Advances in the study of natural small molecules to protect the intestinal epithelial tight junction barrier in inflammatory bowel disease / 中国药理学通报

Inflammatory bowel disease (IBD), as an idiopathic inflammatory disease of the intestinal tract, consisting mainly of Crohns disease and ulcerative colitis, which can involve the rectum, colon and ileum, and whose pathogenesis is still not fully understood. The initiation of intestinal inflammation associated with IBD and its chronieity begins with increased intestinal permeability caused by intestinal epithelial barrier disruption. The anti-permeability of the intestinal epithelial barrier is maintained by tight junction in the apical region of the intestinal epithelial cells, and disruption of the tight junction structure is closely associated with intestinal epithelial barrier damage and the development of IBD. Therefore, it is significant to find drugs for the prevention and treatment of IBD using tight junctions as regulatory targets. In recent years, many small molecules of natural product origin have been reported to improve the effects of IBD. In particular, we review the compounds that have the function of repairing intestinal epithelial barrier and protecting tight junction structure, in order to provide research ideas for the design and development of new drugs for the prevention and treatment of IBD.

Microbial sensing in the intestine

The gut microbiota plays a key role in host health and disease, particularly through their interactions with the immune system. Intestinal homeostasis is dependent on the symbiotic relationships between the host and the diverse gut microbiota, which is influenced by the highly co-evolved immune-microbiota interactions. The first step of the interaction between the host and the gut microbiota is the sensing of the gut microbes by the host immune system. In this review, we describe the cells of the host immune system and the proteins that sense the components and metabolites of the gut microbes. We further highlight the essential roles of pattern recognition receptors (PRRs), the G protein-coupled receptors (GPCRs), aryl hydrocarbon receptor (AHR) and the nuclear receptors expressed in the intestinal epithelial cells (IECs) and the intestine-resident immune cells. We also discuss the mechanisms by which the disruption of microbial sensing because of genetic or environmental factors causes human diseases such as the inflammatory bowel disease (IBD).

Mechanism of Acupuncture and Moxibustion on Promoting Mucosal Healing in Ulcerative Colitis / 中国结合医学杂志

The latest guideline about ulcerative colitis (UC) clinical practice stresses that mucosal healing, rather than anti-inflammation, is the main target in UC clinical management. Current mucosal dysfunction mainly closely relates to the endoscopic intestinal wall (mechanical barrier) injury with the imbalance between intestinal epithelial cells (IECs) regeneration and death, as well as tight junction (TJ) dysfunction. It is suggested that biological barrier (gut microbiota), chemical barrier (mucus protein layer, MUC) and immune barrier (immune cells) all take part in the imbalance, leading to mechanical barrier injury. Lots of experimental studies reported that acupuncture and moxibustion on UC recovery by adjusting the gut microbiota, MUC and immune cells on multiple targets and pathways, which contributes to the balance of IEC regeneration and death, as well as TJ structure recovery in animals. Moreover, the validity and superiority of acupuncture and moxibustion were also demonstrated in clinic. This study aims to review the achievements of acupuncture and moxibustion on mucosal healing and analyse the underlying mechanisms.

Hypoglycemic Mechanism of Gegen Qinliantang： An Exploration Based on GPR119/cAMP/GLP-1 Pathway / 中国实验方剂学杂志

