绞股蓝皂苷体外抗牛病毒性腹泻病毒的作用

doi:10.3969/j.issn.1004-1524.20231161

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (12): 2687-2695.DOI: 10.3969/j.issn.1004-1524.20231161

绞股蓝皂苷体外抗牛病毒性腹泻病毒的作用

才冬杰¹(), 申子凡¹, 左之才¹^,^*(), 田斌², 叶刚¹, 李伟强¹, 卜庆龙³

1.四川农业大学动物医学院,四川成都 611130
2.四川农业大学动物疫病与人类健康四川省重点实验室,四川成都 611130
3.山东畜牧兽医职业学院宠物科技学院,山东潍坊 261061

收稿日期:2023-09-28 出版日期:2024-12-25 发布日期:2024-12-27
作者简介:才冬杰(1987—),女,黑龙江拜泉人,博士,讲师,研究方向为反刍动物腹泻疾病。E-mail:dongjie_cai@sicau.edu.cn
通讯作者: *左之才,E-mail:czyhzzc@126.com
基金资助:
国家重点研发计划(2022YFD1601600);国家重点研发计划(2021YFD1600200);现代农业产业技术体系(肉牛牦牛)(CARS-37);四川省自然科学基金(2022NSFSC1620)

Antiviral effects of gypenoside against bovine viral diarrhea virus in vitro

CAI Dongjie¹(), SHEN Zifan¹, ZUO Zhicai¹^,^*(), TIAN Bin², YE Gang¹, LI Weiqiang¹, BU Qinglong³

1. College of Veterinary Medicine, Sichuan Agricultural University, Chengdu 611130, China
2. Key Laboratory of Animal Diseases and Human Health of Sichuan Province, Sichuan Agricultural University, Chengdu 611130, China
3. School of Pet Technology, Shandong Vocational Animal Science and Veterinary College, Weifang 261061, Shandong, China

Received:2023-09-28 Online:2024-12-25 Published:2024-12-27

摘要/Abstract

摘要：

为研究绞股蓝皂苷对牛病毒性腹泻病毒(bovine viral diarrhea virus, BVDV)的作用,采用体外细胞培养技术、细胞病变效应观察与CCK-8相结合的方法,首先确定绞股蓝皂苷和α-单月桂酸甘油酯的安全浓度,然后采用先加药后加病毒、先加病毒后加药,及药和病毒预先作用这3种加药方式,利用Western blot和定量PCR检测BVDV E2蛋白基因和5'-UTR表达量的变化,探究其在体外对病毒的抑制作用。结果表明,当绞股蓝皂苷和α-单月桂酸甘油酯的浓度分别为16 μmol·L^-1和25 μg·mL^-1时,细胞活性与空白组相比的差异无统计学意义(P>0.05),将其确定为两种药物的最大安全浓度。与对照组相比,绞股蓝皂苷可极显著(P<0.01)抑制BVDV E2蛋白基因和5'-UTR的表达,且其直接灭活作用优于α-单月桂酸甘油酯。此外,绞股蓝皂苷还显示出了显著(P<0.05)的吸附阻断作用,但其复制阻断作用与单独病毒组无显著差异。

关键词: 牛病毒性腹泻病毒, 绞股蓝皂苷, 抗病毒

Abstract:

To investigate the effect of gypenoside on bovine viral diarrhea virus (BVDV), the in vitro cell culture technique combined with cytopathic effect (CPE) observation and CCK-8 assay was employed. The safe concentrations of gypenoside and α-glycerol monolaurate were determined. Three different treatment protocols were used:pre-treatment with compound followed by viral infection, post-treatment with compound after viral infection, and pre-incubation of compound and virus prior to infection. Western blot and quantitative PCR were conducted to evaluate the changes of expression levels of BVDV E2 protein gene and 5'-UTR, to explore the inhibitory effect in vitro. The results showed that the cell viability at the concentration of 16 μmol·L^-1 for gypenoside and 25 μg·mL^-1 for α-glycerol monolaurate was not significantly different from the control group (P>0.05), indicating that these concentrations were the maximum safe concentrations of the two compounds. Compared to the control group, gypenoside exhibited significant (P<0.01) inhibition on the expression levels of BVDV E2 protein gene and 5'-UTR, and its direct virucidal effect was superior to the α-glycerol monolaurate. Gypenoside also showed significant (P<0.05) adsorption inhibition against BVDV, while no significant difference was found in replication inhibition when compared to the virus control group.

