太子参多糖经Let-7d-3p下调伪狂犬病病毒感染小鼠的炎症基因转录水平

doi:10.11843/j.issn.0366-6964.2025.05.040

畜牧兽医学报 ›› 2025, Vol. 56 ›› Issue (5): 2438-2450.doi: 10.11843/j.issn.0366-6964.2025.05.040

太子参多糖经Let-7d-3p下调伪狂犬病病毒感染小鼠的炎症基因转录水平

罗诗师¹^,²(), 陈蓓蕾¹^,², 张蕾¹, 冯启贤¹^,², 吴瑞森¹^,², 陈佳祺¹^,², 王媛¹^,², 简子昕¹, 许丽惠¹^,², 陈秋勇³, 马玉芳¹^,², 王全溪¹^,²^,*()

1. 福建农林大学动物科学学院, 福建省兽医中药与动物保健重点实验室, 福州 350002
2. 福建农林大学中西兽医结合与动物保健福建省高等学校重点实验室, 福州 350002
3. 福建省农业科学院畜牧兽医研究所福州 350013

收稿日期:2024-06-03 出版日期:2025-05-23 发布日期:2025-05-27
通讯作者: 王全溪 E-mail:2272892708@qq.com;wqx608@126.com
作者简介:罗诗师(1999-)，女，四川泸州人，硕士生，主要从事预防兽医学研究，E-mail：2272892708@qq.com
基金资助:
福建省对外合作项目(2024I01010067)

Radix Pseudostellariae Polysaccharide Regulates Let-7d-3p to Alleviates Inflammatory-induced by Pseudorabies Virus Infection in Mice

LUO Shishi¹^,²(), CHEN Beilei¹^,², ZHANG Lei¹, FENG Qixian¹^,², WU Ruisen¹^,², CHEN Jiaqi¹^,², WANG Yuan¹^,², JIAN Zixin¹, XU Lihui¹^,², CHEN Qiuyong³, MA Yufang¹^,², WANG Quanxi¹^,²^,*()

1. Fujian Key Laboratory of Traditional Chinese Veterinary Medicine and Animal Health, College of Animal Science, Fujian Agriculture and Forestry Universities, Fuzhou 350002, China
2. University Key Laboratory for Integrated Chinese Traditional and Western Veterinary Medicine and Animal Healthcare in Fujian Province, Fuzhou 350002, China
3. Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agriculture Sciences, Fuzhou 350013, China

Received:2024-06-03 Online:2025-05-23 Published:2025-05-27
Contact: WANG Quanxi E-mail:2272892708@qq.com;wqx608@126.com

