Mechanism Of MiR-124-3p Regulating Lung Injury In Mice Infected With H1N1 Swine Influenza Virus

Pigs are common susceptible hosts of avian,swine and human influenza viruses,and are"mixers"for gene recombination or redistribution of influenza viruses because of the simultaneous human influenza and avian influenza sialic acid receptors in pigs.The public health significance of swine flu is becoming prominent increasingly.Almost major influenza history is related to swine flu in the history of human influenza.A lot of studies have shown that viruses cause pathological immune response and acute immune injury in host and they are the main cause of severe acute respiratory syndrome,respiratory failure and immune system dysfunction.In the differential expression of micro RNAs in H1N1 swine influenza virus-infected mice,the expression of mi R-124-3p was significantly up-regulated.Bioinformatics analysis showed that mi R-124-3p may play an important role when the host regulate lung injury infected influenza virus.In this study,a mouse model of swine influenza virus infection was used.Adenovirus mediated mi R-124-3p overexpression and inhibition expression were used to study the regulatory mechanism of mi R-124-3p on lung injury in mice infected with H1N1 swine influenza virus by transcriptional analysis.This study not only provides important scientific value for elucidating the mechanism of influenza virus-mediated immune injury,but also has important guiding significance for exploring new targets for preventing and controlling influenza virus-induced lung injury.The SPF chicken embryo was used to proliferate A/Swine/GD/2/12?H1N1?and identification by RT-PCR and determination of EID₅₀.The results showed that the EID₅₀of A/Swine/GD/2/12?H1N1?was 10^-6.83/0.2 m L.Admax system was used to construct micro RNA-124-3p overexpression adenovirus?micro RNA-124-3p-over?,inhibitoryadenovirus?micro RNA-124-3p-sponge?and empty vector adenovirus?micro RNA-124-3p-blank?in 293A cells.The expression of micro RNA-124-3p in each group was detected by q PCR.The results showed that 293A cells showed green fluorescence,indicating that adenovirus plasmid vector was expressed in 293A cells.Overexpression group,inhibition group and empty vector group had significant difference in the expression level of mi R-124-3p in mouse melanoma cells.Overexpression group had the highest expression level,inhibition group had the lowest expression level,and empty vector group had the middle expression level.The results showed that adenovirus expression vector was successfully constructed.Seventy-five 6-week-old female Balb/c mice were randomly divided into mi R-124-3p overexpression group?group A?,micro RNA-124-3p inhibition group?group B?,control group?group C?and homologous group?group D??blank group?group E?,with 15 mice in each group.Mice in each group were injected with mi R-124-3p-over,mi R-124-3p-sponge and mi R-124-3p-blank by tail vein respectively.After 48h,the A,B and C groups were injected with A/Swine/GD/2/12?H1N1?in nose,and the mice in the D group and the E group were instilled with normal saline.The body weight and state of the mice were recorded daily.The lungs were taken for paraffin sections after the mice were dissected.The results were as follows:the body weight of mice in group A was higher than that in group B and group C;while the body weight of mice in group B was lower than that in group C,and all 3 groups had a period of weight loss and recovery,while the body weight of mice in group D and group E without nose dropping influenza virus continued to increase;the lung paraffin section HE staining observation showed that lung injury in group B and group C was more serious than that in group A.Serious,alveolar septum showed thickening,alveolar smaller or even collapse;q PCR detection of lung mi R-124-3p expression level,the highest expression level of mi R-124-3p in group A,the lowest expression level in group B,the middle expression level in group C.These results suggest that mi R-124-3p can alleviate lung injury caused by A/Swine/GD/2/12?H1N1?infection in mice,and exert an anti inflammatory effect and effect body weight.Nine 6-week-old female Babl/c mice were randomly divided into 3 groups:the over-expression group of mi R-124-3p?group A?,the suppression group of mi R-124-3p?group B?and the control group?group C?.Group A,group B and group C were injected with adenoviral vectors of mi R-124-3p-over,mi R-124-3p-sponge and mi R-124-3p-blank,respectively.48 hours later,all mice in group A,B and C were intranasally dripped with A/Swine/GD/2/12?H1N1?.Lungs of mice in group A and C were dissected 24 hours later for transcriptional histological analysis.Transcriptional sequencing revealed that differentially expressed genes were highly enriched in the MAPK signaling pathway,which played a role in the inflammatory response related to the immune system.Referring to the relevant literature,pay attention to p38?,TNF?,IL-6 and IL-1?.The four genes were used to study the regulation mechanism of mi R-124-3p on lung injury induced by H1N1 influenza.The results showed that the expression of four genes was the lowest in A group and the highest in B group.The expression level of genes in C group was in the middle and the difference was significant.These results indicate that mi R-124-3p regulates the expression of p38?gene,then affects the expression levels of TNF?,IL-6 and IL-1?,and finally plays a regulatory role in lung injury caused by A/Swine/GD/2/12?H1N1?.To sum up,through the weight change analysis,pathological observation,combined with transcriptome analysis and q PCR detection of mi R-124-3p overexpression group,mi R-124-3p inhibition group,control group,homologous group and blank group,we found that mi R-124-3p has anti-inflammatory effect in lung injury of A/Swine/GD/2/12?H1N1?infected mice.The mechanism may be that mi R-124-3p acts on p38?in MAPK signaling pathway then lead to the levels of TNF?,IL-6 and IL-1?reduce,thereby alleviated lung injury caused by A/Swine/GD/2/12?H1N1?in mice.