【作者】 张华；

【导师】 李宏全；

【作者基本信息】 山西农业大学 ， 兽医博士（专业学位）， 2021， 博士

【摘要】 猪繁殖与呼吸综合征(Porcine Reproductive and Respiratory Syndrome,PRRS)是持续威胁我国养猪业的重要猪病毒性疾病之一,目前多数市售疫苗无法对猪繁殖与呼吸综合征病毒(Porcine Reproductive and Respiratory Syndrome virus,PRRSV)高遗传多样性的毒株提供完全的保护作用。中药及天然药物活性成分是创新药物研发的重要源泉。但由于缺乏重大理论和核心技术突破,“成分不清、机制不明”成为了制约中兽药发展的“卡脖子”问题。组分中药因其物质基础和作用机理相对清楚,为中兽药现代化研究提供了新的研发策略。本实验室前期筛选出数十种具有抗病毒、抗炎和抗氧化等药理作用的中药单体化合物,本研究在此基础上以PRRSV感染猪肺泡巨噬细胞(porcine alveolar macrophage,PAM)为模型,对13种中药单体化合物进行抗PRRSV作用筛选,将筛选出抗PRRSV作用最优的3个单体化合物分别进行蛋白质组学分析,解析其潜在的作用靶点;结合蛋白质组学结果,以组分组方的思路,按照正交设计,进行组分配伍,筛选最优组分配比。建立PRRSV感染断奶仔猪模型,进行组分复方的体内靶动物安全性和抗PRRSV作用评价,为临床防控PRRSV提供新的策略,为创制新型中兽药提供新的研究范式,推动中兽药现代化的发展。试验结果:1.通过MTT法获得芹菜素、黄芩苷、咖啡酸、千金藤素、姜黄素、莪术醇、绿原酸、甘草酸、苦参碱、蛇床子素、丹皮酚、紫丁香苷、茶皂素共13种中药单体化合物在PAM上的最大安全浓度。以此为起始浓度,二倍倍比稀释三个浓度梯度,q PCR和western blot分别检测它们对PRRSV N基因/蛋白的影响。筛选出了苦参碱、甘草酸和茶皂素呈剂量依赖性抑制PRRSV的复制。2.利用TMT相对定量蛋白质组学技术分别检测PRRSV、苦参碱、甘草酸和茶皂素作用于PAM后差异蛋白质的表达。结果显示:与空白组相比,甘草酸、苦参碱、茶皂素可共同升高14个相同蛋白质的表达,降低23个相同蛋白质的表达;与PRRSV组相比,甘草酸、苦参碱、茶皂素可共同升高5个相同蛋白质的表达,降低36个相同蛋白质的表达。苦参碱、甘草酸和茶皂素可共同抑制28个PRRSV感染上调的蛋白质,共同上调2个PRRSV感染下调的蛋白质。通过对28个差异表达蛋白质的信息进行分析,显著变化排序前4的蛋白质相对应的基因名称分别是CCL8、IFIT3、IFIH1、ISG15。q PCR检测IFIT3、IFIH1、ISG15和IFN-βm RNA的相对表达,结果与蛋白质组学的结果一致。提示,在PRRSV感染PAM 12 h时,可以激活IFN-β通路,而苦参碱、甘草酸和茶皂素均能促进细胞的抗病毒反应。甘草酸组和茶皂素组的差异表达蛋白质的GO功能类别和富集的KEGG通路比较类似,苦参碱组与之相差较大。3.将苦参碱、甘草酸和茶皂素的3个有效浓度进行正交设计,通过MTT法获得4个安全组方并进行抗PRRSV作用评价,结果显示甘草酸、苦参碱和茶皂素具有协同抗PRRSV的作用;将上述有效组方中每个药物的最高剂量进行逐一配伍,q PCR和western blot分别检测其对PRRSV N基因/蛋白的影响,并综合考虑甘草酸、苦参碱和茶皂素的经济因素,最后确定苦甘茶组分复方的最佳浓度比为0.4 mg/m L苦参碱+0.25 mg/m L甘草酸+1.95μg/m L茶皂素。4.体外溶血试验证实0.4 mg/m L苦参碱+0.25 mg/m L甘草酸+1.95μg/m L茶皂素配伍使用无溶血特性。分别在小鼠模型和仔猪模型进行体增重、体温、血细胞、血液生化指标和病理组织学检测,证实10 mg/kg苦参碱+6 mg/kg甘草酸+50μg/kg茶皂素可用于后续抗PRRSV体内活性评价。5.通过颈部肌肉注射和滴鼻成功建立PRRSV感染仔猪模型,q PCR检测不同脏器中PRRSV N基因的表达,结果显示,肺泡灌洗液中PRRSV N基因的表达量最高。因此,选择肺泡灌洗液进行后续抗病毒作用评价。6.与PRRSV感染组相比,高中低剂量的苦参碱、甘草酸和茶皂素组分复方均能显著降低仔猪肺泡灌洗液中的病毒载量(p<0.05),并且改善PRRSV诱导的间质性肺炎和体温升高的症状。与正常对照组相比,PRRSV感染对仔猪血清和肺泡灌洗液中IFN-β的表达没有显著影响(p>0.05);与PRRSV感染组相比,低剂量的苦参碱、甘草酸和茶皂素组分复方显著升高IFN-β的表达(p<0.05),高中剂量的苦参碱、甘草酸和茶皂素组分复方及阳性药物替米考星对IFN-β的表达没有显著影响(p>0.05)。本研究证实:(1)由苦参碱、甘草酸和茶皂素复配组成的组分复方具有抗PRRSV作用,其在PAM上的剂量为0.4 mg/m L苦参碱+0.25 mg/m L甘草酸+1.95μg/m L茶皂素,在仔猪上的最优剂量为10 mg/kg苦参碱+6 mg/kg甘草酸+50μg/kg。(2)苦甘茶组分复方的组方依据是甘草酸和茶皂素针对同一靶点具有叠加作用,复配苦参碱针对不同靶点的协同作用。(3)IFN-β参与了苦甘茶组分复方发挥抗病毒的作用。更多还原

【Abstract】 Porcine Reproductive and Respiratory Syndrome(PRRS)is one of the most important porcine viral diseases that continue to threaten the pig industry in China.At present,most commercial vaccines failed to provide complete protection because of the highly genetic diversity of PRRSV strains.Traditional Chinese medicine and natural compounds are important sources for the development and research of innovative drugs.However,due to the lack of major theoretical and core technological breakthroughs,“complex compositions