Yongtao Xiao

Up-regulation of miR-200b in biliary atresia patients accelerates proliferation and migration of hepatic stallate cells by activating PI3K/Akt signaling.

An increasing body of evidence suggests that miRNAs are involved in fibrotic process of several organs including heart, lung and kidney. It has been observed recently that aberrant expression of miR-200s are associated with hepatic fibrosis. However, the role and underlying mechanism of miR-200s in hepatic fibrogenesis remains unknown. Here, we investigate the role of miR-200b in the activation of immortalized human hepatic stallate cells (HSCs), LX-2 cells. We firstly found that miR-200b significantly enhanced proliferation and migration of LX-2 cells. Secondly, our findings showed that miR-200b enhanced the phosphorylation of Akt, a downstream effector of phosphatidyl-inositol 3-Kinase (PI3K). FOG2, as the targets of fly miR-8 and human miR-200s, directly binds to p85α and inhibits the activation of the PI3K/Akt pathway. Here, we showed that FOG2 protein levels in LX-2 cells were suppressed significantly by miR-200b mimics. FOG2 knockdown by siRNAs activated the PI3K/Akt signaling, which increased cell growth and migration that mimicked the effect of miR-200b. Conversely, LY294002, a highly selective inhibitor of PI3K, could block phosphorylation of Akt and effect of miR-200b. In addition, we showed that miR-200b enhanced the expression of matrix metalloproteinase-2 (MMP-2), which may increase the migration of LX-2 cells. Finally, our results indicated that the expression of miR-200b was unregulated in the biliary atresia (BA) and associated with liver fibrotic progression. These data suggest a potential mechanism for Akt activation through FOG2 down-regulation by miR-200b that can lead to HSC growth and migration. In view of the putative pathogenic role of miR-200b in HSCs, miR-200b may constitute a potential marker for HSC activation and liver fibrosis progression.

Neutralization of IL-6 and TNF-α ameliorates intestinal permeability in DSS-induced colitis.

The cytokines tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) have been implicated as important mediators of the inflammatory reaction in patients with intestinal inflammation. The present study was designed to investigate the roles of these cytokines on mucosal barrier function in a mouse model of acute colitis with using anti-cytokine strategies. Mice received 3% dextran sulfate sodium (DSS) in their drinking water for 7days showed morphological alteration of mucosa and increase of intestinal permeability. Administration of IL-6 monoclonal antibody (mAb) or TNF-α mAb significantly attenuated intestinal permeability. IL-6 mAb and TNF-α mAb treatment also effectively suppressed the expression of claudin-2 and myosin light chain kinase (MLCK). Taken together, we indicated that anti-IL-6 and anti-TNF-α therapy prevent intestinal permeability induced by intestinal inflammation.

The expression of epithelial-mesenchymal transition-related proteins in biliary epithelial cells is associated with liver fibrosis in biliary atresia

Background:The epithelial–mesenchymal transition (EMT) has been implicated as a key mechanism in the pathogenesis of liver fibrosis. The miR-200 family has been shown to inhibit EMT.Methods:Liver fibrosis levels were assessed with Masson’s trichrome staining of liver samples obtained from biliary atresia (BA) patients. The expressions of cytokeratin-7 (CK-7) and α-smooth muscle actin (α-SMA) in the liver sections were detected by immunohistochemical and immunofluorescent staining. EMTs were induced by transforming growth factor (TGF)-β1 in human biliary epithelial cells (BECs) in vitro.Results:We showed that the EMT-related proteins CK-7 and α-SMA colocalized to the intrahepatic BECs in the liver sections of patients with BA. The level of α-SMA expression was related to liver fibrosis stage in BA. EMT in primary human intrahepatic BECs was induced by TGF-β1 in vitro. miR-200b is one member of the miR-200 family and significantly inhibited TGF-β1-mediated EMT in BECs.Conclusion:Together, these data suggest that the occurrence of EMT in BECs might contribute to BA fibrosis. miR-200b significantly affects the development and progression of TGF-β1-dependent EMT and fibrosis in vitro.

