| Mink hemorrhagic pneumonia is an important disease that endangers mink,which has caused great economic losses to the development of mink aquaculture.Early studies suggested that mink hemorrhagic pneumonia was mainly caused by Pseudomonas aeruginosa(P.aeruginosa)infection.However,studies in the past ten years have shown that P.aeruginosa is not very pathogenic when it infects mink alone,and it is difficult to induce mink hemorrhagic pneumonia.As a common respiratory virus,H9N2 influenza A virus(IAV)exists widely in mink,but a single infection only causes mild respiratory disease in mink.P.aeruginosa is a common conditional pathogen,when animals are infected with other pathogens,it is easy to cause P.aeruginosa infection.The results of previous studies in our laboratory showed that co-infection of H9N2 IAV with P.aeruginosa could easily lead to hemorrhagic pneumonia in mink and significantly increase the mortality rate.However,the molecular mechanism of co-infection between H9N2 IAV and P.aeruginosa is still unclear.Lipopolysaccharide(LPS)is a component of the cell wall of P.aeruginosa and other Gramnegative bacteria,and is also an important virulence factor of P.aeruginosa.It has a variety of biological activities and can activate the innate immune response.In order to further explore the molecular mechanism of H9N2 IAV and P.aeruginosa coinfection leading to mink hemorrhagic pneumonia,this study established a Mk/SD/F10/13(H9N2)and P.aeruginosa LPS in vitro co-infection/stimulation model of macrophages.In this study,the hot phenol water method was used to extract LPS.The yield,nucleic acid content,protein content and polysaccharide content of the extracted LPS were detected,and the extract was identified by silver staining test.The results showed that the average yield of extracted LPS was 2.24%,the nucleic acid content was 0.083%,the polysaccharide content was 15.6%,and the protein content was 0.37%.The results of the silver staining test showed that both the extracted LPS and the standard E.coli LPS showed typical polysaccharide-like bands.This indicated that the extracted P.aeruginosa LPS had high yield,high purity and high biological activity.In order to explore the regulation of the immune response of mouse macrophages(MH-S)by co-infection/stimulation of H9N2 IAV and P.aeruginosa LPS,H9N2 IAV single-infected MH-S cells(H9N2/MH-S),P.aeruginosa LPS single-stimulated MH-S cells(LPS/MH-S)and H9N2 IAV coinfected/stimulated MH-S cells with P.aeruginosa LPS(H9N2+LPS/MH-S)were established.The inoculation dose of H9N2 IAV was MOI=1,and the inoculation dose of LPS was 10 ?g/ml.In addition,a blank control was set,and the control cells were inoculated with the same volume of sterile 2% FBS RPMI1640 nutrient solution.The viral nucleic acid level of H9N2 IAV in MH-S cells was detected by qRT-PCR at 1,3,6,9,and 12 h after inoculation with H9N2 IAV and LPS,respectively.The results showed that at 1 h,there was no significant difference in H9N2 IAV nucleic acid levels between H9N2/MH-S and H9N2+LPS/MH-S,but at 3,6,9,and 12 h,the H9N2 IAV nucleic acid levels of H9N2/MH-S were significantly higher than those of H9N2+LPS/MH-S.This suggests that LPS interferes with the proliferation of H9N2 IAV.The apoptosis level of cells was detected by flow cytometry.The results showed that at 1 h,the apoptosis rate of H9N2/MH-S,LPS/MH-S,and H9N2+LPS/MH-S was higher than that of the control,and the difference was significant.There was no significant difference in the apoptosis rate between H9N2/MH-S and H9N2+LPS/MH-S,but the apoptosis rate of the two groups was higher than that of LPS/MH-S,with a significant difference.At 12 h,the apoptosis rate of H9N2/MH-S,LPS/MH-S,H9N2+LPS/MH-S was higher than that of the control,and the difference was significant.The apoptosis rate of H9N2+LPS/MH-S was significantly higher than that of H9N2/MH-S and LPS/MH-S.It can be seen that compared with single infection,coinfection/stimulation has a stronger destructive effect on cells,LPS has a strong toxic effect on cells,and impaired cell function is one reason for the slowdown of H9N2 IAV replication.Cytokine mRNA expression levels were detected using qRT-PCR.The results showed that the overall cytokine mRNA expression levels first increased and then decreased with the increase of infection/stimulation time.The mRNA expression levels of chemokines CCL5,CXCL10 and cytokines IL-1?,IL-6,and IL-10 in H9N2+LPS/MH-S were higher than those in H9N2/MH-S and LPS/MH-S at 3,6,9,and 12 h,the difference is significant.At 6h,the expression level of H9N2+LPS/MH-S TLR4 mRNA reached the highest level.At 9 and 12 h,the expression level was lower than that of H9N2/MH-S.The expression level of H9N2/MHS TLR4 mRNA showed an increasing trend.The expression levels of TLR4 mRNA in H9N2+LPS/MH-S and H9N2/MH-S were higher than those in LPS/MH-S.The excessive activation of TLR4 led to the secretion of a large number of inflammatory factors,resulting in cytokine storm and pathological damage to tissue cells.The protein levels of cytokines TNF-?,IL-1?,IL-10 and IFN-? were detected at 1,6 and 12 h by ELISA.The results showed that with the increase of infection/stimulation time,the protein expression levels of IFN-?,TNF-?,IL-1? and IL-10 increased continuously.The protein levels of IFN-?,TNF-?,IL-1? and IL-10 in H9N2+LPS/MH-S were significantly higher than those in H9N2/MH-S and LPS/MH-S.Thus,coinfection/stimulatory cytokine secretion and immune response were more intense,which caused more severe damage to cells,than mono-infection.In conclusion,both H9N2 IAV and LPS can induce immune responses in macrophages,but coinfection/stimulation with H9N2 IAV and LPS has a synergistic effect,inducing stronger cellular immune responses,higher expression levels of pro-inflammatory factors,and more severe cell damage.This study enriched the research data on the pathogenic mechanism of coinfection between H9N2 IAV and P.aeruginosa,and provided a theoretical basis for the prevention and control of mink hemorrhagic pneumonia and severe human pneumonia. |
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