摘 要

非洲猪瘟是由非洲猪瘟病毒引起的一种急性、烈性、高度接触性传染病，严重危害着全球养猪业。中医药不管在治疗人类病毒性疫病还是畜禽类病毒性疫病中，均发挥着举足轻重的作用，因此，开发能够治疗非洲猪瘟的中药制剂具有重要的现实意义和研究价值。本试验分为三部分，首先对几种单味中药以及中药复方是否具有抗 ASFV 作用进行初步筛选，找出抗 ASFV 效果最好的中药或中药复方；随后对筛选出的中药或者中药复方进行深入研究，探究不同中药浓度以及不同的给药方式对抗 ASFV 效果的影响；最后针对中药或者中药复方发挥的抗 ASFV 作用是否与 cGAS-STING 信号通路的激活产生 I 型 IFN 发挥抗病毒功能有关，作了简单的探讨。本实验共涉及四种中药复方以及两种单味中药，分别以复方 1，复方 2，复方 3，复方 4，连翘和马勃进行表示。

试验一 抗 ASFV 中药的筛选

首先测定了六种类型中药对 PAM 细胞的最大安全浓度范围，挑选出对细胞影响最小中药浓度进行后续实验。接着设置病毒对照组和中药组对细胞内中产生的 ASFV-p72 基因进行检测，找出能够抑制该基因表达效果最佳的中药。

中药最大安全浓度试验结果显示，复方 1 最大安全浓度范围为：7.8~15.6 mg/mL；复方2 最大安全浓度范围为：7.8~15.6 mg/mL；复方 3 最大安全浓度范围为：0.122~0.244 mg/mL；复方 4 最大安全浓度范围为：7.8~15.6 mg/mL；连翘最大安全浓度范围为：0.98~1.95 mg/mL；马勃最大安全浓度范围为：7.8~15.6 mg/mL。ASFV-p72基因检测结果显示，中药复方 1，复方 2，复方 3，复方 4和连翘对 ASFV 毒力大小为 103TCID50 时，对 ASFV-p72 基因表达量的降低效果优于 ASFV 毒力大小为 102TCID50。马勃对 ASFV 毒力大小为 102TCID50 时，对 ASFV-p72 基因表达量的降低效果优于 ASFV 毒力大小为 103TCID50。此外，在 ASFV 毒力大小为 103TCID50，观察中药复方 1，复方 2，复方 3，复方 4，连翘和马勃对 ASFV-p72 基因表达量的降低效果，以病毒对照组相比，发现复方 1和复方 2具有较好的抑制效果（P＜0.01）。结论：中药复方 1和复方 2可以显著降低 ASFV-p72 基因表达。后续实验主要对复方 1产生的抗病毒机制进行深入研究。

实验二 中药复方对猪 cGAS-STING 信号通路的影响

根据试验一的结果，将 PAM 细胞分为空白组，低剂量中药组（3.9 mg/mL），高剂量中药组（7.8 mg/mL）。通过 Western Blot 检测细胞中 IFNα、IFNβ 和 cGAS 蛋白的表达量；ELISA 法检测细胞上清液中 STING、TBK1、IRF3 和 IFITM3 蛋白的表达量。

结果显示，中药复方激活了 c GAS-STING-TBK1-IRF3-IFNβ 这一信号通路，产生 IFITM3，这些可以作为抗病毒储备力量，对 ASFV 感染可以起到良好的预防作用。

试验三 中药复方抗 ASFV 效果实验

根据试验一的结果，不同中药浓度抗 ASFV 效果实验中，将 PAM 细胞分为 CON 组，MOD 组，五个不同浓度梯度中药组。不同给药方式抗 ASFV 效果实验中，将 PAM 细胞分为 CON 组，MOD 组，七种不同给药方式中药组。

试验结果显示，不同中药浓度抗 ASFV 效果实验中，与模型组相比，发现中药复方浓度为7.8 mg/mL时细胞活力最高（P＜0.05），并存在剂量依赖性。不同给药方式抗 ASFV 效果实验中，与模型组相比，发现攻毒后 2 h 给药这种给药方式，细胞活力最高（P＜0.01）。结论：中药复方能够增强 AFSV 导致的 PAM 细胞活力降低，并存在浓度依赖性。发现攻毒后 2 h 给药具有较好的抗病毒效果。

