Mechanism Of The Protective Effect Of Bile Acid On Intestinal Mucosal Barrier In Obstructive Jaundice

Obstructive jaundice(OJ) causes reduced or absent bile in the intestinal lumen, bacteria overgrowth, increased endotoxin release, decreased intestinal epithelial cell proliferation, increased apoptosis and intestinal mucosal tight junction damage, results in intestinal mucosal injury and increased intestinal permeability. Bacteria and endotoxin within the intestine lumen translocate into the bloodstream through the impaired intestinal barrier, causing local and systemic infections, even serious complications such as sepsis, systemic inflammatory response syndrome and multiple organ failure.Bile acids(BAs) are active constituents of bile. The main BAs in humans are the primary BAs cholic acid(CA) and chenodeoxycholic acid(CDCA), and the secondary BAs deoxycholic acid(DCA) and lithocholic acid(LCA), as well as their glycine and taurine conjugates. Bacteria in the intestinal lumen metabolize the primary BAs into the secondary BAs mainly in colon. BAs are amphipathic molecules that facilitate the digestion and uptake of lipids together with the fat-soluble vitamins. Additionally,BAs also control the gut microflora and help to maintain the integrity of the gut barrier. Recent studies have emphasized the role of BAs as signaling molecules, which modulate their own biosynthesis and coordinately regulate a network of metabolic pathways including glucose, lipid, drug and energy metabolism. Furthermore, BAs are involved in the immune response, cell proliferation, apoptosis and cell differentiation.Bile acid membrane receptor TGR5 is a G protein coupled receptor(GPCR). TGR5 is expressed in many different organs and cells and high expression levels of TGR5 have been detected in gallbladder epithelium and in the intestine. TGR5 could mediate bile acid homeostasis and metabolism. TGR5 activation could increase energy expenditure and induce a significant reduction of the body weight of mice fed a high fat diet. TGR5 induces glucagon-like peptide-1(GLP-1) secretion in enteroendocrine cells, increases insulin sensitivity and modulates glucose homeostasis. The total bile acid pool size in TGR5 knockdown mice was decreased as compared to that of wild type mice. TGR5 knockown mice were protected against cholesterol gallstone formation. TGR5 also modulates the immune response and exerts anti-inflammatory function. Recent studies showed TGR5 plays the role in inhibiting apoptosis and promoting cell proliferation, for example, taurocholate can promote the proliferation of cholangiocyte.This paper focuses on the role of the internal and external biliary drainage in the intestinal mucosal barrier of patients with OJ and TGR5 expression in gut mucosa, and investigates whether chenodeoxycholic acid(CDCA) could promote intestinal epithelial cell proliferation through upregulating TGR5 expression in animal and cell models, thereby preserving the gut barrier.Part 1 The role of bile in the intestinal barrier and TGR5 expression in intestinal mucosa in patients with obstructive jaundiceObjectives: To investigate the effect of bile on the intestinal mucosa and TGR5 expression in intestinal mucosa in patients with obstructive jaundice.Materials and methods: Sixteen patients with malignant obstructive jaundice who underwent endoscopic retrograde cholangiopancreatography(ERCP) are divided into two groups. Eight patients underwent internal biliary drainage(ID) through endoscopic retrograde biliary drainage(ERBD). The other eight patients underwent external biliary drainage(ED) through endoscopic nasobiliary drainage(ENBD). Biopsy was done in all participants at the second part of the duodenum, 2-5 cm distal to the ampulla of Vater before treatment and one week after treatment. The morphological and ultrastructure changes of intestinal mucosa were observed. The distributions and expressions of TGR5 and proliferation marker proliferating cell nuclear antigen(PCNA) in intestinal mucosa were evaluated by immunohistochemistry(IHC) and Western blot. Meanwhile, the serum was collected before and after treatment in all patients, intestinal epithelial cell enzyme marker diamine oxidase(DAO) and intestinal fatty acid binding protein(I-FABP), and productions of intestinal bacteria lipopolysaccharides(LPS) and D-lactate levels in serum were detected.Results: ① HE staining was observed under light microscope. Compared with that before ID, after ID, the intestinal mucosal villi were intact and epithelial cell arrayed relatively neat, the pathological score of the intestinal mucosal injury was 0.7 ± 0.63 and significantly lower than that before ID(2.5 ± 0.37, P<0.01). There was no significantly difference in the intestinal mucosal morphology and the pathological score of the intestinal mucosal injury before and after ED(P>0.05). ② After ID, the ultrastructure of intestinal mucosa observed by electron microscopy showed that microvilli increased and arranged neatly and the gap of tight junction became narrow compared with that before ID. After ED, There was no significantly difference in the ultrastructure of intestinal mucosa and the gap of tight junction was still broad compared to that