ObjectiveTo explore the effects of Gegen Qinliantang（GGQL） on the proliferation and apoptosis of intestinal epithelial cells as well as on the expression of cyclic adenosine monophosphate （cAMP）， G protein-coupled receptor 119 （GPR119）， and glucagon-like peptide-1 （GLP-1）， so as to explore its potential hypoglycemic mechanism. MethodTwenty-five Wistar rats were gavaged with GGQL at the dose of 23 g·kg-1 crude drug， twice a day， which meant that 6 mL was administered into each rat per day for preparing the GGQL-containing serum. After seven consecutive times of administration， the intestinal epithelial L （NCI-H716） cells were cultured with different concentrations （1%， 2.5%， 5%， 7.5%， and 10%） of GGQL. The cell proliferation was evaluated using cell counting kit-8 （CCK-8） and the apoptosis by flow cytometry. The GLP-1 and cAMP contents in cell supernatant were determined by enzyme-linked immunosorbent assay （ELISA）. The mRNA and protein GLP-1 and GPR119 levels were assayed by real-time fluorescence quantitative polymerase chain reaction （Real-time PCR） and Western blot， respectively. ResultCompared with the control group， GGQL significantly reduced the proliferation of NCI-H716 cells（P<0.05）. As the GGQL concentration increased， its inhibitory effect became more obvious. GGQL at each concentration significantly promoted the apoptosis of NCI-H716 cells （P<0.05）. Compared with the control group， GGQL significantly up-regulated the expression of cAMP， GLP-1， and GPR119 （P<0.05）. The results showed that the effect of GGQL was positively correlated with its concentration， and 10% GGQL exhibited the best effect. ConclusionGGQL effectively inhibits the proliferation of NCI-H716 cells and promotes their apoptosis， and it may promote the secretion of GLP-1 by up-regulating the expression of cAMP and GPR119.

The protective effect of MANF on intestinal epithelial cell injury induced by endoplasmic reticulum stress / 中国药理学通报

Aim To observe the proteetive effect of meseneephalie astrocyte-de rived neurotrophic faetor (MANF) on intestinal epithelial eells under endoplas- mic reticulum stress (EH stress).Methods Normal human intestinal epithelial cell line FHs74Int was stimulated with EH stress inducers, tunicmycin (TM) and TNF-cx, then the expression of endogenous MANF was observed.The recombinant plasmids MANF-GFP and GFP were transfected into FHs74Int cells individually, the transfection efficiency was observed by fluorescence j j microscopy, and the effect of MANF on EH stress was observed by Western blot.The effect of MANF on the proliferation of intestinal epithelial cells stimulated by TM was detected via CCK-8 assay.The effect of MANF on apoptosis after EH stress was detected by Western blot and flow cytometry.Results EH stress could induce the expression of endogenous MANE in intestinal epithelial cells.Overexpression of MANE significantly inhibited the expression of the EH stress-related proteins, Bip and CHOP, and promoted the proliferation of intestinal epithelial cells.At the same time, it could reduce the production of the proapoptotic proteins cleaved-c a spa se-3 and Bax, increase the expression of the antiapoptotic protein Bcl-2, and inhibit the proportion of early and late apoptosis of intestinal epithelial cells.Conclusions MANF plays a protective role in inhibiting EH stress in intestinal epithelial cells by promoting cell proliferation and reducing apoptosis.

Mechanisms of Gancao Xiexin Decoction on protecting intestinal epithelial cell barrier under stress state by regulating PERK-elF2α-CHOP signaling pathway / 中国药理学通报

Aim To observe the protective effect of Gancao Xiexin Decoction (GXD) on intestinal epithelial cells (IECs) under endoplasmic reticulum stress from the perspective of PERK-elF2α-CH0P signaling pathway. Methods Caco-2 cells were cultured and divided into normal control group (NC), model control group(MC), low-dose GXD group(GXD-L), mediumdose GXD group (GXD-M) and high-dose GXD group (GXD-H). Models of the stress epithelial cells were induced by tunicamycin(Tm), and GXD groups were treated with GXD contained serum at the same time. The cell survival rate was assessed by CCK-8 method, the cell apoptotic rate and cell cycle distribution were determined by flow cytometry, and the cell barrier permeability was determined by TEER and FITC-dextran method; the expression levels of core proteins of PERK-elF2α-CH0P signaling pathway were detected by Western blot. Results Compared with MC group, GXD intervention could improve cell survival rate(P < 0. 05), reduce their apoptotic rate(P <0. 01), relieve cell cycle arrest(P < 0. 01), improve cell barrier permeability by increasing cell TEER value (P < 0. 01) and decreasing FITC-dextran concentration (P < 0.05), and the levels of p-PERK, p-elF2α, ATF4 and CHOP in GXD-M and GXD-H groups all visibly descended (P < 0. 01). Conclusions GXD can reduce the excessive apoptosis of IECs and protect intestinal epithelial cell barrier homeostasis by inhibiting the signal transduction of PERK-elF2α-CH0P apoptotic pathway.

