3) group, 10 in each group.Mice in Lv-GFP and Lv-PON3 group were injected with 2×10⁷ TU of Lv-GFP and Lv-PON3 lentiviruses via the tail vein respectively. After 3 days, they were intraperitoneally injected with a solution of dichlorvos (DDVT) 9 mg/kg.The same dose of DDVT was injected intraperitoneally into the DDVT group, and the same dose of saline was injected into the NC group.In each group, 10 mice were killed by anesthesia 12 hours after DDVT exposure to take liver tissue. ELISA was used to detect serum acetylcholinesterase(AChE), liver function indexes alamine aminotransferase(ALT) and aspartate aminotransferase(AST) and oxidative stress indexes malondialdehyde(MDA), catalase(CAT), superoxide dismutase(SOD) and glutathione(GSH) in liver tissues of each group. HE staining was used to observe the morphological changes of liver tissues. Results Compared with the NC group, the levels of MDA, ALT, AST, hemoglobin oxygenase 1(HO-1) and Nrf2 mRNA expression were significantly increased in DDVT group and Lv-GFP group, and AChE, CAT, SOD, GSH levels and Kelch-like epichlorohydrin-associated protein 1(Keap1)mRNA were significantly decreased (P<0.05). Compared with the DDVT group, the levels of MDA, ALT, AST and Keap1mRNA expression in liver tissue were significantly decreased in Lv-PON3 group, and AChE, CAT, SOD, GSH levels and HO-1 and Nrf2 mRNA expression were significantly increased (P<0.05). The structure of hepatocytes in the normal control group was clear, no hepatocyte necrosis and fat lesions were seen. Pathological changes such as hepatocyte necrosis and fatty degeneration under light microscope in DDVT and Lv-GFP groups, the number of hepatocyte necrosis and steatosis cells in the liver lesions of the Lv-PON3 group were reduced. Conclusion PON3 can alleviate oxidative stress and improve liver function through Keap1-Nrf2/HO-1 pathway, which has a certain protective effect on liver damage induced by acute poisoning of dichlorvos in mice.

Key words: Paraoxonase 3, Acute poisoning, Liver injury, Oxidative stress, Transfection, Real-time PCR, Mouse

伍洋刘英刘济滔尹德锋. 对氧磷酶3基因过表达对急性中毒小鼠肝损伤的保护作用及机制[J]. 解剖学报, 2019, 50(2): 179-184.

WU Yang LIU Ying LIU Ji-tao YIN De-feng.

A preliminary study on the protective effect and mechanism of paraoxonase 3 gene overexpression on hepatic injury in acutely poisoned mice

[J]. Acta Anatomica Sinica, 2019, 50(2): 179-184.

参考文献

［1］ Lee WJ, Ko Y, Cha ES.Acute pesticide poisoning among children in South Korea: findings from National Health Insurance claims data, 2006-2009［J］.J Trop Pediatr, 2014,60(1):4-9.

［2］ Chen CF, Lin HL, Chou HY,et al.Acute pesticide poisoning outcomes: a nationwide study in Taiwan［J］.Emerg Med J,2015,32(3):226-231.

［3］ Jiang XL. Protective effect of paraoxonase 3 transgene expression on liver injury［D］. Nanjing.Nanjing University, 2008.(in Chinese)

姜晓玲.对氧磷酶3转基因表达对肝损伤的保护作用［D］.南京:南京大学,2008.

［4］ Liu YT,Dong GF,Pan JF.Expression of paraoxonase 3 in adipose tissue of diabetic rats and intervention with metformin［J］.Pharmaceutical Journal of Chinese People’s Liberation Army,2017,33(6):532-534.(in Chinese)

刘云涛,董国锋,潘敬芳.对氧磷酶3在糖尿病大鼠脂肪组织的表达及二甲双胍的干预［J］.解放军药学学报,2017,33(6):532-534.

