The Component Formula Of Traditional Chinese Medicine Inhibited PRRSV Infection And Its Mechanisms

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.