Phytoglycoprotein isolated from Dioscorea batatas Decne promotes intestinal epithelial wound healing / 中国天然药物

Dioscorea batatas Decne (DBD) has been used to heal various illnesses of the kidney and intestine as an herbal medicine in Asia. As a source of therapeutic agents, many glycoproteins have been isolated from mushrooms and plants, but the functional role of glycoprotein in intestinal epithelial wound healing has not been understood yet. In the present study, we investigated the wound healing potentials of the 30 kDa glycoprotein (DBD glycoprotein) isolated from DBD in human intestinal epithelial (INT-407) cells. We found that DBD glycoprotein (100 μg·mL) significantly increased the motility of INT-407 cells for 24 h by activating protein kinase C (PKC). DBD glycoprotein stimulated the activation of p38 mitogen-activated protein kinase (MAPK), which is responsible for the phosphorylation of NF-κB inhibitor α (IκBα). DBD glycoprotein increased the level of profilin-1 (PFN1), α-actinin and F-actin expression via activation of transcription factor, nuclear factor-kappa B (NF-κB) during its promotion of cell migration. Experimental mouse colitis was induced by adding dextran sulfate sodium (DSS) to the drinking water at a concentration of 4% (W/V) for 7 days. We figured out that administration of DBD glycoprotein (10 and 20 mg·kg) lowers the levels of disease activity index and histological inflammation in DSS-treated ICR mice. In this regard, we suggest that DBD glycoprotein has ability to promote the F-actin-related migration signaling events via activation of PKC and NF-κB in intestinal epithelial cells and prevent inflammatory bowel disease.

Chang'an II Decoction ( II )-Containing Serum Ameliorates Tumor Necrosis Factor-α-Induced Intestinal Epithelial Barrier Dysfunction via MLCK-MLC Signaling Pathway in Rats / 中国结合医学杂志

OBJECTIVE@#To investigate the effect of Chang'an II Decoction ( II ))-containing serum on intestinal epithelial barrier dysfunction in rats.@*METHODS@#Tumor necrosis factor (TNF)-α-induced injury of Caco-2 monolayers were established as an inflammatory model of human intestinal epithelium. Caco-2 monolayers were treated with blank serum and Chang'an II Decoction-containing serum that obtained from the rats which were treated with distilled water and Chang'an II Decoction intragastrically at doses of 0.49, 0.98, 1.96 g/(kg·d) for 1 week, respectively. After preparation of containing serum, cells were divided into the normal group, the model group, the Chang'an II-H, M, and L groups (treated with 30 ng/mL TNF-α and medium plus 10% high, middle-, and low-doses Chang'an II serum, respectively). Epithelial barrier function was assessed by transepithelial electrical resistance (TER) and permeability of fluorescein isothiocyanate (FITC)-labeled dextran. Transmission electron microscopy was used to observe the ultrastructure of tight junctions (TJs). Immunofluorescence of zonula occludens-1 (ZO-1), claudin-1 and nuclear transcription factor-kappa p65 (NF-κ Bp65) were measured to determine the protein distribution. The mRNA expression of myosin light chain kinase (MLCK) was measured by real-time polymerase chain reaction. The expression levels of MLCK, myosin light chain (MLC) and p-MLC were determined by Western blot.@*RESULTS@#Chang'an II Decoction-containing serum significantly attenuated the TER and paracellular permeability induced by TNF-α. It alleviated TNF-α-induced morphological alterations in TJ proteins. The increases in MLCK mRNA and MLCK, MLC and p-MLC protein expressions induced by TNF-α were significantly inhibited in the Chang'an II-H group. Additionally, Chang'an II Decoction significantly attenuated translocation of NF-κ Bp65 into the nucleus.@*CONCLUSION@#High-dose Chang'an II-containing serum attenuates TNF-α-induced intestinal barrier dysfunction. The underlying mechanism may be involved in inhibiting the MLCK-MLC phosphorylation signaling pathway mediated by NF-κ Bp65.

