circRNA3669 promotes goat endometrial epithelial cells proliferation via miR-26a/RCN2 to activate PI3K/AKT-mTOR and MAPK pathways

The development of receptive endometrium(RE) from pre-receptive endometrium(PE) for successful embryo implantation is a complex dynamic process in which the morphology and physiological states of the endometrial epithelium undergo a series of significant changes, including cell proliferation and apoptosis. However, the molecular mechanisms are not yet fully understood. In this study, a higher circRNA3669 level was observed in PE than in RE of goats. Functional assays revealed that this overexpression promoted the proliferation of goat endometrial epithelial cells(GEECs) by activating the expression of genes related to the PI3K/AKT-mTOR and MAPK pathways,thereby inhibiting apoptosis in vitro. Furthermore, circRNA3669 functioned as a competing endogenous RNA(ceRNA) to upregulate Reticulocalbin-2(RCN2) expression at the post-transcriptional level by interacting with and downregulating miR-26a in GEECs. In addition, RCN2, which is highly expressed in the PE of goats, was found to be regulated by β-estradiol(E2) and progesterone(P4). Our results demonstrated that RCN2 also affected the key proteins PI3K, AKT, mTOR, JNK, and P38 in the PI3K/AKT-mTOR and MAPK pathways, thereby facilitating GEECs proliferation and suppressing their apoptosis in vitro. Collectively, we constructed a new circRNA3669-miR-26aRCN2 regulatory network in GEECs, which further provides strong evidence that circRNA could potentially play a crucial regulatory role in the development of RE in goats.

SIRT1 inhibits apoptosis of human lens epithelial cells through suppressing endoplasmic reticulum stress in vitro and in vivo

AIM:To explore the effect of silent information regulator factor 2-related enzyme 1(SIRT1)on modulating apoptosis of human lens epithelial cells(HLECs)and alleviating lens opacification of rats through suppressing endoplasmic reticulum(ER)stress.METHODS:HLECs(SRA01/04)were treated with varying concentrations of tunicamycin(TM)for 24h,and the expression of SIRT1 and C/EBP homologous protein(CHOP)was assessed using real-time quantitative polymerase chain reaction(RT-PCR),Western blotting,and immunofluorescence.Cell morphology and proliferation was evaluated using an inverted microscope and cell counting kit-8(CCK-8)assay,respectively.In the SRA01/04 cell apoptosis model,which underwent siRNA transfection for SIRT1 knockdown and SRT1720 treatment for its activation,the expression levels of SIRT1,CHOP,glucose regulated protein 78(GRP78),and activating transcription factor 4(ATF4)were examined.The potential reversal of SIRT1 knockdown effects by 4-phenyl butyric acid(4-PBA;an ER stress inhibitor)was investigated.In vivo,age-related cataract(ARC)rat models were induced by sodium selenite injection,and the protective role of SIRT1,activated by SRT1720 intraperitoneal injections,was evaluated through morphology observation,hematoxylin and eosin(H&E)staining,Western blotting,and RT-PCR.RESULTS:SIRT1 expression was downregulated in TMinduced SRA01/04 cells.Besides,in SRA01/04 cells,both cell apoptosis and CHOP expression increased with the rising doses of TM.ER stress was stimulated by TM,as evidenced by the increased GRP78 and ATF4 in the SRA01/04 cell apoptosis model.Inhibition of SIRT1 by siRNA knockdown increased ER stress activation,whereas SRT1720 treatment had opposite results.4-PBA partly reverse the adverse effect of SIRT1 knockdown on apoptosis.In vivo,SRT1720 attenuated the lens opacification and weakened the ER stress activation in ARC rat models.CONCLUSION:SIRT1 plays a protective role against TM-induced apoptosis in HLECs and slows the progression of cataract in rats by inhibiting ER stress.These findings suggest a novel strategy for cataract treatment focused on targeting ER stress,highlighting the therapeutic potential of SIRT1 modulation in ARC development.