Key words: bovine viral diarrhea virus, gypenoside, antivirus

中图分类号:

S853.74

才冬杰, 申子凡, 左之才, 田斌, 叶刚, 李伟强, 卜庆龙. 绞股蓝皂苷体外抗牛病毒性腹泻病毒的作用[J]. 浙江农业学报, 2024, 36(12): 2687-2695.

CAI Dongjie, SHEN Zifan, ZUO Zhicai, TIAN Bin, YE Gang, LI Weiqiang, BU Qinglong. Antiviral effects of gypenoside against bovine viral diarrhea virus in vitro[J]. Acta Agriculturae Zhejiangensis, 2024, 36(12): 2687-2695.

图/表 8

图1 BVDV QL1903株的免疫荧光结果

Fig.1 Indirect immunofluorescence results of BVDV QL1903 strain

图2 不同浓度药物致MDBK细胞病变的结果

Fig.2 Results of drug-induced cytopathic effect in MDBK cells

图3 绞股蓝皂苷和α-单月桂酸甘油酯对MDBK细胞活力的影响空白对照的浓度为0。柱上标“*”或“**”的分别表示与空白对照差异显著(P<0.05)或极显著(P<0.01)。

Fig.3 Effect of gypenoside and α-glycerol monolaurate on MDBK cell viability The concentration in blank control is 0. Bars marked with “*” or “**” indicate significant difference with the blank control at P<0.05 or P<0.01 level, respectively.

图4 绞股蓝皂苷联合牛病毒性腹泻病毒(BVDV)对细胞活力的影响 Control,空白对照;BVDV,全病毒组;L+BVDV,4 μmol·L-1绞股蓝皂苷+BVDV;M+BVDV,8 μmol·L-1绞股蓝皂苷+BVDV;H+BVDV,16 μmol·L-1绞股蓝皂苷+BVDV。

Fig.4 Effects of gypenoside combined with bovine viral diarrhea virus (BVDV) on cell viability Control, Blank control; BVDV, Virus group; L+BVDV, 4 μmol·L-1 gypenoside+BVDV; M+BVDV, 8 μmol·L-1 gypenoside+BVDV; H+BVDV,16 μmol·L-1 gypenoside+BVDV.

图5 绞股蓝皂苷灭活牛病毒性腹泻病毒(BVDV)的效果 Control,空白对照组(不加BVDV);BVDV,病毒对照组(含BVDV);DMSO,溶剂对照组(含BVDV);Y,阳性对照组(α-单月桂酸甘油酯+BVDV);Z:直接杀伤组(绞股蓝皂苷+BVDV)。标“**”的表示差异极显著(P<0.01)。下同。

Fig.5 Inactivation of gypenoside on bovine viral diarrhea virus (BVDV) Control, Blank control without BVDV; BVDV, Virus group (with BVDV); DMSO, Solvent control group (with BVDV); Y, Positive drug group (α-glycerol monolaurate with BVDV); Z, Direct killing group (gypenoside+BVDV). “**” indicates significant difference at P<0.01.The same as below.

图6 绞股蓝皂苷对牛病毒性腹泻病毒(BVDV)复制的影响 F,复制阻断组(绞股蓝皂苷+BVDV)。下同。

Fig.6 Effect of gypenoside on replication of bovine viral diarrhea virus (BVDV) F, Replication inhibition group (gypenoside+BVDV). The same as below.

图7 绞股蓝皂苷对牛病毒性腹泻病毒(BVDV)吸附的影响 X,吸附阻断组(绞股蓝皂苷+BVDV)。下同。

Fig.7 Effect of gypenoside on adsorption of bovine viral diarrhea virus (BVDV) X, Adsorption inhibition group (gypenoside+BVDV). The same as below.

图8 绞股蓝皂苷不同作用方式对牛病毒性腹泻病毒(BVDV)E2蛋白的影响 A,Western blot试验结果;B,不同处理对BVDV E2蛋白表达的影响分析。

Fig.8 Effects of action modes of gypenoside on E2 protein of bovine viral diarrhea virus (BVDV) A, Result of Western blot; B, Effect of treatments on relative expression level of BVDV E2 protein.

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绞股蓝皂苷体外抗牛病毒性腹泻病毒的作用

Antiviral effects of gypenoside against bovine viral diarrhea virus in vitro

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