Glycyrrhizic Acid Protect IEC-6 Cells from Apoptosis Via HuR Mediated Posttranscription of p21 / 中国实验方剂学杂志

Objective:To investigate the protective effect of glycyrrhizic acid (GA) from tumor necrosis factor-α+actinomycetes ketone （TNF-α+CHX） induced apoptosis in rat small intestine crypt epithelial cell line (IEC-6) via AU rich element mRNA binding protein HuR mediated posttranscription of p21 and the potential mechanism. Method:The cultured IEC-6 cells were observed. The experiment was divided into blank group, GA (60 μmol·L-1) group, TNF-α+CHX group and GA+TNF-α+CHX group. Cytoplasmic and nuclear HuR were measured by Western blot. The interaction of HuR and p21 mRNA was detected by biotin pull down and RNA IP. Luciferase activity was measured after transfection with construct with p21 3'-UTR cloned into downstream of luciferase reporter. Cell apoptosis was detected by real-time dynamic cell analyzer, p21 and cysteine proteinas-3 precursor protein（proCaspase-3） association was analysised by CO-IP. Result:After GA treatment for 48 h, cytoplasmic HuR protein expression increased（P<0.05）,the binding between HuR and p21 mRNA expression up regulated(P<0.05), luciferase activity increased(P<0.01), and p21 mRNA and protein expression also increased(P<0.05), while these results were abolished by HuR silencing with siRNA. GA enhanced p21 and procaspase3 interaction(P<0.05), and attenuated TNF-α+CHX induced apoptosis in IEC-6 cells. Conclusion:GA protected IEC-6 cells from TNF-α/CHX induced apoptosis via HuR mediated p21 posttranscription, which due to GA enhanced HuR binding to endogenous and recombinant p21 mRNA and increased p21 interaction with proCaspase3.

The protective effect of water-soluble proteins from selenium-enriched Bifidobacterium longum on lipopolysaccharide-induced injury in rat IEC6 cells / 中国药理学通报

To investigate the protective effect of selenium-enriched Bifidobacterium longum preparation on rat IEC6 cells via the application of lipopolysaccharide (LPS)-induced IEC6 cell injury model. Methods The experiment was divided into five groups; negative control group treated with phosphoric acid equilibrium salt solution, model group treated with LPS, and low, medium, high concentration group in which IEC6 cells were treated with LPS plus 10, 30, 100 mg • L"1 water-soluble proteins from selenium-enriched Bifidobacterium longum respectively. After treatment with LPS for 24 hours, IEC6 cell viability, apoptosis, mitochondria membrane potential, and the expression of zonula occludens 1 (ZO-1) and Occludin were detected. Results LPS induced rat intestinal epithelial cell damage, such as the decrease of cell viability, the increase of cell apoptosis, the collapse of mitochondria membrane potential, and the decrease of cell tight junction protein expression. Water-soluble proteins from selenium-enriched Bifidobacterium longum inhibited LPS-induced IEC6 cell damage, decreased cell apoptosis and increased cell tight junction between cells. Conclusion Water-soluble proteins from selenium-enriched Bifidobacterium longum have protective effect on LPS-induced rat intestinal epithelial cell injury.

Research progress of interaction models between intestinal microflora and host cells / 中华预防医学杂志

Intestinal microbes play an important role in human health. The development of various clinical diseases, such as obesity, diabetes and cardiovascular disease, is closely related to the imbalance of intestinal microflora. With the development of high-throughput sequencing technology, there has been a breakthrough in the understanding of intestinal microorganism. The interaction between intestinal epithelial cells and intestinal microbes has become one of the hotspots and difficulties of current research. Because of the constraints of ethical review and experimental cost, people are more interested in the development of interaction models between the intestinal microflora and the host cells. In this paper, interaction models between intestinal microflora and host cells, and its working principle and application prospect are reviewed, hoping to provide new techniques and new ideas for studying functions of intestinal microbes.

miR￣7 regulates intestinal trefoil factor and affects intestinal epithelial cell proliferation and migration / 中国小儿急救医学

