Preliminary Evaluation Of The Anti-infectious Bronchitis Virus Activity Of Berberine

Infectious bronchitis(IB)is an acute contact respiratory infectious disease of chickens caused by the Infectious Bronchitis Virus(IBV).Chicken is the only natural host of IBV,and chickens of different ages can be infected with IBV,leading to direct death or secondary bacterial infection,resulting in significant losses to the poultry industry.Currently,vaccination is the main method to prevent IBV.However,due to the extreme variability of IBV and the poor cross protection effect of vaccines against different virus strains,IBV infection still occurs after immunization.Therefore,searching for drugs that can effectively control IBV infection is of great significance for the prevention and treatment of IBV.Palmatine is a natural non flavonoid alkaloid that can be extracted from various traditional Chinese medicines.Research has shown that Palmatine has various pharmacological activities such as antiviral,antibacterial,anti-inflammatory,and anticancer.This study established an in vitro infection model by infecting CEK cells with IBV,systematically evaluating the anti IBV activity of Palmatin,providing a theoretical basis for the development and application of Palmatine against IBV.The main research results are as follows:1.Evaluation of the inhibitory activity of berberine on IBVThe CPE of CEK cells was observed under an inverted microscope after exposure to different concentrations of palmatine solutions for 48 hours.The median toxic concentration(CC50)of palmatine is 672.92 μM.Maximum non-toxic concentration is125 μM.After continuous double dilution of palmatin at maximum concentration,different concentrations of palmatine solutions were applied to IBV infected CEK cells.The results showed that the 50% inhibitory concentration(IC50)of palmatine on IBV was 7.76 μM.The selection index SI is 86.74.In order to further evaluate the inhibitory activity of palmatine on IBV,CEK cells were infected with different IBV infection complexes(MOI=1,0.5,0.1,0.05,0.01),and the proliferation of IBV in CEK cells was detected using the absolute fluorescence quantitative detection method of IBV N gene established in the laboratory earlier.The results showed that the inhibitory effect of palmatine on IBV would not be weakened by the increase in the amount of IBV infection.At the same time,the inhibitory effect of palmatine on IBV was tested at different stages of IBV infection with CEK.The results showed that palmatine could inhibit the adsorption,penetration,and replication of IBV,and the co incubation of palmatine with IBV could directly inactivate IBV particles.2.The effect of palmatine on changes in inflammatory signaling pathways associated with IBV infectionUsing absolute fluorescence quantitative PCR,the effect of palmatine on the proliferation of IBV in the host was studied,and the birth length curve was plotted.The results showed that palmatine had a significant inhibitory effect on IBV after 6 hours of infection,which may be related to its ability to directly kill the progeny virus released by IBV into the extracellular space.Furthermore,the effect of palmatine on the expression of m RNA of related genes in the NF-κB/IRF7/JAK-STAT signaling pathway after 24 h of IBV infection was further detected by real-time PCR,and the antiviral effect of palmatine by influencing cytokines was evaluated.The results showed that:(1)Palmatine could significantly upregulate the m RNA expression of TRAF6,TAB1 and IKK-β genes(P <0.001)to promote the expression of inflammatory factors such as IL-1 and TNF-α.(2)After IBV infection,palmatine can significantly upregulate the expression of m RNA(P <0.001)of MDA5 and MAVS genes,and promote the expression of type I interferon by upregulating the expression of IRF7 pathway.(3)After IBV infection,the expression of JAK1 and STAT1 genes of JAK-STAT pathway was upregulated,but there was no significant difference in the expression of downstream antiviral effector proteins,which may be related to IBV inhibiting phosphorylation of STAT1,and although the expression of JAK1 and STAT1 decreased after treatment with palmatine,the expression of downstream antiviral effector protein OAS increased significantly(P <0.001).This indicates that palmatine can inhibit changes in host inflammatory signaling pathways caused by IBV infection and exert antiviral activity.3.Effect of palmatine on apoptosis of IBV-infected cellsFirstly,the effect of Annexin V-FITC/PI double stain on apoptosis caused by IBV infection was observed,secondly,the expression of apoptosis-related genes was detected by fluorescence PCR,and finally the effect of flavanin on the distribution of apoptosis caused by IBV infection was detected by flow cytometry.The results showed that:(1)At 24 h of IBV infection,the number of apoptotic and necrotic cells increased significantly compared with that of normal group cells,and the number of apoptotic cells did not change significantly after treatment with palmatine,but the number of necrotic cells decreased significantly;(2)The expression of anti-apoptotic gene Bcl-2was significantly upregulated during IBV infection;The expression of the pro-apoptotic genes Bax and Caspase-3 was upregulated at 24 h,and the expression of the antiapoptotic gene Bal-2 was downregulated at 24 h,which inhibited the regulation of the above genes by IBV after the treatment of palmatine.(3)The specific situation of apoptosis was detected by flow cytometry,and it was found that after treatment of IBVinfected cells with palmatine,apoptosis was inhibited,and the late apoptosis and necrotic cells in the drug group were less than those in the viral group.