| Mycoplasma gallisepticum(MG)can cause chronic respiratory diseases in chickens,and has a high infection rate.It will lead to lower wei ght,dropping egg production and reduce the hatching rate of breeding eggs,increase the elimination rate of chicks,be susceptible to mixed infection with E.coli,Newcastle disease,and increase the case fatality rate,causing huge economic losses to the poultry industry.Therefore,it is particularly important to develop drugs for treating MG infection.This topic is based on the principle of dialectical treatment of traditional Chinese veterinarians,and through the pre-screening of prescriptions,it is determined that five kinds of Chinese herbal medicines of Scutellaria baicalensis,Indigowoad Root,Herbal Ephedrae,Stemonae Radix and Asteris radix et rhizome are made into a compound oral liquid(named Qinlan oral liquid)Play the role of clearing away heat and detoxification,antibacterial and anti-inflammatory,moisturizing lungs and cough.This study intends to comprehensively optimize the extraction process,alcohol precipitation process,water precipitation process,and molding process of Qinlan oral liquid,and at the same time,conduct a signal mechanism study on its anti-MG infection based on the determination of drug efficacy.The results are as follows:(1)In the extraction process,the baicalin absorption peak area and solid yield were used as detection indicators.A single factor investigation was combined with the L9(34)orthogonal test for comprehensive evaluation.The best extraction process was selected to determine the extraction process of the product:20-fold amount of water is used to cook the ingredients in the prescription twice for 1 hour each time at 95℃.The decoction was collected twice,dried under reduced pressure and concentrated to a relative density between 1.05 and 1.15.In the alcohol precipitation process,the baicalin absorption peak area and solid yield were used as the detection indicators.A single factor inspection was combined with the L9(34)orthogonal test for comprehensive evaluation.Concentrate in the box at 70℃under reduced pressure to a relative density of 1.10,alcohol precipitation concentration of 70%,alcohol precipitation for 15 h,remove the precipitate and recover excess ethanol.In the process of water sedimentation,the clarity of the solution was taken as the inspection index,and L9(34)orthogonal test was used to determine the water-sinking process as follows:the recovered ethanol solution is placed in a vacuum drying oven at 70°C and reduced to a relative density of 1.10,add water to 50%of the total,and sink for 48 h.In the molding test,the pH value and preservative of the solution were investigated.Finally,the following results were selected:pH was 4.0～5.5,and 0.02%Methyl Paraben was added as preservative.(2)80 1-day-old chickens in good condition were randomly divided into four groups,20 in each group,which A were blank control group,drug group,MG i nfection group and qinlan oral liquid treatment group.The chicks were fed to 7 days old.Each chick in the qinlan oral liquid treatment group and the MG infection group was injected with 0.2 m L Mycoplasma gallisepticum with a concentration of 1×10~9 CCU/m L in the left side air bag,and the blank control group and drug group were injected with 0.2 m L of normal saline.On the third day after infection,the drug group and qinlan oral liquid treatment group were mixed to feed qinlan oral liquid for 7consecutive days,once in the morning and once in the evening.At the end of the administration,the thymus was dissected.It was found by light microscope that after MG infection that the thymus lobule shrank obviously,the interval between lobules increased,t he boundary between cortex and medulla was not clear,the number of lymphocytes decreased,and the arrangement was sparse,the epithelial cells were exfoliated.Compared with MG infection group,the pathological observation of qinlan oral liquid treatment group was improved,while that of drug group and blank control group was no significantly affected.The results showed that the drug group did not cause damage to chickens,and qinlan oral liquid could reduce the pathological damage caused by MG infection.Based on the effect of qinlan oral liquid on MG infection through IL-17 pathway,RT-PCR results showed that compared with the blank control group,the m RNA expression levels of IL-17C,MMP1,and CXCL1 in the thymus of the MG infection group were no signi ficant difference(P>0.05).It can be seen from the MG infection group’s m RNA expression levels of IL-17A,IL-17B,IL-17F,CXCL2,AP-1,NF-kB,CIKS,GM-CSF were significantly compared with the blank control group(P<0.01),the qinlan oral liquid treatment group has a very significant difference compared with the MG infection group(P<0.01).From the m RNA expression levels of C/EBPband MMP3,the difference between the attack group and the blank control group is significant(P<0.05),there was also a significant difference between the qinlan oral liquid treatment group and the MG infection group(P<0.05).It is worth noting that the m RNA expression level of MUC5AC was significantly different in the MG infection group compared with the blank control group(P<0.01),and the difference between the qinlan oral liquid treatment group and the MG infection group was very significant(P<0.01),indicating that the Qinlan Oral liquid played a significant role in it.ELISA results showed that compared with the blank control group,the protein expression levels of CXCL1,CXCL2,and MMP1 in the thymus of the MG infection group were significantly increased(P<0.05),and the expression levels of MUC5AC protein were significantly increased(P<0.01);Compared with the blank control group,there was no significant difference in the protein expression levels of CXCL1,CXCL2,MMP1,and MUC5AC in the thymus of the drug group(P>0.05).Compared with the MG infection group,the levels of CXCL1,CXCL2,MMP1,and MUC5AC in the thymus of the qinlan oral liquid treatment group were significantly different(P<0.05),indicating that Qinlan Oral Liquid can inhibit the overexpression of CXCL1,CXCL2,MMP1,and MUC5AC proteins caused by MG infection.In summary,this study optimizes a series of extraction,alcohol precipitation,water sedimentation,and molding processes of Qinlan Oral Liquid and proves that Qinlan Oral Liquid can exert anti-MG infection treatment effects by inhibiting related factors on the IL-17 pathway. |
