The Molecular Mechanism Of Baicalin In Interfering With Inflammatory Injury Caused By Mycoplasma Gallisepticum

Mycoplasma gallisepticum(MG)is a kind of pathogenic microorganism that is widely distributed in the world and can cause respiratory diseases in poultry.It belongs to the genus Mycoplasma.The positive rate of infection is extremely high,and the mortality rate is low without co-infection.After MG infection,the feed utilization rate is generally reduced,the weight of the broiler is decreased,and the culling rate of chicks are increased.These problems have brought a great deal of economic losses to the poultry industry.The outbreak of the disease is often caused by clinically mixed infections with other diseases or poor feeding and management,environmental mutations etc.,which is extremely harmful to the poultry breeding industry.The antibiotics which acting on the cell wall have no obvious effect on the treatment of this disease,because mycoplasma has no cell wall.The drug development of the disease becomes particularly important with the problems of the limited choice and the unreasonable use of antibiotics.Chinese medicine treatment program is based on the main pathogenesis of MG,to achieve the purpose of treating both the symptoms and the cause.Among them,baicalin is an effective active ingredient extracted from the root of traditional Chinese medicine Astragalus membranaceus,and the research on this medicine attaches great importance.Researches in modern medicine show that baicalin has obvious anti-inflammatory effects and can effectively inhibit the occurrence of inflammatory reactions.The study on the action and mechanism of baicalin can promote,further development of the drug and its application in the treatment of MG infection.In this study,four widely-accepted assessment methods(SPA score,Gross's air sac lesion score,tracheal lesion score,and tracheal mucosal thickness)were used to evaluate four different infection models with parallel comparison.The results showed that infection of a 7-day-old chick with 5×10~9CCU/ml MG R strain inoculated with a air sac developed the typical clinical symptoms of MG infection at 5 days after infection.The progression of the disease was moderate.So,it is suitable for follow-up treatment trials.In the treatment trial of baicalin,a well-established model construction method was used.According to the characteristics of the course of the disease,treatment was started after 3 days of infection,and the dose was 1.5 g/day for 7 days.Samples were taken at 1,4,and 7 days after treatment.Advanced molecular biology techniques such as ELISA,transmission electron microscopy,Western Blot,and Real Time-PCR were used to investigate the expression of inflammation related genes caused by baicalin on MG infection and to discuss the role of p65 protein in the TLR2/NF-?B pathway.Pathological and ultra-structural observations:The gross air sac lesion score,tracheal lesion score,and SPA score,which were clinically manifested by pathology,were found to reduce the tissue damage and lesions of the target organ after treatment with baicalin mixed water for 7 days.The degree was significantly reduced and there were statistical differences.Using transmission electron microscopy to observe the ultrastructure of lung and tracheal cells,it can be seen that the cilia cells in the MG-infected group have pyknosis,degeneration,and necrosis.In lung tissue,alveolar type I epithelial cells were severely damaged and the cytoplasm was broken.In the baicalin-treated group,cilia swelled,mushroom-shaped edema bubbles formed on the apex,and cilia fused together.Alveolar type I epithelial cell injury was significantly reduced.This shows that the baicalin treatment test can effectively improve the inflammatory tissue and cell damage caused by MG infection.Inflammation-related factors and p65 protein expression results:4 days after challenge,MG infection group compared with the blank control group by ELISA kit detection of inflammatory cytokines IL-1?,TNF-?expression was significantly increased;Western Blot detection of TLR2and P-p65 protein content was significantly increased(P<0.05)and reached peak at 7 day after challenge.At this time,the expression levels of IL-1?and TNF-?mRNA also showed an obvious increase;10 days after the challenge,the expression of inflammatory factors and related proteins decreased.Compared with the MG-infected group,the levels of inflammatory cytokines IL-1?and TNF-?were significantly decreased(P<0.01)and the corresponding proteins TLR2 and P-p65 were expressed in the baicalin-treated group after 1,4 and 7 days,respectively.The amount was also significantly lower than the MG infection group.The results of this experiment showed that the establishment of a model for infecting 7-day-old chickens with air sac inoculum at a concentration of 5×10~9 CCU/ml of MG R strain 0.2 ml was suitable for the study of the mechanism of drug action.Baicalin can interfere with inflammatory injury caused by MG infection by inhibiting the release of inflammatory cytokines IL-1?and TNF-?during MG infection.At the same time,baicalin can also interfere with the activation of TLR2-NF?B signaling pathway by inhibiting the phosphorylation of p65 protein,thereby affecting the expression of inflammatory factors.