多西环素长期高剂量诱导对滇南小耳猪肝肾功能的影响

doi:10.13560/j.cnki.biotech.bull.1985.2024-0882

生物技术通报 ›› 2025, Vol. 41 ›› Issue (4): 345-354.doi: 10.13560/j.cnki.biotech.bull.1985.2024-0882

• 研究报告 • 上一篇

多西环素长期高剂量诱导对滇南小耳猪肝肾功能的影响

娄亚楠¹^,²(), 熊晶晶¹^,², 娄亚玲¹^,², 王福斌¹^,², 程文杰¹^,³, 陈晓晶¹^,⁴, 李加宇¹, 普少瑕¹, 魏红江¹^,²^,³(), 赵红业¹^,²()

^1.云南农业大学云南省小型猪基因编辑与异种器官移植重点实验室，昆明 650201
^2.云南农业大学动物医学院，昆明 650201
^3.云南农业大学动物科学技术学院，昆明 650201
^4.云南农业大学食品科学技术学院，昆明 650201

收稿日期:2024-09-10 出版日期:2025-04-26 发布日期:2025-04-25
通讯作者: 魏红江，男，博士，教授，研究方向：动物基因编辑与体细胞克隆；E-mail: hongjiangwei@126.com
赵红业，女，博士，教授，研究方向：疾病动物模型构建；E-mail: hyzhao2000@126.com
作者简介:娄亚楠，女，硕士，研究方向：疾病动物模型构建；E-mail: lyn99179388@163.com
基金资助:
云南省重大科技专项计划(202102AA100054)

Effects of Long-term High-dose Induction of Doxycycline on Liver and Kidney Functions of Diannan Miniature Pigs

LOU Ya-nan¹^,²(), XIONG Jing-jing¹^,², LOU Ya-ling¹^,², WANG Fu-bin¹^,², CHENG Wen-jie¹^,³, CHEN Xiao-jing¹^,⁴, LI Jia-yu¹, PU Shao-xia¹, WEI Hong-jiang¹^,²^,³(), ZHAO Hong-ye¹^,²()

^1.Yunnan Provincial Key Laboratory of Gene Editing and Xenotransplantation of Small Pigs, Yunnan Agricultural University, Kunming 650201
^2.College of Veterinary Medicine, Yunnan Agricultural University, Kunming 650201
^3.College of Animal Science and Technology, Yunnan Agricultural University, Kunming 650201
^4.College of Food Science and Technology, Yunnan Agricultural University, Kunming 650201

Received:2024-09-10 Published:2025-04-26 Online:2025-04-25

摘要/Abstract

摘要：

目的探究Tet-On系统中多西环素（doxycycline, Dox）持续高剂量摄入对实验猪健康状态的影响。方法选用9只6月龄滇南小耳猪作为模型动物，分别设置3倍（30 mg/kg）和5倍（50 mg/kg）推荐剂量组及空白对照组。各组实验猪均肌肉注射相应药物或生理盐水，持续30 d，分别采集第0、10、20、28天的血液进行血常规及肝肾功能指标测定，末次给药后2 h采集实验猪肝、肾进行形态学观察、病理学检测及疾病检测。结果与对照组相比，30 mg/kg剂量组Lym#、MPV下降，而PLT上升；50 mg/kg剂量组中这些指标的变化更为显著，且RDW-SD显著降低，同时ALT、MG、P等指标升高，NA、CO2、CYSC等指标下降，显示出较为明显的肝肾功能损伤。组织病理学检查结果显示30 mg/kg和50 mg/kg剂量组实验猪的肝脏和肾脏均出现不同程度的病变，且50 mg/kg剂量组的肝脏炎性细胞和红细胞浸润更为严重，肾脏则显示出更明显的萎缩和组织结构损伤。病原感染检测结果排除病毒感染对实验结果的干扰。结论高剂量Dox长期使用会对实验猪的肝肾功能造成显著损伤，且损伤程度与Dox的剂量呈正相关，为大型基因编辑动物的条件性诱导表达相关研究提供参考。

关键词: 盐酸多西环素, Tet-On系统, 滇南小耳猪, 肝肾损伤

Abstract:

