| Chronic Respiratory Disease(CRD)is caused by Mycoplasma gallisepticum(MG)infection,leading to a substantial reduction in the performance of chickens.The disease is widespread in all poultry-rearing countries of the world and MG is susceptible to mixing with other pathogens or secondary infections,greatly increasing mortality in chickens and causing enormous economic losses to the poultry industry.Currently,antibiotics are primarily used to treat CRD,but antibiotics can lead to safety issues such as the development of resistant strains of bacteria and antibiotic residues in poultry products,hence it is urgent to find safe,effective and low-cost alternatives to antibiotics to combat CRD.Glycyrrhizic acid(GA)is the most vital active component of Glycyrrhiza glabra,which is widely applied in the treatment of pulmonary diseases owning to the outstanding characteristics of antibacterial,anti-inflammatory,antiviral and antioxidant.In this study,we investigated the role and molecular mechanism of GA in MG-infected chickens by constructing cellular and animal models of MG infection,providing a theoretical basis for its clinical studies in the treatment of CRD.The main results are as follows:1.The minimum inhibitory concentration of GA against MG was 50 μg/m L,which effectively depressed the expression of MG adhesion proteins p MGA1.2 and Gap A and inhibited MG proliferation;there was no effect of GA concentration between 0-300 μg/m L on AEC-Ⅱ cell viability.2.Upon MG infection of AEC-Ⅱ cells and AA broilers,there were significantly upregulated levels of IL-1β,TNF-α and MMP-2 and MMP-9 expression in cells and lung tissue,whereas the expression of the above factors were decreased significantly after GA and tiamulin treatment.MG infection increased apoptosis in AEC-Ⅱ cells,lung tissue and spleen,with significant increases in the pro-apoptotic proteins casp3 and casp9 and significant decreases in the anti-apoptotic proteins Bcl-xl and Bcl-2.GA and tiamulin treatment effectively suppressed MG-induced inflammation and apoptosis,indicating that GA could efficiently repair MG-induced inflammatory injury.3.The histopathological results showed that MG infection caused different degrees of inflammatory damage to the trachea,lung and spleen organs of broiler chickens,with a large number of inflammatory cells infiltrating the tissues.The damage to all tissues was recovered to a certain extent after GA and tiamulin treatment,further indicating that GA can effectively alleviate the MG-induced inflammatory damage in chicken tissues.4.The network pharmacology database and analysis platform were constructed for the potential targets of GA action,and the online database was used to screen CRD disease targets.The common targets obtained were analyzed by GO and KEGG online analysis tools,and it was found that GA may inhibit the infection process of MG by modulating the MAPK pathway.Further studies at the cellular and animal levels revealed that the MAPK pathway phosphorylated proteins p-ERK1,p-p38,p-JNK and p-Jun were activated in cells and lung tissue after MG infection,while the expression of the above proteins was significantly reduced after treatment with GA and tiamulin.In order to confirm the direct pathways through which GA acts at the cellular level,MG-infected AEC-Ⅱ cells were treated with p38,Jun and ERK pathway inhibitors,respectively.The results showed that GA reduced the expression levels of MMP-2,MMP-9,IL-1β and TNF-α,and inhibited apoptosis and promoted cell proliferation in AEC-Ⅱ cells by inhibiting the p38 and Jun pathways of the MAPK pathway,thereby alleviating MG-induced inflammatory damage.In conclusion,GA can effectively inhibit the adhesion of MG to host cells and tissues,and alleviate the inflammatory damage induced by MG infection via inhibiting the MAPK/p38/Jun signaling pathway,with therapeutic effects comparable to those of tiamulin,providing theoretical support for the clinical application of GA as an antibiotic alternative in the prevention and treatment of CRD in production practice. |
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