| Mycoplasma gallisepticum(MG)is a group of pathogenic microorganisms commonly found in chickens,ducks,and other birds.MG infection can cause chronic respiratory system diseases in chickens,as well as reduce the immunity of the organism,and easily mix with other pathogens,leading to increased mortality in chickens.Since MG has no cell wall,it is usually controlled with macrolide and quinolone antibiotics,but in recent years,new control methods need to be found due to the increase of drug resistance of MG.Baicalin,the active component of the Chinese herbal medicine Scutellaria baicalensis,has obvious anti-inflammatory,anti-microbial and antioxidant effects and plays an important role in the clinical treatment of MG infection.Macrophages can be polarized into M1 and M2 macrophages after stimulation,which exert pro-inflammatory and anti-inflammatory effects respectively,and the dynamic change of M1/M2 is always throughout the process of disease occurrence and development.Previous studies of our research group have proved that baicalin can alleviate inflammatory damage caused by MG infection,but the effect and mechanism of MG infection on chicken macrophages are still unclear.Therefore,this study started from macrophage polarization and adopted optical microscope,transmission electron microscope,ELISA,RT-QPCR,Western blot and other experimental methods.To investigate the role and mechanism of baicalin in the polarization of chicken macrophages induced by MG infection,and to provide a new idea and direction for the prevention and treatment of MG with Traditional Chinese medicine.(1)Effect of MG infection on the polarization of HD11 cellsIn this study,chicken macrophages(HD11)were selected for in vitro experiments.The effect of MG(1×10~9 CCU/m L)on HD11 cell viability was determined by the CCK-8 method.400 MOI was selected as the infection dose,and cell samples were collected from the normal control group(NC group)and MG-infected group(MG group)at 4,6,8 and 10 hours of MG infection of HD11cells and subsequent experiments were performed.The results of optical microscopy showed that the morphology and number of HD11 cells in the NC group did not change significantly,and the number of M1 macrophages increased at 4 and 6 hours of MG infection;the number of M2macrophages increased at 8 and 10 hours of MG infection.Under transmission electron microscopy,it was observed that at 6 hours of MG infection,the cell membrane was folded,vacuoles appeared in the cytoplasm,mitochondria were swollen,mitochondria were vacuolated,and chromatin was condensed;at 10 hours of MG infection,the cell membrane was ruptured,vacuoles in the cytoplasm were increased and enlarged,and some mitochondria were shrunk.Compared with the NC group,the IL-6 content in the cell supernatant of the MG group increased significantly at 4,6,8,and 10hours of infection(p<0.05),and the IL-10 content increased significantly at 10 hours of infection(p<0.05).The m RNA expression levels of i NOS,TNF-α,and IL-6 in the cells of the MG group increased significantly at 4 and 6 hours of infection(p<0.05),and the m RNA expression levels of IL-10,TGF-β,and VEGFA in cells were significantly increased at 10 hours of infection(p<0.05);the expression of i NOS and TNF-αproteins in cells of MG group were significantly increased at 4and 6 hours of infection(p<0.05).(2)Effect of baicalin on polarization of HD11 cells caused by MG infectionThe optimal intervention time and concentration of baicalin in M1 macrophage polarization model were measured by CCK-8 method for 6 hours and 150μg/m L,respectively.the experiment was divided into normal control group(NC group),MG infection group(MG group),baicalin control group(BC group),and baicalin intervention group(BI group),and the cell samples of each group were collected for subsequent experiments.The results showed that the number of M1macrophages in HD11 cells was reduced in the BI group compared with the MG group;cell pseudopods were reduced,cytoplasmic vacuoles were reduced,and the number of mitochondrial swelling was decreased;the levels of IL-6 and TNF-αin the cell supernatant were significantly reduced(p<0.05)and IL-10 was significantly increased(p<0.05);in the cells i NOS,TNF-α,and IL-6 m RNA expression levels were significantly decreased(p<0.05),IL-10,TGF-β,VEGFA m RNA expression levels were significantly increased(p<0.05);TLR4,P38(MAPK),P65(NF-κB)m RNA expression levels were significantly decreased(p<0.05),TLR4,p-P38/P38,p-P65/P65 protein expression was significantly decreased(p<0.05);TNF-αand i NOS protein expression in cells was significantly decreased(p<0.05).(3)Effect of MG infection on the polarization of lung macrophagesIn this study,7-day-old White Leghorns chickens were selected for in vivo experiments.Sixty chickens were randomly divided into two groups:the normal control group(NC group)and the MG-infected group(MG group),and lung tissues were taken by dissection at 3,5 and 7 day after MG infection.Compared with the NC group,at 3 day of MG infection,some alveoli in the lungs were ruptured and a small amount of bruising was present,at 5 and 7 day,most alveoli in the lungs were ruptured and inflammatory cells were infiltrated;at 5 day of MG infection,cells in the lungs had shrunken mitochondria,malformed nuclei,and some cells were broken,at 7 day,there were more broken cells,lysed contents,and nuclear sequestration;IL-6 content was significantly increased at3 and 5 day of MG infection(p<0.05),and the amount of IL-10 content was significantly increased at 7 day(p<0.05).The m RNA expression levels of i NOS,TNF-α,and IL-6 in the MG group were significantly increased at 3 and 5 day(p<0.05),and the m RNA expression levels of IL-10,TGF-β,and VEGFA m RNA expression levels were significantly increased at 7 day of infection(p<0.05);TNF-αand i NOS protein expressions were significantly increased at 3 and 5 day in lung tissues of the MG group(p<0.05).(4)Effect of baicalin on the macrophage polarization in chicken lungs caused by MG infectionSixty White Leghorns chickens were randomly divided into four groups:normal control group(NC group),MG-infected group(MG group),baicalein control group(BC group),baicalein intervention group(BI group).It was found that compared with the MG-infected group,alveolar structure was intact and inflammatory cell infiltration was reduced in the BI group chickens;cytoplasmic vacuoles were reduced in lung cells,nuclei were slightly restored,and only some cells were fragmented;IL-6 and TNF-αlevels were significantly reduced(p<0.05)and IL-10 levels were significantly increased(p<0.05);i NOS,TNF-α,IL-6 m RNA expression levels were significantly reduced(p<0.05),and the m RNA expression levels of IL-10,TGF-β,and VEGFA were significantly increased(p<0.05);the m RNA expression levels of TLR4,P38,and P65 were significantly reduced(p<0.05),and TLR4,p-P38/P38,and p-P65/P65 protein expression was significantly reduced(p<0.05);TNF-α,i NOS protein expression was significantly reduced in cells(p<0.05).In summary,MG infection induced polarization of HD11 cells and chicken lung macrophages,with HD11 cells mainly undergoing M1 polarization at 6 hours of MG infection and M2 polarization at 10 hours of MG infection;macrophages in the lungs mainly underwent M1 polarization at 5 day of MG infection in chicks and M2 polarization at 7 day.Baicalin could alleviate the M1 polarization of macrophages and promote the M2 polarization of macrophages by inhibiting TLR4-MAPK/NF-кB signaling pathway,and reduce the inflammatory damage caused by MG.This study opens up new ideas and directions for the treatment protocol of MG infection,and also provides a theoretical basis for baicalin in the clinical treatment of MG infection. |
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