氯硝柳胺及其新型口服纳米药物抗猪流行性腹泻病毒活性及作用机制

doi:10.16431/j.cnki.1671-7236.2025.10.041

摘要/Abstract

摘要： 【目的】氯硝柳胺(niclosamide,NIC)最初作为治疗肠道寄生虫的驱虫药使用，近年来研究表明其对多种病毒具有良好的抑制作用。本研究旨在评估NIC对猪流行性腹泻病毒(Porcine epidemic diarrhea virus，PEDV)的体外抗病毒效果，开发新型口服纳米化NIC制剂并评估其体内抗病毒效果，探讨其抑制PEDV感染的潜在分子机制。【方法】通过细胞毒性试验和病毒蚀斑试验评估NIC的体外抗PEDV活性；在前期胆酸盐递送系统的基础上制备增强型氯硝柳胺纳米药物(enhanced niclosamide-delivery nanoparticles，eNDN)，并对其粒径、水溶性和药代动力学特性等进行表征；在APN转基因小鼠模型中验证口服eNDN对PEDV感染的抑制作用。通过文献挖掘及生物信息学综合分析NIC抗PEDV感染的潜在靶点和信号通路，并通过分子对接初步确定NIC抑制PEDV感染的潜在核心靶点。【结果】体外抗病毒试验结果显示，NIC对PEDV具有良好的抑制作用，其半数抑制浓度(IC₅₀)为226.4 nmol/L。与氯硝柳胺纳米药物(NDN)相比，eNDN粒径降低了约30 nm (约为140 nm)，水溶性提高了20%(达到5 mg/mL)，生物利用度提高了约10%(达到47.54%)。毒性试验结果显示，口服eNDN未引起明显临床症状或肝脏病理变化，具有良好的生物安全性。实时荧光定量PCR结果显示，与对照组相比，eNDN处理组小鼠肠道内PEDV滴度极显著降低(P＜0.01)。生物信息学分析共识别出NIC的作用靶点159个，PEDV感染相关靶点1 678个，两者共有靶点22个。分子对接结果表明，MAPK14、NAIP、NLRC4、SERPINE1、ATP2A2、CLTC、MCL1、TMPRSS2、APN和STAT3为NIC抑制PEDV感染的潜在核心靶点。【结论】NIC及其新型口服纳米药物eNDN在体内外均表现出良好的抗PEDV活性，生物信息学预测NIC通过抗炎反应及抑制病毒侵入等多靶点、多通路发挥抗PEDV作用。本研究结果为探明NIC抗PEDV感染的分子机制及其纳米药物eNDN的临床转化提供了理论基础和试验依据。

关键词: 氯硝柳胺; 口服纳米药物; 猪流行性腹泻病毒(PEDV); 抗病毒感染

Abstract: 【Objective】 Niclosamide (NIC),originally developed as an anthelmintic agent,has recently been recognized for its potent inhibitory activity against a broad spectrum of viruses.This study aimed to evaluate the in vitro antiviral activity of NIC against Porcine epidemic diarrhea virus (PEDV),develop and assess a novel oral NIC nanomedicine for antiviral efficacy in vivo,and preliminarily explore the molecular mechanisms underlying its anti-PEDV action.【Method】 The antiviral effect of NIC against PEDV was first evaluated in vitro using cytotoxicity and plaque reduction assays.An enhanced niclosamide-delivery nanoparticle (eNDN) was formulated based on a previously established bile salt-based delivery system,and its physicochemical properties,including particle size,water solubility,and pharmacokinetics,were characterized.The antiviral efficacy of orally administered eNDN was then evaluated in APN-transgenic mice challenged with PEDV.Additionally,literature mining and integrative bioinformatics analyses were performed to identify potential antiviral targets and signaling pathways,and molecular docking was used to predict key targets involved in its anti-PEDV activity.【Result】 The results of in vitro antiviral test showed that,NIC exhibited potent antiviral activity against PEDV in vitro,with a half-maximal inhibitory concentration (IC₅₀) of 226.4 nmol/L.Compared with niclosamide-delivery nanoparticles (NDN),the optimized nanomedicine eNDN exhibited a reduced particle size by approximately 30 nm (average diameter was about 140 nm),a 20% improvement in water solubility (reaching 5 mg/mL),and an approximately 10% increase in oral bioavailability (up to 47.54%).Toxicological assessment showed no apparent clinical symptoms or hepatic pathology following oral administration of eNDN,indicating good biosafety.Real-time quantitative PCR analysis revealed an extremely significant reduction of PEDV titer in intestines of eNDN-treated mice (P＜0.01),compared with control group.Bioinformatics analysis identified 159 NIC-related targets and 1 678 PEDV-associated targets,with 22 overlapping targets.Molecular docking suggested MAPK14,NAIP,NLRC4,SERPINE1,ATP2A2,CLTC,MCL1,TMPRSS2,APN and STAT3 as core targets mediating NIC’s antiviral effects.【Conclusion】 Both NIC and its novel oral nanomedicine eNDN exhibited robust antiviral activity anti-PEDV in vitro and in vivo.Bioinformatics predicted that NIC played an anti-PEDV role through multiple targets and pathways such as anti-inflammatory response and inhibition of virus invasion.The results of this study provided a theoretical and experimental basis for exploring the molecular mechanism of NIC against PEDV infection and the clinical transformation of nanomedicine eNDN.

