A novel avian intestinal epithelial cell line: its characterization and exploration as an in vitro infection culture model for Eimeria species.

BACKGROUND: The gastrointestinal epithelium plays an important role in directing recognition by the immune system, and epithelial cells provide the host's front line of defense against microorganisms. However, it is difficult to cultivate avian intestinal epithelial cells in vitro for lengthy periods, and the lack of available cell lines limits the research on avian intestinal diseases and nutritional regulation. Chicken coccidiosis is a serious intestinal disease that causes significant economic losses in the poultry industry. In vitro, some cell line models are beneficial for the development of Eimeria species; however, only partial reproduction can be achieved. Therefore, we sought to develop a new model with both the natural host and epithelial cell phenotypes. METHODS: In this study, we use the SV40 large T antigen (SV40T) gene to generate an immortalized cell line. Single-cell screening technology was used to sort positive cell clusters with epithelial characteristics for passage. Polymerase chain reaction (PCR) identification, immunofluorescence detection, and bulk RNA sequencing analysis and validation were used to check the expression of epithelial cell markers and characterize the avian intestinal epithelial cell line (AIEC). AIECs were infected with sporozoites, and their ability to support the in vitro endogenous development of Eimeria tenella was assessed. RESULTS: This novel AIEC consistently expressed intestinal epithelial markers. Transcriptome assays revealed the upregulation of genes associated with proliferation and downregulation of genes associated with apoptosis. We sought to compare E. tenella infection between an existing fibroblast cell line (DF-1) and several passages of AIEC and found that the invasion efficiency was significantly increased relative to that of chicken fibroblast cell lines. CONCLUSIONS: An AIEC will serve as a better in vitro research model, especially in the study of Eimeria species development and the mechanisms of parasite-host interactions. Using AIEC helps us understand the involvement of intestinal epithelial cells in the digestive tract and the immune defense of the chickens, which will contribute to the epithelial innate defense against microbial infection in the gastrointestinal tract.

Dietary berberine supplementation improves growth performance and alleviates gut injury in weaned piglets by modulating ileal microbiota and metabolites.

This study investigated the effects of dietary berberine (BBR) supplementation on the growth performance, intestinal health, and ileal microbiome and metabolomic profile in weaned piglets challenged with enterotoxigenic Escherichia coli (ETEC). Dietary BBR supplementation significantly attenuated the reduced average daily gain (ADG) and attenuated the increased feed to gain ratio (F/G) and the incidence of diarrhea induced by ETEC K88 (P < 0.05). Dietary BBR supplementation significantly increased the villus height and the villus height to crypt depth ratio in the ileum (P < 0.05). Moreover, the mRNA expression of ZO-1 and occludin as well as aquaporins (AQP1, AQP3, AQP4, AQP7, and AQP10) and Na+/H+ exchanger 3 (NHE3) in ileal mucosa was significantly upregulated by BBR treatment (P < 0.05). Additionally, BBR treatment significantly inhibited the increase of interleukin-1ß (IL-1ß) in jejunal mucosa caused by ETEC and reduced the levels of tumor necrosis factor-α (TNF-α) and IL-1ß and increased interleukin-10 (IL-10) in colonic mucosa (P < 0.05). Dietary BBR treatment significantly increased the Observed_species, Chao 1, abundance based coverage estimators (ACE), and PD_whole tree in the ileal digesta of weaned piglets challenged with ETEC. At the genus level, the relative abundance of unidentified Clostridiales was decreased, while Weissella, Alloprevotella, unidentified Prevotellaceae, and Catenibacterium were increased in the BBR + ETEC group when compared to the ETEC group (P < 0.05). Spearman correlation analysis showed that the relative abundance of unidentified Clostridiales (genus) was negatively correlated with the ileal villus height but negatively correlated with diarrhea and intestinal IL-1ß and TNF-α concentrations (P < 0.05). The ileal metabolome analysis showed that the metabolic pathways including primary and secondary bile acid biosynthesis and bile secretion were significantly enriched by BBR treatment. Collectively, dietary BBR supplementation effectively improved the growth performance and alleviated the diarrhea and intestinal injury induced by ETEC K88 in weaned piglets, which might closely involve the modulation of ileal microbiota and metabolites.