Objective To investigate the regulation of miR-7 on intestinal trefoil factor (TFF3) and its effect on the proliferation and migration of intestinal epithelial cells. Methods miR-7 mimics and miR-7 inhibitor were transfected into LS174T cells respectively. The experiment was divided into 5 groups,including blank control group,miR-7 mimic negative control group,miR-7 mimic group,miR-7 inhibitor negative con-trol group,miR-7 inhibitor group. MTT assay was used to detect cell proliferation. Cell scratch assay was used to detect the effect of miR-7 on cell migration. Western blot was used to detect the change of TFF3 protein in each group. Results Compared with the blank control group,the miR-7 mimic negative control group and the miR-7 inhibitor negative control group,the OD value of the miR-7 mimic group decreased significantly, the difference was statistically significant(P<0. 05); the cell scratch interval increased,the cell migration rate decreased,the difference was statistically significant( P<0. 05); and the TFF3 protein expression was accompanied(P<0. 05). The OD value of the miR-7 inhibitor group significantly increased,the difference was statistically significant(P<0. 05); the cell scratch gap decreased,the cell migration rate was enhanced, the difference was statistically significant( P <0. 05); and the expression of TFF3 protein increased ( P <0. 05). Conclusion miR-7 can regulate the expression of TFF3 and further inhibit the proliferation and migration of LS174T cells.

Nucleocapsid protein from porcine epidemic diarrhea virus isolates can antagonize interferon-λ production by blocking the nuclear factor-κB nuclear translocation / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Porcine epidemic diarrhea virus (PEDV) is a highly infectious pathogen that can cause severe diseases in pigs and result in enormous economic losses in the worldwide swine industry. Previous studies revealed that PEDV exhibits an obvious capacity for modulating interferon (IFN) signaling or expression. The newly discovered type III IFN, which plays a crucial role in antiviral immunity, has strong antiviral activity against PEDV proliferation in IPEC-J2 cells. In this study, we aimed to investigate the effect of PEDV nucleocapsid (N) protein on type III IFN-λ. We found that the N proteins of ten PEDV strains isolated between 2013 and 2017 from different local farms shared high nucleotide identities, while the N protein of the CV777 vaccine strain formed a monophyletic branch in the phylogenetic tree. The N protein of the epidemic strain could antagonize type III IFN, but not type I or type II IFN expression induced by polyinosinic-polycytidylic acid (poly(I:C)) in IPEC-J2 cells. Subsequently, we demonstrated that the inhibition of poly(I:C)-induced IFN-λ3 production by PEDV N protein was dependent on the blocking of nuclear factor-κB (NF-κB) nuclear translocation. These findings might help increase understanding of the pathogenesis of PEDV and its mechanisms for evading the host immune response.

Research progress on intestinal immunoregulation and mechanism of Sijunzi Decoction polysaccharide / 中草药

Sijunzi Decoction polysaccharide (SJZDP) is the active component contributing to the function of intestinal immunoregulation, which is the highest content in Sijunzi Decoction. SJZDP can activate immunological response in peyer’s patch, mesenteric lymph nodes, intestinal epithelial cells and intestinal intraepithelial lymphocytes, but the mechanism is unknown. The reported mechanisms of SJZDP’s intestinal immunoregulation activity are related to its regulation of intestinal flora and polyamine signaling pathway. This review is to give a comprehensive summary of information regarding the intestinal immunoregulation of SJZDP and mechanism to help us take the action for reasonable clinical utilization and further researches.

Primary cultures of mouse small intestinal epithelial cells using the dissociating enzyme type I collagenase and hyaluronidase

