| Enterococcus faecalis is one of the important components of normal flora in humans and animals.With the continuous use of antibiotics,the resistance of Enterococcus faecalis has also been improved,becoming an opportunistic pathogen and causing human and animal infectivity.Therefore,the occurrence of these diseases threatens human health and affects the development of animal husbandry.Nowadays,cases of meningitis caused by Enterococcus faecalis infection have increased year by year,but there is little report on how Enterococcusfaecalis can pass through the blood-brain barrier with selective permeability to the brain and cause damage to brain tissue.Therefore,based on the preliminary work,this study selected two strains of Enterococcus faecalis,namely XJ3 and XJ8 isolated from the brain tissue of lambs with meningitis as the research objects,and explored its specific damage to brain tissue and its mechanism of action on p38MAPK signaling pathways,which aims to provide reference for further studies.Purposes:(1)To established 2 brain models of mice infected with Enterococcus faecalis and evaluated.(2)The expression levels of AQP4,GFAP,MMP-2 and Claudin-5 in blood-brain barrier of mice were detected,and the damage caused by two strains of Enterococcus faecalis to blood-brain barrier was analyzed.(3)To study the changes of SOD and MDA in the oxidative stress response of the 2 strains of Enterococcus faecalis in mice,and to determine the damage of Enterococcus faecalis to brain tissue.(4)To study the correlation between the Enterococcus faecalis and the p38MAPK signaling pathway in the brain of mice,and to lay the foundation for studying the infection mechanism of Enterococcus faecalis crossing the blood-brain barrier.Methods:(1)The median lethal dose(LD50)was calculated by the Karber method,and the mice were infected at a dose of 2/3 LD50.The condition of the mice was observed,and Evans blue was injected at the tail 2 hours before sampling.The sampling time was taken at 2 h,8 h,12 h,24 h,36 h,48 h,60 h and 72 h.After sampling,frozen sections were made and the distribution of Evans blue in brain tissue was observed under laser confocal microscope;(2)In the experiment,110 male and female mice were randomly divided into XJ3 strain group(22),XJ8 strain group(22)and blank control group(6).The model of brain injury in mice was established by using 2/3 LD50 dose of each strain,and samples were taken at 8 time points of 2 h,8 h,12 h,24 h,36 h,48 h,60 h and 72 h.The qRT-PCR and immunohistochemistry methods were used to detect of changes in gene levels and protein levels of AQP4,GFAP,MMP-2 and Claudin-5;(3)The mouse brain injury model was established by the 2/3 LD50 dose of each strain,and the blood samples of the mouse heart were separated at the corresponding time points.The ELISA method was used to detect the activity of SOD and the content of MDA in serum of mice infected with different strains;(4)The mouse model of blood-brain barrier injury was established by using 2/3 LD50 of each strain as the infection dose.The samples were taken at the corresponding time points,and the expression of phosphorylated p38MAPK in the brain tissue of the two strains was detected by Western Bloting method.Results:(1)XJ3 LD50=3.12×109 CUF,XJ8 LD50=2.53×1010 CUF,the mortality rate of mice infected with 2/3 LD50 was 24.0%and 8.0%,respectively.Both strains of Enterococcus faecalis could open the blood-brain barrier of mice,and the amount of EB in brain tissue was higher than that of the blank control group,and the time change trend was the same,both peaked at 24 h and 48 h.There were significant differences between the two strains in each group and the blank group,and the time difference between the two strain groups was significant(P<0.05).(2)The expression levels of AQP4,GFAP and MMP-2 were higher in the 2 strains than in the control group,and reached the maximum at 24 h and higher expression at 48 h.The expression of Claudin-5 was lower than that of the control group,the expression level was the lowest at 24 h,and the expression at 48 h was slightly lower.The comparison between the two strains and the comparison between the groups were significant(P<0.05).(3)After the two strains were infected with mice,the activity of SOD in serum and the content of MDA were significantly increased in the time points of each group,and the difference was statistically significant(P<0.05),at the same time,the changes were obvious at 24 h,and the changes at 48 h were more obvious;the 2 strains were compared between the two groups at each time point.It was found that the changes of SOD and MDA of XJ3 strain were higher than those of XJ8 strain.(4)After the 2 strains were infected with mice,the p38MAPK signaling pathway was activated to the form of p-p38.By detecting the protein expression of p-p38,it was found that there was no expression of p-p38 in the blank control group.The protein expression was higher than that of the blank control group,and there was significant difference(P<0.05),and reached the peak at 24 h,and had a higher expression at 48 h;Similarly,significant differences(P<0.05)were found between the two strain groups when comparing the two groups.Conclusions:(1)The fluorescence of Evans Blue can be used to visually observe the openness of the blood-brain barrier,indicating that the two strains were successfully infected with a 2/3 LD50 dose.(2)Both strains of Enterococcus faecalis can damage the structure and function of the blood-brain barrier of mice,impairing their structure and increasing permeability.(3)Enterococcus faecalis with different pathogenicity and different virulence factors can cause oxidative stress in the brain,thereby aggravating the damage of the blood-brain barrier.(4)Enterococcusfaecalis can affect the brain through the blood-brain barrier and is directly related to the activation of the p38 MAPK signaling pathway. |
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