Research Note: Dietary resveratrol supplementation improves the hepatic antioxidant capacity and attenuates lipopolysaccharide-induced inflammation in yellow-feathered broilers.

This experiment investigated the protective effect of resveratrol (RES) on the hepatic antioxidant status and systemic inflammation in yellow-feathered broilers challenged with lipopolysaccharide (LPS). A total of 240 healthy 1-day-old yellow-feathered broilers were randomly divided into 4 groups (control, LPS, RES, and RES+LPS), with 5 replicates of 12 chickens per replicate. The experiment lasted 21 d. The broilers were fed with either the basal diet or the basal diet supplemented with 400 mg/kg RES followed by intraperitoneal challenge with LPS (1 mg/kg body weight) or the same amount of saline at d 16, 18, and 20. The results showed that dietary RES supplementation could improve the activities of total antioxidant capacity (T-AOC) and superoxide dismutase (SOD) in the liver of yellow-feathered broilers challenged with LPS (P < 0.05). Furthermore, LPS challenge increased the plasma interleukin-17 (IL-17) concentration, the hepatic interleukin-6 (IL-6) and interleukin-1ß (IL-1ß) concentrations, as well as the concentrations of tumor necrosis factor (TNF-α), IL-6, and IL-1ß in the spleen (P < 0.05), and decreased the transforming growth factor-ß (TGF-ß) concentrations in the plasma, liver, and spleen (P < 0.05). However, dietary RES supplementation could reduce the increased TNF-α levels in the plasma, liver, and spleen induced by LPS, and increased TGF-ß level in the liver and spleen (P < 0.05). Collectively, these results suggest that dietary RES supplementation could effectively improve the hepatic antioxidant capacity and attenuate LPS-induced inflammation in yellow-feathered broilers during the starter stage.

Umbilical cord mesenchymal stem cells promote the repair of trochlear groove reconstruction in dogs.

Trochlear groove reconstruction (TGR) is a common treatment for patellar luxation (PL) in dogs. Nevertheless, the prognosis of TGR is poor due to the cartilage damage and secondary inflammation. To study the repair effect of canine umbilical cord mesenchymal stem cells (UC-MSCs) after TGR, 10 experimental dogs were given TGR surgery and then randomized into two groups: Treatment group (1 ml suspension allogeneic UC-MSCs (106 cells/kg) was injected into the cavum articulare on days 0, 7, and 14 after TGR); and the Model group (injected with 1 ml of physiological saline as negative control). The therapeutic effect of UC-MSCs was studied by blood routine examination, inflammatory factor index detection, double-blind knee score, histopathology, and computed tomography (CT) scans. The results showed that the total number of white blood cells and neutrophils in the model group were significantly higher than those in the treatment group on both 7 days and 21 days, postoperatively (P < 0.05); there were no significant changes in the levels of IL-6, MMP-13, and TGF-ß1 between the model group and the treatment group throughout the days of testing. The double-blind knee scores of the treatment group were significantly lower than the model group on 1st, 4th, and 5th days postoperatively (P < 0.05). The treatment group showed low-pain sensation, stable gait, and fast recovery of muscle strength in the knee score, and the wound healing of the treatment group returned to normal on the 5th day after surgery; CT scans and gross observation showed that the cartilage growth in the treatment group was faster than that in the model group. Histological observation of cases showed that fibro chondrocytes were predominantly found in the treatment group, and the distribution of chondrocytes was uneven, while the model group showed a large number of fibrous tissue hyperplasia, fissures, and unequal matrix staining. Intra-articular injection of UC-MSCs after TGR has the effect of relieving pain and promoting the repair of bone defects, making the operative limb recover function earlier, making up for the deficiency of TGR, and improving the effect of PL treatment. Future studies should furthermore explore the dose and frequency of therapy based on the multiple advantages of UC-MSCs and the mechanism of cartilage repair in dogs.

Transcriptional regulation of nuclear miRNAs in tumorigenesis (Review).