Key words: niclosamide; oral nanomedicine; Porcine epidemic diarrhea virus (PEDV); antiviral infection

中图分类号:

S859.7

翟崇凯, 毛福超, 田文静, 王聪慧, 王迎鲜, 张贺伟. 氯硝柳胺及其新型口服纳米药物抗猪流行性腹泻病毒活性及作用机制[J]. 中国畜牧兽医, 2025, 52(10): 4964-4976.

ZHAI Chongkai, MAO Fuchao, TIAN Wenjing, WANG Conghui, WANG Yingxian, ZHANG Hewei. Niclosamide and Its Novel Oral Nanomedicine:Antiviral Efficacy and Potential Molecular Mechanisms Against Porcine Epidemic Diarrhea Virus[J]. China Animal Husbandry & Veterinary Medicine, 2025, 52(10): 4964-4976.

参考文献

[1] ZHANG S,WANG J,LIU X,et al.Pemetrexed alleviates piglet diarrhea by blocking the interaction between Porcine epidemic diarrhea virus nucleocapsid protein and Ezrin[J].Journal of Virology,2024,98(1):e01625-23.
[2] XU J,SHI P Y,LI H,et al.Broad spectrum antiviral agent niclosamide and its therapeutic potential[J].ACS Infectious Diseases,2020,6(5):909-915.
[3] BRAGA L,ALI H,SECCO I,et al.Drugs that inhibit TMEM16 proteins block SARS-CoV-2 spike-induced syncytia[J].Nature,2021,594(7861):88-93.
[4] JAGTAP P,MEENA V K,SAMBHARE S,et al.Exploring niclosamide as a multi-target drug against SARS-CoV-2:Molecular dynamics simulation studies on host and viral proteins[J].Molecular Biotechnology,2024.Doi:10.1007/s12033-024-01296-2.Online ahead of print.
[5] DE ALMEIDA L,DA SILVA A L N,RODRIGUES T S,et al.Identification of immunomodulatory drugs that inhibit multiple inflammasomes and impair SARS-CoV-2 infection[J].Science Advances,2022,8(37):eabo5400.
[6] ZHAI C,WANG M,JIN Y,et al.Oral delivery of a host-directed antiviral,niclosamide,as a cholate-coated nanoformulation[J].International Journal of Antimicrobial Agents,2023,62(5):106973.
[7] HUANG Z X,ZHOU S T,WANG J,et al.Remdesivir inhibits Porcine epidemic diarrhea virus infection in vitro[J].Heliyon,2023,9(11)：e21468.
[8] YUAN C,HUANG X,ZHAI R,et al.In vitro antiviral activities of salinomycin on Porcine epidemic diarrhea virus[J].Viruses,2021,13(4):580.
[9] KO S,GU M J,KIM C G,et al.Rapamycin-induced autophagy restricts Porcine epidemic diarrhea virus infectivity in porcine intestinal epithelial cells[J]. Antiviral Research,2017,146:86-95.
[10] WANG Y,QIN P,ZHAO C,et al.Evaluating anti-viral effect of ivermectin on Porcine epidemic diarrhea virus and analyzing the related genes and signaling pathway by RNA-Seq in vitro[J].Virology,2023,587:109877.
[11] LU Y,LIANG X,SONG J,et al.Niclosamide modulates phenotypic switch and inflammatory responses in human pulmonary arterial smooth muscle cells[J].Molecular and Cellular Biochemistry,2024,480(3):1583-1593.
[12] WANG Y,HUANG H,LI D,et al.Identification of niclosamide as a novel antiviral agent against Porcine epidemic diarrhea virus infection by targeting viral internalization[J].Virologica Sinica,2023,38(2):296-308.
[13] YANG C W,PENG T T,HSU H Y,et al.Repurposing old drugs as antiviral agents for Coronaviruses[J].Biomedical Journal,2020,43(4):368-374.
[14] DEVARAKONDA B,HILL R A,LIEBENBERG W,et al.Comparison of the aqueous solubilization of practically insoluble niclosamide by polyamidoamine (PAMAM) dendrimers and cyclodextrins[J].International Journal of Pharmaceutics,2005,304(1-2):193-209.
[15] BASAVARAJ S,BETAGERI G V.Can formulation and drug delivery reduce attrition during drug discovery and development—Review of feasibility,benefits and challenges[J].Acta Pharmaceutica Sinica B,2014,4(1):3-17.
[16] CHOI G,REJINOLD N S,PIAO H,et al.The next generation COVID-19 antiviral;Niclosamide-based inorganic nanohybrid system kills SARS-CoV-2[J].Small,2024,20(39):2305148.