试验四 中药复方抗 ASFV 机制的探讨

根据试验三的结果，将 PAM 细胞分为 CON 组，MOD 组，低剂量中药组，中剂量中药组，高剂量中药组。通过 Western Blot 检测细胞中 IFNα、IFNβ 和 cGAS 蛋白的表达量；ELISA 法检测细胞上清液中 STING、TBK1、IRF3和 IFITM3 蛋白的表达量。

试验结果显示，与模型组相比，中药组增加了细胞内 IFNα、IFNβ和cGAS 蛋白表达（P＜0.05），增加了细胞培养液内 STING、TBK1、IRF3 和 IFITM3 蛋白的表达（P＜0.05）。结论：中药复方发挥抗 ASFV 作用可能与 cGAS-STING-TBK1-IRF3-IFNβ-IFITM3 信号通路的激活有关。

本试验结果表明，中药复方可以增强 PAM 细胞活力，降低 ASFV-p72 基因表达，浓度依赖性发挥抗病毒效果，并且中药复方可能通过激活 PAM 细胞内 cGAS-STING 信号通路产生 I 型 IFN，表达抗病毒蛋白 IFITM3 干扰 ASFV 的复制过程。

关键词：非洲猪瘟；cGAS-STING 信号通路；中药复方

Abstract

African Swine Fever (ASF) is an acute,virulent and highly contagious disease caused by ASF virus, which seriously harms the global pig industry. Traditional Chinese medicine (TCM) plays an important role in the treatment of both human viral diseases and livestock and poultry viral diseases. Therefore, it is of great practical significance and research valueto develop TCM preparations for the treatment of African Swine Fever. This experiment is divided into three parts.Firstly, the anti-ASFV effect of several single Chinese medicine and Chinese medicine compound is preliminarily screened to find out the best anti-ASFV effect of Chinesemedicine or Chinese medicine compound; Then,in-depth study was conducted on the screenedChinese medicine or Chinese medicine compound to explore the effects of different concentrations of Chinese medicine and different ways of administration against ASFV. Finally, a simple discussion was made on whether the anti-ASFV effect of traditionalChinese medicine or traditional Chinese medicine compound was related to the antiviral function of type I IFN produced by the activation of cGAS-STING signaling pathway. This experiment involved a total of four TCM compounds and two single TCM compounds, which were respectively represented by Compound 1, Compound 2, Compound 3, Compound 4, Forsythia and Mabu.

Experiment 1:Screening of traditional Chinese medicine against ASFV

Firstly, the maximum safe concentration range of six types of Chinesemedicine on PAM cells was determined,and the minimum concentration of Chinese medicine on cells was selected for subsequent experiments. Then, the virus control group and the Chinese medicine group were setto detect the ASFV-P72 gene produced in cells,to find out the best Chinese medicine which could inhibit the expression of the gene.

The results showed that the maximum safe concentration of compound 1 was 7.8~15.6 mg/mL.The maximum safe concentration range of compound 2 was 7.8~15.6 mg/mL.The maximum safe concentration range of compound 3 was 0.122~0.244 mg/mL.The maximum safe concentration range of compound 4 was 7.8~15.6 mg/mL.The maximum safe concentration range of forsythia is 0.98~1.95 mg/mL.The maximum safe concentration range of MAB is 7.8~15.6 mg/mL.The results of ASFV-p72 gene test showed that when the virulence of Chinese herbalcompounds 1, 2, 3, 4 and forsythia on ASFV was 103TCID50,the effect of reducing ASFV-p72 gene expression was better than that of the virulence of ASFV was 102TCID50. When the virulence of MAB to ASFV was 102TCID50,the decrease of ASFV-p72 gene expression was better than that when the virulence of MAB to ASFV was 103TCID50. In addition, when the virulence of ASFV was 103TCID50, we observed the effect of compound 1,compound 2, compound 3, compound 4, forsythia and Mabu on reducing the expression of ASFV-p72 gene. Compared with the virus control group,compound 1 and compound 2 showed better inhibition effect (P < 0.01).Conclusion: Chinese medicine compound 1 and compound 2can significantly reduce the expression of ASFV-p72 gene. Subsequent experiments mainly studied the antiviral mechanism produced by compound 1.

Experiment 2: Effect of Chinese herbal compound on cGAS-STING signaling pathway in pigs