| With the large-scale development of cattle farms,the increasing frequency of bovine respiratory diseases(BRD)has caused huge economic losses in industrial cattle production worldwide.Dairy cattle pneumonia is one of the most common BRD,whose increasing frequency seriously threatens the healthy long-term development of animal husbandry.There are many pathogenic microorganisms that cause dairy cattle pneumonia.Especially,bacterial pathogens have the characteristics of fast onset,strong infectivity,pathogenicity,and great harm,which are the main infectious pathogens to induce the occurrence of pneumonia in cows and serious consequences.Although the research on bacterial pneumonia in dairy cows in China has been deepened in recent years,the characteristics of pathogenic bacteria,the pathogenic mechanism of bacterial infection and the pathogenesis of pneumonia are still not compeletly clear until now.As based on this circumstances,the treatment of dairy cattle pneumonia has major limitations,and clinically,high use of antimicrobials is required for therapy.Excessive use of antibiotics can easily cause a large number of antibiotic residues and the rapid appearance of drug-resistant genes and multidrug resistant strains.Now many countries have strictly restricted or banned the use of veterinary antibiotics.Micro RNA is a type of small molecule RNA that plays an important role in the regulation of post-transcriptional gene expression,and participates in the occurrence and development of various biological processes and diseases.mi R-182,belonging to the mi R-183 gene cluster,plays an important role in acute lung injury(ALI),atherosclerosis,and various inflammatory reactions.However,the function and regulatory mechanism of mi R-182 in dairy cattle bacterial pneumonia is not clear.In this study,inflammatory lung tissue of dairy cattle was chosed as the research object.Starting from the verification of the target gene of mi R-182,the role and mechanism of mi R-182 in the inflammatory response of the cow’s lungs were explored in depth to provide new methods and ideas for the prevention and treatment of cow’s pneumonia in clinic.The main research contents and test results are as follows:(1)Lung tissues of healthy and cows with obvious respiratory symptoms and inflammatory lesions on the lungs can be selected and collected through clinical diagnosis.The results of histopathological analysis,MPO activity and ELISA assay showed that the lung tissue in the disease group presented lung injury characterized by diffuse interstitial and alveolar edema,and a large number of inflammatory cells exudate in the alveolar cavity;MPO activity and the expression levels of the inflammatory cytokines(including TNF-α,IL-6 and IL-1β)were significantly increased.It was indicated that there was severe inflammatory damage in the lung tissues of dairy cattle.The analysis of RT-q PCR revealed that TLR4 was significantly increased in inflammatory lung tissues of dairy cattle,while mi R-182 was significantly reduced.Addtionally,16 S r RNA sequence analysis confirmed that all 10 strains were routinely isolated and identified as E.coli.10 groups of mice were artificially infected with the isolated E.coli,and the results showed that 8 groups of them died within a short time after infection,and the other 2 groups showed different degrees of pathogenicity.The lung of dead mice were had pulmonary hyperemia and local necrotic foci by autopsy.It was proved that the E.coli isolated in this experiment was highly pathogenic.These results suggested that mi R-182 played an important role in the development of extraintestinal pathogenic E.coli pneumonia and may be related to the TLR4/NF-κB signaling pathway.(2)Biological information softwares were performed to predict the possible targets and the base pairing of mi R-182,and TLR4 was preliminarily identified as the target gene of mi R-182.The results of duel-luciferase reporter assay found that mi R-182 significantly inhibited TLR4 3’-UTR luciferase activity.And over-expression of mi R-182 significantly reduced TLR4 transcription and translation levels in RAW264.7 cells.It was confirmed TLR4 was the target gene of mi R-182,and mi R-182 was negatively correlated with the expression of TLR4.(3)In order to further explore the role of mi R-182 in the inflammatory response,CCK-8 assay was used to detect the cytotoxicity of LPS in RAW264.7 cells.It was found that cell viability was not affected by LPS.Then,an inflammation model of macrophage cell line in vitro was established using LPS,and the related genes were measured by RT-q PCR.The results indicated that mi R-182 was down-regulated in a time-and dose-dependent manner.Overexpression or inhibition of mi R-182 showed that transfection of mi R-182 mimics in RAW264.7 cells could significantly reduce the expression of proinflammatory cytokines such as IL-1β,IL-6 and TNF-α;in contrast,transfection with mi R-182 inhibitor could obviously increase the expression of proinflammatory cytokines.In addition,the founding of western blot and immunofluorescence staining displayed that NF-κB signaling pathway activity,IκBα phosphorylation level,and phosphorylated p65 nucleus were significantly inhibited in LPS stimulated-macrophages which overexpressed mi R-182.These data suggested that mi R-182 had a negative regulatory or inhibitory effect on LPS-induced inflammatory responses.To further clarify whether mi R-182 extered anti-inflammatory effects by inhibiting TLR4,si RNA interference technology was used in RAW264.7 cells for in vitro validation.The results showed that si RNA interference with TLR4 significantly inhibited the levels of proinflammatory factors and LPS-induced phosphorylation of NF-κB p65,which were consistent with the results of overexpression of mi R-182.These results suggested that mi R-182 could reduce the LPS-stimulated inflammatory response by regulating TLR4.(4)To further verify the effect of mi R-182 on the excessive inflammatory damage of pneumonia tissue,LPS-induced ALI model in mice was successfully constructed in present study.The results found that mi R-182 expression was significantly down-regulated in the lung tissue of mice with ALI,which was consistent with the results in inflammatory lung tissue of dairy cattle.It is suggested that mi R-182 has a protective effect on acute inflammatory lung injury in mice.In summary,all results of this study indicate that mi R-182 inhibits the cascade reaction triggered by the NF-κB signaling pathway through targeting TLR4,thereby protecting and reducing the damage of inflammatory response to the lung of dairy cattle. |
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