Overexpression of TRPV1 activates autophagy in human lens epithelial cells under hyperosmotic stress through Ca^(2+)-dependent AMPK/mTOR pathway

●AIM:To explore whether autophagy functions as a cellular adaptation mechanism in lens epithelial cells(LECs)under hyperosmotic stress.●METHODS:LECs were treated with hyperosmotic stress at the concentration of 270,300,400,500,or 600 mOsm for 6,12,18,24h in vitro.Polymerase chain reaction(PCR)was employed for the mRNA expression of autophagyrelated genes,while Western blotting detected the targeted protein expression.The transfection of stub-RFP-sens-GFPLC3 autophagy-related double fluorescence lentivirus was conducted to detect the level of autophagy flux.Scanning electron microscopy was used to detect the existence of autolysosome.Short interfering RNA of autophagy-related gene(ATG)7,transient receptor potential vanilloid(TRPV)1 overexpression plasmid,related agonists and inhibitors were employed to their influence on autophagy related pathway.Flow cytometry was employed to test the apoptosis and intracellular Ca^(2+)level.Mitochondrial membrane potential was measured by JC-1 staining.The cell counting kit-8 assay was used to calculate the cellular viability.The wound healing assay was used to evaluate the wound closure rate.GraphPad 6.0 software was utilized to evaluate the data.●RESULTS:The hyperosmotic stress activated autophagy in a pressure-and time-dependent manner in LECs.Beclin 1 protein expression and conversion of LC3B II to LC3B I increased,whereas sequestosome-1(SQSTM1)protein expression decreased.Transient Ca^(2+)influx was stimulated caused by hyperosmotic stress,levels of mammalian target of rapamycin(mTOR)phosphorylation decreased,and the level of AMP-activated protein kinase(AMPK)phosphorylation increased in the early stage.Based on this evidence,autophagy activation through the Ca^(2+)-dependent AMPK/mTOR pathway might represent an adaptation process in LECs under hyperosmotic stress.Hyperosmotic stress decreased cellular viability and accelerated apoptosis in LECs and cellular migration decreased.Inhibition of autophagy by ATG7 knockdown had similar results.TRPV1 overexpression increased autophagy and might be crucial in the occurrence of autophagy promoted by hyperosmotic stress.●CONCLUSION:A combination of hyperosmotic stress and autophagy inhibition may be a promising approach to decrease the number of LECs in the capsular bag and pave the way for improving prevention of posterior capsular opacification and capsular fibrosis.

Umbilical cord mesenchymal stem cell exosomes alleviate necrotizing enterocolitis in neonatal mice by regulating intestinal epithelial cells autophagy

BACKGROUND Necrotizing enterocolitis(NEC)is a severe gastrointestinal disease that affects premature infants.Although mounting evidence supports the therapeutic effect of exosomes on NEC,the underlying mechanisms remain unclear.AIM To investigate the mechanisms underlying the regulation of inflammatory response and intestinal barrier function by umbilical cord mesenchymal stem cell(UCMSCs)exosomes,as well as their potential in alleviating NEC in neonatal mice.METHODS NEC was induced in 5-d-old C57BL/6 pups through hypoxia and gavage feeding of formula containing lipopolysaccharide(LPS),after which the mice received human UCMSC exosomes(hUCMSC-exos).The control mice were allowed to breastfeed with their dams.Ileal tissues were collected from the mice and analyzed by histopathology and immunoblotting.Colon tissues were collected from NEC neonates and analyzed by immunofluorescence.Molecular biology and cell culture approaches were employed to study the related mechanisms in intestinal epithelial cells.RESULTS We found that autophagy is overactivated in intestinal epithelial cells during NEC,resulting in reduced expression of tight junction proteins and an increased inflammatory response.The ability of hUCMSC-exos to ameliorate NEC in a mouse model was dependent on decreased intestinal autophagy.We also showed that hUCMSC-exos alleviate the inflammatory response and increase migration ability in intestinal epithelial cells induced by LPS.CONCLUSION These results contribute to a better understanding of the protective mechanisms of hUCMSC-exos against NEC and provide a new theoretical and experimental foundation for NEC treatment.These findings also enhance our understanding of the role of the autophagy mechanism in NEC,offering potential avenues for identifying new therapeutic targets.