[17] LI M,WANG Q,LI Y,et al.Apical sodium-dependent bile acid transporter,drug target for bile acid related diseases and delivery target for prodrugs:Current and future challenges[J].Pharmacology & Therapeutics,2020,212:107539.
[18] CHATTERJEE S,HUI P C,KAN C,et al.Dual-responsive (pH/temperature) pluronic F-127 hydrogel drug delivery system for textile-based transdermal therapy[J].Scientific Reports,2019,9(1):11658.
[19] SAKHI M,KHAN A,KHAN I,et al.Effect of polymeric stabilizers on the size and stability of PLGA paclitaxel nanoparticles[J].Saudi Pharmaceutical Journal,2023,31(9):101697.
[20] DAHMANI F Z,YANG H,ZHOU J,et al.Enhanced oral bioavailability of paclitaxel in pluronic/LHR mixed polymeric micelles:Preparation,in vitro and in vivo evaluation[J].European Journal of Pharmaceutical Sciences,2012,47(1):179-189.
[21] JIA W,LI Y,CHEUNG K C P,et al.Bile acid signaling in the regulation of whole body metabolic and immunological homeostasis[J].Science China Life Sciences,2024,67(5):865-878.
[22] LI W,LUO R,HE Q,et al.Aminopeptidase N is not required for Porcine epidemic diarrhea virus cell entry[J].Virus Research,2017,235:6-13.
[23] JI C M,WANG B,ZHOU J,et al.Aminopeptidase-N-independent entry of Porcine epidemic diarrhea virus into Vero or porcine small intestine epithelial cells[J].Virology,2018,517:16-23.
[24] PARK J E,PARK E S,YU J E,et al.Development of transgenic mouse model expressing porcine aminopeptidase N and its susceptibility to Porcine epidemic diarrhea virus[J].Virus Research,2015,197:108-115.
[25] FENG Z,FU Y,YANG S,et al.Siglec-15 is a putative receptor for Porcine epidemic diarrhea virus infection[J].Cellular and Molecular Life Sciences,2025,82(1):136.
[26] THI TRAN U,KITAMI T.Niclosamide activates the NLRP3 inflammasome by intracellular acidification and mitochondrial inhibition[J].Communications Biology,2019,2(1):2.
[27] REJINOLD N S,JIN G,CHOY J H.Insight into preventing global dengue spread:Nanoengineered niclosamide for viral infections[J].Nano Letters,2024,24(46):14541-14551.
[28] HIGGINS C A,NILSSON-PAYANT B E,BONAVENTURE B,et al.SARS-CoV-2 hijacks p38β/MAPK11 to promote virus replication[J].mBio,2023,14(4):e01007-23.
[29] MATICO R E,YU X,MILLER R,et al.Structural basis of the human NAIP/NLRC4 inflammasome assembly and pathogen sensing[J].Nature Structural & Molecular Biology,2024,31(1):82-91.
[30] SHENG J,LI L,LV X,et al.Integrated interactome and transcriptome analysis reveals key host factors critical for SARS-CoV-2 infection[J].Virologica Sinica,2023,38(4):508-519.
[31] LI Y,LI J,WANG X,et al.Role of intestinal extracellular matrix-related signaling in Porcine epidemic diarrhea virus infection[J].Virulence,2021,12(1):2352-2365.
[32] DE ANTONELLIS P,FERRUCCI V,BIBBO F,et al.Targeting ATP2B1 impairs PI3K/Akt/FOXO signaling and reduces SARS-COV-2 replication in vivo[J].EMBO Reports,2024,25(7):2974-3007.
[33] FAN B,ZHU L,CHANG X,et al.Mortalin restricts Porcine epidemic diarrhea virus entry by down regulating clathrin-mediated endocytosis[J].Veterinary Microbiology,2019,239:108455.
[34] PAN P,GE W,LEI Z,et al.SARS-CoV-2 N protein enhances the anti-apoptotic activity of MCL-1 to promote viral replication[J].Signal Transduction and Targeted Therapy,2023,8(1):194.
[35] SHI W,FAN W,BAI J,et al.TMPRSS2 and MSPL facilitate trypsin-independent Porcine epidemic diarrhea virus replication in Vero cells[J].Viruses,2017,9(5):114.
[36] WANG S,XU C,SHI J,et al.Regulatory effect and mechanism of APN gene on Porcine epidemic diarrhea virus resistance[J].Gene,2021,775:145448.
[37] HUANG H,LI Y,LI D,et al.The tyrosine phosphatase PTPN14 inhibits the activation of STAT3 in PEDV infected Vero cells[J].Veterinary Microbiology,2022,267:109391.