The epithelium is a highly dynamic system, which plays a crucial role in the homeostasis of the intestinal tract. However, studies on the physiological and pathophysiological functions of intestinal epithelial cells (IECs) have been hampered due to lack of normal epithelial cell models. In the present study, we established a reproducible method for primary culture of mouse IECs, which were isolated from the viable small intestinal crypts of murine fetuses (on embryonic day 19), using type I collagenase and hyaluronidase in a short span of time (≤20 min). With this method, continuously growing mouse IECs, which can be subcultured over a number of passages, were obtained. The obtained cell lines formed a tight cobblestone-like arrangement, displayed long and slender microvilli, expressed characteristic markers (cytokeratin 18 and Notch-1), and generated increasing transepithelial electrical resistance and low paracellular permeability during in vitro culture. The cells also had enzymatic activities of alkaline phosphatase and sucrase-isomaltase, and secreted various cytokines (IL-1β, IL-6, IL-8, and monocyte chemoattractant protein-1), responding to the stimulation of Escherichia coli. These results show that the primary-cultured mouse IECs obtained by the method established here had the morphological and immunological characteristics of IECs. This culture system can be a beneficial in vitro model for studies on mucosal immunology and toxicology.

Establishment of a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8] / 中国实验动物学报

Objective To explore the proliferation characteristics of primary small intestinal epithelial cells of tree shrews and the characteristics of human rotavirus(RV) G1P[8] infection to these cells,and establish a model of tree shrew primary small intestinal epithelial cells infected with human rotavirus G1P[8].Methods The primary small intestinal epithelial cells were obtained by collagenase Ⅺ and dispase I digestion from tree shrew.After purification and identification,the obtained primary small intestinal epithelial cells were infected with RV.Then,culture supernatants of infected cells were collected every 12 hours after infection.Viral titer and viral load were subsequently determined.Western blot and indirect immunofluorescence observation were used to detect the expression of RV protein VP6 in the primary cells.The infectivity of RV to the tree shrew primary cells was finally evaluated.Results After purification and identification of primary epithelial cells from the tree shrew,high purity above 90% primary tree shrew small intestinal epithelial cells was obtained.These primary small intestinal epithelial cells could be infected with RV virus by comparing the virus infectivity to primary renal cells,HCT116 cells and MA104 cells.The virus titer reached to 2.0×105TCID 50/mL at 72 h after infection.Using Western blot and indirect immunofluorescence observation,the specific viral protein of VP6 was determined to be expressed in the tree shrew primary small intestinal epithelial cells,and were located in the cytoplasm from days 1 to 5.Conclusions The separation,purification and cultivation methods of tree shrew primary small intestinal epithelial cells are successful,and the tree shrew model of RV-infected the tree shrew primary small intestinal epithelial cells is successfully established.

Research progress on mechanism of autophagy in intestinal mucosal barrier function / 基础医学与临床

Autophagy is a biological process in which cells maintain homeostasis through degradation of cytoplasmic macromolecules and damaged organelles by membrane vesicle structure .Autophagy plays a critical role in maintai-ning survival of intestinal epithelial cells during intestinal mucosal barrier dysfunction .A negative regulator of auto-phagy may lead to intestinal inflammation and tumorigenesis .

Regulatory effect of vitamin D on the expression of occludin in the intestinal epithelial cells of neonatal ;rats with necrotizing enterocolitis / 中国小儿急救医学

Objective To investigate the expression of occludin in the intestinal epithelial cells of neonatal rats with necrotizing enterocolitis ( NEC) . The influence of vitamin D on NEC and the expression of occludin was also explored. Methods Sixty neonatal Wistar rats from different litters were divided into four groups 48 hours after birth:10 in the breast-fed+ control group,10 in the breast-fed +vitamin D group,20 in the NEC+ control group and 20 in the NEC+ vitamin D group. In groups with NEC,neonatal rats were sep-arated from their mothers and treated with rat milk substitute+hypoxia+hypothermia. In groups with vitamin D treatment,paricalcitol was given through intraperitoneal injection 30 minutes before,one day and two days after the induction of NEC. The tissue was harvested from the ileum of the rats 72 hours after induction of NEC. Microscopically,the tissue was assessed according to the Nadler scoring system. The expression and location of occludin was observed after immunofluorescence staining. Western blotting was performed and quantified to compare the expression of occludin among groups. Results In NEC rats,the intestinal structure was destructed. Separation of the submucosal or muscular layer,discharge or disappearance of the villa was accompanied by necrosis of the intestinal cells. Vitamin D treatment could significantly alleviate those chan-ges,with the Nadler score 1. 70 ± 0. 21 compared to 2. 90 ± 0. 23 without vitamin D treatment ( P<0. 01 ) . The expression of occludin was decreased in NEC rats and vitamin D could partly rescue this decrease (P<0. 01). The distribution of occludin was sparse and discontinuous in NEC rats while after vitamin D treatment,the distribution was maintained even and continuous. Conclusion The expression of occludin in intestinal epithelial cells is significantly reduced in NEC rat. Vitamin D treatment may inhibit the development of NEC by maintaining the expression of occludin.