Objective To explore the impact of continuous high-dose intake of doxycycline (Dox) in the Tet-On system on the health status of experimental pigs. Method Nine 6-month-old Diannan miniature pigs were selected as model animals, and the recommended dose groups of 3 (30 mg/kg) times and 5 (50 mg/kg) times and blank control group were set up. The experimental pigs in each group were intramuscularly injected with corresponding drugs or physiological saline for 30 d. During the experiment, blood was collected on the day 0, 10, 20, and 28 for blood routine and liver and kidney function index measurements. The experimental pigs’ livers and kidneys at 2 h after last injection were subjected to morphological observation, pathological examination and disease detection. Result Compared with the control group, Lym# and MPV decreased, while PLT increased in the 30 mg/kg dose group; while the changes in these indicators were more significant in the 50 mg/kg dose group, and RDW-SD significantly reduced, meanwhile ALT, MG, P and other indicators increased, and NA, CO2, CYSC and other indicators decreased, showing more obvious damages to liver and kidney function. Histopathological examination results showed that the livers and kidneys of the experimental pigs in the 30 mg/kg dose group and the 50 mg/kg dose group had varying degrees of lesions, and the liver inflammatory cells and red blood cell infiltration in the 50 mg/kg dose group were more severe. The kidneys showed more obvious atrophy and structural tissue damage. The pathogenic infection detection results excludes the interference of viral infection on the experimental results. Conclusion This study preliminarily proves that long-term use of high-dose Dox can cause significant damage to experimental pigs' liver and kidney functions, and the degree of damage is positively correlated with the dose of Dox. This study may provide references for the research of conditional induced expression in large gene editing animals.

Key words: doxycycline hydrochloride, Tet-On system, Diannan miniature pigs, liver and kidney injury

娄亚楠, 熊晶晶, 娄亚玲, 王福斌, 程文杰, 陈晓晶, 李加宇, 普少瑕, 魏红江, 赵红业. 多西环素长期高剂量诱导对滇南小耳猪肝肾功能的影响[J]. 生物技术通报, 2025, 41(4): 345-354.

LOU Ya-nan, XIONG Jing-jing, LOU Ya-ling, WANG Fu-bin, CHENG Wen-jie, CHEN Xiao-jing, LI Jia-yu, PU Shao-xia, WEI Hong-jiang, ZHAO Hong-ye. Effects of Long-term High-dose Induction of Doxycycline on Liver and Kidney Functions of Diannan Miniature Pigs[J]. Biotechnology Bulletin, 2025, 41(4): 345-354.

图/表 11

表1 实验猪分组情况

Table 1 Grouping situation of experimental pigs

组别 Group	序号 Serial number	耳号 label	体重 Weight/kg	剂量 Dose/mL
对照 Control	1	2A016S3	17.00	17.00
	2	3072T1	14.80	27.80
	3	2793T1	29.00	29.00
30 mg/kg	1	3152PA3	12.00	7.20
	2	3045T1	22.80	13.68
	3	3057Q1	13.00	7.80
50 mg/kg	1	3149PA3	13.00	13.00
	2	2A051T1	27.80	27.80
	3	2A073P1	18.00	18.00

表2 病原感染检测引物序列

Table 2 Primer sequences for pathogenic infection detection

引物名称	引物序列
Primer name	Primer sequence（5'-3'）
PEDV-F	GCGTAGTTGAGATTGTTG
PEDV-R	CCTCTGTTGTTACTTGGA
TGEV-F	GATGGAGTTGTCTGGGTTGC
TGEV-R	GTGACCTTGAATTGTCCCTT
RV-F	TATTTCCGCAAGCRCCRCCATT
RV-R	ACCHGGTGGAAATACYGGT-CCT
MP-1-F	AGCCTAGCTACCGATTGGCT
MP-1-R	CTGGGTAACTGACTTTGCAA

图1 实验猪生存曲线

Fig. 1 Experimental pig survival curve

图2 Dox对实验猪血液生理指标的影响A：实验猪血液Lym#计数；B：实验猪血液MPV；C：实验猪血液PLT计数；D：实验猪血液RDW-SD；E：实验猪血液Eos#计数。 *P<0.05， **P<0.01， ##P<0.01

Fig. 2 Effects of Dox on the blood physiological indicators of experimental pigsA: Experimental pig blood Lym# count. B: Experimental pig blood MPV. C: Experimental pig blood PLT count.D: Experimental pig blood RDW-SD. E: Experimental pig blood EOS # count. *P<0.05, **P<0.01, ##P<0.01

图3 Dox对实验猪肝功能相关指标的影响A：前白蛋白；B：胆碱酯酶；C：白蛋白和球蛋白含量比值；D：丙氨酸氨基转移酶；E：天冬氨酸氨基转移酶；F：谷丙和谷草含量比值。 *P<0.05， **P<0.01

