| Mycoplasma gallisepticum(MG)is a kind of pathogenic microorganism that is widely distributed in the world and can cause respiratory diseases in poultry.After being infected with MG,the utilization rate of feed by poultry is usually reduced,the weight of broilers is reduced,the elimination rate of chicks is increased,and the egg production rate of adult hens is reduced,which brings huge economic losses to the poultry industry in various countries.Because there is no cell wall in the structure of mycoplasma,the antibiotics acting on the cell wall have no effect on the treatment of MG,narrowing the scope of antibiotics that can treat this disease,and the abuse of antibiotics and other issues,making drug development for this disease extremely important.Starting from the main pathogenesis of MG,the traditional Chinese medicine group for the treatment of this disease is based on the righting and eliminating the evil,in order to achieve the goal of both symptoms and cure,which is currently the hot direction of MG prevention and treatment drugs.The curative effect has been well exerted in clinical practice,and it is the hot research direction of MG prevention and treatment drugs.This article selects Chinese medicines that have been proven by modern medical research to have a significant therapeutic effect on infectious diseases,11 kinds of traditional Chinese medicines,such as Scutellaria,isatis root,aster,Stemona,liquorice,etc.,were selected by the method of double dilution,Finally,it was determined that the extract of Scutellaria baicalensis Georgi 35 g,Isatis indigotica 35 g,aster 20 g,Stemona 20 g and liquorice 20 g was prepared and named as "Qinlan extract" with two main drugs for further research and application.Infected with the chicken MG infection model established in previous studies.According to the characteristics of its disease development,the Qinland extract was mixed with water for feeding treatment on the 3rd day after infection.The test was divided into high,medium and low dose groups(concentrations were 3.0,1.5,and 0.5 g / m L),the doses were 3.0,1.5,and 0.75 g / d,which were administered continuously for 7 days.Tylosin was used as a positive drug control group,and body weight records and feed records were continuously performed to calculate the average weight gain and feed-to-meat ratio of each group of chickens during feeding and treatment.The trachea and lungs were dissected at 1,3,and 7 days after treatment with drinking water.The molecular biological techniques such as ELISA,TUNEL green fluorescence,transmission scanning electron microscopy,and Western Blot,Real Time-PCR were used to explore the extract of Qinlan,Effect on the expression of apoptosis-related genes caused by MG infection.Histopathological observations showed that: The gross airbag lesion score of pathological clinical manifestations showed that the tissue damage of target organs could be reduced after 7 days of treatment with Qinlan extract mixed with water,and the degree of lesions was significantly reduced,and there were statistical differences.Transmission electron microscope observation of the lung and scanning electron microscope observation of the ultrastructure of tracheal cells were visible.Scanning electron microscope observation of the tracheal site of the MG infection group showed that the ciliated cells contracted and degenerated,and fell off and died.Transmission electron microscopy showed that alveolar type I epithelial cells were severely damaged,the cytoplasm was broken,and the cells showed significant apoptosis in the lung tissue.In the treatment group of Qinlan extract,the cilia were swollen,mushroom-shaped edema formed at the top,and the cilia were fused together.The alveolar type I epithelial cell injury and apoptosis were alleviated in a dose-dependent manner.It can be seen that the treatment test of Qinlan extract can effectively improve the damage and apoptosis caused by MG infection.The expression results of oxidative stress-related factors and apoptosis-related proteins show that: On the 7th day of treatment with Qinlan extract,the expression levels of SOD,GSH-PX,and CAT increased significantly(p<0.05)and the expression levels of MDA decreased significantly(p<0.05)compared with the MG infection group,the increase and decrease are concentrationdependent.Western Blot test results showed that the levels of pro-apoptotic proteins Caspase-3,Caspase-9,Bax and Cycle-C in the model group were significantly increased(p<0.05),and the levels of anti-apoptotic protein Bcl-2 were significantly decreased(p<0.05).At this time,the m RNA expression of Caspase-3,Caspase-9,Bax and Cycle-C also showed a significant increase(p<0.05),and the expression of Bcl-2 m RNA showed a significant decrease(p<0.05).Compared with the MG infection group,the expression of proapoptotic factors Caspase-3,Caspase-9,Bax and Cycle-C decreased significantly after 1,3,and 7 days of treatment with Qinlan extract treatment group(p <0.05)The expression of anti-apoptotic factor Bcl-2 was significantly increased(p<0.05)and the effect was concentration-dependent.And the corresponding pro-apoptotic proteins Caspase-3,Caspase-9,Bax and Cycle-C were significantly lower than the MG infection group(p<0.05),and the content of anti-apoptotic protein Bcl-2 was significantly higher than the MG infection group(p<0.05),and the same effect was concentration-dependent.The results showed that the high-dose group(concentration:3.0 g/m L)of Qinlan extract on day 7 of treatment had the best treatment effect.In the model test of MG-induced HD11 cell damage,the optimal concentration range of Qinlan extract for cells was 15?30 mg / m L,and it had no significant toxicity to it.The optimal action time was 12 hours;After MG stimulated HD11 cells,the m RNA expression levels of pro-apoptotic cytokines Caspase-3,Caspase-9,and Bax were significantly increased in cell culture(p<0.05),and the m RNA expression levels of anti-apoptotic cytokines Bcl-2 Were significantly reduced(p<0.05),when the extract of Qinlan extract was applied to MG-treated HD11 cells,the m RNA expression levels of the above pro-apoptotic cytokines were significantly down-regulated and concentrationdependent compared to the MG group(p<0.05).The m RNA expression level of anti-apoptotic cytokine Bcl-2 was significantly up-regulated and concentration-dependent(p<0.05)compared with the MG group.In the detection of enzyme activity,the enzymatic activities of pro-apoptotic cytokines Caspase-3 and Caspase-9 in the group of HD11 cells treated with MG significantly increased.After MG-treated HD11 cells were treated with Qinlan extract,the apoptosis cytokines Caspase-3.Caspase-9's enzyme activity was significantly down-regulated and concentrationdependent.By TUNEL fluorescence staining,it can be seen that compared with the MG group,the treatment group of Qinlan extract significantly reduced the apoptosis of HD11 cells in a concentration-dependent manner.In summary,this test was based on the successful screening of the Chinese herbal medicine prescription Qinlan extract,the MG R strain bacterial solution(0.2m L/piece)was used to infect 7-day-old chicks by balloon inoculation to establish MG infection model,and study the mechanism and mechanism of Qinlan extract against injury and apoptosis caused by MG infection.Experiments have shown that Qinlan extract can reduce the damage caused by MG-induced oxidative stress by increasing the expression of oxidative stress factors SOD,GSH-PX,and CAT,and inhibiting the expression of MDA.Meanwhile,Qinlan extract can inhibit the release of apoptosis factors Caspase-3,Caspase-9,Bax,and Cycle-C and increase the release of Bcl-2 in MG infection in vivo and in vitro tests,which can interfere with MG infection and cause apoptosis. |
PDF Full Text Request