before ED. ③ The results of serum measurement showed the levels of DAO, I-FABP, LPS and D-lactate after ID were 57.23 ± 8.6 U/L, 149.6 ± 29.16 pg/ml, 0.2 ± 0.02 EU/ml, 1.0 ± 0.2 m M respectively and significantly decreased compared with that before ID(96.12 ± 12.51 U/L, 302.5 ± 6103 pg/ml, 0.39 ± 0.04 EU/ml, 1.68 ± 0.18 m M, P<0.05). The levels of DAO, I-FABP, LPS and D-lactate in serum had no significantly difference before and after ED(P>0.05). ④ The result of IHC showed that PCNA was expressed in the cell nucleus and TGR5 was expressed in the cell membrane. After ID, the expressions of PCNA and TGR5 were significantly increased compared with that before ID. There was no significantly difference in the expressions of PCNA and TGR5 before and after ED. The results of Western blot were consistent with IHC. After ID, the protein levels of PCNA and TGR5 were significantly increased compared with that before ID(P<0.05, P<0.01). The protein levels of PCNA and TGR5 before and after ED had no significantly difference(P>0.05).Conclusions: In patients with OJ, bile could promote the intestinal epithelial cell proliferation, improve the intestinal morphology and ultrastructure, decrease the permeability of the intestinal mucosal barrier, and upregulate the expression of TGR5. TGR5 may be contributed to the intestinal epithelial cell proliferation.Part 2 The effect of bile acid on the intestinal mucosal barrier and TGR5 expression in intestinal mucosa in rat with obstructive jaundiceObjectives: To establish OJ rat model and investigate the effect of CDCA on the intestinal mucosal barrier and TGR5 expression in intestinal mucosa.Materials and methods: Thirty Wistar rats were randomly divided into three groups: sham operation(SHAM), bile duct ligation + Vehicle(BDL + Vehicle) and BDL + CDCA. After one week,the terminal ileum tissues were harvested. The morphological and ultrastructure changes of terminal ileum were observed. The distributions and expressions of TGR5 and PCNA in terminal ileum were evaluated by IHC and Western blot. Meanwhile, the DAO, I-FABP, LPS and D-lactate levels in serum were detected.Results: ① The results of HE staining showed that the intestinal mucosal villi and epithelial cells arrayed neatly in SHAM. The integrity of intestinal mucosa was damaged in BDL + Vehicle, the villi was short and thick, subepithelial space at the tip of the villi was extended, and the epithelial layer moderately lifted from the lamina propria. In BDL + CDCA, the intestinal mucosal villi and epithelial cells arrayed relatively neatly, the pathological score of the intestinal mucosal injury was 1.8 ± 0.2 and significantly decreased compared with BDL + Vehicle(2.6 ± 0.16, P<0.01). ② The ultrastructure of intestinal mucosa observed by electron microscopy showed that in the SHAM group, microvilli arranged neatly and tightly; in the BDL + Vehicle group, microvilli arranged loosely, the gap of tight junction became broad and there was vacuolization in the cytoplasm; in the BDL + CDCA group, microvilli arranged relatively neatly and there was not vacuolization in the cytoplasm. ③ The levels of DAO, I-FABP, LPS andD-lactate in serum in the BDL + Vehicle group were significantly increased compared with that in the SHAM group. The levels of these indexes in the BDL + CDCA group were significantly decreased compared with that in the BDL + Vehicle group(P<0.001). ④ The result of IHC showed that PCNA was expressed in the cell nucleus and TGR5 was expressed in the cell membrane. The expressions of PCNA and TGR5 in intestinal mucosa were decreased in BDL + Vehicle compared with that in SHAM. The expressions of PCNA and TGR5 in intestinal mucosa were increased in BDL + CDCA compared with that in the BDL + Vehicle group(P<0.001). The results of Western blot were consistent with IHC. The protein levels of PCNA and TGR5 were significantly decreased in BDL + Vehicle compared with that in SHAM(P<0.01). The protein levels of PCNA and TGR5 were significantly increased in BDL + CDCA compared with that in BDL + Vehicle(P<0.05).Conclusions: The bile acid CDCA could promote the intestinal epithelial cell proliferation, improve the intestinal morphology, decrease the permeability of the intestinal mucosal barrier, and upregulate the expression of TGR5 in the intestinal mucosa. TGR5 may be involved in the intestinal epithelial cell proliferation.Part 3 Bile acid promotes the intestinal epithelial cell proliferation through upregulating the expression of TGR5Objectives: To explore whether CDCA promotes the intestinal epithelial cell proliferation through upregulating the expression of TGR5.Materials and methods: We induced FHs 74 Int cell injury with LPS as cell model of intestinal mucosal injury in obstructive jaundice and observed the effect of CDCA on human small intestinal epithelial cell line FHs 74 Int cell injury caused by LPS. There were four groups: control, CDCA, LPS and LPS + CDCA. We treated FHs 74 Int cell with LPS(1 mg/ml) and/or CDCA(100 μM) for 48 h, then collected the cells. We downregulaed and overexpressed the expression of TGR5 in FHs 74 Int by lentiviral vector and observed the relationship between TGR5 and cell proliferation. The efficacy of knockdown