and uncleared mechanisms” are the stranglehold problems which restricts the development of traditional Chinese veterinary medicines.Based on the relatively clear material and mechanism of action,component-based Chinese medicine provides a new R & D strategy for the modernization of traditional Chinese veterinary medicine.Our previous studies have screened dozens of monomer compounds which possessed antiviral,anti-inflammatory and antioxidant effects.In this study,13 natural compounds were used to investigate the anti-PRRSV activity in porcine alveolar macrophages(PAM).Three compounds with the best anti-PRRSV effects were selected for proteomic analysis to analyze their potential targets.Combined with the results of proteomics,orthogonal design was carried to screen the optimal proportion and to check the component compatibility.PRRSV infected weaned piglet model was established to evaluate the safety and effectiveness of the component formula.This study provided new strategies for clinical prevention and control of PRRSV as well as a new research paradigm for the development of novel traditional Chinese veterinary medicine to promote the development of modernization of traditional Chinese veterinary medicine.MTT method was used to check the maximum safe concentrations of apigenin,baicalin,caffeic acid,cepharanthine,curcumin,curcumol,chlorogenic acid,glycyrrhizic acid,matrine,osthol,paeonol,syringin and tea saponin on PAM.QPCR and western blot were used to investigate the PRRSV N gene and protein expression,respectively.The results showed that matrine,glycyrrhizic acid and tea saponin inhibited PRRSV replication in a dose-dependent manner.The TMT technique of relative quantitative proteomics was used to detect the differential protein expression of PAM infected with PRRSV,matrine,glycyrrhizic acid and tea saponin.Compared with the normal control,the results showed that glycyrrhizic acid,matrine and tea saponin increased and decreased the expression of 14 and 23 identical proteins,respectively.Compared with PRRSV group,glycyrrhizic acid,matrine and tea saponin increased and decreased the expression of 5 and 36 identical proteins,respectively.Matrine,glycyrrhizic acid and tea saponin inhibited 28 PRRSV up-regulated proteins and induced 2 PRRSV down regulated proteins.By analyzing the information of 28 differentially expressed proteins,CCL8,IFIT3,IFIH1 and ISG15 were the top four proteins with significant changes.The relative expression of IFIT3,IFIH1,ISG15 and IFN-β m RNAs were consistent with the results of proteomics.These results suggested that PRRSV infection activated the IFN-β pathway and matrine,glycyrrhizic acid and tea saponin promoted the antiviral response.Based on the GO enrichment analyses and KEGG pathway