摘要/Abstract

摘要：

本研究旨在探究太子参多糖(Radix pseudostellariae polysaccharide, RPP)调控微小核糖核酸(microRNA, miRNA)Let-7d-3p对伪狂犬病病毒(pseudorabies virus, PRV)感染引起炎症基因表达的影响及其机理。体外培养猪肾细胞(porcine kidney 15, PK-15)，使用不同浓度RPP(0.5~32.0 mg·mL^-1)处理PK-15细胞，通过细胞增殖与活性检测试剂盒(cell counting kit-8, CCK8)法检测细胞存活率。接着将PK-15细胞分为空白组、PRV组、0.5 mg·mL^-1 RPP组、1.0 mg·mL^-1 RPP组、2.0 mg·mL^-1RPP组，通过实时荧光定量PCR(quantitative real-time PCR, qPCR)检测各组细胞中炎症因子白细胞介素(interleukin, IL)-6、肿瘤坏死因子-α(tumor necrosis factor-α, TNF-α)、IL-17的mRNA转录水平。转染Let-7d-3p抑制物至PK-15细胞，经0或0.5 mg· mL^-1RPP干预后，采用qPCR检测Let-7d-3p和炎症因子mRNA转录水平。选择15只健康小鼠随机分为空白组、PRV组、RPP(10 mg·kg^-1)组(每组5只)，qPCR检测小鼠肾组织中Let-7d-3p和炎症因子mRNA转录水平。结果显示，在0~4.0 mg·mL^-1 浓度RPP无细胞毒性。与空白组相比，PRV感染PK-15细胞会显著上调炎症因子IL-6、TNF-α、IL-17的mRNA转录水平(P < 0.01)，显著下调Let-7d-3p转录水平(P < 0.01)。与PRV组相比，三个浓度的RPP作用细胞后均显著上调Let-7d-3p的转录水平(P < 0.01)，同时显著下调3个炎症因子的mRNA转录水平(P < 0.01)。抑制Let-7d-3p后，与PRV组相比Let-7d-3p抑制组的IL-6、TNF-α、IL-17的mRNA转录水平呈现显著上调(P < 0.01)。而与PRV+RPP组相比，抑制Let-7d-3p后加入RPP干预，细胞炎症因子的mRNA转录水平显著上调(P < 0.01)。体内试验结果表明，与空白组相比，PRV感染组小鼠肾中Let-7d-3p的转录水平显著下降(P < 0.01)，且炎症因子IL-6、TNF-α、IL-17的mRNA转录水平显著上调(P < 0.01)。与PRV组相比，RPP处理小鼠后肾脏中Let-7d-3p转录水平显著上升(P < 0.01)，炎症因子IL-6、TNF-α、IL-17的转录水平显著下降(P < 0.01)。体内体外结果表明，RPP可通过Let-7d-3p下调PRV感染引起的炎症基因转录水平，在mRNA水平上发现RPP具有一定的抗炎效用，为后续研究太子参多糖缓解PRV引起的炎症提供思路。

关键词: 太子参多糖, 伪狂犬病病毒, Let-7d-3p, 炎症

Abstract:

The study aims to explore the impact and mechanism of Radix pseudostellariae polysaccharide (RPP) in regulating miRNA Let-7d-3p on the inflammation caused by pseudorabies virus (PRV) infection. PK-15 cells were cultured in vitro and treated with various concentrations of RPP (0.5-32.0 mg·mL^-1). The cell viability was then assessed using the CCK-8 assay. And PK-15 cells were divided into the control group, PRV group, 0.5 mg·mL^-1 RPP group, 1.0 mg·mL^-1 RPP group, and 2.0 mg·mL^-1 RPP group. The expression levels of inflammatory cytokines IL-6, TNF-α, and IL-17 in cells from each group were detected using real-time fluorescent qPCR. Let-7d-3p inhibitors were transfected into PK-15 cells and then subjected to intervention with 0 or 0.5 mg·mL^-1 RPP. The expression levels of Let-7d-3p and inflammatory factors were examined using qPCR. In the vivo experiment, 15 healthy mice were randomly assigned to the control group, PRV group, and RPP (10.0 mg·kg^-1) group, with 5 mice in each group. The expression levels of Let-7d-3p and inflammatory factors in mouse kidney tissues were detected using qPCR. The results showed that within the concentration range of 0 to 4.0 mg·mL^-1, RPP has no cytotoxicity. Compared to the control group, the transcription levels of the inflammatory factors IL-6, TNF-α, and IL-17 mRNA in PK-15 cells infected by PRV are significantly up regulated (P < 0.01), while the transcription level of Let-7d-3p is significantly down regulated (P < 0.01). In comparison with the PRV group, the expression of Let-7d-3p was significantly up regulated (P < 0.01) after the cells were treated with three concentrations of RPP, while the transcription levels of the three inflammatory factors were concurrently and significantly down regulated (P < 0.01). After the inhibition of Let-7d-3p, compared with the PRV group, the transcription levels of IL-6, TNF-α, and IL-17 in the Let-7d-3p inhibition group were significantly up regulated (P < 0.01). Compared with the PRV+RPP group, after the suppression of Let-7d-3p and subsequent intervention with RPP, the transcription levels of cellular inflammatory factors were significantly up regulated (P < 0.01). In vivo results indicate that, in comparison with the control group, the transcriptional level of Let-7d-3p in the kidneys of mice in the PRV group was significantly decreased (P < 0.01), and the transcriptional levels of inflammatory factors IL-6, TNF-α, and IL-17 were significantly up regulated (P < 0.01). Compared with the PRV group, the expression of Let-7d-3p in the kidneys of mice treated with RPP was significantly increased (P < 0.01), and the transcription levels of the inflammatory factors IL-6, TNF-α, and IL-17 were significantly decreased (P < 0.01). Results from both in vivo and in vitro experiments indicate that RPP can downregulate the transcription levels of inflammatory genes induced by PRV infection through Let-7d-3p, providing a rationale for subsequent studies on the alleviation of PRV-induced inflammation by RPP.

