Preliminary Studies On The Antiviral And Antitumor Mechanism Of Taishan Pinus Massoniana Pollen Polysaccharide On Acute Tumorigenic ALV-J And Its-related Tumor

Subgroup J avian leucosis virus?ALV-J?is one of the most important viruses endangering chicken industry.Immunosuppression and malignant tumors occur in infected chickens,which seriously hinder the healthy development of chicken industry and cause huge economic losses.In recent years,acute tumorigenic ALV-J virus carrying v-fps oncogene has become more and more prevalent,which has brought new challenges to chicken industry.Taishan Pinus Massoniana pollen polysaccharide?TPPPS?,natural plant macromolecule,demonstrates immune enhancement and antiviral activity potential.In this sthudy,the mechanism of TPPPS against acute tumorigenic ALV-J and its-related tumor was studied.Fu-J?SDAU1005?strain of acute tumorigenic ALV-J was used to establish animal model in vitro and in vivo.Then we detected and analyzed the inhibitory effects of TPPPS on the replication of Fu-J?SDAU1005?and tumor growth,as well as the immunoregulatory activity of splenic lymphocytes.Finally,the immunoregulatory,antiviral and antitumor mechanism of TPPPS and were discussed.1.Study on antiviral activity of TPPPS in vitroIn this study,50 ug/mL,100 ug/mL and 200 ug/mL concentration of TPPPS were used to treat DF-1 cells inoculated with Fu-J?SDAU1005?.ELISA and qRT-PCR were used to detect the viral quantities and we analyze the inhibitory effect of TPPPS on Fu-J?SDAU1005?.The results showed that different concentrations of TPPPS had antiviral effect,and the effect was dose-dependent.200 ug/mL TPPPS had the most significant effect,but it's not significantly different from 100 ug/mL.Furthermore,in order to analyze the antiviral effect of TPPPS on Fu-J?SDAU1005?infected cells at different stages,and explore the effect of TPPPS on the growth and migration of DF-1 cells infected with virus,100 ug/mL TPPPS was applied to DF-1 cells inoculated with Fu-J?SDAU1005?,and then the quantities of Fu-J and SDAU1005 viruses was detected by qRT-PCR and IFA.The results showed that TPPPS could inhibit virus by affecting the adsorption and release of virus.It can also inhibit the growth and migration of DF-1 cells infected by virus.2.Study on immunoregulatory activity of TPPPS on splenic lymphocyteInorder to study the immunoregulatory activity of TPPPS on splenic lymphocytes,different concentrations of TPPPS and Fu-J?SDAU1005?were applied to splenic lymphocyte simultaneously.Then,the lymphocyte proliferation,cell subsets,and intracellular second messenger Ca²⁺,cAMP,cGMP,PKC,PKA,Calcineurin and NFAT were detect and analyze by MTT,flow cytometry and ELISA.The results showed that TPPPS could promote T and B lymphocyte proliferation,increase the ratio of and CD4⁺/CD8⁺,and increase the contents of CD3⁺,CD4⁺,CD8⁺,IL-2,IL-4,IFN-?,IgG,Ca²⁺,cAMP,cGMP,PKC,PKA,Calcineurin and NFAT.The results demostrated that TPPPS could activate signal transduction pathways such as NO-cGMP,Camp-PKA,Ca²⁺-PKC-Calcineurin-NFAT in splenic lymphocyte,and had good immunoregulatory activity on splenic lymphocyte.3.Study on antiviral and antitumor activity of TPPPS in vivoTo study the antiviral and antitumor activity of TPPPS on acute tumorigenic ALV-J and its-related tumor,fristly,1-day-old SPF chickens were randomly divided into 5 groups,20chickens in each grou,1-4 groups were orally administered 1 mg,3 mg,5 mg and 10 mg TPPPS and 5 group as PBS group were orally administered 200?L of PBS every day.After that 10⁴ TCID₅₀0 virus was inoculated subcutaneously on the legs of each group at 7 day-old.The clinical symptoms and histopathological changes of experimental animals were observed and detected every day after exposure,and the results were continuously observed for 20days.The results showed that different concentrations of TPPPS had antitumor effect in vivo,which was dose-dependent,and 10 mg TPPPS had the best effect,but it's not significantly different from 5 mg.In further study,90 1-day-old SPF chickens were randomly divided