［5］ Chen XY,Guo RJ,Wang F,et al.Study on the protective effect of paraoxonase 1 overexpression on hepatic injury in acute poisoning mice with dichlorvos［J］.Zhejiang Medical Journal,2016,38(15):1230-1233，1245，1312.(in Chinese)

陈晓宇,郭瑞娟,王菲,等.对氧磷酶1过表达对敌敌畏急性中毒小鼠肝损伤保护作用的研究［J］.浙江医学,2016,38(15):1230-1233，1245，1312.

［6］ Botha CJ, Coetser H, Labuschagne L, et al.Confirmed organophosphorus and carbamate pesticide poisonings in South African wildlife (2009-2014)［J］.J S Afr Vet Assoc,2015,86(1):1329.

［7］ Bird SB, Krajacic P, Sawamoto K, et al.Pharmacotherapy to protect the neuromuscular junction after acute organophosphorus pesticide poisoning［J］.Ann N Y Acad Sci,2016,1374(1):86-93.

［8］ Guo JP,Li Y,Sang ZhZh.Effects of fat emulsion on liver injury caused by acute severe organophosphorus pesticide poisoning［J］.Chinese Journal of Clinical Emergency,2015,16(5):358-361.(in Chinese)

郭晋平,李勇,桑珍珍.脂肪乳用于急性重度有机磷农药中毒所致肝损伤的疗效分析［J］.临床急诊杂志,2015,16(5):358-361.

［9］ Peng W.Protection of transgenic expression of paraoxonase 3 against CCl4-induced acute liver injury in mice［D］. Nanjing:Nanjing University, 2009.(in Chinese)

彭薇.转基因表达对氧磷酶3对CCl4诱导的小鼠急性肝损伤的保护作用［D］.南京:南京大学,2009.

［10］Wang HH,Shen XH,Xiong XD.Influence of Yantiaofang on myeloperoxidase and malondialdehyde in lung tissue of rats with acute lung injury induced by sepsis［J］.Drug Evaluation Research, 2016,39(3):394-397.(in Chinese)

汪海慧,沈晓红,熊旭东.炎调方对脓毒症急性肺损伤大鼠肺组织髓过氧化物酶和丙二醛水平的影响［J］.药物评价研究,2016,39(3):394-397.

［11］Guo RJ,Chen LW,Lian J,et al.The protective effect of paraoxonase 1 gene on liver oxidative stress injury induced by dichlorvos poisoning mice［J］.Chinese Critical Care Medicine,2015,27(4):285-290.(in Chinese)

郭瑞娟,陈隆望,连洁,等.调控对氧磷酶1基因对急性敌敌畏中毒小鼠肝氧化损伤的保护作用［J］.中华危重病急救医学,2015,27(4):285-290.

［12］ Pearson JN, Patel M.The role of oxidative stress in organophosphate and nerve agent toxicity［J］.Ann N Y Acad Sci,2016,1378(1):17-24

［13］ Sun WN,Wang M. Tang shenan decoction on ROS levels and Nrf2, HO-1 expression in experimental type 2 diabetic rats kidney［J］.Jilin Journal of Traditional Chinese Medicine,2016,36(12):1247-1250.(in Chinese)

孙伟娜,王镁.中药复方糖肾安对实验性2型糖尿病大鼠肾脏ROS水平及Nrf2、HO-1表达的影响［J］.吉林中医药,2016,36(12):1247-1250.

［14］ Hu LF,Wang Y,Ren RJ,et al. Anti-oxidative stress actions and regulation mechanisms of Keap1-Nrf2/ARE signaling pathway［J］.International Journal of Pharmaceutical Sciences, 2016,43(1):146-152,166.(in Chinese)

胡流芳,王迎,任汝静,等. Keap1-Nrf2/ARE信号通路的抗氧化应激作用及其调控机制［J］.国际药学研究杂志,2016,43(1):146-152,166.

［15］ Lu ZhG,Huang JB,Liu Y,et al.Effects of Xingnaojing injection on Keap1-Nrf2/ARE oxidative stress pathway in acute cerebral infarction［J］.Guangdong Medical Journal,2016,37(20):3127-3129.(in Chinese)

卢志刚,黄家彬,刘芸,等.醒脑静注射液对急性脑梗死Keap1-Nrf2/ARE氧化应激通路的影响［J］.广东医学,2016,37(20):3127-3129.