Primary Culture of Bovine Mammary Epithelial Cells

[ Objective] To investigate the feasibility of the primary culture of bovine mammary epithelial cells in biochemical incubator. [ Method] In vitro, bovine mammary epithelial cells were isolated and cultured by the tissue explant method in order to investigate the optimal culture conditions. The morphology observation and identification of the cultured cells were performed by inverted microscope observation, Giemsa staining and cytokeratin immunohistochemistry. [ Result] Observed with inverted microscope, most of the bovine mammary epithelial cells were polygonal and displayed typical slabstone-like appearance. As it can be seen from cell staining results, the cell body was big and the nucleus was stained dark blue and was round or oval in shape, with clearly visible nucleoli, generally 2 -4 nucleoli. The tissue-specific expression of cytokeratin 14 and cytokeratin 18 genes in mammary epithelial cells was identified by cytokeratin immunohistochemistry. [ Conclusion] Primary bovine mammary epithelial cells were successfully cultured in biochemical incubator.

Acellular porcine corneal matrix as a carrier scaffold for cultivating human corneal epithelial cells and fibroblasts in vitro

AIM：To investigate the feasibility of corneal anterior lamellar reconstruction with human corneal epithelial cells and fibroblasts,and an acellular porcine cornea matrix（APCM） in vitro.·METHODS：The scaffold was prepared from fresh porcine corneas which were treated with 0.5%sodium dodecyl sulfate（SDS）solution and the complete removal of corneal cells was confirmed by hematoxylin-eosin（HE）staining and 4’,6-diamidino-2-phenylindole（DAPI）staining.Human corneal fibroblasts and epithelial cells were cultured with leaching liquid extracted from APCM,and then cell proliferative ability was evaluated by MTT assay.To construct a human corneal anterior lamellar replacement,corneal fibroblasts were injected into the APCM and cultured for 3d,followed by culturing corneal epithelial cells on the stroma construction surface for another 10d.The corneal replacement was analyzed by HE staining,and immunofluorescence staining.·R ESULTS：Histological examination indicated that there were no cells in the APCM by HE staining,and DAPI staining did not detect any residual DNA.The leaching liquid from APCM had little influence on the proliferation ability of human corneal fibroblasts and epithelial cells.At 10d,a continuous 3 to 5 layers of human corneal epithelial cells covering the surface of the APCM was observed,and the injected corneal fibroblasts distributed within the scaffold.The phenotype of the construction was similar to normal human corneas,with high expression of cytokeratin 12 in the epithelial cell layer and high expression of Vimentin in the stroma.·CONCLUSION：Corneal anterior lamellar replacement can be reconstructed in vitro by cultivating human corneal epithelial cells and fibroblasts with an acellular porcine cornea matrix.This laid the foundation for the further transplantation in vitro.

Differentiation of human amniotic epithelial cells into corneal epithelial-like cells in vitro

·AIM: To explore the feasibility that human amniotic epithelial cells (hAECs) have the potential to differentiate into corneal epithelial -like cells under the microenvironment replicated by spontaneously immortalized human corneal epithelial cells (S-ihCECs). ·METHODS: hAECs were isolated by enzyme digestion, and flow cytometry was used to analysis the expression of CD29/90/166/73/34 and HLA -DR. Recovered and cultured S -ihCECs, immunocytochemistry was used to detect the expression of CK3/12. The proliferation of S - ihCECs handled by different concentrations of mitomycin was detected by CCK -8. The proliferation of hAECs cultured by S-ihCECs culture media collected at different time was analyzed by CCK -8. After filtered out the optimal conditions, we collected S-ihCECs culture media for 5 days, then prepared conditioned medium to incubate hAECs, inverted phase contrast microscope and scanning electron microscope were used to observe the change of morphology in hAECs. Quantitative real -time reverse transcription -polymerase chain reaction (QRT - PCR) was carried out to evaluate the expression of Oct - 4, NANOG, PAX6, and CK12 in the differentiation period. Immunocytochemistry and western bloting were used to detect the expression of CK3/12. ·RESULTS: The culture media collected every 12h, from 20μg/mL mitomycin pretreatment S -ihCECs could significantly promote the proliferation of hAECs. In the period of differentiation, the morphology of differentiated hAECs was obviously different compared with the control group, and the distinctive CK3/12 for corneal epithelial cells was detected.·CONCLUSION: This study showed that hAECs can differentiate into corneal epithelial -like cells by replication of the corneal epithelial microenvironment, using the culture media collected from S -ihCECs, and it is possible that S -ihCECs culture media could be used in corneal tissue engineering. ·

