| Swine influenza is a highly contagious disease caused by swine influenza virus infection in pigs.Its clinical symptoms are mainly cough,fever,dyspnea and so on.Pigs of different breeds,genders and ages are susceptible.It is one of the most common and difficult diseases to eradicate in pig farms.Because of the sialic acid receptor of avian influenza virus and human influenza virus in pig respiratory tract,it is considered to be a"mixer"for the recombination of new influenza virus.The four human influenza pandemics in human history are closely related to swine influenza virus,which seriously endangers human and animal health.Influenza virus infection of the host can cause significant changes in the miRNA expression profile.The virus may achieve immune escape and enhance its ability to infect the host by regulating changes in the expression of certain miRNAs.At the same time,the host can also regulate the expression of certain miRNAs to change Initiate a response mechanism against the virus.In the previous study of our group,we found that the expression of miR-124-3p was significantly increased（about 3.8times）in mice infected with H1N1 swine influenza virus compared with that in the control group.One of the target genes regulated by miR-124-3p is STAT3,which was also one of the key transcription factors for Th17 cell differentiation.Therefore,it is suggested that miR-124-3p may reduce the inflammatory response induced by influenza virus infection by regulating the differentiation of Th17 cells.In this project,we investigated the regulatory effect of miR-124-3p on Th17 cell immune response in mice infected with H1N1 swine influenza virus.The results showed that the EID50of A/Swine/GD/2/12（H1N1）swine influenza virus is 10-7.2/200μL.Eighteen four-week-old C57BL/6 mice were randomly divided into two groups,nine in each group.They were swine influenza virus infection group and control group.Three groups of mice were vaccinated with swine influenza virus at a dose of106.9EID50（100μL）at three time points,0h,24h,and 48h.The infection was recorded as infection group 3,infection group 2,and infection group 1,respectively.The same amount of chicken embryo allantoic fluid was inoculated at the same time point and recorded as control group 3,control group 2 and control group 1,respectively.Mice were sacrificed 24 hours after the last challenge,lung lavage fluid cells and appropriate spleen single cell suspensions were obtained,and the proportion of Th17cells was detected by flow cytometry,and RT-qPCR Detection and analysis of mRNA expression levels of inflammatory cytokines and key transcription factors of Th17cells in lung tissues of infection group 3 and control group 3.The results showed that there was no significant difference between the groups of Th17 cells induced by H1N1 swine influenza virus in the spleen tissue.Among the cells of lung lavage fluid,the Th17 cells of H1N1 influenza virus-induced infection group 1 and infection group2 were different from the control group and control group 2 respectively.The difference between the groups was not significant,and the proportion of Th17 cells in the infected group 3 increased relative to the control group 3,and the difference between the two groups was significant（P<0.05）,compared with the control group 3,the proinflammatory factors IL-1βand IL detected by the infected group 3 The mRNA expressions of-6 and IL-17A were both up-regulated,and IL-6 was significantly up-regulated（P<0.05）;the mRNA expressions of STAT3 and RORγt were up-regulated,but the difference was not significant（P>0.05）.The single lymphocyte suspension of mouse spleen was obtained by density gradient centrifugation.The na（?）ve T cells were selected by immunomagnetic bead method.Transfection of na（?）ve T cells with adenovirus expression vector to mediate miR-124-3p overexpression,inhibit expression and set a blank control group,add Th17 cell differentiation-related cytokines to induce na（?）ve T cells to polarize to Th17cells,and the proportion of Th17 cells after polarization were detected and analyzed by flow cytometry and also the IL-17A concentration in the cell culture supernatant by ELISA method.The results showed that the purity of na（?）ve T cells purified by immunomagnetic bead method was>90%.Transfection of na（?）ve T cells with miR-124-3p overexpression adenovirus vector significantly increased the expression of miR-124-3p（P<0.05）.The results of cytometry and ELISA showed that compared with the blank control group,overexpression of miR-124-3p significantly inhibited Th17 cell differentiation in vitro,while inhibition of miR-124-3p expression significantly promoted Th17 cell differentiation in vitro.In summary,H1N1 swine influenza virus-infected mice can promote a significant increase in Th17 cells in mouse lung lavage cells,but have no significant effect on spleen tissue.In vitro studies have shown that increased expression of miR-124-3p can inhibit Th17 cell differentiation.Therefore,miR-124-3p significantly up-regulated in swine influenza virus-infected mice can reduce the inflammatory response caused by swine influenza virus infection by inhibiting Th17 cell differentiation. |
