| Porcine Extraintestinal pathogenic Escherichia coli(Ex PEC)is a class of pathogenic bacteria that can cause a variety of organ and tissue lesions outside the intestinal tract.It often infects pigs mixed with other pathogens,causing meningitis,pneumonia,arthritis and sepsis in pigs.In China,porcine Ex PEC is characterized by high separation rate,wide drug resistance and great zoonotic potential,which lead to difficult antibiotic treatment and high mortality rate of sick pigs,and cause huge economic losses to breeding industry and endangering human public safety.This experiment initially tested the effect of baicalin on the growth characteristics of porcine Ex PEC PCN033 and its role in vitro infection of porcine Ex PEC PCN033 with host cells were investigated.The protective effect of baicalin on porcine Ex PEC PCN033infected mice was further investigated by comparing survival rate,weight change,clinical lesion,tissue bacteria load and histopathological changes of mice.Finally,the activation of NF-κB,MAPK signaling pathway and NLRP3 inflammasome in porcine Ex PEC PCN033infection host as well as the regulatory mechanism of baicalin were explored.1.Protective effect of baicalin on porcine Ex PEC infected cells.In this study,the minimum inhibitory concentration and growth characteristics of baicalin on porcine Ex PEC PCN033 were determined to determine whether baicalin had an inhibitory effect on the growth of porcine Ex PEC PCN033 and the optimal concentration of baicalin.Secondly,the ability of porcine Ex PEC PCN033 to attach to and invade porcine small intestine epithelial cells(IPEC-J2)and porcine kidney epithelial cells(PK-15)and proliferate in porcine alveolar macrophages(3D4/21)was determined to further determine whether baicalin had a protective effect on porcine Ex PEC PCN033 invasion of host cells.Finally,lactate dehydrogenase test(LDH)was used to determine whether baicalin had a protective effect on porcine Ex PEC PCN033 mediated cell damage.The results showed that the minimum inhibitory concentration of baicalin on porcine Ex PEC PCN033 was over 1600μg/m L.In addition,baicalin concentration of 25,50,100μg/m L had no effect on the growth characteristics of porcine Ex PEC PCN033,and had no effect on the proliferation of bacteria in host cells.However,baicalin can inhibit the adhesion and invasion of porcine Ex PEC PCN033 to host cells and protect the cell damage caused by porcine Ex PEC PCN033.2.Protective effect of baicalin on mice infected with porcine Ex PEC.Four-week-old female Kunming mice were randomly grouped.Porcine Ex PEC PCN033 infection was established by intraperitoneal injection of mice in the treatment group and the infection group.Each mouse was infected with 1×10~7 CFU PCN033.Mice in the treatment group were injected subcutaneously with 50 mg/kg baicalin at multiple points.After the first administration,baicalin was administered twice a day with an interval of 6 h until the day of autopsy.After challenge,the clinical status,weight change and death of mice were observed and recorded.After the 6 h challenge,the mice were anesthetized,and the heart,liver,spleen,lung,kidney,brain and blood were extracted,fully ground and diluted to record the bacterial load in each tissue.At the same time,the tissues were soaked in 4%paraformaldehyde and fixed for pathological section observation.The results showed that after infection,the mice were depressed,with decreased feed intake and body weight in both the infected group and the treatment group,besides the mortality and death rate of mice in the infection group were higher than those in the treatment group.After autopsy,there were no obvious lesions in the blank control group,and different degrees of lesions appeared in both the infection group and the treatment group,but the degree of lesions in the treatment group was relieved compared with that in the infection group.The histopathological results showed that the degree of pathology changes in the treatment group was less than that in the infection group.The results of tissue bacterial load showed that the amount of bacteria in blood and tissues of the treatment group was significantly lower than that of the infection group.3.Effect of baicalin on activation of host inflammatory signaling pathway by porcine Ex PEC.Firstly,cytotoxicity of baicalin on 3D4/21 was preliminary investigated through cytotoxicity test,so as to select the optimal drug concentration for subsequent tests.Secondly,the gene expression level of inflammatory genes IL-1β,IL-6,IL-8 and nuclear transcription factors AP-1(c-jun and c-fos)were determined by Q-RT PCR after 3 h co-culture.The release of inflammatory factors IL-1β,IL-6 and IL-8 in 3 h was determined by ELISA.The expression levels of NF-κB signaling pathway protein(P65,p-P65,IκBα,p-IκBα)and NLRP3 inflammasome protein(NLRP3,ASC,caspase-1)in cells and mice were determined by Western blot.And the expression levels of MAPK signaling pathway proteins(JNK,p-JNK,ERK,p-ERK,P38,p-P38)in cells were determined by Western blot.Cell test groups were divided into blank control group,PCN033 modeling group and PCN033+baicalin treatment group(25,50,100μg/m L)and mouse test groups were divided into blank control group,PCN033 modeling group and 50mg/kg baicalin treatment group,respectively.The cells were treated with baicalin for 1 h and then co-cultured with 10:1 MOI.The infection method of mice was the same as experiment 2,and the infection volume was 5×10~6 CFU.The results of cytotoxicity test showed that baicalin treate d with 25,50,100 and 200μg/m L had no obvious toxicity to cells.Therefore,baicalin was selected 25,50,100μg/m L for subsequent cell tests.Q-RT PCR results showed that the transcription levels of IL-1β,IL-6 and IL-8 were significantly up-regulated after porcine Ex PEC PCN033 infection with 3D4/21,and baicalin could significantly inhibit the transcription levels of these inflammatory factors.Moreover the transcription level of nuclear transcription factors c-jun and c-fos were extremely significantly up-regulated after porcine Ex PEC PCN033 infection with 3D4/21,and the transcription level of nuclear transcription factors was significantly inhibited by baicalin.The ELISA results showed that the release levels of IL-1β,IL-6 and IL-8 were significantly up-regulated after porcine Ex PEC PCN033 infection with 3D4/21,and the release levels of these inflammatory factors can be significantly inhibited after treatment with baicalin.Western blot results showed that the protein expression levels of p-P65,p-IκBα,p-JNK,p-ERK,p-P38,NLRP3,ASC and caspase-1 were significantly up-regulated after infection of porcine Ex PEC PCN033 with host cells.The expression levels of above group proteins were significantly down-regulated in baicalin treatment.The protein expression levels of p-P65,p-IκBα,NLRP3,ASC and caspase-1 were significantly up-regulated after infection of porcine Ex PEC PCN033 with mice,while the expression levels of above proteins were significantly down-regulated in 50 mg/kg baicalin treatment group.In conclusion,under the premise of not affecting the growth and cellular proliferation of porcine Ex PEC PCN033,baicalin down-regulated the expression of host NF-κB and MAPK signaling pathways,inhibited the activation of NLRP3 inflammasome,reduced the cell damage and pathogenicity caused by porcine Ex PEC PCN033 infection,and played a protective role in vitro and in vivo.The experimental results provide a scientific basis for the use of baicalin in the treatment of porcine Ex PEC infection. |
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