Adherent properties of Helicobacter pylori to human epithelial cells *

AIM To study the properties and factors of Helicobacter pylori adherence to human epithelial cells. METHODS The adherent properties of human epithelial cells were studied by using a group of isolated H. pylori strains, anti H. pylori monoclonal antibodies and varied pH environment in in vitro adherence model with HEp 2 cell.

Uptake of bacterial lipopolysaccharide and expression of tumor necrosis factor α mRNA in isolated rat intrahepatic bile duct epithelial cells *

AIM To study the uptake of bacterial lipopolysaccharides (LPS) and expression of tumor necrosis factor α mRNA (TNF α mRNA) with cultured rat intrahepatic bile duct epithelial cells.

Isolation, Cuture and Biological Characteristic Analysis of Stromal and Glandular Epithelial Cells of Buffalo (Bubalus bubalis) Endometrium

[Objective] The experiment aimed to set up a method for isolating and culturing endometrial stromal cells (BESC) and endometrial glandular epithelial cells(BEGEC) of buffalo as well as laid foundation for studying biological mechanism of embryo implantation and uterine diseases. [Method] The enzymatic digestion method, scraping method, serial filtration and differential velocity adherent technique were used to isolate BESC and BEGEC, then immunocytochemical method and TRYPAN-Blue assay were used to determine the purity and survival rate of isolated cells. [Result] The BESC and BEGEC were successfully isolated and cultured while immunocytochemical method and cell count method demonstrated that the purity was over 90%. The result of TRYPAN-Blue assay shown that survival rate of BESC and BEGEC was 91% and 78% respectively. [Conclusion] The enzymatic digestion method, scraping method, serial filtration and differential velocity adherent technique could isolate BESC and BEGEC with high purity.

Effect of Helicobacter pylori on gastric epithelial cells

The gastrointestinal epithelium has cells with features that make them a powerful line of defense in innate mucosal immunity. Features that allow gastrointestinal epithelial cells to contribute in innate defense include cell barrier integrity, cell turnover, autophagy, and innate immune responses. Helicobacter pylori (H. pylori) is a spiral shape gram negative bacterium that selectively colonizes the gastric epithelium of more than half of the world’s population. The infection invariably becomes persistent due to highly specialized mechanisms that facilitate H. pylori’s avoidance of this initial line of host defense as well as adaptive immune mechanisms. The host response is thus unsuccessful in clearing the infection and as a result becomes established as a persistent infection promoting chronic inflammation. In some individuals the associated inflammation contributes to ulcerogenesis or neoplasia. H. pylori has an array of different strategies to interact intimately with epithelial cells and manipulate their cellular processes and functions. Among the multiple aspects that H. pylori affects in gastric epithelial cells are their distribution of epithelial junctions, DNA damage, apoptosis, proliferation, stimulation of cytokine production, and cell transformation. Some of these processes are initiated as a result of the activation of signaling mechanisms activated on binding of H. pylori to cell surface receptors or via soluble virulence factors that gain access to the epithelium. The multiple responses by the epithelium to the infection contribute to pathogenesis associated with H. pylori.

