天然小分子药物小檗胺影响牛肠道病毒复制的研究

Study on the effect of natural small molecule drug berbamine on bovine enterovirus replication

为分析天然小分子药物小檗胺(BBM)是否影响牛肠道病毒(BEV)的复制,本研究以不同浓度的BBM与牛肾细胞(MDBK)共孵育后,采用MTT法检测BBM对MDBK细胞的影响,结果显示,与对照浓度相比,1μmol/L、5μmol/L、10μmol/L BBM对细胞增殖抑制作用均较小(P>0.05)。因此选择上述3种浓度的BBM分别处理MDBK 12 h后以103TCID50BEV感染,24 h后采用间接免疫荧光试验(IFA)检测BEV双链RNA(dsRNA),以确定BBM对BEV的最佳抑制浓度。MDBK细胞经10μmol/L BBM处理后感染BEV,观察48 h内BEV致细胞病变效应(CPE),采用荧光定量PCR检测BEV 5'UTR m RNA转录水平及测定子代病毒滴度,以确定BBM体外对BEV的抑制效果。IFA结果显示,与对照组相比,10μmol/L BBM对BEV ds RNA具有极显著性抑制作用(P<0.01);细胞形态观察结果显示,BBM对BEV引起的细胞大面积脱落具有一定的抑制效果;荧光定量PCR结果显示,与对照组相比,在感染48 h的细胞中BEV 5'UTR m RNA转录水平极显著降低(P<0.01);子代病毒滴度测定结果显示,在BEV感染24 h和36 h时子代病毒滴度极显著降低(P<0.01)。小鼠预防试验时将24只BALB/c小鼠均分为3组,分别为对照组(0)、低剂量组(50 mg/kg)、高剂量组(100 mg/kg),每日将BBM灌胃1次。给药后3 d滴鼻感染1015TCID50BEV,间隔48 h重复感染一次。感染后分别于5 d、10 d剖杀各组小鼠并采集各组织,采用荧光定量PCR检测BEV 5'UTR m RNA的转录水平。小鼠感染治疗试验时将48只BALB/c小鼠均分为BEV阳性对照组(BEV/DMSO)、感染给药组(BEV/BBM)、给药对照组(BBM),BEV阳性对照组和BEV/BBM组小鼠滴鼻感染1015TCID50BEV后,各组每天按原剂量灌胃BBM一次,感染后0、3 d、6 d和10 d剖杀各组小鼠并采集各组织,检测BEV 5'UTR m RNA的转录水平,并制备病理切片观察各组织的病变。BALB/c小鼠预防试验结果显示,各浓度BBM均具有显著或极显著抑制BALB/c小鼠肝、脾、肾、小肠组织中BEV 5'UTR m RNA转录水平升高的作用(P<0.05或P<0.01);与对照组相比,各浓度BBM均具有减轻各组织病变作用。其中高剂量组预防效果最明显,且随给药时间延长预防效果也最好。小鼠治疗试验结果显示,感染BEV后灌胃BBM当日即可抑制BEV在小鼠组织中的复制;病理学观察结果显示,对感染对照组相比,BBM可减轻小鼠各组织充血、出血、炎性细胞浸润等情况。综上所述,本研究首次证实BBM在体内外均具有抑制BEV复制的作用,为牛腹泻病的治疗及开发BBM抗病毒药物提供参考依据。

To investigate the effect of Berbamine(BBM)on bovine enterovirus(BEV)replication.MDBK cells were treated with different concentrations of BBM,and MTT assay was used to detect the effect of BBM on the cells.To detect the optimal concentration on the inhibition of BEV replication,MDBK cells,treated with different concentrations of BBM for eight hours,were infected with 103 TCID50 of BEV.The BEV double-strand RNA(dsRNA)expression level was detected by immunofluorescence assay(IFA).Reverse transcription-quantitative real-time PCR(RT-qPCR)and cytopathic effect were used to detect the impact of BBM on inhibition of BEV replication in vitro.Results showed that 1μmol/L,5μmol/L,and 10μmol/L of BBM inhibited virus replication compared to the control group(P>0.05).Microscopic examination of the BEV infection caused cell death,and BBM-inhibited cell death was performed.In addition,IFA showed that 10μmol/L of BBM significantly reduced the accumulation of BVDV dsRNA(P<0.01).Besides,RT-qPCR showed that the level of BEV 5'-UTR mRNA reduced considerably(P<0.01)after BBM treatment for 72 hours.Moreover,viral titers decreased significantly after BBM treatment for 24 hours and 36 hours(P<0.01).Subsequently,the prevention and therapeutic of BEV infection in BALB/c mice were investigated.Twenty-four mice were randomly divided into three different groups and treated with varying concentrations of BBM,named control group(0),low dose group(50mg/kg),high dose group(100mg/kg)by oral administration every 24 hours,followed by infection with 1015 TCID50/μL BEV by nasal drip,and re-infection after 48 hours.Tissues were collected for RT-qPCR to determine the BEV 5'-UTR mRNA replication level,and an inverted microscope observed histopathologic characteristics at 5 days and 10 days.Another forty-eight mice were randomly divided into three different concentration groups,named positive control group(0),high dose group(100mg/kg),and un-infection control group(100mg/kg).The control and high-dose groups were infected with 1015 TCID50/μL of BEV by nasal drip and given the indicated concentration BBM by oral administration every 24 hours.Tissues were collected for RT-qPCR to determine the effect of BEV 5'-UTR mRNA replication level,and an inverted microscope observed histopathologic characteristics at 0,3 days,6 days,and 10 days.The prevention experiment showed that BBM inhibited the BEV at different concentrations,significantly inhibiting the BEV 5'-UTR mRNA level in the liver,spleen,kidney,and small intestine(P<0.01 or P<0.05).The histopathologic results showed that BBM significantly inhibited the histopathologic changes in tissue,particularly in the high-dose group.The therapeutic results showed that BBM inhibits BEV inflection in tissue from one day after infection.The histopathological result showed that hyperemia,bleeding,and inflammatory cell infiltration were recovered.In conclusion,we confirmed that BBM significantly inhibited BEV replication in vivo and in vitro,provided a new method for preventing and treating BEV,and provided a reference for treating bovine diarrhea and developing BBM as an antiviral drug.