Effects Of Brucella LPS On Endoplasmic Reticulum Stress And Inflammatory Factors In Goat Endometrial Epithelial Cells

Brucella spp are Gram-negative facultative intracellular bacteria that cause brucellosis,a wide spread zoonosis that affects mainly domestic animals and humans.Brucellosis is a chronic disease and may last several weeks or months.If it is not treated effectively,the disease can lead to pathologies in the liver,spleen,lymph nodes,bone marrow,reproductive tract,and skeletal system.Endometrial cells is one of the main target cells of Brucella,Previous study confirmed that Brucella survival in association with the endoplasmic reticulum(ER)of the target cells.as an important pathogenic factor of Brucella in intracellular survival,The LPS(lipopolysaccharide)of Brucella plays a key role in the replication process.After bacterial releaseing LPS,LPS of vertebrates(especially susceptible animal)the main function produce immune response of the organism through the combination of specific receptors immune cells.The extraction of Brucella LPS infect goat endometrial epithelial cells(EEC),(1)to detect the expression changes of endoplasmic reticulum stress markers of molecular GRP78 and CHOP protein,and UPR pathway in IRE1 and ATF6 expression,the effect of apoptosis,autophagy and inflammation;(2)Add the endoplasmic reticulum stress inhibitor 4 phenyl butyric acid(4-PBA)to verify the relationship between endoplasmic reticulum stress and apoptosis,autophagy and inflammation by inhibition of endoplasmic reticulum stress.The results are as follows:1.The brucella LPS was extracted and purified by thermophenol method,and the brucella LPS was extracted by silver dye identification.The concentration of unknown LPS solution was determined by phenol-sulfuric acid method.By method of CCK8 determined the optimal concentration of LPS function cells,the results show that the LPS unknown concentration is 131 ug/mL,when the concentration of LPS reaches 1 ug/m L,cell vitality has reached around 85%,cells begin to change.2.The expression of GRP78 and CHOP was significantly increased in 12 h(P<0.01)by Western Blot test,indicating that the brucella LPS could stimulate the stress response of EEC.RT-PCR and Western Blot further detected the UPR related molecules,and the results showed that the IRE1 and ATF6 pathways in UPR were activated by brucella LPS to induce endoplasmic retral stress.Flow cytometry and fluorescence quantitative detection of apoptosis and autophagy related molecules showed that the LPS of brucellae caused EEC apoptosis and autophagy.ELISA detected inflammatory cytokines in the cells,and the results showed that the LPS caused EEC to secrete IL-1? and IL-8.3.Add the endoplasmic reticulum stress inhibitors 4-PBA,through flow cytometry,RT-PCR,Western Blot and ELISA to detect autophagy,apoptosis and cell inflammation related factors,and the results show that the endoplasmic reticulum inhibitors decrease autophagy,apoptosis and cell inflammatory factor expression...