Detection of H.pylori DNA in gastric epithelial cells by in situ hybridization

AIM: To investigate the presence of H.pylori DNA within gastric epithelial cells in patients with H.pylori infection and its possible carcinogenic mechanism. METHODS: Total 112 patients, with pathologically confirmed chronic superficial gastritis, chronic atrophic gastritis, intestinal metaplasia, atypical hyperplasia or gastric cancer were studied. Among them, 28 were H.pylori negative and 84 H.pylori positive. H.pylori DNA in gastric epithelial cells was detected by GenPoint catalyzed signal amplification system for in situ hybridization. RESULTS: In the H.pylori positive group, zero out of 24 chronic superficial gastritis (0.0%), four out of 25 precancerous changes (16.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the nucleus of gastric epithelial cells, the positive rates of H.pylori DNA in the nucleus of gastric epithelial cells were progressively increased in chronic superficial gastritis, precancerous changes and gastric cancer groups (chi(2)=12.56, P=0.002); One out of 24 chronic superficial gastritis (4.2%), eleven out of 25 precancerous changes (44.0%) and thirteen out of 35 gastric cancers (37.1%) showed H.pylori DNA in the cytoplasm of gastric epithelial cells (chi(2)=10.86, P=0.004). In the H.pylori negative group, only one patient with gastric cancer was found H.pylori DNA in the nucleus of gastric epithelial cells; Only two patients, one patient with precancerous changes and another with gastric cancer, showed H.pylori DNA in the cytoplasm of gastric epithelial cells. Furthermore, H.pylori DNA must have been in the cytoplasm as long as it existed in the nucleus of gastric epithelial cells. CONCLUSION: H.pylori DNA exists both in the nucleus and the cytoplasm of gastric epithelial cells in patients with H.pylori infections. The pathological progression from chronic superficial gastritis, precancerous changes to gastric cancer is associated with higher positive rates of H.pylori DNA presence in the nucleus of gastric epithelial cells.

Intestinal epithelial cells in inflammatory bowel diseases

The pathogenesis of inflammatory bowel diseases (IBDs) seems to involve a primary defect in one or more of the elements responsible for the maintenance of intestinal homeostasis and oral tolerance. The most important element is represented by the intestinal barrier, a complex system formed mostly by intestinal epithelial cells (IECs). IECs have an active role in producing mucus and regulating its composition; they provide a physical barrier capable of controlling antigen traff ic through the intestinal mucosa. At the same time, they are able to play the role of non-professional antigen presenting cells, by processing and presenting antigens directly to the cells of the intestinal immune system. On the other hand, immune cells regulate epithelial growth and differentiation, producing a continuous bi-directional cross-talk within the barrier. Several alterations of the barrier function have been identif ied in IBD, starting from mucus features up to its components, from epithelial junctions up to the Toll-like receptors, and altered immune responses. It remains to be understood whether these defects are primary causes of epithelial damage or secondary effects. We review the possible role of the epithelial barrier and particularly describe the role of IECs in the pathogenesis of IBD.

Parthenolide protects human lens epithelial cells from oxidative stress-induced apoptosis via inhibition of activation of caspase-3 and caspase-9

The apoptosis of lens epithehal cells has been proposed as the common basis of cataract formation, with oxidative stress as the major cause. This study was performed to investigate the protective effect of the herbal constituent parthenolide against oxidative stress-induced apoptosis of human lens epithelial （HLE） cells and the possible molecular mechanisms involved. HLE cells （SRA01-04） were incubated with 50 μM H2O2 in the absence or presence of different doses of parthenolide （10, 20 and 50 μM）. To study apoptosis, the cells were assessed by morphologic examination and Annexin V-propidium iodide double staining flow cytometry; to investigate the underlying molecular mechanisms, the expression of caspase-3 and caspase-9 were assayed by Western blot and quantitative RT-PCR, and the activities of caspase-3 and caspase-9 were measured by a Chemicon caspase colorimetric activity assay kit. Stimulated with H202 for 18 h, a high fraction of riLE cells underwent apoptosis, while in the presence ofparthenolide of different concentrations, dose-dependent blocking of HLE cell apoptosis was observed. The expression of caspase-3 and caspase-9 induced by H202 in HLE cells was significantly reduced by parthenolide both at the protein and mRNA levels, and the activation ofcaspase-3 and caspase-9 was also suppressed by parthenolide in a dose-dependent manner. In conclusion, parthenolide prevents HLE cells from oxidative stress-induced apoptosis through inhibition of the activation ofcaspase-3 and caspase-9, suggesting a potential protective effect against cataract formation.