and overexpression of TGR5 in FHs 74 Int cells were analyzed in the level of m RNA and protein by RT-real time PCR and Western blot. The TGR5 knockdown FHs 74 Int cells were treated with LPS(1 mg/ml) for 48 h, there were four groups: Lv-sh Con, Lv-sh TGR5, Lv-sh Con + LPS and Lv-sh TGR5 + LPS. The TGR5 overexpression FHs 74 Int cells were treated with LPS(1 mg/ml) for 48 h, there were four groups: Lv-Flag, Lv-TGR5, Lv-Flag + LPS and Lv-TGR5 + LPS. In order to further demonstrate that CDCA could promote cell proliferation through TGR5, We treated the TGR5 knockdown FHs 74 Int with LPS(1 mg/ml) and/or CDCA(100 μM) for 48 h, there were four groups: Lv-sh Con, Lv-sh Con + LPS, Lv-sh Con + LPS + CDCA and Lv-sh TGR5 + LPS + CDCA. The cells of every group were collected. In order to reflect the level of cell proliferation, the cell viability was measured by CCK-8, the cell cycle was determined by flow cytometry and the DNA synthesis was measured through Ed U incorporation. The expression of PCNA and TGR5 were detected by Western blot.Results: ① CCK-8 analysis showed that compared with the LPS group, the cell viability was significantly enhanced. The results of cell cycle showed that in LPS + CDCA the proportion of cells in S phase was increased and the proportion in G0/G1 phase was decreased compared to the LPS group. The result of Ed U was consistent with that of CCK-8 and cell cycle. DNA synthesis was increased in LPS + CDCA compared with the LPS group. These results demonstrated that CDCA could attenuate the inhibited role of LPS in cell proliferation. The result of Western blot showed that LPS inhibited the expressions of PCNA and TGR5, after treatment with CDCA, the expressions of PCNA and TGR5 were significantly upregulated compared with the LPS group. This result showed that bile acid could promote intestinal epithelial cell proliferation and the expression of TGR5, both had a positive relationship. ② CCK-8 analysis suggested that after treatment with LPS, the cell viability was decreased, and when TGR5 was downregulated by Lv-sh TGR5 in FHs 74 Int cell, the cell viability decreased more significantly. The results of cell cycle showed that after treatment with LPS the proportion of cells in S phase was decreased and the proportion in G0/G1 phase was increased, this suggested that the cells were blocked in G0/G1 phase. In Lv-sh TGR5 + LPS group, more cells were blocked in G0/G1 phase compared to the Lv-sh Con + LPS group. The result of Ed U showed that DNA synthesis was inhibited after treatment with LPS. When TGR5 was downregulated in FHs 74 Int cell, DNA synthesis was further inhibited. The result of Western blot showed that LPS could significantly inhibit the expression of proliferation marker protein PCNA, and knockdown of TGR5 further downregulated the level of PCNA. These results suggested that knockdown of TGR5 aggravated the inhibited effect of LPS on FHs 74 Int cell proliferation and TGR5 could promote the cell proliferation. ③ CCK-8 analysis suggested that after treatment with LPS, the cell viability was decreased in Lv-Flag and Lv-TGR5 group, when TGR5 was overexpressed in FHs 74 Int cell, the cell viability increased in Lv-TGR5 + LPS compared with that in Lv-Flag + LPS. The results of cell cycle showed that after treatment with LPS the proportion of cells in S phase was decreased and the proportion in G0/G1 phase was increased, this suggested that the cells were blocked in G0/G1 phase. In Lv-TGR5 + LPS group, less cells were blocked in G0/G1 phase compared to the Lv-Flag + LPS group. The result of Ed U showed that DNA synthesis was inhibited after treatment with LPS, when TGR5 was overexpressed in FHs 74 Int cell, DNA synthesis was increased. The result of Western blot showed that LPS could significantly inhibit the expression of proliferation marker protein PCNA, and overexpression of TGR5 inhibited the downregulation of PCNA casued by LPS. These results suggested that overexpression of TGR5 attenuated the inhibited effect of LPS on FHs 74 Int cell proliferation and TGR5 could promote the cell proliferation. ④ After treatment with LPS, the cell viability decreased, the proportion of cells in S phase was decreased and the proportion in G0/G1 phase was increased, DNA synthesis decreased and the expression of PCNA downregulated. These suggested that LPS significantly inhibited the cell proliferation. In the Lv-sh Con + LPS + CDCA group, the cell viability increased, the proportion of cells in S phase was increased and the proportion in G0/G1 phase was decreased, DNA synthesis increased and the expression of PCNA upregulated. These suggested that CDCA attenuated the inhibited effect of LPS on the cell proliferation. However, in TGR5 knockdown cells, CDCA could not attenuate the inhibited effect of LPS on the cell proliferation, for example, compared with the Lv-sh Con + LPS + CDCA group, the cell viability decreased, the proportion of cells in S phase was decreased and the proportion in G0/G1 phase was increased, DNA synthesis decreased and the expression of PCNA downregulated in the Lv-sh TGR5 + LPS + CDCA group.Conclusions: Bile acid promoted intestinal epithelial cell prolifertation through upregulating the expression of TGR5.