IEC-6 Cells Research Articles

Citric Acid Promotes Immune Function by Modulating the Intestinal Barrier.

Amidst increasing concern about antibiotic resistance resulting from the overuse of antibiotics, there is a growing interest in exploring alternative agents. One such agent is citric acid, an organic compound commonly used for various applications. Our research findings indicate that the inclusion of citric acid can have several beneficial effects on the tight junctions found in the mouse intestine. Firstly, the study suggests that citric acid may contribute to weight gain by stimulating the growth of intestinal epithelial cells (IE-6). Citric acid enhances the small intestinal villus-crypt ratio in mice, thereby promoting intestinal structural morphology. Additionally, citric acid has been found to increase the population of beneficial intestinal microorganisms, including Bifidobacterium and Lactobacillus. It also promotes the expression of important protein genes such as occludin, ZO-1, and claudin-1, which play crucial roles in maintaining the integrity of the tight junction barrier in the intestines. Furthermore, in infected IEC-6 cells with H9N2 avian influenza virus, citric acid augmented the expression of genes closely associated with the influenza virus infection. Moreover, it reduces the inflammatory response caused by the viral infection and thwarted influenza virus replication. These findings suggest that citric acid fortifies the intestinal tight junction barrier, inhibits the replication of influenza viruses targeting the intestinal tract, and boosts intestinal immune function.

Radix Sanguisorbae Improves Intestinal Barrier in Septic Rats via HIF-1 α/HO-1/Fe2+ Axis.

To investigate whether Radix Sanguisorbae (RS, Diyu) could restore intestinal barrier function following sepsis using a cecal ligation and puncture (CLP)-induced septic rat model and lipopolysaccharide (LPS)-challenged IEC-6 cell model, respectively. Totally 224 rats were divided into 4 groups including a control, sham, CLP and RS group according to a random number table. The rats in the control group were administrated with Ringer's lactate solution (30 mL/kg) with additional dopamine [10 µ g/(kg·min)] and given intramuscular injections of cefuroxime sodium (10 mg/kg) 12 h following CLP. The rats in the RS group were administrated with RS (10 mg/kg) through tail vein 1 h before CLP and treated with RS (10 mg/kg) 12 h following CLP. The rats in the sham group were only performed abdominal surgery without CLP. The rats in the CLP group were performed with CLP without any treatment. The other steps were same as control group. The effects of RS on intestinal barrier function, mesenteric microvessels barrier function, multi-organ function indicators, inflammatory response and 72 h survival window following sepsis were observed. In vitro, the effects of RS on LPS-challenged IEC-6 cell viability, the expressions of zona occludens-1 (ZO-1) and ferroptosis index were evaluated by cell counting kit-8, immunofluorescence and Western blot analysis. Bioinformatic tools were applied to investigate the pharmacological network of RS in sepsis to predict the active compounds and potential protein targets and pathways. The sepsis caused severe intestinal barrier dysfunction, multi-organ injury, lipid peroxidation accumulation, and ferroptosis in vivo. RS treatment significantly prolonged the survival time to 56 h and increased 72-h survival rate to 7/16 (43.75%). RS also improved intestinal barrier function and relieved intestinal inflammation. Moreover, RS significantly decreased lipid peroxidation and inhibited ferroptosis (P<0.05 or P<0.01). Administration of RS significantly worked better than Ringer's solution used alone. Using network pharmacology prediction, we found that ferroptosis and hypoxia inducible factor-1 (HIF-1 α) signaling pathways might be involved in RS effects on sepsis. Subsequent Western blot, ferrous iron measurements, and FerroOrange fluorescence of ferrous iron verified the network pharmacology predictions. RS improved the intestinal barrier function and alleviated intestinal injury by inhibiting ferroptosis, which was related in part to HIF-1 α/heme oxygenase-1/Fe2+ axis.

Argininyl-Fructosyl-Galactose: A Novel Amino Acid Derivative improves Cisplatin-induced Intestinal Toxicity via Reactive Oxygen Species- mediated Apoptosis Pathway.

