Research On The Effect Of COX-2 Inhibitors On Reducing BLV Proviral Load In Dairy Cows

Bovine leukemia virus（BLV）belongs to the deltaretrovirus genus of the retrovirus family and primarily infects peripheral blood lymphocytes and monocytes,resulting in the continuous proliferation of bovine lymphocytes.Despite the lack of obvious clinical symptoms,BLV is frequently overlooked.However,the worldwide prevalence of BLV is on the rise,and there are no effective commercialized BLV vaccines.Therefore,it is crucial to investigate and discover compounds that can reduce the BLV proviral load（PVL）to control its infection and transmission.Prostaglandin E₂（PGE₂）,an inflammatory mediator formed from arachidonic acid metabolism by cyclooxygenase-2（COX-2）.Related studies have shown that during viral infection,the organism secretes and produces PGE₂.PGE₂has an immunosuppressive effect and can promote immune evasion during viral infection through various pathways.At the same time,PGE₂also participates in and regulates viral replication,and using COX-2 inhibitors to reduce PGE₂synthesis can activate host antiviral immune responses in vitro and in vivo.Therefore,COX-2inhibitors are potential new methods to treat chronic infectious diseases.However,the involvement of PGE₂in BLV infection remains uncertain.The goal of this study is to examine the relationship between PGE₂and BLV PVL,clarify the impact of PGE₂on the immune function and replication of BLV in dairy cows,and provide new strategies for treating BLV infection in dairy cows,as well as experimental evidence to reduce the risk of BLV transmission and maintain the health of the bovine immune system.This study gathered blood samples from 107 lactating dairy cows on a large-scale ranch in Heilongjiang Province.First,BLV-positive cows were screened by ELISA,and then BLV PVL in BLV-positive dairy cows samples were measured by absolute quantitative PCR.Virus copy numbers over 1,000 copies/10 ng DNA were defined as high proviral load（H-PVL）,otherwise were designated as low proviral load（L-PVL）.Meanwhile,lymphocyte counts in BLV-positive dairy cows blood were analyzed using an automatic blood routine analyzer for correlation assessments.Results showed that out of the 107 dairy cows,21 cows tested positive for BLV antibodies with a positivity rate of 19.62%.Among the 21 dairy cows,5 cows had H-PVL,and16 cows had L-PVL.BLV PVL was positively and correlated with antibody titers and lymphocyte counts（r=0.66,P<0.01 and r=0.88,P<0.01）.To investigate the effects of PGE₂on PBMC function and BLV replication,PBMC from healthy dairy cows and BLV-positive cows were co-cultured with BLV antigen（BLV Ag）to measure the concentration of PGE₂in the supernatant and the transcription level of COX-2m RNA in PBMC via ELISA and q RT-PCR,respectively.Different concentrations of PGE₂were co-cultured with healthy dairy cows PBMC to measure PBMC proliferation by CCK-8 assay,and m RNA transcription levels of cytokines and immunosuppressive molecules in PBMC were determined by q RT-PCR.Furthermore,PGE₂,EP2 agonist,and EP4 agonist were co-cultured with BLV-positive dairy cows PBMC,and the transcriptional and protein expression levels of BLV genes were detected by q RT-PCR and Western blot,respectively.Results indicated a significant increase in COX-2 m RNA transcription levels and PGE₂production（P<0.01）in BLV-positive dairy cows PBMC stimulated with BLV Ag,while healthy dairy cows PBMC remained unaffected.500 n M PGE₂inhibited PBMC proliferation（P<0.01）and downregulated IL-2（P<0.05）,IFN-γ（P<0.01）,and TNF-α（P<0.01）,but upregulated m RNA transcription levels of IL-10,PD-L1,and Tim-3（P<0.01）compared to the control group.Moreover,PGE₂and EP2/EP4 agonists stimulated BLV-gp51 and