［16］ Guo Y, Yu S, Zhang C,et al.Epigenetic regulation of Keap1-Nrf2 signaling［J］.Free Radic Biol Med,2015,88(Pt B):337-349.

[1]	洪晓敏李三强崔钦奕郑润月杨孟利罗仁利李前辉 . 酒精性肝纤维化核因子κB信号通路与性别的差异 [J]. 解剖学报, 2024, 55(1): 55-61.
[2]	谢惠兰方芳林毅. Chir99021调控Wnt/β-catenin 信号通路抑制大鼠牙髓干细胞成骨诱导分化的机制[J]. 解剖学报, 2024, 55(1): 67-72.
[3]	袁蕾杨智伟杨惠刘政海何洁万炜. 三七总皂苷抑制小鼠星形胶质细胞活化缓解完全弗氏佐剂所致炎性痛 [J]. 解剖学报, 2024, 55(1): 25-31.
[4]	马婷婷陈建红刘爱翠李海宁. 抑制lncRNA TUG1下调核苷酸结合寡聚结构域样受体蛋白1炎症小体在延缓阿尔茨海默病进展的作用 [J]. 解剖学报, 2024, 55(1): 32-42.
[5]	邹成功冯浩陈兵唐辉邵川孙谋杨荣何家全. 创伤性颅脑损伤中神经功能障碍与认知功能损伤的动态变化 [J]. 解剖学报, 2024, 55(1): 43-48.
[6]	郑丽平刘霞杜晓华吴亚娟刘珊珊 . 脑源性神经营养因子在牦牛与黄牛端脑不同区域的表达与分布 [J]. 解剖学报, 2024, 55(1): 10-16.
[7]	张琴杨俊霞蒋青松. 福辛普利对糖尿病视网膜病变小鼠血管紧张素转化酶2和血管内皮生长因子的影响[J]. 解剖学报, 2023, 54(6): 628-634.
[8]	陈子璇司丽娜舒薇菡张欣魏晨阳程露阳杨松鹤乔跃兵. 褪黑素调控下丘脑PI3K/Akt/mTOR信号延缓雌性小鼠青春期启动的机制 [J]. 解剖学报, 2023, 54(6): 644-651.
[9]	刘哲川李坤马帅男孟家琪王艳芹. 利拉鲁肽对百草枯诱导的小鼠帕金森病模型炎症及线粒体融合/分裂的影响 [J]. 解剖学报, 2023, 54(6): 676-681.
[10]	刘叶楚亚楠徐岑璐何佳澄苏炳银太颢然. Roscovitine挽救帕金森病小鼠相关脑区神经元丢失和神经炎症[J]. 解剖学报, 2023, 54(6): 635-643.
[11]	魏纯纯王平林方兴麻献华章卫平谢志芳 . 小鼠棕色脂肪透射电子显微镜样品固定方法的改良 [J]. 解剖学报, 2023, 54(6): 738-743.
[12]	阎锟武筱林刘英娟葛科立任雷鸣李红云 . 脑蛋白水解物-Ⅰ对帕金森病小鼠肠道菌群的调节作用[J]. 解剖学报, 2023, 54(5): 497-504.
[13]	顾玲张萍宋科昕李娜陈虹宇张婕丛敬翟效月 . 发生发育小鼠肾输尿管芽分支的形态特点[J]. 解剖学报, 2023, 54(5): 593-598.
[14]	何可可李远迪文廷浩朱婕高杰胡蓉韩锋苏敏. 红细胞膜相关蛋白通过IL-6/STAT3/ROR-γt 信号通路抑制小鼠实验性自身免疫性脑脊髓炎中Th17细胞分化[J]. 解剖学报, 2023, 54(5): 538-545.
[15]	范文娟陈旭东陈永芳杨旭光金少举赵志军邓锦波. 体外培养大脑皮质类器官与活体大脑皮质发育的比较[J]. 解剖学报, 2023, 54(4): 383-391.