Fig. 3 Effects of Dox on related indicators of experimental pig liver functionA: Prealbumin (PA). B: Cholinesterase (PCHE). C: Ratio of albumin and globulin (A/G). D: Serum alanine aminotransferase (ALT). E: Aspartate aminotransferase (AST). F: Ratio of serum glutamic propyl to glutamic and grass content (L/S). *P<0.05, **P<0.01

图4 Dox对实验猪肾功能相关指标的影响A：胱抑素C；B：肌酐。 *P<0.05， #P<0.05

Fig. 4 Effect of Dox on the related indicators of renal function in experimental pigsA: Cystatin C (CYSC); B: Creatinine (CREA). *P<0.05, #P<0.05

图5 Dox对实验猪血液电解质相关指标的影响A：镁离子；B：无机磷；C：碳酸氢盐；D：钠离子。 *P<0.05， #P<0.05

Fig. 5 Effects of Dox on the blood electrolyte related indicators of experimental pigsA: Magnesium ion (MG) . B: Inorganic phosphorus (P). C: Bicarbonate (CO2). D: Sodium ion (NA). *P<0.05, #P<0.05

图6 Dox对实验猪肝、肾器官的影响A：30 mg/kg组实验猪左耳后；B：30 mg/kg组实验猪肝脏；C：30 mg/kg组实验猪肾脏；D：30 mg/kg组实验猪肾脏横截面；E：50 mg/kg组实验猪左耳后；F：50 mg/kg组实验猪肝脏；G：50 mg/kg组实验猪肾脏；H：50 mg/kg组实验猪肾脏横截面

Fig. 6 Effects of Dox on experimental pig liver and kidney organsA: Behind the left ear of experimental pigs in 30 mg/kg group. B: Experimental pig liver in 30 mg/kg group. C: 30 mg/kg experimental pig kidney. D: Cross-section of pig kidney in 30 mg/kg group. E: Behind the left ear of experimental pigs in 50 mg/kg group. F: 50 mg/kg experimental pig liver. G: Pig kidney in 50 mg/kg group. H: Kidney cross-section of experimental pigs in the 50 mg/kg group

图7 实验猪肝、肾组织病理学检查A：对照组实验猪肝脏；B：30 mg/kg组实验猪肝脏；C：50 mg/kg组实验猪肝脏；D：对照组实验猪肾脏；E：30 mg/kg组实验猪肾脏；F：50 mg/kg组实验猪肾脏

Fig. 7 Histopathological examination of experimental pig's liver and kidneyA: Experimental pig liver in control group. B: Experimental pig liver in 30 mg/kg group. C: 50 mg/kg experimental pig liver. D: Control group experimental pig kidney. E: Pig kidney in 30 mg/kg group. F: Experimental pig kidney in 50 mg/kg group

图8 30 mg/kg组实验猪给药第28天时相关疾病检测A：流行性腹泻（459 bp）；B：传染性胃肠炎（517 bp）；C：轮状病毒（289 bp）；D：支原体肺炎（457 bp）。下同

Fig. 8 Detection of related diseases in experimental pigs in the 30 mg/kg group on the 28 th day after administrationA: Epidemic diarrhea (459 bp). B: Transmissible gastroenteritis (517 bp). C: Rotavirus (289 bp). D: Mycoplasma pneumonia (457 bp). The same below

图9 30 mg/kg组实验猪死亡时相关疾病检测

Fig. 9 Detection of related diseases at the death of experimental pigs in the 30 mg/kg group