MicroRNAs (miRNAs/miRs) are a type of endogenous non­coding small RNA that regulates gene expression. miRNAs regulate gene expression at the post­transcriptional level by targeting the 3'­untranslated region (3'UTR) of cytoplasmic messenger RNAs (mRNAs). Recent research has confirmed the presence of mature miRNAs in the nucleus, which bind nascent RNA transcripts, gene promoter or enhancer regions, and regulate gene expression via epigenetic pathways. Some miRNAs have been shown to function as oncogenes or tumor suppressor genes by modulating molecular pathways involved in human cancers. Notably, a novel molecular mechanism underlying the dysregulation of miRNA expression in cancer has recently been discovered, indicating that miRNAs may be involved in tumorigenesis via a nuclear function that influences gene transcription and epigenetic states, elucidating their potential therapeutic implications. The present review article discusses the import of nuclear miRNAs, nucleus­cytoplasm transport mechanisms and the nuclear functions of miRNAs in cancer. In addition, some software tools for predicting miRNA binding sites are also discussed. Nuclear miRNAs supplement miRNA regulatory networks in cancer as a non­canonical aspect of miRNA action. Further research into this aspect may be critical for understanding the role of nuclear miRNAs in the development of human cancers.

Arginine Promotes the Expression of Aquaporin-3 and Water Transport in Porcine Trophectoderm Cells Through NO- and cAMP-Dependent Mechanisms.

BACKGROUND: Dietary supplementation with L-arginine (Arg) has been shown to increase the volume of fetal fluids in gestating swine. Aquaporins (AQPs), known as water channel proteins, are essential for embryonic growth and development. It was not known if Arg mediates water transport through AQPs in porcine conceptus trophectoderm (pTr2) cells. METHODS: pTr2 cells derived from pregnant gilts on day 12 of gestation were cultured in customized Arg-free Dulbecco's modified Eagle's Ham medium (DMEM) supplemented with either 0.00, 0.25, or 0.50 mM Arg. RESULTS: Arg treatment increased water transport and the expression of AQP3, which was abundantly expressed in pTr2 cells at both the mRNA and protein levels. Arg also increased the expression of iNOS and the synthesis of nitric oxide (NO) in pTr2 cells. The presence of Nω-nitro-L-arginine methyl ester hydrochloride (L-NAME; an inhibitor of NO synthase) significantly attenuated the Arg-induced expression of AQP3. Furthermore, 0.50 mM Arg increased the concentrations of cAMP and the abundances of phosphorylated cAMP-dependent protein kinase A (PKA), phosphorylated PKA α/ß/γ, and phosphorylated CREB. These effects of Arg were mimicked by Forskolin (a cell-permeable activator of adenylyl cyclase), but inhibited by H-89 (an inhibitor of cAMP-dependent protein kinase). CONCLUSIONS: The results of this study demonstrate that Arg regulates AQP3 expression and promotes water transport in pTr2 cells through NO- and cAMP-dependent signaling pathways.

Protegrin-1 inhibits porcine ovarian granulosa cell apoptosis from H2O2-induced oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro.

In mammals, oxidative stress-induced apoptosis of granulosa cells is one of the major causes of follicular atresia, affecting ovarian physiological function. Protegrin-1 (PG-1) is an antimicrobial peptide with effective antimicrobial activity, immunomodulatory function, and porcine growth-promoting effects. PG-1 has been detected in porcine ovaries follicles. This study aimed to investigate the effect of PG-1 on oxidative stress-induced apoptosis of porcine ovarian granulosa cells and the underlying molecular mechanism. Granulosa cells were obtained from porcine follicles and treated with H2O2 to establish the oxidative stress model, and then treated with or without PG-1 (10 µg/mL). PG-1 significantly suppressed H2O2-induced apoptosis in granulosa cells after 24 h of treatment. Furthermore, these results revealed that PG-1 increased the mRNA and protein expression of anti-apoptotic B cell lymphoma/leukemia 2 (BCL2) and the BCL2/Bcl-2-associated X protein (BAX) ratio while decreasing the expression of pro-apoptotic BAX and active caspase-3. Using Western blot analysis, it was found that PG-1 decreased the phosphorylation of RNA-like endoplasmic reticulum kinase (PERK) and the α-subunit of eukaryotic initiation factor 2 (eIF2α) as well as the protein expression level of CCAAT enhancer-binding protein homologous protein (CHOP), all of which were increased by H2O2. Moreover, inhibitors against PERK and phospho-eIF2ɑ both suppressed the H2O2-induced granulosa cells apoptosis and enhanced the anti-apoptosis effect of PG-1. Taken together, our findings demonstrated that PG-1 inhibited porcine ovarian granulosa cell apoptosis from oxidative stress via the PERK/eIF2α/CHOP signaling pathway in vitro, which suggests the novel regulatory function of the antimicrobial peptide in the ovary.

