The Investigation Regarding The Anti-PRRSV Proliferation Mechanism Of Irps In Vitro Based On TLR4 Signaling Pathway

Porcine reproductive and respiratory syndrome(PRRS)is a highly contagious disease caused by porcine reproductive and respiratory syndrome virus(PRRSV).The disease mainly causes abortion of sows in the middle and late stages of pregnancy,increased body temperature and dyspnea of weaned piglets.The mortality rate of weaned piglets is high,which can reach 100 %.Since PRRSV was introduced into China,it has caused huge economic losses to the pig industry.PRRSV infection can activate the innate immune response,induce the expression of pattern recognition receptors and their downstream signaling molecules,and then cause a large number of inflammatory cytokines such as IL-1β,IL-6,and TNF-α secretion,and may even cause “cytokine storm”,causing severe interstitial pneumonia and viremia.Radix Isatidis Polysaccharide(IRPS)is the most abundant active substance in Radix Isatidis,which has anti-inflammatory,antibacterial,antiviral,immuneenhancing and antioxidant effects.Previous laboratory studies have found that IRPS can inhibit the proliferation of PRRSV in vitro,while IRPS was found to reduce the secretion of inflammatory cytokines after PRRSV infection of 3D4/21/CD163 cells.At present,the main polysaccharide receptors found are TLR2 and TLR4.TLR4 is a pattern recognition receptor mainly distributed on the surface of the cell membrane and can induce the secretion of inflammatory cytokines.Therefore,we speculate that IRPS can affect the secretion of inflammatory cytokines through the TLR4 signaling pathway and play an indirect antiviral role.1.IRPS binding to 3D4/21/CD163 cellsAccording to the test requirements,IRPS was extracted by water extraction and alcohol precipitation,and the polysaccharide content and protein content of the extract were determined.IRPS was fluorescently labeled with tyramine reduction method to obtain the labeled product IRPS-FITC.The maximum safe concentration was detected by MTT method,and it was found that the maximum safe concentration after labeling was consistent with that before labeling,both 0.0625 mg/m L.After combining IRPSFITC with 3D4/21/CD163 cells,it was found that IRPS could bind to 3D4/21/CD163 cells and interact with TLR4 receptors on the surface,which provided a theoretical basis for subsequent experiments.2.The effect of IRPS on TLR4 and its downstream signaling molecules in 3D4/21/CD163 cells infected with PRRSVTo investigate the effect of IRPS on TLR4 and its downstream signaling molecules in PRRSV-infected 3D4/21/CD163 cells,we used fluorescence quantitative PCR and western blot assays to detect the transcription and protein expression levels of signaling molecules such as TLR4,My D88,NF-κB,TRIF,and IRF3.The results showed that IRPS could reduce the increase of TLR4 transcriptional and protein levels in 3D4/21/CD163 cells infected with PRRSV.Examination of transcriptional and protein expression levels of downstream signaling molecules such as My D88,TRIF,TRAF6,NF-κB,and IRF3 revealed that IRPS was able to reduce the phenomenon of increased protein expression levels of signaling molecules such as My D88,TRIF,TRAF6,NF-κB,p38 MAPK,and IRF3 after PRRSV infection of3D4/21/CD163 cells at 6 h and 12 h,similar to TLR4 protein expression.These results indicated that IRPS can indirectly affect the activation of My D88-dependent signaling pathway and TRIF-dependent signaling pathway by inhibiting the secretion of TLR4 at 6 h and 12 h,and inhibit the expression of its downstream signaling molecules.3.The effect of IRPS on the secretion of inflammatory cytokines and virus proliferation in PRRSV infection in vitro when TLR4 signaling pathway was blocked.In Experiment 2,we detected the m RNA and protein expression of TLR4 and its downstream signaling molecules,and found that IRPS could reduce the secretion of TLR4 and its downstream signaling molecules after PRRSV infection.Therefore,we conclude that IRPS can influence cytokine secretion by PRRSV-infected3D4/21/CD163 cells via TLR4 signaling pathway,thereby indirectly affecting viral proliferation within cells.To verify the above results,we used specific TAK-242(TLR4 inhibitor)to inhibit/block TLR4,and used ELISA and fluorescence quantitative PCR to detect the effect of IRPS on the secretion of inflammatory cytokines and virus proliferation during PRRSV infection in vitro when TLR4 signaling pathway was blocked.The results showed that when the TLR4 signaling pathway was blocked,the regulatory effect of IRPS on the secretion of inflammatory cytokines after PRRSV infection of 3D4/21/CD163 cells was inhibited,indicating that IRPS can affect the secretion of inflammatory cytokines when PRRSV infected3D4/21/CD163 cells through TLR4 and its downstream signaling pathways.When the TLR4 signaling pathway was blocked,the inhibitory effect of IRPS on PRRSV proliferation was reduced,but there was still a certain degree of inhibition.When the TLR4 signaling pathway was blocked,there was no significant difference in the copy number between the virus control group and the normal virus control group.These results indicate that the TLR4 signaling pathway did not directly affect the proliferation of PRRSV in cells.Combined with cytokine secretion analysis,IRPS can reduce the secretion of inflammatory cytokines after PRRSV infection in3D4/21/CD163 cells through the TLR4 signaling pathway,and enhance cell stability,thereby reducing PRRSV proliferation in cells.