复方中药对禽流感病毒的防治研究

Study of the Compound Chinese Medicine Against the Anti-avian Influenza Virus

【目的】探讨自行研制的由贯众、板蓝根、黄芪、柴胡、连翘、黄连、金银花、黄芩及甘草配制的复方中药在体内、体外对禽流感病毒的抑制作用、药物代谢动力学、急性毒性试验及对SPF鸡免疫功能的影响。【方法】在鸡胚、MDCK细胞上进行复方中草药对H5N1和H9N2亚型禽流感病毒的直接灭活作用、治疗作用和预防作用;在体内通过药物累计法研究复方中药在昆明鼠体内的药物代谢动力学;采取口服和腹腔注射两种给药途径研究复方中药对昆明鼠的急性毒性试验;通过免疫器官的指数、在FAC上测量CD3+、CD4+、CD8+及TCR+的含量及采用血凝抑制(HI)方法检测血清中H9亚型禽流感抗体滴度研究复方中草药的对SPF鸡免疫功能的影响;通过体外试验研究其对H5N1禽流感病毒的治疗作用。【结果】该复方药物在鸡胚内对H5N1亚型禽流感病毒的有效灭活浓度、治疗浓度及预防浓度分别是31.25mg·ml-1、125mg·ml-1及250mg·ml-1,对H9N2亚型禽流感病毒的有效灭活浓度、治疗浓度及预防浓度分别是31.25mg·ml-1、15.625mg·ml-1及62.5mg·ml-1;在MDCK上对H5N1亚型禽流感病毒的有效灭活浓度及预防浓度分别是31.25mg·ml-1、125mg·ml-1,治疗浓度未测出。对H9N2亚型禽流感病毒的有效灭活浓度、治疗浓度及预防浓度分别是15.625mg·ml-1、31.25mg·ml-1及31.25mg·ml-1;其在小白鼠体内的经时过程为一室模型。其药-时方程式为:C=650.66e-4.9078t;口服复方中药后对昆明小鼠无毒;腹腔注射复方中药后对昆明小鼠的半数致死量(LD50)为425.6mg·kg-1;给药后21d,CD3+、CD4+、CD8+及TCR+的含量分别为64.01%、27.39%、45.05%和32.19%;免疫后半个月,给药-免疫组的禽流感抗体滴度比免疫组高1.6个滴度;同群感染组中,该复方中药预防组的保护率达30%,而治疗组为20%;【结论】体外试验表明,复方中药不仅对禽流感病毒有直接灭活作用,而且对吸附于细胞表面和进入细胞内的禽流感病毒都有抑制作用;其药物代谢动力学特征是吸收快、消除慢;该复方中药不仅安全、而且能增强鸡的体液免疫、细胞免疫功能、增加免疫器官指数。体内试验表明,该复方中药能延长鸡的平均死亡时间,预防效果强于治疗效果。总之,自行研制的复方中药具有预防、安全、延长感染鸡的存活时间、增强机体免疫力的作用。

[Objective] To research the inhibiting effects of compound Chinese medicine against the avian influenza virus in vivo and in vitro, its pharmacokinetics and acute toxicity test, its effects on immune function of SPF chicken. [Method] The effects of direct inactivation, preventive and therapeutic effects of compound Chinese medicine on the anti-avian influenza virus were studied on chicken embryo and MDCK cell. Its pharmacokinetics were studied by drug residual accumulation. The acute toxicity test of the compound Chinese medicine was studied by oral administration and intraperitoneal injection on Kunming mice. The effects of the compound Chinese medicine on immune function of SPF chicken was studied by means of the indexes of immune organs in chickens, the contents of CD3, CD4, CD8 and γδTCR by FAC and antibody titer of avian influenza virus detected in HI method. [Result] The effective inactivate concentration, therapeutic concentration and preventive concentration of the compound Chinese medicine against the H5NI and H9N2 avian influenza virus was 31.25 mg·kg^-1, 125 mg·kg^-1and 250 mg·kg^-1, 31.25 mg·kg^-1, 15.625 mg·kg^-1 and 62.5 mg·kg^-1 on chicken embryo, respectively. The effective inactivate concentration, preventive concentration of the compound Chinese medicine against the H5NI avian influenza virus was 31.25 mg·kg^-1, 125 mg·kg^-1, but the therapeutic concentration was not detected, and 15.625 mg·kg^-1, 31.25 mg·kg^-1 and 31.25 mg·kg^-1 against H9N2 on the MDCK, respectively. The concentration-time curves fitted the one compartment model and its equation was C=650.66e^-4.9078t. The study of acute toxic experiments on mouse was as follows： After oral administration the compound medicine was safe. After intraperitoneal injection administration, the LDso was 425.6mg·kg^-1bw. The contents of CD3, CD4, CD8 and γδTCR was 64.01%, 27.39%, 45.05% and 32.19% after 21 days, respectively. The antibody titre of administration-immunity group was 1.6 titre higher than that of immunity group. The protective rate of preventive group was 30%, and 20% in the therapeutic group.[ Conclusion ]The compound medicine has a strong action of direct inactivation, preventive and therapeutic effects on the anti-avian influenza virus in vitro. Furthermore, it inhibited avian influenza virus adsorbed in cell surface and entered into intracellular. The pharmacokinetics parameter showed that its elimination was slow. It had a long time and it had long effect in the mouse. It enhanced cellular and humoral immune function and the indexes of immune organs in chickens. It could prolong the mean death time of chicken and took effect on the chicken challenged the subtype H5N1 of highly avian influenza virus in vivo. The compound Chinese medicine is preventive and safe, prolongs the mean death time of chicken and strengthens the immunity of chickens.