CHD1L augments autophagy-mediated migration of hepatocellular carcinoma through targeting ZKSCAN3.

Autophagy is an important biological process in normal cells. However, how it affects tumor progression still remains poorly understood. Herein, we demonstrated that the oncogenic protein Chromodomain-helicase-DNA-binding-protein 1-like gene (CHD1L) might promote HCC cells migration and metastasis through autophagy. CHD1L could bind to the promotor region of Zinc finger with KRAB and SCAN domain 3 (ZKSCAN3), a pivotal autophagy suppressor, and inhibit its transcription. We established inducible CHD1L conditional knockout cell line (CHD1L-iKO cell) and found that the deletion of CHD1L significantly increased ZKSCAN3 expression both at mRNA and protein level. Deletion of CHD1L impaired the autophagic flux and migration of HCC cells, while specifically inhibiting ZKSCAN3 blocked these effects. Further exploration demonstrated that the enhanced tumor cell migration and metastasis induced by CHD1L was mediated through ZKSCAN3-induced autophagic degradation of Paxillin. In summary, we have characterized a previously unknown function of CHD1L in regulating tumor migration via ZKSCAN3-mediated autophagy in HCC. Further inhibition of CHD1L and its downstream autophagy signaling might shed new light on cancer therapeutics.

MicroRNA 195-5p Targets Foxo3 Promoter Region to Regulate Its Expression in Granulosa Cells.

Forkhead box O3 (Foxo3) is a member of the FOXO subfamily within the forkhead box (FOX) family, which has been shown to be essential for ovarian follicular development and maturation. Previous studies have shown the abundant expression of miR-195-5p in the nuclei of porcine granulosa cells (GCs), suggesting its potential role during ovarian follicle growth. In this study, a conditional immortalized porcine granulosa cell (CIPGC) line was used to determine whether the expression of Foxo3 could be regulated by the nuclear-enriched miR-195-5p. Through silico target prediction, we identified a potential binding site of miR-195-5p within the Foxo3 promoter. The over-expression of miR-195-5p increased Foxo3 expression at both mRNA and protein levels, while the knockdown of miR-195-5p decreased the expression of Foxo3. Furthermore, driven by the Foxo3 promoter, luciferase reporter activity was increased in response to miR-195-5p, while the mutation of the miR-195-5p binding site in the promoter region abolished this effect. In addition, the siRNA knockdown of Argonaute (AGO) 2, but not AGO1, significantly decreased Foxo3 transcript level. However, miR-195-5p failed to upregulate Foxo3 expression when AGO2 was knocked down. Moreover, chromatin immunoprecipitation (CHIP) assay showed that anti-AGO2 antibody pulled down both AGO2 and the Foxo3 promoter sequence, suggesting that AGO2 may be required for miR-195-5p to regulate Foxo3 expression in the nucleus. Additionally, Foxo3 expression was significantly increased by valproic acid (VPA), the inhibitor of deacetylase, as well as by methyltransferase inhibitor BIX-01294, indicating the involvement of histone modification. These effects were further enhanced in the presence of miR-195-5p and were decreased when miR-195-5p was knocked down. Overall, our results suggest that nuclear-enriched miR-195-5p regulates Foxo3 expression, which may be associated with AGO2 recruitment, as well as histone demethylation and acetylation in ovarian granulosa cells.

Proteomic alteration of porcine intestinal epithelial cells after pretreatment with Lactobacillus plantarum followed by infection with enterotoxigenic Escherichia coli F4.

