| Objective: Obstructive jaundice is a common disease and occurs frequently in general surgery.Mainly due to partial or complete mechanical obstruction of extrahepatic or intrahepatic bile ducts caused by inflammation,stones,tumors and other causes,the process of bile discharging into the intestine from the bile duct is hindered,resulting in bile stasis and jaundice caused by the reverse inflow of ester bilirubin into the blood.Obstructive jaundice often causes changes in liver function and biliary tract infection,which require prompt surgical treatment.At present,surgical operations have achieved minimal invasiveness,with the characteristics of small wounds,light pain,and quick recovery after surgery,which are in line with the concepts of "Minimally invasive and precise" and "fast track" in contemporary medicine.However,there are still many patients who need to use external bile drainage techniques for a long time,including surgically indwelling T tubes and puncture drainage tubes by various interventional procedures.It is clinically observed that the incidence of intestinal infections in these patients will increase and threatens the lives of patients.Alkaline phosphatase is a monolipid phosphohydrolase that is widely distributed in various tissues and organs of the human body and it is a homodimer.It is located on the brush-like edge of the intestinal mucosa and is capable of hydrolyzing monophosphate.Under physiological conditions,lysolecithin can specifically promote the release of IAP into the intestinal lumen.IAP has the function of maintaining tight junction proteins between intestinal mucosal cells.Studies have demonstrated that IAP can decrease the expression levels of tight junction proteins ZO-1,ZO-2,claudin and Occludin.Occludin,claudin and ZO participate in the formation of tight junction complexes and play a role in maintaining the normal intestinal mucosal barrier function.While the decrease in tight junction protein expression can cause changes in the intestinal mucosal barrier function,which in turn affects intestinal mucosal permeability.Intestinal bacteria,endotoxin,inflammatory mediator,and other factors are able to enter the internal environment through the damaged intestinal mucosal barrier,leading to the release of more inflammation mediator,which would activate the process of systemic inflammatory response syndrome,and SIRS.This process would further damage the intestinal mucosal barrier function,and ultimately lead to the occurrence of multiple organ failure.This study aims at exploring the effect of IAP on the intestinal mucosal barrier function after bile loss,and to further clarify the effect of IAP on the MLCK/pMLC signaling pathway.Method Part 1: We selected SD rats,aged 8-10 weeks,and established experimental rat animal models by surgical methods.The five groups were sham group(n=10),obstructive jaundice group(n=10),obstructive jaundice group + IAP feeding group(n=10),external bile drainage group(n=10),and external bile drainage + IAP feeding group(n=10).Animals were sacrificed 14 days after modeling,and specimens were taken.Detection method: 1.Observe the general condition of the rat and record the weight change.2.HE staining to observe the morphological changes of the intestinal mucosa.3.Observe the changes in the ultrastructure of intestinal mucosal cells under an electron microscope.4.Intestinal mucosal permeability test to detect changes in intestinal mucosal permeability of rats.5.Collect the intestinal mucosal cells of rats at the same time,and detect the change of oxidative stress indicators malondialdehyde(MDA),reactive oxygen species(ROS),antioxidant indicators of superoxide dismutase(SOD)and reduced glutathione(GSH).6.Detect the expression of IAP and tight junction proteins ZO-1,Occludin,and Claudin-1 in rat intestinal mucosa.7.Observe the distribution of ZO-1 in the tissue by immunohistochemical staining.Part 2: Caco-2 cells are morphologically similar to human small intestinal epithelial cells,with the same tight junctions and cell polarity.Therefore,we chose Caco-2 cell line to establish an intestinal barrier model in vitro.Detection method: 1.CCK-8experiment detected the effect of different concentrations of IAP(0,100,200,500,1000 U/ml)on the viability of Caco-2 cells.2.IAP(200u/ml,500u/ml,1000u/ml)was added on the basis of TNF-a(100ng/mL),and the transepithelial cell resistance(TEER)was measured at 3h,6h,12 h,and 24 h.3.On the basis of TNF-a(100ng/mL),IAP(200u/ml,500u/ml,1000u/ml)was added separately and FITC-dextran was used for intestinal mucosal permeability test.4.Western blot was used to detect the expression of tight junction proteins ZO-1,Occludin and Claudin-1 in Caco-2monolayer cells under the action of IAP(500u/ml)and TNF-a(100ng/mL).5.Immunofluorescence to detect the expression and distribution of ZO-1,Occludin and Claudin-1.6.Caco-2 monolayer cells are measured for malondialdehyde(MDA),reactive oxygen species(ROS),superoxide dismutase(SOD)and reduced glutathione(GSH)under the action of IAP(500u/ml)and TNF-a(100ng/mL).7.Caco-2 cells were pretreated with IAP inhibitor L-PHE,and then IAP(500u/ml)and TNF-a(100ng/mL)were given to treat Caco-2 monolayer cells,and Western blot was used to detect the MLCK/pMLC pathway and Changes in tight junction proteins.Results:Part 1: 1.SH group: The rats showed signs of poor appetite,decreased activity,poor fur color and irritability in the early stage.The rats in this group recovered the fastest from the above symptoms and achieved weight gain.OJ group:The sclera and urine of the rats were yellow,the proximal end of the common bile duct was enlarged,the liver was swollen and yellowish.And some rats had a feeling of sand on the liver surface.The weight gain of rats in this group was slow.ED group:a large amount of golden bile flowed out from the biliary drainage tube,and the local fur was stained yellow.The rat's hair is rough and dull,with little activity,accompanied by diarrhea.The weight of the rats decreased significantly,and the weight of the rats increased slowly