| There have been no reports on the expression of prostaglandins and apoptosis-related genes in chicken erythrocytes treated with H9N2 and E.coli.This study is of great significance to evaluate the in vitro and in vivo expression changes of prostaglandins and apoptosis-related genes in chicken erythrocytes.In this study,prostaglandins and apoptosisrelated genes were selected to evaluate their expression changes in chicken erythrocytes treated with avian influenza virus H9N2 subtype and E.coli.The expression of target genes can provide new evidence and detection target for diseases caused by avian influenza and E.coli.For in vitro H9N2 study,specific pathogen-free(SPF)chickens were purchased and 4mL fresh blood samples were taken from SPF chickens,and take 2:1:1(8 mL:4 mL:4 mL)lymphocyte separation solution: PBS: blood,centrifuge at 2000 r/min for 20 min to remove the supernatant,and collect and Separate the precipitated erythrocytes.After the erythrocytes were separated,50 μL of the erythrocytes(3×108)were put into 16 centrifuge tubes containing DMEM maintenance medium,and randomly divided into 4 parts for treatment at four time points at 0,2,6,and 10 h.In each group,four tubes were divided into control and experimental sub-groups.The H9N2 virus A/chicken/Shanxi/1.23 TGRL003-O/2019 was saved in Laboratory.H9N2 virus solution(100 μL)was added to the test subgroups of all 4groups,and 900 μL DMEM was added to make the final volume 1050 μL.The erythrocytes were collected to extract RNA,and the relative mRNA expression of related genes was evaluated by qRT-PCR.Western blot was done to determine the expression level of related proteins.Indirect immunofluorescence was done to check the interaction of erythrocytes with H9N2 and transmission electron microscopy was used to compare the ultra-structural changes in H9N2 treated chickens.For in vivo H9N2 expression 3 groups of chickens were arranged and H9N2 treatment was introduced at day 3,7 and 14 of H9N2 treatment by following the other procedures as followed for in vitro interaction of H9N2 and chicken erythrocytes.Secondly,according to the same method as in vitro and in vivo H9N2 expression levels,in vitro and in vivo expression of chicken erythrocyte at different time intervals treated with E.coli was evaluated.mRNA expression of same genes were detected by qRT-PCR,Indirect immunofluorescence was done to check the interaction of erythrocytes with E.coli,and transmission electron microscopy were used to detect the structural difference in control and experimental E.coli samples.Immunohistochemistry was done to check the expression changes of prostaglandins and apoptosis-related genes at different time intervals of treatment.In the first stage,expression of 6 prostaglandin-related genes(COX-2,GSTA3,PTGDS,PTGES,PTGER3 and PTGER4)and 5 genes related to apoptosis(Bax,Bcl-2,BAG-1,BAG-3and STAT3)were observed in vitro and in vivo separately in chicken erythrocytes treated with H9N2 subtype of avian influenza virus.The results showed that the expression of PG-related genes in chicken erythrocytes was up-regulated in the early stage of H9N2 treatment,and the expression of PG-related genes in chicken erythrocytes was down-regulated in the later stages.The COX-2 protein was selected as the target,and the results of qRT-PCR were verified by Western blotting,and the results were consistent with the COX-2 mRNA expression level.The expression of all genes related to apoptosis was significantly up-regulated(P < 0.05)except Bcl-2.Instead of in vivo study,Bcl-2 was significantly down-regulated in vitro at 6 and10 h after treatment(P < 0.05).Bax protein was selected as the target,and the results are consistent with the changes in Bax mRNA expression.H9N2 and chicken erythrocyte interaction was confirmed by indirect immunofluorescence.Ultra-structural analysis by transmission electron microscopy also revealed the edges difference in the control and experimental group in H9N2 treated chickens.Immunohistochemistry findings also supported the H9N2 and chicken erythrocytes interaction at protein level.The above results indicate that H9N2 AIV can affect the expression of prostaglandins and apoptosis-related genes in chicken erythrocytes.In the second phase of the study,the expression of prostaglandin and apoptosis-related genes were detected in vitro(0.5,1 and 2 h of time interval)and in vivo(1,3 and 7 days of treatment)in chicken erythrocytes treated with E.coli.We selected several genes including 6prostaglandins-related genes(COX-2,GSTA3,PTGDS,PTGES,PTGER3 and PTGER4)and 5apoptosis-related genes(Bax,Bcl-2,STAT3,BAG-1,and BAG-3).All these apoptosis and PGrelated genes were significantly up-regulated(P < 0.05)at different time intervals of E.coli treatment in vitro.In vivo expression of apoptosis-related genes were significantly upregulated(P < 0.05)at different stages of study.In case of in vivo expression of PG-related genes were significantly up-regulated(P < 0.05)at different stages of time,except at day 3 in case of PTGES and PTGER4,where it was significantly down-regulated(P < 0.05).E.coli and chicken erythrocyte interaction was also confirmed by indirect immunofluorescence.Ultra-structural changes can also be observed in E.coli treated chicken erythrocyte in comparison with the control group.Relative genes expression of different apoptosis and prostaglandins-related genes in erythrocytes of chickens was checked at different time intervals of 1,3 and 7 days of E.coli treatment.The transcriptome sequencing analysis evaluated total of 2,657 DEGs identified in EC3 d vs ET3 d in-vivo group,among which 638 were up-regulated and 2,019 were down-regulated.Moreover at 0.5 h in-vitro E.coli challenged data set contains total 255 DEGs of which 108 were up-regulated and 147 were down-regulated.In addition to enriching the existing transcriptome data of E.coli,this study also clarified the immune response of chicken erythrocytes against E.coli in-vivo and in-vitro treatment.The results of RNA-seq and qRT-PCR revealed that,apoptosis and prostaglandinsrelated genes were highly correlated and showed a consistency with each other.These results support the reliability of differential expression identified by RNA-Seq.In short,prostaglandins and apoptosis-related genes in chicken erythrocytes are closely related to H9N2 and E.coli treatment,providing a new biomarker for the detection of these pathogens.The current research provides a new method for targeting H9N2 and E.coli treatments.Further research is ongoing to understand that host factors may control the level of gene expression during treatment in chickens. |
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