Enterotoxigenic Escherichia coli (ETEC) F4 causes diarrhea in infants and weaned piglets. The technique of isobaric tags for relative and absolute quantitation (iTRAQ) was used in this study to determine the differentially expressed proteins in porcine intestinal epithelial cells (IPEC-J2) after pretreatment with Lactobacillus plantarum (LP) followed by challenge with ETEC F4. A total of 4771 proteins were identified in IPEC-J2 cells, with 90, 105, and 134 differentially expressed proteins in cells exposed to ETEC, LP, and LP + ETEC, respectively. The COG analysis divided the identified proteins into 20 categories. The GO and KEGG annotation indicated that most of the differentially expressed proteins were enriched in various biological metabolism including cell cycle control, cell division and differentiation. Additionally, western blotting analyses confirmed the reduced abundance of selected proteins of the mTOR and MAPK signal pathways affected by ETEC F4. Moreover, LP pretreatment increased JNK activation in IPEC-J2 cells infected with ETEC F4. These results may provide further insights into the mechanisms involved in the interaction between ETEC F4 and intestinal epithelial cells, and broaden the understanding of the protective effects of LP in alleviating ETEC-provoked diarrhea of piglets.

Establishment of A Reversibly Inducible Porcine Granulosa Cell Line.

Granulosa cells (GCs) are the key components of ovarian follicles for regulating oocyte maturation. Previous established GC lines have allowed prolonged proliferation, but lost some physiological features owing to long-term immortalization. This study was to establish an induced immortal porcine GC line with reversible proliferation status by the tetracycline inducible (Tet-on) 3G system. Our conditional immortal porcine GCs (CIPGCs) line steadily propagated for at least six months and displayed primary GC morphology when cultured in the presence of 50 ng/mL doxycycline [Dox (+)]. Upon Dox withdrawal [Dox (-)], Large T-antigen expression, reflected by mCherry fluorescence, gradually became undetectable within 48 h, accompanied by less proliferation and size increase. The levels of estradiol and progesterone, and the expression of genes associated with steroid production, such as CYP11A1 (cytochrome P450 family 11), 3ß-HSD (3ß-hydroxysteroid dehydrogenase), StAR (steroidogenic acute regulatory protein), and CYP19A1 (cytochrome P450 family 19 subfamily a member 1), were all significantly higher in the Dox (-) group than Dox (+) group. The CIPGCs could switch into a proliferative state upon Dox induction. Interestingly, the expression of StAR and CYP19A1 in the CIPGCs (-Dox) was significantly increased by adding porcine follicular fluid (PFF) to mimic an ovary follicle environment. Moreover, PFF priming the CIPGCs in Dox (-) group resulted in similar estradiol production as that of primary GC, and enabled this cell line to respond to gonadotrophins in estradiol production. Collectively, we have established an inducible immortal porcine GC line, which offers a unique and valuable model for future research on the regulation of ovarian functions.

A Comparative Study of Biological Characteristics and Transcriptome Profiles of Mesenchymal Stem Cells from Different Canine Tissues.

Mesenchymal stem cells (MSCs) are the most promising seed cells for cell therapy. Comparing the biological and transcriptome gene characteristics of MSCs from different sources provides an important basis for the screening of clinically used cells. The main purpose of this experiment was to establish methods for the isolation and culture of MSCs from five different canine sources, including adipose tissue, bone marrow, umbilical cord, amniotic membrane, and placenta, and compare biological and transcriptome characteristics of MSCs, in order to provide a basis for the clinical application of canine MSCs. MSCs were isolated from Chinese pastoral dogs, and the following experiments were performed: (1) the third, sixth, and ninth generations of cells were counted, respectively, and a growth curve was plotted to calculate the MSC population doubling time; (2) the expression of CD34 and CD44 surface markers was studied by immunofluorescence; (3) the third generation of cells were used for osteogenetic and adipogenic differentiation experiments; and (4) MSC transcriptome profiles were performed using RNA sequencing. All of the five types of MSCs showed fibroblast-like adherent growth. The cell surface expressed CD44 instead of CD34; the third-generation MSCs had the highest proliferative activity. The average population doubling time of adipose mesenchymal stem cells (AD-MSCs), placenta mesenchymal stem cells (P-MSCs), bone marrow mesenchymal stem cells (BM-MSCs), umbilical cord mesenchymal stem cells (UC-MSCs), and amniotic mesenchymal stem cells (AM-MSCs) were 15.8 h, 21.2 h, 26.2 h, 35 h, and 41.9 h, respectively. All five types of MSCs could be induced to differentiate into adipocytes and osteoblasts in vitro, with lipid droplets appearing after 8 days and bone formation occurring 5 days after AD-MSC induction. However, the multilineage differentiation for the remaining of MSCs was longer compared to that of the AD-MSCs. The MSC transcriptome profiles showed that AD-MSC and BM-MSCs had the highest homology, while P-MSCs were significantly different compared to the other four types of MSCs. All the isolated MSCs had the main biological characteristics of MSCs. AD-MSCs had the shortest time for proliferation, adipogenesis, and osteogenic differentiation.

