空心莲子草乙酸乙酯部位体内外抗H9N2亚型禽流感病毒活性的研究

Study on the Anti-AIV-H9N2 Activity of the Ethyl Acetate Extract from Alternanthera philoxeroides in vitro and in vivo

[目的]探究空心莲子草乙酸乙酯部位(ethyl acetate extract from Alternanthera philoxeroides,AETAC)体内外抗H9N2亚型禽流感病毒(H9N2 subtype Avian influenza virus, AIV-H9N2)活性及其作用机制。[方法]利用CCK-8法检测不同浓度AETAC作用不同时间对鸡胚成纤维细胞(DF-1)的抑制作用,通过预防感染、影响复制、阻止吸附及直接杀灭4种方式作用后检测AETAC对DF-1细胞的毒性,选择体外最佳抗AIV-H9N2作用方式。尿囊腔接种不同浓度AETAC确定其对鸡胚的最大安全浓度;将AETAC以3种不同给药方式作用于AIV-H9N2感染后鸡胚,选择体内最佳给药方式。经尿囊腔同时接种AETAC和AIV-H9N2,统计各组鸡胚存活数,测定血凝效价,并观察胚胎的发育情况和鸡胚法氏囊病理组织变化。[结果]在一定范围内,随着AETAC浓度升高或作用时间延长,AETAC对DF-1细胞的抑制率升高。与空白对照组相比,4种抗AIV-H9N2作用方式下,病毒对照组和药物组细胞存活率均显著降低(P<0.05);与病毒对照组相比,AETAC浓度为5~30μg/mL时,4种抗AIV-H9N2作用方式下,药物组细胞存活率均显著升高(P<0.05)。4种作用方式下,DF-1细胞存活率分别为8.15%~64.96%(预防感染)、8.56%~83.83%(影响复制)、1.82%~5.90%(阻止吸附)和6.90%~40.12%(直接杀灭)。AETAC对鸡胚的最大安全浓度为16.41 mg/mL,对感染鸡胚的最佳给药方式为AETAC和病毒混合后接种。以最佳体内作用方式给药后,AETAC中、低浓度组鸡胚存活率为40%~60%,高浓度组鸡胚全部存活,胚胎喙部发育明显,鸡胚法氏囊淋巴滤泡发育完整,滤泡大小和数量正常,充血量少,组织病理状态改善。[结论]AETAC在体内外对AIV-H9N2均具有显著的抑制作用,且主要与影响病毒复制作用方式相关。

[Objective]The objective of this study was to investigate the activity and mechanism of ethyl acetate extract from Alternanthera philoxeroides(AETAC)against H9N2 subtype Avian influenza(AIV-H9N2)virus in vitro and in vivo.[Method]The inhibitory effect of AETAC at different concentrations on chicken embryo fibroblasts(DF-1)at different time was detected by CCK-8 method.The toxicity of AETAC to DF-1 cells was detected by preventing infection,influence replication,preventing adsorption and directly kill,and the best anti-AIV-H9N2 mode was selected in vitro.The maximum safe concentration of AETAC was determined by inoculation of different concentrations in allantoic cavity.AETAC was administered to AIV-H9N2 infected chicken embryo by three different administration methods,and the best administration method was selected in vivo.AETAC and AIV-H9N2 were simultaneously inoculated through allantoic cavity,the survival number of chicken embryos in each group was counted,the hemagglutination titer was determined,and the development of embryo and pathological changes of the bursa of Fabricius of chicken embryos were observed.[Result]Within a certain range,as the concentration of AETAC increases or the duration of action prolongs,the inhibitory rate of AETAC on DF-1 cells increased.Compared with blank control group,the cell survival rate of virus control and drug groups was significantly decreased under four anti-AIV-H9N2 modes(P<0.05).Compared with virus control group,the cell survival rate of the four anti-AIV-H9N2 modes was significantly increased when AETAC concentration was 5-30μg/mL(P<0.05).The survival rates of DF-1 cells were 8.15%-64.96%(prevent infection),8.56%-83.83%(influence replication),1.82%-5.90%(prevent adsorption)and 6.90%-40.12%(directly kill),respectively.The maximum safe concentration of AETAC for chicken embryos was 16.41 mg/mL,and the best administration method for infected chicken embryos was mixed AETAC and virus inoculation.After the optimal in vivo administration,the survival rate of embryos in medium and low AETAC concentration groups was 40%-60%and those in high concentration group were all alive.In high concentration group,the beak development of embryos was obvious,the lymphoid follicles of the bursa of Fabricius of chicken embryos were intact,the size and number of follicles were normal,the amount of congestion was less,and the histopathological status was improved.[Conclusion]AETAC had significant inhibitory effect on AIV-H9N2 both in vivo and in vitro,and was mainly related to affecting the mode of viral replication.