Influence on Cellular Signal Transduction Pathway in Dairy Cow Mammary Gland Epithelial Cells by Galactopoietic Compound Isolated from Vaccariae segetalis

The galactopoietic mechanism of Vaccaria segetalis is still unknown. Understanding dibutyl phthalate （DBP） separated from Vaccaria segetalis on the expression of lactation signal transduction genes of mammary gland epithelial cells, including prlr, erα, akt1, socs2, pparγ and elf5, will be helpful to reveal the molecular mechanism. Western blot and qRT- PCR were used to study the change of prlr, erα, akt, socs2, pparγ, and elf5 expression at mRNA and protein level. Co- localization expression of prolactin receptor （PRLR） and estrogen receptor α （ERα） was observed by immunofluorescence; the expression changes of miRNAs （21, 125b, 143, and 195） and the secretion of β-casein and lactose were detected by qRT-PCR and RP-HPLC. The results showed that Vaccaria segetalis active compound had similar fuctions as estrogen and/or prolactin （PRL） in dairy cow mammary gland epithelial cells （DCMECs）, increased the expressions of prlr, erα, akt1, and elf5 genes, while repressed pparγ expressions. DBP promoted socs2 mRNA expression, but its protein expressions were repressed. Furthermore, both DBP and PRL could repress the expressions of miRNA-125b, miRNA-143 and miRNA- 195 in DCMECs. DBP could repress the expression of miRNA-21, while the influence of PRL on miRNA-21 was not certain. DBP could promote the lactation ability of DCMECs by regulating the ER and PRLR cellular signal transduction pathway.

Potential role of chitinase 3-like-1 in inflammation-associated carcinogenic changes of epithelial cells

The family of mammalian chitinases includes members both with and without glycohydrolase enzymatic activity against chitin, a polymer of N-acetylglucosamine. Chitin is the structural component of fungi, crustaceans, insects and parasitic nematodes, but is completely absent in mammals. Exposure to antigens containing chitin- or chitin-like structures sometimes induces strong T helper type-I responses in mammals, which may be associated with the induction of mammalian chitinases. Chitinase 3-like-1 （CHI3L1）, a member of the mammalian chitinase family, is induced specifically during the course of inflammation in such disorders as inflammatory bowel disease, hepatitis and asthma. In addition, CHI3L1 is expressed and secreted by several types of solid tumors including glioblastoma, colon cancer, breast cancer and malignant melanoma. Although the exact function of CHI3L1 in inflammation and cancer is still largely unknown, CHI3L1 plays a pivotal role in exacerbating the inflammatory processes and in promoting angiogenesis and remodeling of the extracellular matrix. CHI3L1 may be highly involved in the chronic engagement of inflammation which potentiates development of epithelial tumorigenesis presumably by activating the mitogen-activated protein kinase and the protein kinase B signaling pathways. Anti-CHI3L1 antibodies or pan-chitinase inhibitors may have the potential to suppress CHI3Ll-mediated chronic inflammation and the subsequent carcinogenic change in epithelial cells.

Probiotic modulation of dendritic cells co-cultured with intestinal epithelial cells

