The Expression Of Water Channel Protein 3 And Tight Junction Proteins And Its Role In Intestinal Barrier Function

Chapter 1 This chapter summarized the structure and function and its classification of the protein in the body. The chapter introduced the methods, principles, advantages and experimental optimization of testing specific proteins based on the theory of immune markers in biomedical field. In this chapter, the structure and research progress of water channel protein 3 and tight junction protein are reviewed. Water channel protein 3 and tight junction protein are the important specific proteins of maintaining the intestinal barrier function.Chapter 2 Water channel protein 3 is an important protein in the intestinal regulating of water and small molecules in-and-out of the colon. Lipopolysaccharide(LPS) is the main pathogen of the intestinal tract and impairs the barrier function of the intestinal tract. By RT-PCR and Western blot methods, mRN A and the protein expression of water channel protein 3 in intestinal epithelial cell were determined after the lipopolysaccharide impacting the intestinal epithelial cells. The results showed that LPS interference group than the control group of aquaporin 3 expression was down-regulated markedly, The down-regulation level decreased with the increase of LPS concentration and time. Further experiments showed that the activity of total p38 and phosphorylation of p38, JNK and phosphorylated JNK was significantly increased by Western blot methods determination after the lipopolysaccharide impacting the intestinal epithelial cells. The activity of total p38 and phosphorylation of p38, JNK and phosphorylated JNK elevated to was blocked by the p38 inhibitor SB203580 and JNK inhibitor sp600125 after the lipopolysaccharide impacting the intestinal epithelial cells. It proves that the p38 and JNK signaling pathways involved in the regulation of lipopolysaccharide induced downregulation of the water channel protein 3 in colonic epithelial cells.Chapter 3 Clostridium butyricum is one of the important intestinal probiotics, maintain the intestinal barrier function. We added the supernatant of Clostridium butyricum after effect of lipopolysaccharide on intestinal epithelial cells, mRNA and the protein expression of water channel protein 3 in intestinal epithelial cell were determined by RT-PCR and Western blot methods. The results showed that Clostridium butyricum can improve the down-regulated effect of lipopolysaccharide on the expression of water channel protein 3. Improvement was strengthened with the increased of Clostridium butyricum concentration and time. Further experiments showed that the activity of total p38 and phosphorylation of p38, JNK and phosphorylated JNK was significantly increased by Western blot methods determination after the Clostridium butyricum impacting the intestinal epithelial cells. The activity of total p38 and phosphorylation of p38, JNK and phosphorylated JNK elevated to was blocked by the p38 inhibitor SB203580 and JNK inhibitor sp600125 after the Clostridium butyricum impacting the intestinal epithelial cells. It verified that p38 and JNK signaling pathway involved in the up-regulated the expression of aquaporin 3 in colonic epithelial cells by Clostridium butyricum.Chapter 4 Tight junction proteins are another important protein which protects the gut barrier function. By RT-PCR and Western blot methods, mRNA and the protein expression of tight junction proteins in intestinal epithelial cell were determined after the lipopolysaccharide impacting the intestinal epithelial cells. The permeability of intestinal epithelial cells was determined by tranwells method. The results showed that the proteins expression and mRNA expression of tight junction proteins were down regulated and the permeability of the intestinal epithelial cells was increased after LPS acting intestinal epithelial cells. With the increase of the concentration and time of lipopolysaccharide, the expressions of tight junction proteins were down-regulated gradually and the intestinal permeability increased gradually. It reveals that lipopolysaccharide damage of the gut barrier function by reducing the espression of tight junction proteins.Chapter 5 On the basis of LPS down-regulated the expression of intestinal epithelial tight junction proteins, We added the supernatant of Clostridium butyricum after effect of lipopolysaccharide on intestinal epithelial cells, mRNA and the protein expression of tight junction proteins in intestinal epithelial cell were determined by RT-PCR and Western blot methods. The permeability of intestinal epithelial cells was determined by tranwells method. The results showed that Clostridium butyricum improved the down-regulated effect of lipopolysaccharide on the expression of tight junction proteins. Improvement was strengthened with the increased of Clostridium butyricum concentration and time. Clostridium butyricum improved the increased permeability of intestinal epithelial cells induced by lipopolysaccharide. It reveals Clostridium butyricum maintains the intestinal barrier function by upregulating the expression the tight junction proteins.