In vitro anti-influenza A virus effects and mechanisms of non-toxic dried toad and Bufonis Venenum extracts

QIN Xinxin; YU Huiyong; WANG Mingzhe; ZHANG Shan; GUO Yufei; NIE Weicheng; WANG Chengxiang

doi:10.16025/j.1674-1307.2025.03.011

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In vitro anti-influenza A virus effects and mechanisms of non-toxic dried toad and Bufonis Venenum extracts

更新时间：2025-04-29

- In vitro anti-influenza A virus effects and mechanisms of non-toxic dried toad and Bufonis Venenum extracts
- Beijing Journal of Traditional Chinese Medicine Vol. 44, Issue 3, Pages: 314-324(2025)
- 作者机构：
  
  1.北京中医药大学第三附属医院呼吸科，北京 100029
  2.北京中医药大学东直门医院呼吸科，北京 100007
  3.河南省中医院肿瘤科，郑州 450002
- 作者简介：
- 基金信息：
- DOI：10.16025/j.1674-1307.2025.03.011
  CLC：
- Received：26 February 2024，
  
  Published：25 March 2025
- 稿件说明：
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秦欣欣，于会勇，王明哲，等.无毒干蟾及蟾酥提取物体外抗甲型流感病毒作用及机制［J］.北京中医药，2025，44（3）：314-324.

QIN Xinxin,YU Huiyong,WANG Mingzhe,et al.In vitro anti-influenza A virus effects and mechanisms of non-toxic dried toad and Bufonis Venenum extracts[J]. Beijing Journal of Traditional Chinese Medicine,2025,44(03):314-324.
秦欣欣，于会勇，王明哲，等.无毒干蟾及蟾酥提取物体外抗甲型流感病毒作用及机制［J］.北京中医药，2025，44（3）：314-324. DOI： 10.16025/j.1674-1307.2025.03.011.

QIN Xinxin,YU Huiyong,WANG Mingzhe,et al.In vitro anti-influenza A virus effects and mechanisms of non-toxic dried toad and Bufonis Venenum extracts[J]. Beijing Journal of Traditional Chinese Medicine,2025,44(03):314-324. DOI： 10.16025/j.1674-1307.2025.03.011.

摘要

目的

探讨无毒干蟾及蟾酥提取物的体外抗甲型流感病毒（influenza A virus，IAV）的作用及机制。

方法

制备无毒干蟾水提物、无毒干蟾醇提物、蟾酥水提物、蟾酥醇提物以及磷酸奥司他韦的稀释液，同时设置空白组、病毒组，以感染流感病毒亚洲甲型鼠肺适应株A/PR/8/34（H1N1）的犬肾（MDCK）细胞为载体，细胞活力检测（CCK8）法测定药物对MDCK细胞的最大无毒浓度（TC

）及半数中毒浓度（TC

），检测在不同浓度的各药物作用下PR8诱导后的细胞存活率、抗IAV的有效率（ER）、半数有效浓度（EC50）和治疗指数（TI）。选择ER和TI较高者对应的治疗方式，分别在处理后12、24、48 h，脱氧核糖核苷酸末端转移酶介导的原位末端标记法（TUNEL）检测细胞凋亡率，实时荧光定量PCR（qPCR）法检测内质网应激通路相关基因及凋亡基因的表达。

结果

无毒干蟾水提物、无毒干蟾醇提物、蟾酥水提物、蟾酥醇提物对MDCK细胞的TC

分别为39.06、195.31、1.22、4.88 mg/L，TC

分别为4 191.40、2 573.90、19.12、59.49 mg/L。各药物在3种给药方式下均有一定的体外抗IAV作用，并呈现出浓度依赖性，以直接灭活作用方式下的中药提取物体外抗IAV的ER、TI最高；给药后12、24、48 h，蟾酥水提物组的细胞凋亡率均低于其余3组中药提取物组；与病毒组比较，无毒干蟾醇提物、蟾酥水提物均能明显下调IAV感染后细胞的葡萄糖调节蛋白78（GRP78）、C/Ebp-同源蛋白质（CHOP）、半胱氨酸天冬氨酸蛋白水解酶-12（Caspase-12）、Caspase-3，上调B淋巴细胞瘤-2（Bcl-2）mRNA表达。

结论

无毒干蟾水提物、无毒干蟾醇提物、蟾酥水提物、蟾酥醇提物均对IAV有直接灭活作用，无毒干蟾醇提物、蟾酥水提物的抗病毒机制可能与其抑制内质网应激，诱导细胞凋亡有关。

Abstract

Objective

To explore the in vitro anti-influenza A virus （IAV） effects and mechanisms of non-toxic dried toad and Bufonis Venenum extracts.

Methods

Dilutions of non-toxic dried toad water extract， non-toxic dried toad alcohol extract， Bufonis Venenum water extract， Bufonis Venenum alcohol extract， and oseltamivir phosphate were prepared. A blank group and a virus group were also set up. Madin-Darby canine kidney （MDCK） cells infected with influenza virus A/PR/8/34 （H1N1） were used as carriers. The maximum non-toxic concentration （TC0） and median toxic concentration （TC50） of the drugs were determined using the CCK-8 method. In three different administration methods，

i.e.

， direct inactivation， prophylaxis， and therapeutic administration， cell su

rvival rate， anti-IAV effective rate （ER）， half-effective concentration （EC50）， and therapeutic index （TI） were assessed. The treatment methods corresponding to higher ER and TI were selected. Apoptosis rates were detected by TUNEL assay at 12， 24， and 48 hours after treatment， and the mRNA expression levels of genes related to the endoplasmic reticulum stress pathway and apoptosis were detected by real-time quantitative PCR （qPCR）.

Results

The TC0 values of non-toxic dried toad water extract， non-toxic dried toad alcohol extract， Bufonis Venenum water extract， and Bufonis Venenum alcohol extract against MDCK cells were 39.06 mg/L， 195.31 mg/L， 1.22 mg/L， and 4.88 mg/L， respectively. The TC50 values were 4 191.40 mg/L， 2 573.90 mg/L， 19.12 mg/L， and 59.49 mg/L， respectively. Each drug demonstrated in vitro anti-IAV effects in all three administration methods， showing a concentration-dependent relationship. The highest ER and TI for anti-IAV effects were observed under the direct inactivation method. At 12， 24， and 48 hours after treatment， the apoptosis rate was lowest in the Bufonis Venenum water extract group， followed by the other three Chinese medicine extract groups. Compared to the virus group， non-toxic dried toad alcohol extract and Bufonis Venenum water extract significantly downregulated the mRNA expression of glucose-regulated protein 78 （GRP78）， C/EBP homologous protein （CHOP）， cysteinyl aspartate-specific protease （Caspase）-12， and Caspase-3， and upregulated B-cell lymphoma 2 （Bcl-2） mRNA expression after IAV infection.

Conclusion

Non-toxic dried toad water extract， non-toxic dried toad alcohol extract， Bufonis Venenum water extract， and Bufonis Venenum alcohol extract exhibit direct virucidal effects against IAV. The anti-viral mechanisms of non-toxic dried toad alcohol extract and Bufonis Venenum water extract may be related to their inhibition of endoplasmic reticulum stress and induction of cell apoptosis.

关键词

Keywords

references

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