AIM： To investigate cytokine production and cell surface phenotypes of dendritic cells （DC） in the presence of epithelial cells stimulated by probiotics.METHODS： Mouse DC were cultured alone or together with mouse epithelial cell monolayers in normal or in- verted systems and were stimulated with heat-killed probiotic bacteria, Bifidobacterium lactis ADO 11 （BL）, Bifidobacterium bilfidum BGN4 （BB）, Lactobacillus casei IBS041 （LC）, and Lactobacillus acidophilus AD031 （LA）, for 12 h. Cytokine levels in the culture supernatants were determined by enzyme-linked immunosorbent as say and phenotypic analysis of DC was investigated by flow cytometry.RESULTS： BB and LC in singlecultured DC increased the expression of I-Ad, CD86 and CD40 （I-Ad, 18.51 vs 30.88, 46.11, CD86, 62.74 vs 92.7, 104.12; CD40, 0.67 vs 6.39, 3.37, P 〈 0.05）. All of the experimental probiot-ics increased the production of inflammatory cytokines, interleukin （IL）-6 and tumor necrosis factor （TNF）-α. However, in the normal coculture systems, LC and LA decreased the expression of I-A^α （39.46 vs 30.32, 33.26, P 〈 0.05）, and none of the experimental probiotics increased the levels of IL-6 or TNF-α. In the inverted coculture systems, LC decreased the expression of CD40 （1.36 vs -2.27, P 〈 0.05）, and all of the experimental probiotics decreased the levels of IL-6. In addition, BL increased the production of IL-10 （103.8 vs 166.0, P 〈 0.05） and LC and LA increased transforming growth factor-13 secretion （235.9 vs 618.9, 607.6, P 〈 0.05）.CONCLUSION： These results suggest that specific pro- biotic strains exert differential immune modulation mediated by the interaction of dendritic cells and epithelial cells in the homeostasis of gastrointestinal tract.

Quercetin exerts anti-inflammatory effects via inhibiting tumor necrosis factor-α-induced matrix metalloproteinase-9 expression in normal human gastric epithelial cells

BACKGROUND Gastric injury is the most common digestive system disease worldwide and involves inflammation,which can lead to gastric ulcer or gastric cancer(GC).Matrix metallopeptidase-9[MMP-9(gelatinase-B)]plays an important role in inflammation and GC progression.Quercetin and quercetin-rich diets represent potential food supplements and a source of medications for treating gastric injury given their anti-inflammatory activities.However,the effects and mechanisms of action of quercetin on human chronic gastritis and whether quercetin can relieve symptoms remain unclear.AIM To assess whether tumor necrosis factor-α(TNF-α)-induced MMP-9 expression mediates the anti-inflammatory effects of quercetin in normal human gastric mucosal epithelial cells.METHODS The normal human gastric mucosa epithelial cell line GES-1 was used to establish a normal human gastric epithelial cell model of TNF-α-induced MMP-9 protein overexpression to evaluate the antiinflammatory effects of quercetin.The cell counting Kit-8 assay was used to evaluate the effects of varying quercetin doses on cell viability in the normal GES-1 cell line.Cell migration was measured using Transwell assay.The expression of proto-oncogene tyrosine-protein kinase Src(cSrc),phospho(p)-c-Src,extracellular-signal-regulated kinase 2(ERK2),p-ERK1/2,c-Fos,p-c-Fos,nuclear factor kappa B(NF-κB/p65),and p-p65 and the effects of their inhibitors were examined using Western blot analysis and measurement of luciferase activity.p65 expression was detected by immunofluorescence.MMP-9 m RNA and protein levels were measured by quantitative reverse transcription polymerase chain reaction(q RT–PCR)and gelatin zymography,respectively.RESULTS q RT-PCR and gelatin zymography showed that TNF-αinduced MMP-9 m RNA and protein expression in a dose-and time-dependent manner.These effects were reduced by the pretreatment of GES-1 cells with quercetin or a TNF-αantagonist(TNFR inhibitor)in a dose-and timedependent manner.Quercetin and TNF-αantagonists decreased the TNF-α-induced phosphorylation of c-Src,ERK1/2,c-Fos,and p65 in a dose-and time-dependent manner.Quercetin,TNF-αantagonist,PP1,U0126,and tanshinone IIA(TSIIA)reduced TNF-α-induced c-Fos phosphorylation and AP-1–Luciferase(Luc)activity in a dose-and time-dependent manner.Pretreatment with quercetin,TNF-αantagonist,PP1,U0126,or Bay 11-7082 reduced TNF-α-induced p65 phosphorylation and translocation and p65–Luc activity in a dose-and timedependent manner.TNF-αsignificantly increased GES-1 cell migration,and these results were reduced by pretreatment with quercetin or a TNF-αantagonist.CONCLUSION Quercetin significantly downregulates TNF-α-induced MMP-9 expression in GES-1 cells via the TNFR-c-Src–ERK1/2 and c-Fos or NF-κB pathways.

