Effect Of Lithocholic Acid On Intestinal Mucosal Barrier Function And Its Molecular Mechanism

Objective: Choledocholithiasis is a common disease in biliary surgery,which can cause obstructive jaundice.With the development of the laparoscopic technique,laparoscopic common bile duct exploration(LCBDE)has become the main treatment of choledocholithiasis.Postoperative biliary drainage can relieve biliary obstruction,reduce biliary pressure,reduce inflammatory bile reflux,and relieve local inflammation,but there are still complications that can not be ignored.Obstructive jaundice and bile drainage can lead to bile deficiency in the intestine,damage of intestinal mucosal barrier function,displacement of bacteria and endotoxin,bacteremia and endotoxemia,production of proinflammatory cytokines(TNF-?,IL-6),systemic inflammatory response syndrome and multiple organ dysfunction syndromes.Intestinal epithelial barrier mainly includes mechanical barrier,chemical barrier,biological barrier,and immune barrier.Machinery is the most important defense line of the intestinal epithelial barrier.The mechanical barrier is mainly composed of epithelial cells and TJs.Tight junctions are a group of transmembrane proteins,which are composed of claudins,Occludin,Zos,and E-cadherin.They interact with adjacent cells to form a selective barrier.Studies have shown that vitamin D can maintain intestinal mucosal barrier function by activating vitamin D receptor(VDR).Vitamin D receptor is also the sensor of bile acid.The lithocholic acid(LCA)in bile is the activator of vitamin D receptor,which plays an important role in regulating the metabolism of bile acid and promoting the detoxification metabolism of lithocholic acid.However,the effect of vitamin D receptor activated by lithocholic acid on intestinal mucosal barrier function has not been reported.Silencing SIRT1 can deacetylate Nrf2 and NF-?B,and affect the physiological functions of oxidative stress and inflammation.When Nrf2 is activated,it transfers from cytoplasm to nucleus and mediates the transcription of target genes,such as heme oxygenase-1(HO-1)and superoxide dismutase-1(SOD1).The purpose of this study was to determine the effect of lithocholic acid on the intestinal barrier function and the role of vitamin D receptor in the regulation of intestinal barrier function by lithocholic acid.Furthermore,the effects of lithocholic acid on SIRT1 / Nrf2 and NF-?B signaling pathways were further clarified.Methods: Part One: male Sprague Dawley(SD)rats of SPF grade aged 8-10 weeks were studied.One week after adaptive feeding,30 SD rats were randomly divided into three groups: sham operation group(SH group),bile drainage group(ED group),obstructive jaundice group(OJ group).After 7 days of modeling,the animals were killed,and the specimens were taken.Test method: general state and weight change of rats were recorded.HE staining and electron microscopy were used to detect the morphological and ultrastructural changes of the intestinal mucosa.FITC-dextran was used to detect the permeability of intestinal mucosa.At the same time,the changes of MDA,ROS,SOD,and GSH were detected.The expression of VDR,ZO-1,E-cadherin,Occludin,and Claudin-1 were detected by Western blot.The second part: the subjects were male Sprague Dawley(SD)rats of SPF grade between 8-10 weeks old.One week after adaptive feeding,50 SD rats were randomly divided into five groups: sham operation group(SH group),external bile drainage group(ED group),external bile drainage + lithocholic acid group(ED + 30mg/kg LCA group),obstructive jaundice group(OJ group),obstructive jaundice + lithocholic acid group(OJ + 30mg/kg LCA group).After 7 days of modeling,the animals were killed,and the specimens were taken.Test method: general state and weight change of rats were recorded.HE staining and electron microscopy were used to detect the morphological and ultrastructural changes of the intestinal mucosa.FITC-dextran was used to detect the permeability of intestinal mucosa in rats.At the same time,the contents of MDA,ROS,SOD,and GSH were measured.The expression and distribution of VDR,ZO-1,E-cadherin,Occludin,and Claudin-1 were detected by immunohistochemistry and Western blot.The third part: Caco-2 cell model is a kind of human colonic adenocarcinoma cell.Its structure and function are similar to differentiated intestinal epithelial cells,with microvilli and other structures.So we choose Caco-2 cells to establish the monolayer cell barrier model in vitro.Methods:(1)CCK-8 was used to detect the effects of different concentrations of LCA(5,10,20,50?M)on the activity of Caco-2 cells.(2)Western blot was used to detect the expression of VDR protein in Caco-2 monolayer cells at different concentrations of LCA(5,10,20,50?M)at different times(3,6,12,24h).The best time and dose of LCA treatment cells were selected.(3)After treatment of Caco-2 monolayer cells with different concentrations of LCA(5,10,20,50?M)and TNF-?(100ng/m L),the permeability of Caco-2 monolayer cell barrier was detected by transepithelial electrical resistance(TEER)and FITC-dextran,and the expression of Caco-2 monolayer tight junction proteins ZO-1,E-cadherin,Occludin,and Claudin-1 were detected by Western blot.(4)After treated with LCA(20?M)and TNF-?(100ng/m L)for 24 h,the changes of MDA,ROS,SOD,and GSH were detected;the changes of SIRT1,Nrf2,HO-1,and NF-?B signaling pathways were detected by Western blot;the location of tight junction protein,Nrf2,and NF-?b p65 was detected by immunofluorescence.(5)After pretreated with SIRT1 inhibitor ex527 for 3h,Caco-2 monolayer cells were treated with LCA(20?M)and TNF-?(100ng/m L).The changes of tight junction protein,SIRT1 / Nrf2 signaling pathway,and NF-?B signaling pathway were detected by Western blot.(6)In addition,we used LCA(20?M)and TNF-?