Distinct Plasma Bile Acid Profiles of Biliary Atresia and Neonatal Hepatitis Syndrome.

Biliary atresia (BA) is a severe chronic cholestasis disorder of infants that leads to death if not treated on time. Neonatal hepatitis syndrome (NHS) is another leading cause of neonatal cholestasis confounding the diagnosis of BA. Recent studies indicate that altered bile acid metabolism is closely associated with liver injury and cholestasis. In this study, we systematically measured the bile acid metabolome in plasma of BA, NHS, and healthy controls. Liver bile acids were also measured using biopsy samples from 48 BA and 16 NHS infants undergoing operative cholangiography as well as 5 normal adjacent nontumor liver tissues taken from hepatoblastoma patients as controls. Both BA and NHS samples had significantly elevated bile acid levels in plasma compared to normal controls. BA patients showed a distinct bile acid profile characterized by the higher taurochenodeoxycholic acid (TCDCA) level and lower chenodeoxycholic acid (CDCA) level than those in NHS patients. The ratio of TCDCA to CDCA in plasma was significantly higher in BA compared to healthy infants (p < 0.001) or NHS (p < 0.001). The area under receiver operating characteristic curve for TCDCA/CDCA to differentiate BA from NHS was 0.923 (95% CI: 0.862-0.984). These findings were supported by significantly altered expression levels of bile acid transporters and nuclear receptors in liver including farnesoid X receptor (FXR), small heterodimer partner (SHP), bile salt export pump (BSEP), and multidrug resistant protein 3 (MDR3) in BA compared to NHS. Taken together, the plasma bile acid profiles are distinct in BA, NHS, and normal infants, as characterized by the ratio of TCDCA/CDCA differentially distributed among the three groups of infants.

Elevated Bile Acids in Newborns with Biliary Atresia (BA)

Biliary Atresia (BA), a result from inflammatory destruction of the intrahepatic and extrahepatic bile ducts, is a severe hepatobiliary disorder unique to infancy. Early diagnosis and Kasai operation greatly improve the outcome of BA patients, which encourages the development of early screening methods. Using HPLC coupled tandem mass spectrometry, we detected primary bile acids content in dried blood spots obtained from 8 BA infants, 17 neonatal jaundice and 292 comparison infants at 3–4 days of life. Taurocholate (TC) was significantly elevated in biliary atresia infants (0.98±0.62 µmol/L) compared to neonatal jaundice (0.47±0.30 µmol/L) and comparison infants (0.43±0.40 µmol/L), with p = 0.0231 and p = 0.0016 respectively. The area under receiver operating characteristic (ROC) curve for TC to discriminate BA and comparison infants was 0.82 (95% confidence interval: 0.72–0.92). A cutoff of 0.63 µmol/L produced a sensitivity of 79.1% and specificity of 62.5%. The concentrations of total bile acids were also raised significantly in BA compared to comparison infants (6.62±3.89 µmol/L vs 3.81±3.06 µmol/L, p = 0.0162), with the area under ROC curve of 0.75 (95% confidence interval: 0.61–0.89). No significant difference was found between the bile acids of neonatal jaundice and that of comparison infants. The early increase of bile acids indicates the presentation of BA in the immediate newborn period and the possibility of TC as newborn screening marker.

Common Genetic Variations in Patched1 (PTCH1) Gene and Risk of Hirschsprung Disease in the Han Chinese Population

Hirschsprung disease (HSCR) is the most frequent genetic cause of congenital intestinal obstruction with an incidence of 1:5000 live births. In a pathway-based epistasis analysis of data generated by genome-wide association study on HSCR, specific genotype of Patched 1 (PTCH1) has been linked to an increased risk for HSCR. The aim of the present study is to examine the contribution of genetic variants in PTCH1 to the susceptibility to HSCR in Han Chinese. Accordingly, we assessed 8 single nucleotide polymorphisms (SNPs) within PTCH1 gene in 104 subjects with sporadic HSCR and 151 normal controls of Han Chinese origin by the Sequenom MassArray technology (iPLEX GOLD). Two of the eight genetic markers were found to be significantly associated with Hirschsprung disease (rs357565, allele P = 0.005; rs2236405, allele P = 0.002, genotype P = 0.003). Both the C allele of rs357565 and the A allele of rs2236405 served as risk factors for HSCR. During haplotype analysis, one seven-SNP-based haplotype was the most significant, giving a global P = 0.0036. Our results firstly suggest common variations of PTCH1 may be involved in the altered risk for HSCR in the Han Chinese population, providing potential molecular markers for early diagnosis of Hirschsprung disease.