Based on the Maillard reaction principle of red ginseng, this study innovatively synthesized a new amino acid derivative by combining arginine with lactose through simulated synthesis and was separated and purified through repeated silica gel and polyacrylamide gel (Bio-gel P-II) column chromatography. The work was aimed at elucidating the synthesis of a novel amino acid derivative and investigating the intestinal protective activity of the novel amino acid derivative and possible molecular mechanism by establishing the intestinal injury model induced by cisplatin in mice. The purity and molecular weight of the amino acid derivatives were determined to be by electrospray ionization mass spectrometry (ESI-MS). Subsequently, by establishing cisplatin (20 mg/kg)-induced intestinal injury in vivo for 10 days and IEC-6 cell model. The biochemical indexes and histopathological analysis were used to evaluate the oxidative stress and inflammatory and pathological changes of intestinal tissue in mice. The protein expression levels of p-Nuclear transcription factor-κB (p-NF-κB), cleaved caspase 3/caspase 3, cleaved caspase 9/caspase-9, Bcl-2, Bax, cytochrome C, phosphatidylinositol 3-kinase (PI3K), Protein Kinase B (Akt), p-PI3K, p-Akt were quantified through immunofluorescence staining and western blot analysis. The new amino acid derivatives of chemical structure were identified to be 1- (arginine-Nαgroup)-1-deoxy-4-O-(β-D-galactopyranosyl)-D-fructose, named Argininylfructosyl- galactose (AFGA, C18H34N4O12). The results showed that pretreatment with a single AFGA dose remarkably alleviated cisplatin-evoked intestinal oxidative stress injury, and the levels of reactive oxygen species (ROS) were lessening in IEC-6 cells (p<0.05, p<0.01) and could effectively reduce the secretion of TNF-α and IL-1β in serum and the expression level of NF-κB protein in intestinal tissues (p<0.01). Meantime, AFGA also significantly suppressed the caspase 3, caspase 9, cytochrome C and Bax protein expression in intestinal tissue in mice (p<0.01), and regulated the PI3K/Akt pathway (p<0.05, p<0.01). Importantly, the molecular docking results of AFGA also suggested a better binding ability with the above-mentioned related target proteins. The results clearly revealed AFGA as a potential multifunctional therapeutic agent with a clear protective effect against cisplatin-induced intestinal injury may be related to the PI3K/Akt signaling pathway.

PI3K/Akt signaling pathway mediates the effect of low-dose boron on barrier function, proliferation and apoptosis in rat intestinal epithelial cells

Boron is an essential trace element with roles in growth, development, and physiological functions; however, its mechanism of action is still unclear. In this study, the regulatory roles of the PI3K/Akt signaling pathway on boron-induced changes in barrier function, proliferation, and apoptosis in rat intestinal epithelial cells were evaluated. Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein expression levels of PCNA were higher, while the proportions of cells in the G0/G1 and S phases, apoptosis rate, and caspase-3 mRNA and protein expression levels were lower in cells treated with 0.8 mmol/L boron than in control IEC-6 cells (P < 0.01 or P < 0.05). However, 40 mmol/L boron decreased ZO-1 and Occludin levels, the proportion of cells in the G2/M phase, cell proliferation rate, and mRNA and protein levels of PCNA and increased the apoptosis rate and caspase-3 mRNA expression (P < 0.01 or P < 0.05). After specifically blocking PI3K and Akt signals (using LY294002 and MK-2206 2HCL), 0.8 mmol/L boron had no effects on Occludin, PCNA level, apoptosis rates, and caspase-3 levels (P < 0.05); however, the proliferation rate and PCNA levels decreased significantly (P < 0.01 or P < 0.05). The addition of 40 mmol/L boron did not affect ZO-1 and Occludin levels and did not affect the apoptosis rate or PCNA and caspase-3 levels. These results suggested that the PI3K/Akt signaling pathway mediates the effects of low-dose boron on IEC-6 cells.

The in vitro anti-inflammatory mechanism of Porphyra haitanensis oligosaccharides on lipopolysaccharide-induced injury in IEC-6 cells

Porphyra haitanensis, with its rich nutritional value and distinctive flavour, has become a research hotspot in food field. P. haitanensis oligosaccharides (PHO) are oligosaccharides with potential anti-inflammatory properties that ameliorate lipopolysaccharide-induced inflammatory responses in the in vitro IEC-6 cell line model. Here, the structure of PHO was investigated, and LPS–induced injury mechanisms in IEC-6 were determined by cellular, molecular, and transcriptome analyses. After PHO exposure, LPS-induced IEC-6 cells showed reduced apoptosis, decreased oxidative stress levels, improved cell membrane integrity, and paracellular permeability. This study shows that PHO attenuates injury in the IEC-6 cell line by increasing the expression of tight junctions. PHO also inhibited the TLR4/NF-κB pathway and caused downregulation of intracellular pro-inflammatory cytokines, which attenuates LPS-induced injury in an in vitro model, and further offers the possibility of restoring the intestinal barrier. This study provides insight into the role of PHO in alleviating the intestinal epithelial barrier damage.

Carvacrol improves blood lipid and glucose in rats with type 2 diabetes mellitus by regulating short-chain fatty acids and the GPR41/43 pathway.

Type 2 diabetes mellitus (T2DM) is characterized by hyperglycemia and dyslipidemia. Carvacrol (CAR) has demonstrated the potential to mitigate dyslipidemia. This study aims to investigate whether CAR can modulate blood glucose and lipid levels in a T2DM rat model by regulating short-chain fatty acids (SCFAs) and the GPR41/43 pathway. The T2DM rat model was induced by a high-fat diet combined with low-dose streptozocin injection and treated with oral CAR and/or mixed antibiotics. Fasting blood glucose, oral glucose tolerance, and insulin tolerance tests were assessed. Serum lipid parameters, hepatic and renal function indicators, tissue morphology, and SCFAs were measured. In vitro, high glucose (HG)-induced IEC-6 cells were treated with CAR, and optimal CAR concentration was determined. HG-induced IEC-6 cells were treated with SCFAs or/and GPR41/43 agonists. CAR significantly reduced blood lipid and glucose levels, improved tissue damage, and increased SCFA levels in feces and GPR41/43 expression in colonic tissues of T2DM rats. CAR also attenuated HG-induced apoptosis of IEC-6 cells and enhanced GPR41/43 expression. Overall, these findings suggest that CAR alleviates blood lipid and glucose abnormalities in T2DM rats by modulating SCFAs and the GPR41/43 pathway.