Role of CD56-expressing immature biliary epithelial cells in biliary atresia

AIM: To analyze the clinical and pathological parameters and expression of the neural cell adhesion molecule(CD56) in patients with biliary atresia(BA).METHODS: Established clinical laboratory markers of hepatic function, including enzyme activity, protein synthesis, and bilirubin metabolism, were evaluated in patients with BA and compared with those in patients with choledochal cysts and neonatal hepatitis. Pathological changes in tissue morphology and fibrosis were examined by histological and tissue collagen staining. Immunohistochemical staining for the biliary epithelial cell markers CD56 and CK19 together with the Notch signaling related molecules Notch1 and Notch2 was performed in the context of alterations in the structure of intrahepatic biliary ducts.RESULTS: Differences in some clinical laboratoryparameters among the three diseases examined were observed, but they did not correlate with the pathological classification of fibrosis in BA. Immunohistochemical staining showed the presence of CD56-positive immature bile ducts in most patients(74.5%) with BA but not in patients with choledochal cysts or neonatal hepatitis. The number of CD56-expressing cells correlated with disease severity, with more positive cells present in the later stages of liver damage(81.8% vs 18.2%). Furthermore, bile plugs were mainly found in CD56-positive immature biliary ducts. Notch signaling was a key regulatory pathway in biliary duct formation and played a role in tissue fibrosis. Notch1 was co-expressed in CD56-positive cells, whereas Notch2 was found exclusively in blood vessels in the portal area of patients with BA. CONCLUSION: The maturation of biliary epithelial cells and the expression of Notch may play a role in the pathogenesis of BA.

Bile salts inhibit growth and induce apoptosis of culture human normal esophageal mucosal epithelial cells

AIM： To investigate the effect of six bile salts： glycocholate （GC）, glycochenodeoxycholate （GCDC）, glycodeoxycholate （GDC）, taurocholate （TC）, taurochenodeoxycholate （TCDC）, taurodeoxycholate （TDC）, and their mixture on cultured human normal esophageal rnucosal epithelial cells. METHODS： Human normal esophageal mucosal epithelial cells were cultured with serum-free keratinocyte medium. 3-[4,5-Dimethylthiaolyl]-2,5- diphenyl-tetrazolium bromide assay was applied to the detection of cell proliferation. Apoptotic morphology was observed by phase-contrast video microscopy and terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling （TUNEL） assay. Sub-G1 DNA fragmentations and early apoptotic cells were assayed by flow cytometry （FCI） with propidium iodide （PI） staining and annexin V-FITC conjugated with PI staining. Apoptotic DNA ladders on agarose gel electrophoresis were observed. RESULTS： Except for GC, GCDC, GDC, TC, TCDC, TDC and their mixture could initiate growth inhibition of esophageal mucosal epithelial cells in a dose- and time-dependent manner. TUNEL and FCM assays demonstrated that the bile salts at 500 μmol/L and their mixture at 1 500 μmol/L induced apoptosis except for GC. The percentage of sub-G1 detected by FCM with PI staining was 83.5% in cells treated with 500 μmol/L TC for 2 h, and 19.8%, 20.4%, 25.6%, 13.5%, and 75.8% in cells treated with 500 μmol/L GCDC, TCDC, GDC, TDC, and 1 500 μmol/L mixture for 24 h, respectively, which were higher than that of the control （1.5%）. The percentage was 1.4% in cells with 500 μmol/L GC for 24 h. DNA ladders on agarose gel electrophoresis were seen in cells treated with 500 μmol/L TC for 2 h and i 500 μmnol/L mixture for 24 h. CONCLUSION： All GCDC, GDC, TC, TCDC, TDC and their mixture can inhibit growth and induce apoptosis of cultured human normal esophageal mucosal epithelial cells, but GC is well tolerated by the cells.