Low doses of CMV induce autoimmune-mediated and inflammatory responses in bile duct epithelia of regulatory T cell-depleted neonatal mice

Recent studies have indicated that perinatal infection with cytomegalovirus (CMV) may promote bile duct damage in biliary atresia (BA) and that the decreased regulatory T cell (Treg) percentage associated with BA may further amplify the bile duct damage. Although a majority of BA patients have had previous CMV infections and lower percentages of Tregs, it is unknown whether an initial exposure to a low dose of CMV could induce exaggerated and progressive biliary injury. A Treg-depleted neonatal mouse was infected with low-dose CMV (LD-CMV) as a model to study BA patients. LD-CMV infection in Treg-depleted mice induced extensive inflammation in both the intrahepatic and extrahepatic bile ducts, accompanied with injury to and atresia of intrahepatic bile ducts and partial obstruction of the extrahepatic bile ducts. Serum total and direct bilirubin amounts were also elevated. Evidence for the involvement of cellular and humoral autoimmune responses in LD-CMV-infection of Treg-depleted mice was also obtained through detection of increased percentages of CD3 and CD8 mononuclear cells and serum autoantibodies reactive to bile duct epithelial proteins, one of which was identified as α-enolase. Depletion of Tregs that can lead to the decreased inhibition of aberrantly activated hepatic T-lymphocytes and generation of autoantibodies may lead to further injury. Increased hepatic expression of Th1-related genes (TNF-α), IFN-γ-activated genes (STAT-1) and Th1 cytokines (TNF-α, lymphotactin, IL-12p40 and MIP −1γ) were also identified. In conclusion, autoimmune-mediated and inflammatory responses induced by LD-CMV infection in Treg-depleted mice results in increased intrahepatic and extrahepatic bile duct injury and contributed to disease progression.

Inhibition of Autophagic Degradation Process Contributes to Claudin-2 Expression Increase and Epithelial Tight Junction Dysfunction in TNF-α Treated Cell Monolayers

Tight junction dysfunction plays a vital role in some chronic inflammatory diseases. Pro-inflammatory cytokines, especially tumor necrosis factor alpha (TNF-α), act as important factors in intestinal epithelial tight junction dysfunction during inflammatory conditions. Autophagy has also been shown to be crucial in tight junction function and claudin-2 expression, but whether autophagy has an effect on the change of claudin-2 expression and tight junction function induced by TNF-α is still unknown. To answer this question, we examined the expression of claudin-2 protein, transepithelial electrical resistance (TER), and permeability of cell monolayers, autophagy flux change, and lysosomal pH after TNF-α with or without PP242 treatment. Our study showed that claudin-2 expression, intestinal permeability, microtubule-associated protein 1 light chain 3B II (LC3B-II) and sequestosome 1 (P62) expression largely increased while TER values decreased in TNF-α treated cell monolayers. Further research using 3-methyladenine (3-MA), bafilomycin A1, and ad-mCherry-GFP-LC3B adenovirus demonstrated that LC3B-II increase induced by TNF-α was attributed to the inhibition of autophagic degradation. Moreover, both qualitative and quantitative method confirmed the increase of lysosomal pH, and mammalian target of rapamycin (mTOR) inhibitor PP242 treatment relieved this elevation. Moreover, PP242 treatment also alleviated the change of autophagy flux, TER, and claudin-2 expression induced by TNF-α. Therefore, we conclude that increase of claudin-2 levels and intestinal epithelial tight junction dysfunction are partly caused by the inhibition of autophagic degradation in TNF-α treated cell monolayers.