Effect of leukocyte inhibitory factor on neuron differentiation from human induced pluripotent stem cell-derived neural precursor cells.

Direct derivation of human induced pluripotent stem cells into neural precursor cells and differentiation of these into neurons holds great promise in the cell therapy of neurodegenerative diseases. However, the availability and survival rate of neurons requires improvement. In the present study, it was found that the addition of 5 ng/ml leukocyte inhibitory factor (LIF) during the process of differentiation significantly improved the expression of neuron­specific class III ß­tubulin (TUJ1) and microtubule­associated protein 2 (MAP2), as detected by immunofluorescence and western blotting. In addition, LIF improved the cell viability, increased the expression of phosphorylated­protein kinase B (AKT), downregulated the expression of proinflammatory cytokines, including interleukin­1α (IL­1α) and tumor necrosis factor­α (TNF-α), and upregulated the expression of anti­inflammatory cytokines, including interleukin­10 (IL­10) and transforming growth factor­ß (TGF-ß). After adding the phosphatidylinositol 3-kinase (PI3K)/AKT signaling inhibitor LY294002 or wortmannin to the LIF differentiation group, LIF-induced changes in the protein expression of TUJ1 and MAP2 were reversed, but this effect could not be prevented by rapamycin, a mechanistic target of rapamycin signaling inhibitor. The expression of cytokines associated with inflammation and cell viability was reversed by LY294002 and wortmannin, but not by rapamycin. In conclusion, LIF could improve neuronal differentiation and survival through the activation of PI3K/AKT signaling and the anti­inflammatory effect. The anti­inflammatory effect may be mediated by the activation of PI3K/AKT.

Artesunate attenuates glioma proliferation, migration and invasion by affecting cellular mechanical properties.

Glioma is one of the most common malignant brain tumors. Current chemotherapy is far from providing satisfactory clinical outcomes for patients with glioma. More efficient drugs are urgently needed. Artesunate (ART) is clinically used as an anti-malarial agent and exhibits potent antiproliferative activity as a traditional Chinese medicine. In addition, ART has been shown to exert a profound cytotoxic effect on various tumor cell lines, presenting a novel candidate for cancer chemotherapy. However, its anticancer effect on glioma by altering cell biomechanical properties remains unclear. The present study aimed to identify the anticancer effects of ART on human glioma SHG44 cells by assessing cell proliferation, migration/invasion, the expression of claudin-1 and the biomechanical properties of ART-treated SHG44 cells. The proliferation of the SHG44 cells was assessed by MTT assay. The cell apoptosis was detected by flow cytometry. For cell migration and invasion assays, the Transwell was used. The expression of the gene claudin-1 was detected by polymerase chain reaction. The cell membrane and biomechanical properties, as targets of ART action, were investigated by atomic force microscopy (AFM). ART significantly inhibited the proliferation of SHG44 cells in a dose- and time-dependent manner. After treatment with 30 mg/l ART, the level of cell apoptosis was significantly increased (from 6.88±0.062 to 23.7±4.16%). Furthermore, the cell migration and invasion abilities of the SHG44 cells were markedly inhibited after treatment with 30 mg/l ART. Compared with the control group (0 mg/l ART), the SHG44 cells treated with 30 mg/l ART exhibited upregulated expression of claudin-1, increased adhesive force (from 2,400±300 to 3,600±500 pN), increased high connection among SHG44 cells, increased cytomembrane roughness (from 0.118±0.011 to 0.269±0.015 µm) and reduced elasticity (from 23±8 to 3.5±1.1 MPa). The present study demonstrated that ART could alter the biomechanical properties of the glioma cells to inhibit cell proliferation, migration and invasion.