Effects And Molecular Mechanisms Of Bile Salt Dependent Lipase On Intestinal Adaptation To Massive Small Bowel Resection In Rats

Intestinal adaptation is important for the short bowel syndrome(SBS)patients.Bile salt dependent lipase(BSDL)is a Le X-carrying glycoprotein secreted by the pancreas and activated by bile salts in the intestine.To date,growing evidence has suggested that BSDL not only has the lipolytic activity,but also the antiviral,immune-modulating and pro-proliferative activities.The purpose of this study was to investigate the effects of BSDL on intestinal adaptive growth and gut barrier function in a rat model of SBS.ObjectiveThe purpose of this study was to construct a prokaryotic expression system for m BSDL in E.coli BL21(DE3)cells and prepare the active m BSDL protein and investigate the effects of BSDL on intestinal adaptive growth and gut barrier function in a rat model of SBS.MethodsChapter 1m BSDL c DNA was amplifed via PCR and inserted into the prokaryotic expression vector p ET-28 b.The expression of recombinant protein was induced by IPTG in E.coli BL21 cells.Then it was purified via His Trap FF crude purification system,and refolded via dialysis system.The specificity of m BSDL protein was analyzed by Western Blot.Chapter 2Twenty-four rats were randomly divided into 3 experimental groups: sham group,SBS group(rats underwent 80% bowel resection),SBS-BSDL group(rats underwent 80% bowel resection and were orally administered 0.045 g/kg BSDL per day).The intestinal morpho-histochemical changes were determined 14 days after the operation.Epithelial cell proliferation and apoptosis were also analyzed by immunohistochemistry and immunofluorescence staining.Chapter 3Twenty-four rats were randomly divided into 3 experimental groups: sham group,SBS group(rats underwent 80% bowel resection),SBS-BSDL group(rats underwent 80% bowel resection and were orally administered 0.045 g/kg BSDL per day).The intestinal barrier function was determined 14 days after the operation.The expressions of tight-junction proteins were also analyzed by immunohistochemistry and Western blot.Chapter 4Twenty-four rats were randomly divided into 3 experimental groups: sham group,SBS group(rats underwent 80% bowel resection),SBS-BSDL group(rats underwent 80% bowel resection and were orally administered 0.045 g/kg BSDL per day).The expressions of Wnt signaling molecules in enterocytes were also analyzed by immunohistochemistry and Western blot 14 days after the operation.ResultsChapter 1The prokaryotic expression vector p ET-28b-m BSDL expressing m BSDL protein was successfully constructed and transformed into E.coli BL21(DE3)cells.The active m BSDL protein was successfully prepared.Western Blot revealed that the purification of this recombinant protein was successful.Chapter 2The body weight of the SBS-BSDL group showed significant increases from postoperative day 5 to day 14,in comparison to that of the SBS/untreated group.SBS only rats had a significant increase in intestinal permeability,as measured by serum D-xylose concentration,as compared to the sham group.However,the administration of BSDL reduced the serum D-xylose concentration by 43%,as compared to the SBS group.The rats treated with BSDL had significantly greater villus height,crypt depth,and enterocyte proliferation in their residual intestines,as compared to the sham-control group and the SBS/untreated groupChapter 3The BT rate of the SBS and SBS-BSDL groups was higher than that of the sham group.Only 12.5% sham rats exhibited BT to the MLN(level I)and none in either spleen or liver.The expressions of ZO-1 and claudin-1 proteins tight-junction proteins were increased in the SBS rats treated with BSDL.Chapter 4The number of P-β-catenin(Ser33/37/Thr41)positive cells showed the highest in the SBS only rats.Treatment with BSDL resulted in a decreased number of P-β-catenin(Ser33/37/Thr41)positive cells in rat intestinal tissues as compared to SBS only rats in the residual ileum by immunohistochemistry.In the western blot analysis,the expression level of total β-catenin was unchanged within three experimental groups.However,the total GSK-3β accumulation was increased in the SBS and SBS-BSDL groups as compared to the sham groups.The phosphorylation analysis showed higher expression levels of both P-GSK-3β(Ser9)and P-β-catenin(Ser552)and lower levels of P-β-catenin(Ser33/37/Thr41)in SBS-BSDL group as compared to those in SBS only rats.ConclusionThe active m BSDL protein was successfully prepared in E.coli prokaryotic expression system.The study provides some foundation for further analysis of m BSDL protein.Enteral BSDL supplementation up-regulates proliferation,reduces intestinal permeability,and induces Wnt/β-catenin signaling in intestinal epithelium,which exhibits a significant effect on the gut adaptive process after m SBR in rats.These findings raised the possibility that BSDL-based therapies could be used to enhance adaptive intestinal growth in patients with SBS.However,further research on this topic is still needed.The optimal dosage of enteral BSDL administration should be identified,and the ways via which BSDL might activate the Wnt/β-catenin pathway should be investigated.