into three groups,V-TPPPS group,V-PBS group and PBS group,30 chickens in each group.V-TPPPS group took 5 mg TPPPS orally every day.V-PBS group took 200?L of PBS;V-PBS group and V-TPPPS were injected with 10⁴ TCID₅₀ Fu-J?SDAU1005?subcutaneously into the legs at 7 day-old,respectively,and the PBS group was injected with the same volume of PBS.After challenge,the tumor tissues,liver,thymus,bursa of Fabricius,spleen and blood were collected every 5 days.Then,the contents of Fu-J and SDAU1005 virus in tumor tissues and plasma were detected by qRT-PCR,and the pathological changes of various organs were detected by HE staining.The results showed that compared with V-PBS group,the viral quantities in tumor tissues and plasma in V-TPPPS group was significantly reduced,and the pathological tissue damage was mitigated.The results demostrated that TPPPS had significant antiviral and antitumor effects in vivo,and could alleviate organ damage caused by virus.4.Transcriptionomic analysis the antiviral and antitumor effects of TPPPS in vivoTranscriptome sequencing was performed on the tumor tissues and leg muscle tissues of experimental animals in V-TPPPS group and V-PBS group on the 10 th day after challenge,and then analyse the differentially expressed RNA and microRNAs and related signaling pathways.The results showed that there were 6201 DEGs in V-PBS group were differentially expressed more than twice as much as those in PBS group,of which 3183 were up-regulated and 3018 were down-regulated.The differentially expressed genes were mainly concentrated in the pathogenicity-related pathways of viruses such as P13K-AKT signaling pathway,MAPK signaling pathway,P53 signaling pathway,Rap1 signaling pathway,NF-kappa B signaling pathway,oxytocin signaling pathway and adhesion spot signaling pathway.Compared with V-PBS group,there were 560 DEGs in V-TPPPS group with a difference of more than 1.5 times,of which 321 were up-regulated and 239 were down-regulated.Immune-related genes were up-regulated,while tumor-related genes were down-regulated.The signaling pathways involved in the regulation of different genes include T cell receptor signaling pathway,cytokine-cytokine receptor interaction,chemokine signaling pathway,MAPK signaling pathway,estrogen signaling pathway,P13K-Akt signaling pathway,Ras signaling pathway,JAK-STAT signaling pathway,NF-?B signaling pathway and protein processing in endoplasmic reticulum.Compared with V-PBS group,V-TPPPS had only two differentially expressed microRNAs,one up-regulated and one down-regulated.There were149 differentially expressed microRNAs in V-PBS group compared with PBS group,of which 82 up-regulated and 49 down-regulated.The predictions of differentially expressed microRNAs target genes are basically consistent with the results of gene enrichment pathways.The results showed that TPPPS could increase the expression of immune-related genes,down-regulate the expression of tumor-related genes,and participate in the regulation of immune regulation-related and tumor-related signaling pathways in vivo.On the other hand,microRNAs are involved in the pathogenicity regulation of viruses,but TPPPS is hardly involved in the regulation of microRNAs in vivo.For further study,thirteen differentially expressed genes related to immune regulation and 9 tumor-related were selected to detection in tumors and cells.The results showed that TPPPS could promote the expression of TLR2,TLR3,TLR4?CLEC2D receptor genes,RF1,GZMA,CCL5 and other immune related genes in vivo and in vitro,and down-regulate the expression of HSP2,BAG3,DNAJN4,and other tumor genes.Through the above study and analysis,it is preliminarily determined that TPPPS can inhibit tumor-related signaling pathways by binding with receptors to activate immune-related signaling pathways in vivo,thus playing an antiviral and antitumor role.However,the specific relationship and interaction mechanism between them should be further validated by gene knockout,siRNA interference,over-expression,immunocoprecipitation and other experiments.