enrichment analyses,the differentially expressed target proteins were similar for glycyrrhizic acid and tea saponin treatments,while was different for matrine treatment.The three effective concentrations of matrine,glycyrrhizic acid and tea saponin were used for orthogonal design.Four safe prescriptions were screened by MTT method and their anti-PRRSV effect were evaluated.The results indicated that glycyrrhizic acid,matrine and tea saponin showed synergistic anti-PRRSV effect.The highest dose of each compound in the above effective prescriptions was mixed one by one,and the anti-PRRSV effect was detected by q PCR and Western blot.Considering the economic factors of glycyrrhizic acid,matrine and tea saponin,0.4 mg/m L matrine + 0.25 mg/m L glycyrrhizic acid+ 1.95 μg/m L tea saponin were the optimal concentration ratio in the component formula.This optimal concentration showed no hemolytic activity using in vitro hemolysis test.Body weight gain,body temperature,blood cells,blood biochemical indexes and histopathology were detected both in mouse and piglet models to confirm that 10 mg/kg matrine + 6 mg/kg glycyrrhizic acid + 50 μg/kg tea saponin could be used for subsequent evaluation of anti-PRRSV activity in vivo.PRRSV infected piglet model was established by intramuscular injection and nasal drip.The expression of PRRSV N gene in different organs was detected by q PCR and the results showed that expression of PRRSV N gene was the highest in BALF which was chosen to evaluate the antiviral effect of BALF.Compared with the PRRSV infected group,the high,middle and low dose of component formula significantly reduced the viral load in the bronchoalveolar lavage fluid of piglets(P <0.05)and improve the interstitial pneumonia and body temperature rise induced by PRRSV.Compared with the normal control group,PRRSV infection had no significant effect on the expression of IFN-β in serum and bronchoalveolar lavage fluid(P > 0.05);Compared with PRRSV infection group,low dose of component formula significantly increased the expression of IFN-β(P < 0.05),while the high and middle dose of component formula and timicosin had no significant effect on the expression of IFN-β(P > 0.05).In conclusion,the component formula(matrine,glycyrrhizic acid and tea saponin)possessed anti-PRRSV activity both in vitro and in vivo.The optimal dosage on PAM was 0.4mg/m L matrine + 0.25 mg/m L glycyrrhizic acid + 1.95 μg/m L tea saponin and in piglet was10 mg/kg matrine + 6 mg/kg glycyrrhizic acid + 50 μg/kg tea saponin.The compatibility of the formula was the superposition of the same target with glycyrrhizic acid and tea saponin,while different targets of matrine.IFN-β may be one of the targets of the component formula possessed anti-PRRSV activity.更多还原