参考文献 37

1	Guo CT, Ma XT, Gao F, et al. Off-target effects in CRISPR/Cas9 gene editing [J]. Front Bioeng Biotechnol, 2023, 11: 1143157.
2	Lewandoski M. Conditional control of gene expression in the mouse [J]. Nat Rev Genet, 2001, 2(10): 743-755.
3	Das AT, Zhou X, Metz SW, et al. Selecting the optimal Tet-On system for doxycycline-inducible gene expression in transiently transfected and stably transduced mammalian cells [J]. Biotechnol J, 2016, 11(1): 71-79.
4	Saunderson SC, Hosseini-Rad SMA, McLellan AD. Noise-reduction and sensitivity-enhancement of a sleeping beauty-based Tet-on system [J]. Genes, 2022, 13(10): 1679.
5	Hsiao PF, Chang SK, Hsu TH, et al. Pharmacokinetics and tissue depletion of doxycycline administered at high dosage to broiler chickens via the drinking water [J]. Acta Vet Hung, 2016, 64(4): 472-481.
6	Ziółkowski H, Jasiecka-Mikołajczyk A, Madej-Śmiechowska H, et al. Comparative pharmacokinetics of chlortetracycline, tetracycline, minocycline, and tigecycline in broiler chickens [J]. Poult Sci, 2020, 99(10): 4750-4757.
7	Ma HX, Lu YQ, Lowe K, et al. Regulated hAAT expression from a novel rAAV vector and its application in the prevention of type 1 diabetes [J]. J Clin Med, 2019, 8(9): 1321.
8	Navarro-Triviño FJ, Pérez-López I, Ruiz-Villaverde R. Doxycycline, an antibiotic or an anti-inflammatory agent? the most common uses in dermatology [J]. Actas Dermosifiliogr, 2020, 111(7): 561-566.
9	Herrling T, Jung K. The radical status factor (RSF): a novel metric to characterize skin products [J]. Int J Cosmet Sci, 2012, 34(4): 285-290.
10	Plotzki E, Heinrichs G, Kubícková B, et al. Microbiological characterization of a newly established pig breed, Aachen Minipigs [J]. Xenotransplantation, 2016, 23(2): 159-167.
11	Wareham-Mathiassen S, Pinto Glenting V, Bay L, et al. Characterization of pig skin microbiome and appraisal as an in vivo subcutaneous injection model [J]. Lab Anim, 2023, 57(3): 304-318.
12	Zhao YQ, Xiang L, Liu YQ, et al. Atherosclerosis induced by a high-cholesterol and high-fat diet in the inbred strain of the Wuzhishan miniature pig [J]. Anim Biotechnol, 2018, 29(2): 110-118.
13	Klymiuk N, Böcker W, Schönitzer V, et al. First inducible transgene expression in porcine large animal models [J]. FASEB J, 2012, 26(3): 1086-1099.
14	Jin YX, Jeon Y, Lee SH, et al. Production of pigs expressing a transgene under the control of a tetracycline-inducible system [J]. PLoS One, 2014, 9(1): e86146.
15	Jin Q, Yang XY, Gou SX, et al. Double knock-in pig models with elements of binary Tet-On and phiC31 integrase systems for controllable and switchable gene expression [J]. Sci China Life Sci, 2022, 65(11): 2269-2286.
16	Jin Q, Shi H, Liu XY, et al. Genome editing pig models with elements for controllable gene expression [J]. Anim Res One Health, 2023, 1(2): 242-258.
17	萧惠来. VICH兽药靶动物安全性研究指导原则概述 [J]. 中国兽药杂志, 2009, 43(12): 33-37.
	Xiao HL. Overview of VICH guideline of target animal safety for veterinary pharmaceutical products [J]. Chin J Vet Drug, 2009, 43(12): 33-37.
18	梁霄, 孙萌, 王鹏程, 等. 猪流行性腹泻病毒和猪传染性胃肠炎病毒双重荧光定量PCR检测方法的建立与应用[J]. 畜牧与兽医, 2022, 54(6): 119-125.
	Liang X, Sun M, Wang PC, et al. Establishment and application of dual fluorescence quantitative PCR method for detection of Porcine epidemic diarrhea virus and transmissible gastroenteritis virus [J]. Animal Husbandry and Veterinary Medicine, 2022, 54(6): 119-125.
19	张羽欣, 王树茂, 段宏勇, 等. 猪传染性胃肠炎病毒TaqMan实时荧光定量PCR检测方法的建立与应用 [J]. 中国兽医科学, 2024, 54(4): 479-484.
	Zhang YX, Wang SM, Duan HY, et al. Establishment and application of TaqMan real-time quantitative PCR for detection of porcine transmissible gastroenteritis virus [J]. Chin Vet Sci, 2024, 54(4): 479-484.
