The dissertation presents an experimental research on the pathogenic mechanism of meningitis of the streptococcal infection of pigs based on study of the pathogenetic processes of pyroptosis through the expression of mRAN of pyroptosis-related genes and proteins and changes in morphologyof endothelial cells of brain microvessels of white mice infected with Streptococcus suis serotype 2.
Streptococcus suis (S. suis) is an important zoonotic pathogen that can cause many diseases in pigs, such as sepsis, arthritis, endocarditis and meningitis, of which meningitis is the most serious. There are 35 serotypes of, and serotype 2 is the most virulent.
This paper described several common virulence factors, such as CPS, SLY, MRP, EF, SAO, Srt, FBPS, SadP and Eno.
In this study, S. suis 2 was used to infect mouse brain microvascular endothelial cell (bEnd.3). First, the infection conditions were screened. The results showed that the optimal infection number was 100:1 and the optimal infection time was 12 hours. Brain microvascular endothelial cells were divided into 4 groups: control group, LPS+ Adenosine triphosphate (ATP) infection group, S. suis 2 infection group and S. suis 2+ Ac-YVAD-CMK (CMK, caspase-1 inhibitor) infection group. The following three groups were infected with Streptococcus suis type 2 according to the above conditions, and multiple replicates were performed simultaneously. After 12 hours, the cells and cell supernatants were collected for different tests. Total RNA was extracted, RNA concentration was measured, and cDNA was obtained by reverse transcription. cDNA was detected by qPCR for mRNA expression of cytokines. Compared with the control group, the mRNA expression levels of caspase-1, il-18 and il-1beta in LPS+ATP group and S. suis 2 group were higher than those in the control group, indicating that a large number of cytokines related to pyroptosis were secreted. At the same time, mRNA expression levels of caspase-1, il-18 and il-1beta in S. suis 2 +CMK group were significantly lower than those in S. suis 2 +CMK group, indicating that CMK inhibitor played an inhibitory role. At the same time, protein was extracted from the collected cells, the protein concentration was measured, and then WB test was carried out to detect the protein expression level of related genes. Compared with the control group, the protein expressions of IL-18, IL-1β,caspase-1,GSDMD and GSDME in LPS+ATP group were significantly up-regulated, increasing by 0.477, 0.088, 0.378, 1.118 and 3.05 times, respectively. The protein expressions of IL-18, IL-1β,caspase-1,GSDMD and GSDME in S. SUIS 2 group were significantly up-regulated, increasing by 1.024, 0.066, 0.376, 0.453 and 1.654 times, respectively. Compared with S. SUIS 2 group, protein expression in S. suis 2+CMK group was significantly decreased by 0.6, 0.396, 0.298, 0.743 and 0.586 times, respectively. The results showed that S. suis 2 infection of brain microvascular endothelial cells caused intense inflammatory reaction and even pyroptosis of cells. At the same time, the inhibitor CMK played a good inhibitory effect. In addition, the protein content of IL-6, IL-10, IL-18, IL-1beta, caspase-1 in the cell culture supernatant was also detected.
Compared with control group, the relative expression of TNF-α, IL-6, IL-10, IL-18, IL-1β in cell supernatant of SS group and LPS+ATP group was significantly increased, and the difference was statistically significant (P < 0.01). Compared with S. suis 2 group and LPS+ATP group, the relative expression level of TNF-α, IL-6, IL-10, IL-18, IL-1βin S. suis 2 + CMK group was significantly decreased, and the difference was statistically significant (P < 0.01), indicating that coke death caused by S. suis 2 and LPS+ATP could be inhibited by CMK. At the same time, At the same time, the release rate of lactate dehydrogenase in the cell supernatant was also measured, and the results showed that the release rate of lactate dehydrogenase in LPS+ATP group and S. suis 2 group was much higher than that in the control group, and the difference was extremely significant. In S. suis 2 + CMK group, the release rate of lactate dehydrogenase decreased sharply under the action of the inhibitor. In order to observe the morphology of infected cells, the collected cells were also observed by transmission electron microscopy. The results of electron microscopy showed that the cell membrane of LPS+ATP group and S. suis 2 group was broken and the cell contents were leaked out. Combined with the above other results, it can be concluded that under the infection of S. suis 2, brain microvascular endothelial cells produced an inflammatory response and pyroptosis occurred.
In the dissertation work on the basis of research are substantiated the pathogenic mechanism of Streptococcus suis, inhibitor protection to develop target drugs and vaccine for meningitis, reduce losses of pig industry and promote the healthy development of animal husbandry.