in the later period.ED group + IAP group: The general condition of the rats in this group was basically the same as that of the ED group,but the rats had milder diarrhea and the weight loss trend was slower than that of the ED group.The weight of the rats was higher than that of the ED group one week after the operation(p<0.05).2.In both OJ group and ED group,rat small intestinal mucosal integrity was damaged,small intestinal villi were shortened and dulled,villi apical space enlarged,glands were arranged irregularly,goblet cells in mucosa decreased,and inflammatory cells infiltrated amina propria,mucosal layer and the lamina propria separated.The damage of the small intestinal mucosa of the rats in the ED+IAP group was more serious,and the corresponding chiu's score was significantly lower than that in the ED group(p<0.05).3.The brush border microvilli of the intestinal mucosa cells in the ED group and OJ group were arranged neatly,and the microvilli became shorter.The junction bodies between cells were enlarged,the junctions between cells were no longer tight.the cytoplasm was uneven,and mitochondria appeared vacuolar degeneration.The intestinal epithelial cells of rats in the ED+IAP group were arranged tightly,and the length of microvilli was longer than that in the ED group,and the microvilli were arranged densely and neatly(p<0.05).4.Compared with SH group,the FITC-D concentration in the serum of ED group and OJ group increased significantly,and the difference was statistically significant(p<0.05).Compared with the ED group(ED group: 26.24±3.42?g/ml),the FITC-D concentration in the serum of the ED+IAP group was significantly reduced(ED+IAP group: 20.52±1.29?g/ml,p<0.05).Compared with the OJ group(OJ group:28.1±1.8?g/ml),the serum FITC-D concentration(OJ+IAP group: 23.3±4.95?g/ml)in the OJ+IAP group was higher(p<0.05).5.Compared with the SH group,the MDA and ROS in the intestinal mucosal tissue of the OJ group and the ED group were significantly increased,and the contents of SOD and GSH were significantly decreased.The MDA and ROS levels of the IAP treatment group were lower than the corresponding non-treatment group(p<0.05),and the SOD and GSH levels were higher than the corresponding non-treatment group(p<0.05).6.Compared with the SH group,the expression of IAP,ZO-1,Occludin and Claudin-1 protein in the intestinal mucosa of the ED group and OJ group was reduced.Compared with the ED group,the expression of ZO-1,Occludin,and Claudin-1 proteins in the intestinal mucosa of the ED+IAP group increased(p<0.05).Compared with the OJ group,the expression of ZO-1,Occludin and Claudin-1 protein in the intestinal mucosa of the OJ+IAP group increased(p<0.05).7.The immunohistochemical results showed that the ZO-1 protein in the SH group evenly distributed in the intestinal mucosa.The expression of ZO-1 protein in ED group and OJ group was lower than that in SH group(p<0.05).Part 2: 1.The results of CCK-8 experiment showed that IAP had no effect on cell viability when Caco-2 cells were treated with 0-1000 U/ml IAP for 24 h.2.When Caco-2 monolayer cells were incubated with 100 ng/mL TNF-?,the TEER value of the cells continued to decrease over time,and reached the lowest resistance value after24 h.The 200-1000U/ml IAP pretreated cells significantly improved the decrease in resistance value induced by TNF-?(p<0.05),and the improvement value was related to the concentration of IAP.3.The dose of 100 ng/mL TNF-? significantly increased the permeability of the Caco-2 monolayer cell barrier.While IAP could alleviate the increase in cell barrier permeability induced by TNF-?.4.The dose of 100ng/mL TNF-? significantly reduced the expression of ZO-1,Occludin and Claudin-1.The dose of 500U/ml IAP could partially alleviate the damage of TNF-? to the tight junction protein of Caco-2 cells,and its optical density value was statistically different compared with that in the TNF-? group(p<0.01).5.Immunofluorescence method: 24 hours after adding 100ng/mL TNF-?,the tight junction protein between Caco-2 cells appeared jagged protrusions,and their distribution was sparse and discontinuous.Intervention with IAP(500U/ml)significantly improved the destruction of tight junction proteins induced by TNF-?(p<0.05).6.TNF-? could induce the expression of MDA and ROS to increase,while IAP could significantly reduce the increase.TNF-? inhibited the expression of SOD and GSH(p<0.05).However,IAP significantly alleviated the inhibition of TNF-? on the expression of SOD and GSH(p<0.05).7.IAP treatment reduced the expression of MLCK/pMLC induced by TNF-?,and increased the expression of ZO-1,Occludin and Claudin-1.IAP inhibitor L-PHE can up-regulate the expression of MLCK/pMLC pathway proteins,and reduce the expression of three tight junction proteins,ZO-1,Occludin and Claudin-1(p<0.05).Conclusions:1.IAP can significantly improve the intestinal mucosal barrier damage caused by the lack of bile,such as intestinal villi atrophy,inflammatory cell infiltration,decreased tight junction proteins,and increased intestinal mucosal permeability.Bile loss can lead to lipid peroxidation,increased levels of malondialdehyde(MDA)and reactive oxygen species(ROS),superoxide dismutase(SOD)and reduced glutathione(GSH)levels decrease,and reduce antioxidant capacity.IAP can inhibit lipid peroxidation caused by the loss of bile in the intestine and enhance the body's antioxidant capacity.The loss of bile can reduce the expression of tight junction protein in small intestinal mucosal cells and damage the intestinal mucosal barrier function.IAP can increase the expression of tight junction protein in rats with bile loss.2.IAP can reduce the increase in intestinal mucosal permeability induced by TNF-?.IAP can improve the decreased expression of tight junction protein caused by TNF-?.IAP can inhibit lipid peroxidation caused by TNF-? and improve the antioxidant capacity.IAP can maintain the barrier function of the intestinal mucosa by inhibiting the MLCK/pMLC signaling pathway. |
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