Key words: Radix pseudostellariae polysaccharide, pseudorabies virus, Let-7d-3p, inflammatory

中图分类号:

S853.7

罗诗师, 陈蓓蕾, 张蕾, 冯启贤, 吴瑞森, 陈佳祺, 王媛, 简子昕, 许丽惠, 陈秋勇, 马玉芳, 王全溪. 太子参多糖经Let-7d-3p下调伪狂犬病病毒感染小鼠的炎症基因转录水平[J]. 畜牧兽医学报, 2025, 56(5): 2438-2450.

LUO Shishi, CHEN Beilei, ZHANG Lei, FENG Qixian, WU Ruisen, CHEN Jiaqi, WANG Yuan, JIAN Zixin, XU Lihui, CHEN Qiuyong, MA Yufang, WANG Quanxi. Radix Pseudostellariae Polysaccharide Regulates Let-7d-3p to Alleviates Inflammatory-induced by Pseudorabies Virus Infection in Mice[J]. Acta Veterinaria et Zootechnica Sinica, 2025, 56(5): 2438-2450.

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基因 Genes	引物序列(5′→3′) Primer sequences	基因库登录号 GenBank accession No.
IL-6-pig	F: CAGTCCAGTCGCCTTCT	NM_214399.1
IL-6-pig	R: CACCTTTGGCATCTTCTT	NM_214399.1
TNF-α-pig	F: TTCCAGCTGGCCCCTTGAGC	JF831365.1
TNF-α-pig	R: GAGGGCATTGGCATACCCAC	JF831365.1
IL-17-pig	F: CTGTCACTGCTGCTTCTGCTGAG	KF646141.1
IL-17-pig	R: TGGAGTAATCTGAGGGCCGTCTG	KF646141.1
β-Actin-pig	F: CAGGTCATCACCATCGGCAACG	NC_010445.4
β-Actin-pig	R: GACAGCACCGTGTTGGCGTAGAGGT	NC_010445.4
Let-7d-3p	F: AACGCTATACGACCTGCTGC	NR_128407
	R: AGTGCAGGGTCCGAGGTATT
	茎环：GTCGTATCCAGTGCAGGGT CCGAGGTATTCGCACTGGATACGACAGAAAG
U6	F: GACCCGCAATAGTGAGAGAAGC	EU520423.1
U6	R: CATGGGAAATTGGCCCTCTGC	EU520423.1
PRV-gB	F: GCGGTCACCTTGTGGTTGTT	MN718167.1
PRV-gB	R: AACGTCATCGTCACGACCGT	MN718167.1
IL-6-mouse	F: CACAGAAGGAGTGGCTAA	XM_032905335.1
IL-6-mouse	R: GCATAACGCACTAGGTTT	XM_032905335.1
TNF-α-mouse	F: CTGTCTTAACTAACCTCCTTCT	M20155.1
TNF-α-mouse	R: ATGTCTAACTGCCCTTCC	M20155.1
I L-17-mouse	F: GGTCCAGCTTTCCCTCC	XM_021176489.1
I L-17-mouse	R: AGACTACCTCAACCGTTCCA	XM_021176489.1
β-Actin-mouse	F: TACGAGTTGCCTGATGGTC	KC215183.1
β-Actin-mouse	R: ATGATGGAGTTGAAGGTGGT	KC215183.1

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太子参多糖经Let-7d-3p下调伪狂犬病病毒感染小鼠的炎症基因转录水平

Radix Pseudostellariae Polysaccharide Regulates Let-7d-3p to Alleviates Inflammatory-induced by Pseudorabies Virus Infection in Mice

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