20	柳佳佳, 梁海英, 曾智勇, 等. 猪轮状病毒TaqMan实时荧光定量RT-PCR检测方法的建立与应用 [J]. 中国兽医科学, 2024, 54(2): 162-168.
	Liu JJ, Liang HY, Zeng ZY, et al. Establishment and application of TaqMan real-time fluorescence quantitative RT-PCR for detection of porcine rotavirus [J]. Chin Vet Sci, 2024, 54(2): 162-168.
21	汪志恒, 王景成, 佘志成, 等. 猪支原体肺炎RT-PCR诊断方法的建立和应用 [J]. 现代畜牧科技, 2024(1): 6-9.
	Wang ZH, Wang JC, She ZC, et al. Establishment and application of RT-PCR diagnostic method for Mycoplasma pneumonia of swine [J]. Mod Anim Husb Sci Technol, 2024(1): 6-9.
22	张景锋, 朱宽佑, 徐秋良, 等. 基于Tet-on系统的RFP基因表达载体构建及功能验证 [J]. 西北农林科技大学学报: 自然科学版, 2017, 45(8): 36-41, 55.
	Zhang JF, Zhu KY, Xu QL, et al. Construction and functional verification of Tet-on system of RFP gene expression vector [J]. J Northwest A F Univ Nat Sci Ed, 2017, 45(8): 36-41, 55.
23	Bacanlı M, Başaran N. Importance of antibiotic residues in animal food [J]. Food Chem Toxicol, 2019, 125: 462-466.
24	Ludhiadch A, Sulena, Singh S, et al. Genomic variation affecting MPV and PLT count in association with development of ischemic stroke and its subtypes [J]. Mol Neurobiol, 2023, 60(11): 6424-6440.
25	Snarska A, Wysocka D, Rytel L. Effect of simvastatin on thrombopoiesis in porcine bone marrow [J]. J Vet Res, 2019, 63(1): 117-121.
26	Xu Y, Liu H, Jiang Y, et al. Genome-wide association studies reveal genetic loci associated with plasma cholinesterase activity in ducks [J]. Anim Genet, 2019, 50(3): 287-292.
27	Huang L, Li J, Yan JJ, et al. Prealbumin is predictive for postoperative liver insufficiency in patients undergoing liver resection [J]. World J Gastroenterol, 2012, 18(47): 7021-7025.
28	Lopez-Giacoman S, Madero M. Biomarkers in chronic kidney disease, from kidney function to kidney damage [J]. World J Nephrol, 2015, 4(1): 57-73.
29	Yuan RS, Tao X, Liang S, et al. Protective effect of acidic polysaccharide from Schisandra chinensis on acute ethanol-induced liver injury through reducing CYP2E1-dependent oxidative stress [J]. Biomed Pharmacother, 2018, 99: 537-542.
30	Varma S, Nathanson J, Dowlatshahi M, et al. Doxycycline-induced cholestatic liver injury [J]. Clin J Gastroenterol, 2021, 14(5): 1503-1510.
31	Pan JJ, Promrat K. Doxycycline-induced autoimmune hepatitis [J]. ACG Case Rep J, 2020, 7(8): e00440.
32	阮国安, 吴凤鸣, 郭凯, 等. 犬强力霉素过量服用诊疗病例分析 [J]. 中国兽医杂志, 2018, 54(9): 52-53.
	Ruan GA, Wu FM, Guo K, et al. Analysis of diagnosis and treatment cases of doxycycline overdose [J]. Chin J Vet Med, 2018, 54(9): 52-53.
33	俞雷. 警惕四环素族药物引起的肾损害 [J]. 中国临床医生杂志, 2007, 35(7): 59-60.
	Yu L. Vigilance against kidney damage induced by tetracycline drugs [J]. Chin J Clin, 2007, 35(7): 59-60.
34	Corum O, Uney K, Terzi E, et al. Effects of temperature on the pharmacokinetics, tissue residues, and withdrawal times of doxycycline in rainbow trout (Oncorhynchus mykiss) following oral administration [J]. Vet Sci, 2023, 10(6): 401.
35	Coté GA, Buchman AL. Antibiotic-associated diarrhoea [J]. Expert Opin Drug Saf, 2006, 5(3): 361-372.
36	Costello A, Lao NT, Gallagher C, et al. Leaky expression of the TET-on system hinders control of endogenous miRNA abundance [J]. Biotechnol J, 2019, 14(3): e1800219.
37	Szulc J, Wiznerowicz M, Sauvain MO, et al. A versatile tool for conditional gene expression and knockdown [J]. Nat Methods, 2006, 3(2): 109-116.

多西环素长期高剂量诱导对滇南小耳猪肝肾功能的影响

Effects of Long-term High-dose Induction of Doxycycline on Liver and Kidney Functions of Diannan Miniature Pigs

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 11

参考文献 37

相关文章 0

编辑推荐

Metrics

本文评价