BLV-p24 m RNA transcription and protein expression levels（P<0.01）.These findings reveal that PGE₂inhibits immune function in dairy cows PBMC and promotes BLV replication by activating downstream signaling pathways through EP2 and EP4 receptor binding.To investigate the regulatory effect of COX-2 inhibitors on PBMC function and the impact of BLV replication,as well as their pathways of action in vitro.BLV-positive dairy cows PBMC were incubated with Meloxicam.PBMC proliferation was measured by CCK-8 assay,and PBMC cytokines,immunosuppressive molecules,COX-2,BLV-gp51,and BLV-p24 m RNA transcription levels were measured by q RT-PCR.COX-2,EP2,EP4,BLV-gp51,BLV-p24,and cAMP/PKA/CREB pathway-related protein expression levels were detected by Western blot.Results demonstrated that Meloxicam promoted PBMC proliferation（P<0.01）,upregulated IL-2,IFN-γ,and TNF-α（P<0.01）,and downregulated IL-10,PD-L1,Tim-3,COX-2,BLV-gp51,and BLV-p24 m RNA transcription levels（P<0.01）.Meloxicam also inhibited COX-2,EP2,and EP4protein expression（P<0.01）,inhibited PKA and CREB phosphorylation（P<0.01）,and inhibited BLV-gp51 and BLV-p24 protein expression（P<0.001）.These results indicate that Meloxicam can activate BLV-specific Th1 responses in vitro,block the COX-2/PGE₂pathway,downregulate the EP2 and EP4 receptor expression,block the cAMP/PKA/CREB pathway,and efficiently inhibit BLV replication.To investigate the therapeutic effect of COX-2 inhibitors on reducing the BLV PVL in dairy cows,5 H-PVL dairy cows were selected for clinical research on COX-2 inhibitors Meloxicam,Flunixin meglumine,and volatile oil of H.cordata.The injection period was once a week for a total of 5 times,and the injection doses were:Meloxicam 0.5 mg/kg,Flunixin meglumine 2.5mg/kg,and injection solution of H.cordata 120 m L/time.Samples were collected on days 0,7,14,21,28,35 and 70 for BLV PVL,cytokines transcription and secretion,and immunosuppressive molecules transcription level detection.The results showed that Meloxicam injection could reduce the BLV PVL in dairy cows（P<0.05）,promote Th1 cytokines secretion（P<0.05）,and inhibit Th2 cytokine secretion（P<0.05）,but had no effect on IL-4 secretion.Meanwhile,it can downregulate PD-L1 and Tim-3 m RNA transcription levels（P<0.05）.The changes in cytokines m RNA transcription levels in PBMC were basically consistent with their changes in secretion levels in dairy cows.Flunixin meglumine also showed antiviral activity,but its effect on reducing BLV PVL,inducing Th1 immune response,and down-regulating immunosuppressive molecule m RNA transcription levels was inferior to that of Meloxicam.Volatile oil of H.cordata can also significantly reduce the BLV PVL in dairy cows（P<0.05）,and the therapeutic effect is between Meloxicam and Flunixin meglumine.In addition,volatile oil of H.cordata can significantly reduce the secretion levels of inflammatory cytokines IL-6,IL-8,and TNF-α,and alleviate the inflammation level of dairy cows.These results indicate that all three COX-2 inhibitors have the effect of reducing the BLV PVL in dairy cows and are potential commercial drugs for controlling BLV infection and reducing BLV transmission risk.In summary,a significant positive correlation was found between PGE₂and BLV PVL.PGE₂has an inhibitory effect on the immune function of bovine PBMC and promotes BLV replication by activating downstream signaling pathways through binding to EP2 and EP4.In the vitro experiments,COX-2 inhibitors blocked the COX-2/PGE₂pathway,down-regulated EP2and EP4 receptor expression,blocked the cAMP/PKA/CREB pathway,and inhibited BLV replication.Clinical research further established the potential of COX-2 inhibitors as commercial drugs to control BLV infection and reduce transmission risk.