Study On The Anti-PRRSV JXA1 Proliferation And Mechanism Of Radix Isatidis Polysaccharide In Vitro

Porcine reproductive and respiratory syndrome is a disease caused by porcine reproductive and respiratory syndrome virus(PRRSV)that seriously endangers the swine industry.Since the virus was first discovered in the western United States in the late 1980s,many positive case of PRRSVwere reportted in European countries almost at the same time,and it has been spreading to many countries on all continents with global trade.Vaccination with attenuated or inactivated vaccines are the mainstream measures to prevent the disease.in the present.However,due to the poor immunity of inactivated vaccines and the risk of spreading virus of the attenuated vaccines,vaccination is not enough to control this disease.Finding another way may have far-reaching significance for preventing the disease.Traditional Chinese medicine has a long history of anti-virus,and it has far-reaching significance for the inheritance of Chinese civilization for thousands of years.Since porcine alveolar macrophages are the natural target cells of PRRSV,it is more practical to select these cells to study the mechanism of anti-PRRSV action of Radix Isatidis polysaccharide in vitro.However,the production cost of primary porcine PAM cells is high and difficult to obtain,and it is easy to cause a lot of waste to study the dynamic expression of TLRs and cytokines.Therefore,it is more appropriate to use the immortalized porcine alveolar macrophages 3D4/21/CD163 successfully transfected and stably expressing the CD 163 receptor as the research object for this experimen.In this paper,antiviral effect of Radix Isatidis polysaccharide(IRPS)extracted from Radix Isatidis was tested in vitro,IRPS acts on PRRSV JXA1-infected 3D4/21/CD163 cells with the optimal concentration and mode of action screened by antiviral tests to detect the effects of drugs on the expression of TLRs and their downstream related cytokines,Finally,according to the above experimental results,the effect of TLRs on IRPS inhibiting the proliferation of PRRSV JXA1 in vitro was verified.The trial was divided into four parts,described below:1 Anti-PRRSV JXA1 effect of Radix isatidis polysaccharide(IRPS)in vitroIn this study,immortalized cells of porcine alveolar macrophages(3D4/21/CD163)stably expressing CD 163 were used as target cells to study the optimal drug concentration,administration method and inhibition of replication step of IRPS against PRRSV JXA1 infection.The administration methods were divided into prevention group,direct killing group and treatment group to study the optimal concentration and administration method of IRPS,according to the order of IRPS and PRRSV JXA1 acting on cells.The inhibition of IRPS on replication step of PRRSV JXA1 were explored from adsorption,invasion and intracellular proliferation.The final anti-virus effect of IRPS was verified by fluorescence quantitative PCR and indirect immunofluorescence in cells with the optimal drug concentration,mode of administration and inhibition of virus replication.The results showed that:The optimal dose of IRPS to inhibit the proliferation of PRRSV-JXA1 was 0.015625 mg/mL.The optimal administration method and optimal blocking link were the preventive effect and adsorption link,reduced viral load by 91.34%(P<0.0001)and 88.83%(P<0.0001),respectively.0.015625 mg/mL IRPS acted on the adsorption step of the virus in a preventive way and finally reduced the viral load by 94.97%.2 Effects of IRPS on the transcription and translation levels of TLRs in 3D4/21/CD163 cells infected with PRRSV JXA1The experiment was divided into cell control group,virus control group and administration group,with the optimal doses in preventive mode to anti-adsorption.Fluorescence quantitative PCR and Western blotting were used to detect the transfection and expression levels of TLRs at 6 h,12 h,18 h,24 h,30 h,36 h,and 48 h after virus infection,respectively.The results showed that PRRSV JXA1 promoted the transcription of TLR3,TLR4,TLR7,TLR9 at 3,4,0,and 5 time points,respectively,and showed inhibitory effect at other time points,From the overall results,IRPS bidirectionally regulates the transcription of TLRs;The results of Western blotting combined with protein grayscale analysis showed that IRPS had an overall up-regulation effect on the translation of TLR3,TLR4 and TLR9.Conclusion:IRPS bidirectionally regulates TLRs mRNA transcription level and promotes TLRs protein translation level.3 Effects of IRPS on cytokine secretion by 3D4/21/CD163 cells infected with PRRSV JXA1The experiment was divided into cell control group,drug administration group and virus control group.0.015625 mg/mL IRPS acting on the adsorption step of PRRSV JXA1 to target cells in a preventive manner,cells were collected at the 6 h,12 h,18 h,24 h,30 h,36 h,and 48 h after treatment,and the transcription level of cytokines was detected by relative fluorescence quantitative PCR;the supernatant was collected,and ELISA kits were used to detect the protein translation of cytokines at different time points.The results showed that the drug group exhibited different degrees of bidirectional regulation in 27 of the 28 time points of the four inflammatory factors(IL-1β,IL-6,IL-8 and TNF-α)detected;18 out of 21 time points of the three interferons(IFN-α,IFN-β and IFN-γ)showed bidirectional regulation;The immunosuppressive factor(IL-10)showed bidirectional regulation at all time points.IRPS promotes the expression of antiviral factor(IFN-α,IFN-β,IFN-γ,TNF-α),inhibits the overexpression of some inflammatory factors(IL-1β,IL-8),and promotes immunosuppressive factors(IL-10)expression in the early stage inhibits the expression in the later stage.Overall,IRPS has a significant bidirectional regulation of cytokines produced by 3D4/21/CD163 cells infected with PRRSV JXA1.4 The function of TLR4 and CD163 in IRPS anti-PRRSV JXA1 processIn this experiment CCK8 was used to detect the maximum safe concentration of TLR4 inhibitor TAK-242;Western blotting was used to detect the protein expression level of NF-κB,a key downstream molecule of TLR4,to determine the optimal concentration of TAK-242 to inhibit the TLR4/NF-κB pathway;and the effect of IRPS on the viral copy of PRRSV JXA1 was detected by fluorescence quantitative PCR when TLR4/NF-κB pathway was blocked with the optimal concentration of TAK-242;The effect of IRPS on CD163 protein expression was detected by Western blotting.The grayscale results of NF-κB protein showed that 40 μmol/L of TAK-242 could effectively block the activity of TLR4/NF-κB pathway without causing toxicity to cells.When the TLR4/NF-κB pathway signaling pathway was blocked,the copy number of PRRSV was significantly increased(P<0.001),indicating that the cell resists PRRSV infection through this pathway.When the TLR4 signaling pathway is not inhibited,IRPS has a great anti-PRRSV effect(P<0.0001),but when the TLR4/NF-κB signaling pathway is blocked,IRPS not only loses its anti-PRRSV effect,but promotes the proliferation of PRRSV,which indicates that This pathway is also the key pathway for IRPS to resist PRRSV proliferation in vitro.The detection results of CD 163 protein translation level showed that IRPS promoted CD 163 protein translation comparing with the virus control,which may be one of the reasons why IRPS promoted PRRSV replication when TLRs were blocked.