敲低多房棘球蚴let-7-5p可抑制BALB/c小鼠腹腔巨噬细胞极化和虫体生长

doi:10.11843/j.issn.0366-6964.2024.06.033

畜牧兽医学报 ›› 2024, Vol. 55 ›› Issue (6): 2619-2628.doi: 10.11843/j.issn.0366-6964.2024.06.033

敲低多房棘球蚴let-7-5p可抑制BALB/c小鼠腹腔巨噬细胞极化和虫体生长

王立群¹(), 吴易璇¹^,²(), 蒲桂婷¹, 曹珊菱¹, 王得先¹, 刘婷丽¹, 李红¹, AMUDATharheer Oluwashola¹, 郭小腊¹, 殷宏¹^,³, 骆学农¹^,³^,*()

1. 中国农业科学院兰州兽医研究所动物疫病防控全国重点实验室/甘肃省动物寄生虫病重点实验室, 兰州 730046
2. 西北民族大学生命科学与工程学院, 兰州 730030
3. 扬州大学江苏省动物重要疫病与人兽共患病防控协同创新中心, 扬州 225009

收稿日期:2023-09-12 出版日期:2024-06-23 发布日期:2024-06-28
通讯作者: 骆学农 E-mail:wlq1282690114@163.com;wyx2211591074@163.com;luoxuenong@caas.cn
作者简介:王立群(1994-)，女，甘肃成县人，博士生，主要从事病原与宿主相互作用研究，E-mail: wlq1282690114@163.com
吴易璇(1999-)，女，甘肃兰州人，硕士生，主要从事病原与宿主相互作用研究，E-mail: wyx2211591074@163.com
第一联系人：
王立群和吴易璇为同等贡献作者
基金资助:
国家自然科学基金(32072889)

Knockdown of let-7-5p from Echinococcus multilocularis Inhibited the Peritoneal Macrophages Polarization and Worm Growth in BALB/c Mice

Liqun WANG¹(), Yixuan WU¹^,²(), Guiting PU¹, Shanling CAO¹, Dexian WNAG¹, Tingli LIU¹, Hong LI¹, Tharheer Oluwashola AMUDA¹, Xiaola GUO¹, Hong YIN¹^,³, Xuenong LUO¹^,³^,*()

1. State Key Laboratory for Animal Disease Control and Prevention/Key Laboratory of Veterinary Parasitology of Gansu Province, Lanzhou Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Lanzhou 730046, China
2. Life Science and Engineering College of Northwest University of Nationalities, Lanzhou 730030, China
3. Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou 225009, China

Received:2023-09-12 Online:2024-06-23 Published:2024-06-28
Contact: Xuenong LUO E-mail:wlq1282690114@163.com;wyx2211591074@163.com;luoxuenong@caas.cn

摘要/Abstract

摘要：

旨在明确敲低多房棘球蚴emu-let-7-5p对小鼠腹腔巨噬细胞极化和虫体生长的影响。构建表达多房棘球蚴emu-let-7-5p海绵序列的重组腺相关病毒载体，并在293T细胞上验证其抑制效果和特异性。70只清洁级BALB/c小鼠分别经尾静脉注射AAV8-si-let-7-5p、AAV8-control和PBS后两周，每组随机选取2只小鼠分别用Western blot和qRT-PCR分析肝组织中GFP、let-7-5p及其靶基因C/EBP δ的表达。随后，所有小鼠每只腹腔接种1 000个原头蚴，3个月后qRT-PCR检测腹腔巨噬细胞emu-let-7-5p、C/EBP δ、M1型(IL-1β、iNOS和IL-6)和M2型(Arg-1、IL-4和IL-10)相关分子的转录水平，并统计各组小鼠多房棘球蚴的包囊重量和原头蚴数量。结果表明，注射后第15天，AAV8-si-let-7-5p成功靶向小鼠肝脏，并引起emu-let-7-5p的敲低和C/EBP δ的上调表达。感染后3个月，AAV8-si-let-7-5p组小鼠腹腔巨噬细胞emu-let-7-5p和M2型相关分子Arg-1均显著下调，而C/EBP δ和M1型相关分子IL-1β和iNOS均显著上调。此外，AAV8-si-let-7-5p组IL-4和IL-6下调表达，而IL-10上调表达，且小鼠肝脏包囊重量和原头蚴数量显著低于对照组。敲低多房棘球蚴感染小鼠体内emu-let-7-5p，可促进C/EBP δ的表达，进而抑制M2型极化和虫体生长。