Primary culture of cat intestinal epithelial cell and construction of its cDNA library / 中国血吸虫病防治杂志

Objective To establish the primary cat intestinal epithelial cells(IECs)culture methods and construct the cD-NA library for the following yeast two-hybrid experiment,so as to screen the virulence interaction factors among the final host. Methods The primary cat IECs were cultured by the tissue cultivation and combined digestion with collagenase XI and dispase I separately. Then the cat IECs cultured was identified with the morphological observation and cyto-keratin detection ,by using goat anti-cyto-keratin monoclonal antibodies. The mRNA of cat IECs was isolated and used as the template to synthesize the first strand cDNA by SMARTTM technology,and then the double-strand cDNAs were acquired by LD-PCR,which were subsequently cloned into the plasmid PGADT7-Rec to construct yeast two-hybrid cDNA library in the yeast strain Y187 by homologous recom-bination. Matchmaker?Insert Check PCR was used to detect the size distribution of cDNA fragments after the capacity calcula-tion of the cDNA library. Results The comparison of the two cultivation methods indicated that the combined digestion of colla-genase XI and dispase I was more effective than the tissue cultivation. The cat IECs system of continuous culture was established and the cat IECs with high purity were harvested for constructing the yeast two-hybrid cDNA library. The library contained 1.1× 106 independent clones. The titer was 2.8 × 109 cfu/ml. The size of inserted fragments was among 0.5-2.0 kb. Conclusion The yeast two-hybrid cDNA library of cat IECs meets the requirements of further screen research,and this study lays the foundation of screening the Toxoplasma gondii virulence interaction factors among the cDNA libraries of its final hosts.

Artemisinin attenuates intestinal epithelial barrier damage induced by LPS / 中国病理生理杂志

[ ABSTRACT] AIM:To investigate the effect of artemisinin on lipopolysaccharide ( LPS)-induced intestinal epi-thelial barrier damage in IEC-6 cells and its molecular mechanism.METHODS:Cultured IEC-6 cells were divided to 5 groups:control group, LPS (100 mg/L) group and LPS +Artemisinin (30, 50 and 100μmol/L) groups.The cytotoxici-ty was detected by MTT assay.The releases of TNF-α, IL-1βand IL-6 in the IEC-6 cells were measured by ELISA.The transepithelial electrical resistance ( TER) was detected by electrical resistance tester, and the horseradish peroxidase (HRP) flux permeability were analyzed by a microplate reader.The expression of tight junction proteins, ZO-1, claudin-1 and occludin, and the expression of TLR4/MyD88/NF-κB at mRNA and protein levels were determined by RT-qPCR and Western blot.RESULTS:Artemisinin alone (up to 100 μmol/L) or in combination with LPS (100 mg/L) was not toxic to IEC-6 cells.Compared with control group, the releases of TNF-α, IL-1βand IL-6 in the culture supernatant of IEC-6 cells significantly increased after treatment with LPS.The expression of TLR4/MyD88/NF-κB was activated by LPS.LPS down-regulated the protein expression of ZO-1, claudin-1 and occludin.However, artemisinin treatment decreased the re-leases of TNF-α, IL-1βand IL-6 in the culture supernatant of IEC-6 cells.The expression of TLR4/MyD88/NF-κB at mR-NA and protein levels was gradually reduced after treatment with artemisinin.In addition, artemisinin upregulated the pro-tein expression of ZO-1, claudin-1 and occludin significantly (P<0.01) in a dose-dependent manner.CONCLUSION:Artemisinin attenuates LPS-induced intestinal epithelial barrier damage by inhibiting TLR4/MyD88/NF-κB activation in the IEC-6 cells.