Glucocorticoid treatment alters systemic bile acid homeostasis by regulating the biosynthesis and transport of bile salts

BACKGROUND Dysregulation of systemic bile acid homeostasis can lead to cholestatic liver diseases and metabolic syndromes. As important anti-inflammatory and immunosuppressive drugs, synthetic glucocorticoids (GCs) are used to treat several cholestatic disorders, including biliary atresia (BA), because of their effects on the regulation of bile acid metabolism. However, the molecular mechanisms that underlie GCs regulation of bile acid homeostasis remain unclear. AIMS To provide a mechanistic basis for the effects of GCs on bile acid homeostasis. METHODS Male rats were treated with methylprednisolone for 7 days with slow-release osmotic pumps under physiological and cholestatic status that was induced by bile duct ligation (BDL). Expression of glucocorticoid receptor (GR) and genes related to bile acid metabolism was investigated using western blotting, qRT-PCR and immunohistochemistry. RESULTS We show here that sustained treatment with GCs in rats disrupts the normal changes in systemic bile acid distribution by elevating plasma bile acid levels and reducing faecal bile acid loss. Treatment with GCs stimulated bile acid absorption in the ileum by increasing expression of the apical sodium-dependent bile acid transporter (Asbt). Concomitantly, administration of GCs enhanced liver bile acid uptake by increasing the expression of the major hepatocyte basolateral bile transporter (Ntcp). The reduced expression of a bile acid synthesis rate-controlling enzyme, Cyp7a1, suggests that treatment with GCs suppressed hepatic bile acid synthesis. CONCLUSION Our study provides evidence that GCs can increase enterohepatic bile acid circulation through regulation of the biosynthesis and transport of bile salts, which suggests that plasma bile acid levels should be monitored during treatment with GCs in patients with BA.

miR-200b inhibits TNFα-induced IL-8 secretion and tight junction disruption of intestinal epithelial cells in vitro.

Inflammatory bowel diseases (IBDs) are chronic, inflammatory disorders of the gastrointestinal tract with unclear etiologies. Intestinal epithelial cells (IECs), containing crypt and villus enterocytes, occupy a critical position in the pathogenesis of IBDs and are a major producer of immunoregulatory cytokines and a key component of the intact epithelial barrier. Previously, we have reported that miR-200b is involved in the progression of IBDs and might maintain the integrity of the intestinal epithelial barrier via reducing the loss of enterocytes. In this study, we further investigated the impact of miR-200b on intestinal epithelial inflammation and tight junctions in two distinct differentiated states of Caco-2 cells after TNF-α treatment. We demonstrated that TNF-α-enhanced IL-8 expression was decreased by microRNA (miR)-200b in undifferentiated IECs. Simultaneously, miR-200b could alleviate TNF-α-induced tight junction (TJ) disruption in well-differentiated IECs by reducing the reduction in the transepithelial electrical resistance (TEER), inhibiting the increase in paracellular permeability, and preventing the morphological redistribution of the TJ proteins claudin 1 and ZO-1. The expression levels of the JNK/c-Jun/AP-1 and myosin light chain kinase (MLCK)/phosphorylated myosin light chain (p-MLC) pathways were attenuated in undifferentiated and differentiated enterocytes, respectively. Furthermore, a dual-luciferase reporter gene detection system provided direct evidence that c-Jun and MLCK were the specific targets of miR-200b. Collectively, our results highlighted that miR-200b played a positive role in IECs via suppressing intestinal epithelial IL-8 secretion and attenuating TJ damage in vitro, which suggested that miR-200b might be a promising strategy for IBD therapy. NEW & NOTEWORTHY This was the first time that the inhibitory role of miR-200b on intestinal epithelial inflammation and paracellular permeability has been reported. Moreover, we further divided the intestinal epithelial cells (IECs) into two differentiated conditions and investigated the distinct impacts of miR-200b. Finally, we put forward and proved that myosin light chain kinase (MLCK) was a novel target of miR-200b.