关键词: 多房棘球蚴, emu-let-7-5p, 腹腔巨噬细胞极化, 虫体生长

Abstract:

The purpose of this study was to determine the effects of knockdown of Echinococcus multilocularis emu-let-7-5p on mouse peritoneal macrophages polarization and worm growth. A recombinant adeno-associated virus vector expressing emu-let-7-5p sponge sequence of E. multilocularis was constructed, and its inhibitory effect and specificity were validated in 293T cells. Seventy clean-grade BALB/c mice were injected intravenously (tail vein) with AAV8-si-let-7-5p, AAV8-control, and PBS, respectively. After two weeks, two mice in each group were randomly selected, and the expression of GFP, emu-let-7-5p and its target C/EBP δ in liver were detected by Western blot and qRT-PCR. At 3 months after intraperitoneal inoculation of 1 000 protoscolexs in each mouse, the transcriptional levels of emu-let-7-5p, C/EBP δ, M1 type (IL-1β, iNOS and IL-6) and M2 type (Arg-1, IL-4 and IL-10) related molecules in peritoneal macrophages were detected by qRT-PCR, and cysts weight and protoscolexs number of E. multilocularis in each group were analyzed. The results showed that, on the 15th day after injection, AAV8-si-let-7-5p successfully targeted the mouse liver and caused knockdown of emu-let-7-5p and up-regulation of C/EBP δ. At 3 months after infection, the expressions of emu-let-7-5p and M2 related molecule (Arg-1) in peritoneal macrophages were significantly down-regulated in AAV8-si-let-7-5p group, while C/EBP δ and M1 related molecules (IL-1β and iNOS) were significantly up-regulated. In addition, in AAV8-si-let-7-5p group, the expressions of IL-4 and IL-6 were down-regulated, while the expression of IL-10 was up-regulated, and cysts weight and protoscolexs number in AAV8-si-let-7-5p group were significantly lower than those in the control group. The above results suggested that knockdown of emu-let-7-5p in mice infected with E. multilocularis can promote the expression of C/EPB δ, and thus inhibit M2 macrophage polarization and worm growth.

Key words: Echinococcus multilocularis, emu-let-7-5p, peritoneal macrophages polarization, worm growth

中图分类号:

S852.734

王立群, 吴易璇, 蒲桂婷, 曹珊菱, 王得先, 刘婷丽, 李红, AMUDATharheer Oluwashola, 郭小腊, 殷宏, 骆学农. 敲低多房棘球蚴let-7-5p可抑制BALB/c小鼠腹腔巨噬细胞极化和虫体生长[J]. 畜牧兽医学报, 2024, 55(6): 2619-2628.

Liqun WANG, Yixuan WU, Guiting PU, Shanling CAO, Dexian WNAG, Tingli LIU, Hong LI, Tharheer Oluwashola AMUDA, Xiaola GUO, Hong YIN, Xuenong LUO. Knockdown of let-7-5p from Echinococcus multilocularis Inhibited the Peritoneal Macrophages Polarization and Worm Growth in BALB/c Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2024, 55(6): 2619-2628.

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引物名称 Primer name	上游引物(5′→3′) Forward primer	下游引物(5′→3′) Reverse primer
U6	CTCGCTTCGGCAGCACA	AACGCTTCACGAATTTGCGT
let-7-5p	AGCTGGACTGAGGTAGTGTTTCG	ATCCAGTGCAGGGTCCGAGG
GAPDH	AGGTCGGTGTGAACGGATTTG	TGTAGACCATGTAGTTGAGGTCA
C/EBP δ	CGACTTCAGCGCCTACATTGA	CTAGCGACAGACCCCACAC
IL-1β	AGAGCATCCAGCGTCAAATC	TCATCTCGGAGCCTGTAGTG
IL-6	CTTCTTGGGACTGATGCTGGTGAC	TCTGTTGGGAGTGGTATCCTCTGTG
iNOS	CGAAACGCTTCACTTCCAA	TGAGCCTATATTGCTGTGGCT
IL-4	GCTATTGATGGGTCTCACCC	CAGGACGTCAAGGTACAGGA
IL-10	GCTCTTACTGACTGGCATGAG	CGCAGCTCTAGGAGCATGTG
Arg-1	TCATGGAAGTGAACCCAACTCTTG	TCAGTCCCTGGCTTATGGTTACC

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敲低多房棘球蚴let-7-5p可抑制BALB/c小鼠腹腔巨噬细胞极化和虫体生长

Knockdown of let-7-5p from Echinococcus multilocularis Inhibited the Peritoneal Macrophages Polarization and Worm Growth in BALB/c Mice

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