| Endotoxin,also known as bacterial lipopolysaccharide(LPS),is an important component in the outer membrane of the Gram-negative bacteria.LPS plays an important role in enhancing the stability of the bacterial plasma membrane and protecting the cell from adverse external factors.However,LPS in the plasma membrane is released when bacteria undergo autolysis,artificial methods destroy cell structure,or cells actively gr ow and multiply.Also,as a pathogenic component,LPS can cause toxemia,in severe cases,septic shock,multi-organ failure and diffuse intravascular coagulation.As the largest digestive organ of the body,the gut is also considered to be the largest reservoir of LPS.The intestinal tract is home to trillions of commensal bacteria and when the intestinal environment is affected by adverse factors,such as the widespread use of antibiotics,it effectively kills pathogenic bacteria,but at the same time releases large amounts of LPS and allows them to accumulate in the intestine.The intestinal mucosal barrier plays an important role in maintaining the homeostatic balance of the intestinal environment by protecting the intestinal epithelium from adverse c onditions and thus separating the body?s internal environment from that of the intestinal lumen,as an important defense against the invasion of intestinal contents and pathogenic microorganisms.However,it is not clear how LPS affects the structure and function of the intestinal mucosal barrier and its possible mechanism.In this study,mice administrated with 0,200,600 and 1000 ?g/kg LPS solution and ileal epithelial cells treated with 400 ?g/m L LPS medium were used as test models.Manipulations of HE staining,PAS staining,immunohistochemical staining,ELISA,flow cytometry were used to explain the effects of LPS on the morphological structure and function of the intestinal epithelial barrier.The results of this studies that have been obtained are as follows.1.At 16 d and 32 d,the ileal tissues of mice from different dose groups were collected to observe the changes of the tissue morphology and MUC2 content in the mice mucus layer.HE staining showed that the height of ileal villus was significant shortened in the middle and high dose groups at 32 d(P<0.05),but there was no significant difference in the low dose group(P>0.05);the depth of the crypt was significantly deepened in the low,middle and high dose groups(P<0.05).MUC2 content in the ileal mucus layer showed a significant increase in the low and middle dose groups by immunohistochemical staining and ELISA(P<0.05),but not in the high dose group(P>0.05);The number of Goblet cells in the low and middle dose groups showed a significant increase with PAS staining(P<0.05),but not in the high dose group(P>0.05).Meanwhile,observation of the ileal mucosa by transmission electron microscopy revealed that high doses of LPS increased the number of lysosomes,the mucus layer in the intestinal lumen became loose,and the number of lymphocytes in the lamina propria increased with increasing dose.2.Blood,ileal mesenteric lymph nodes and Peyer?s patch from mice were collected at 16 and 32 d to examine the effect of LPS on the immune response of the mouse ileal mucosa.Routine blood results showed that the number of leukocytes,neutrophils,monocytes and lymphocytes decreased significantly with increasing doses of LPS.HE staining showed that the morphology of Peyer?s patch and mesenteric lymph node was impaired by LPS at 32 d.The results of flow cytometry showed that LPS caused a decrease in the proportion of CD4+T cells,an increase in the number of CD8+ T cells and a decrease in the proportion of CD19+CD38+ B lymphocytes.Meanwhile,the levels of antibodies and inflammatory factors in the blood were also measured using ELISA kits.The results showed that the levels of Ig G and Ig A antibodies decreased(P<0.05),the levels of IFN-γ and IL-6 decreased significantly and the levels of IL-10 increased significantly in the high dose group(P<0.05),while there were no significant differences in the changes in the levels of TNF-α,IL-2 and IL-4 cytokines(P>0.05).3.Ileal epithelial cells were cultured in vitro with 400 ?g/m L LPS medium for 12 h,24 h and 36 h.The expression of protein and m RNA in Occludin and TLR4/NF-κB pathwayrelated factors were examined by Western blotting and q PCR.The results showed that LPS caused a significant decrease in Occludin expression at 24 h(P<0.05)and a highly significant decrease at 36 h(P<0.01).The expression of TLR4 was significantly increased at 24 h and 36h(P<0.01),but not at 12 h(P>0.05).The expression NF-κB was significantly increased at 24 h and 36 h(P<0.05),but not at 12 h(P>0.05).Phosphorylation levels of IκBα were significantly higher at 12 h(P<0.05),whereas there was no significant difference at 24 and 36h(P>0.05).In addition,LPS was able to significantly increase the m RNA expression of Occludin,TLR4,My D88,IκBα and NF-κB(P<0.01).In conclusion,LPS can cause changes in the morphological structure of mouse ileum villi,change the differentiation ratio of lymphocytes in mesenteric lymph nodes and Peyer?s patch,and then lead to changes in related inflammatory factors and decreased levels of antibodies.After LPS contact with epithelial cells,it may affect the expression of tight junction protein by affecting the expression of TLR4/NF-κB pathway related factors in epithelial cells,leading to the destruction of epithelial cell barrier,and then aggravating intestinal injury. |
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