(100ng/m L)to treat the single-layer cells of Caco-2.Western blot was used to detect the changes of tight junction protein,SIRT1/Nrf2 signaling pathway,and NF-? B signaling pathway.Results: Part one:(1)compared with the sham operation group,the rats in the external bile drainage group showed a large amount of bright bile from the drainage tube,rough and lusterless hair,less activity,ulceration of the skin of the neck drainage tube,poor appetite,no significant weight gain,and obvious emaciation of some rats;the integrity of the intestinal mucosa was damaged,the intestinal villi were short and sparse,and the height and thickness of the intestinal villi.Intestinal mucosa homogenate increased MDA,ROS content,SOD and GSH content decreased,while the contents of VDR,ZO-1,E-cadherin,Occludin,and Claudin-1 in the intestinal mucosa decreased.(2)Compared with the sham operation group,the skin and urine of the rats in the obstructive jaundice group were obviously yellow,the bile duct near the heart was swollen,the liver was swollen and yellow stained,and some of the liver surfaces of the rats showed granular nodules.The hair was coarse,disordered and lusterless,and the activity was reduced,some rats' temperament was changed greatly,there was a phenomenon of eating companion,poor appetite,slow weight gain,but no rats with significant weight loss;the integrity of intestinal mucosa was damaged,the intestinal villi were short and sparse,the height of intestinal villi,the thickness of mucosa and the depth of crypt were significantly reduced compared with the sham operation group;FITC-dextran in the serum of rats in the bile external drainage group contained.Intestinal mucosa homogenate increased MDA,ROS content,SOD and GSH content decreased,while the contents of VDR,ZO-1,E-cadherin,Occludin,and Claudin-1 in the intestinal mucosa decreased.The second part:(1)Compared with the simple bile drainage group,the general situation of the rats in the group of bile drainage and 30 mg/kg lithocholic acid was not significantly improved,and the weight was significantly reduced compared with the sham operation group;the morphology of intestinal mucosa was improved,the microvilli were dense and arranged orderly,the height of intestinal villi was increased,but the thickness of mucosa and the depth of crypt was not improved;the content of FITC-dextran in the serum of rats was decreased;the content of MDA and ROS in intestinal mucosa homogenate was decreased,the content of SOD and GSH was increased;the expression of VDR,ZO-1,E-cadherin,Occludin,and Claudin-1 in intestinal mucosa was showed by Western blot all increase.(2)Compared with the group of simple obstructive jaundice,the rats in the group of 30mg/kg lithocholic acid at the same time of establishing obstructive jaundice had no significant improvement in general conditions and no significant increase in body weight;the morphology of intestinal mucosa was improved,the microvilli were dense and arranged orderly,the height of intestinal villi and the depth of crypt were increased,but the thickness of mucosa was not significantly improved;the content of FITC-dextran in the serum of rats was decreased;the content of MDA and ROS in intestinal mucosa homogenate was decreased,the content of SOD and GSH was increased;the expression of VDR,ZO-1,E-cadherin,Occludin and Claudin-1 protein in intestinal mucosa was increased.The third part:(1)The results of CCK-8 showed that LCA had no effect on the cell viability after treated with 0-50?M LCA for 24 h.(2)LCA alleviates TNF-? induced increase in cell barrier permeability in a dose-dependent manner.(3)LCA promotes the expression of VDR protein in a time-dependent and dose-dependent manner.(4)LCA alleviates the down-regulation of TNF-? induced expression of tight junction proteins ZO-1,E-cadherin,Occludin,and Claudin-1 in Caco-2 cells in a dose-dependent manner.(5)The results of immunofluorescence showed that after TNF-? treatment of Caco-2 monolayer cells,the tight junction protein appeared zigzag process,and its distribution was irregular and discontinuous.However,LCA significantly improved TNF-? induced changes in the distribution and destruction of tight junction proteins.(6)Compared with TNF-? group,LCA treatment significantly increased the expression levels of SIRT1,Nrf2,and HO-1 proteins and inhibited the up-regulation of NF-?B p-p65 and p-I?B-? in TNF-? mediated Caco-2 monolayer cells.Immunofluorescence results showed that compared with the TNF-? group,LCA treatment significantly induced Nrf2 activation and nuclear transfer.At the same time,LCA effectively inhibited TNF-? induced NF-?B p65 transport to the nucleus.(7)SIRT1 inhibitor EX527 partially inhibited the expression of LCA induced tight junction protein and SIRT1,Nrf2,and HO-1.Ex527 promoted the expression of NF-?B p-p65 and p-I?B-?.(8)After knocking down Caco-2 cells,LCA ultimately failed to up-regulate tight junction protein,SIRT1,Nrf2,and HO-1 expressions and inhibit the expression of NF-?Bp-p65 and p-I?B-?.Conclusions:(1)The loss of bile in the intestine can lead to atrophy of villi,infiltration of inflammatory cells,increase of intestinal permeability,down-regulation of tight junction protein expression,and destruction of intestinal barrier function.(2)The lack of bile in the intestine can lead to lipid peroxidation,a decrease of antioxidant content,and a reduction of antioxidant capacity.(3)Bile deficiency in the gut reduces VDR protein expression.(4)LCA can improve the intestinal mucosal permeability of bile loss and the destruction of tight junction proteins by bile loss,thereby maintaining the intestinal mucosal barrier's integrity.(5)LCA can alleviate lipid peroxidation caused by bile deficiency in the intestine and enhance the antioxidant capacity.(6)LCA can increase the expression of VDR protein in bile deficient rats.(7)LCA can improve the TNF-? induced permeability of Caco-2 monolayer cells and the destruction of tight junction protein structure.(8)LCA can further activate SIRT1/Nrf2 and inhibit the NF-?B signaling pathway by activating VDR to maintain the integrity of the intestinal mucosal barrier function.