Therapeutic Effects of Qingchang Tongluo Decoction on Intestinal Fibrosis in Crohn's Disease: Network Pharmacology, Molecular Docking and Experiment Validation.

Qingchang Tongluo Decoction (QTF) is clinically used for the treatment of intestinal fibrosis in Crohn's Disease (CD). However, the role of QTF in CD-associated fibrosis and its potential pharmacological mechanism remains unclear. The objective of this study was to elucidate the potential mechanism of QTF in treating CD-associated fibrosis, employing a combination of bioinformatics approaches - encompassing network pharmacology and molecular docking - complemented by experimental validation. To investigate the material basis and potential protective mechanism of QTF, a network pharmacology analysis was conducted. The core components and targets of QTF underwent molecular docking analysis to corroborate the findings obtained from network pharmacology. In vitro, a colon fibrotic model was established by stimulating IEC-6 cells with 10 ng/mL of transforming growth factor(TGF-β1). In vivo, an intestinal fibrosis model was induced in BALB/c mice by TNBS. The role of QTF in inhibiting the TGF-β1/Smad signaling pathway was investigated through RT-qPCR, Western blotting, immunohistochemistry staining, and immunofluorescence staining. Network pharmacology analysis revealed that QTF could exert its protective effect. Bioinformatics analysis suggested that Flavone and Isoflavone might be the key components of the study. Additionally, AKT1, IL-6, TNF, and VEGFA were identified as potential therapeutic targets. Furthermore, experimental validation and molecular docking were employed to corroborate the results obtained from network pharmacology. RT-qPCR, Immunofluorescence, and Western blotting results demonstrated that QTF significantly improved colon function and inhibited pathological intestinal fibrosis in vivo and in vitro. Through the application of network pharmacology, molecular docking, and experimental validation, QTF could be confirmed to inhibit the proliferation of intestinal fibroblasts associated with CD and reduce the expression of Collagen I and VEGFA. This effect is achieved through the attenuation of ECM accumulation, primarily via the inhibition of the TGF-β1/Smad signaling pathway.

Pinobanksin ameliorated DSS-induced acute colitis mainly through modulation of SLC7A11/glutathione-mediated intestinal epithelial ferroptosis.

Inhibition of ferroptosis in intestinal epithelial cells serves as an attractive target for the development of therapeutic strategies for colitis. Pinobanksin, one of the main flavonoids derived from propolis, possesses significant anti-inflammatory effects and inhibits the cell death of several cell lines. Here, we evaluated whether pinobanksin influenced colitis by modulation of epithelial ferroptosis. Mice treated with 2.5% DSS dissolved in sterile distilled water were established for an acute colitis model. The mitochondrial morphology, colonic iron level, lipid peroxidation products MDA/4-HNE, and lipid reactive oxygen species levels were measured to assess ferroptosis in epithelial cells. RNA-seq and functional analyses were performed to reveal key genes mediating pinobanksin-exerted modulation of ferroptosis. We found that pinobanksin, at different doses, induced significant anti-colitis effects and inhibited the elevated ferroptosis in colonic epithelial cells isolated from DSS-treated mice largely by activating GPX4 (negative regulator of ferroptosis). Furthermore, RNA-seq assays indicated that pinobanksin significantly increased the cystine transporter SLC7A11 in colonic tissues from mice with colitis. Depletion of SLC7A11 largely blocked pinobanksin-induced promotion of cystine uptake/glutathione biosynthesis and suppression of ferroptosis in epithelial cells from mice with colitis or IEC-6 cells pretreated with RSL3. Altogether, pinobanksin alleviated DSS-induced colitis largely by inhibition of ferroptosis in epithelial cells. Activation of SLC7A11 by pinobanksin resulted in the promotion of cystine uptake and enhancement of glutathione biosynthesis. This work will provide novel guidance for the clinical use of pinobanksin to treat colitis through inhibition of epithelial ferroptosis.

RIP1/3-dependent programmed necrosis induces intestinal injury in septic rats.

The regulation of various types of cell death may help to restore the normal physiological function of cells and play a protective role in sepsis. In the current study, we explore the role of programmed cell necrosis in sepsis and the underlying mechanisms. The septic rat model is established by Cecal-ligation and perforation (CLP), and the in vitro model is established by LPS in IEC-6 cells. Our results demonstrate that receptor-interacting protein 1 (RIP1) is significantly upregulated in the ileum of septic rats and LPS-treated IEC-6 cells at both the mRNA and protein levels. Nec-1, an inhibitor of RIP1, reduces the protein levels of RIP1, p-RIP3, and phosphorylated mixed-lineage kinase domain-like (MLKL) (serine 358) and relieves intestinal injury in CLP-induced septic rats with decreased IL-6 and TNF-α levels. The in vitro experiments further reveal that LPS induces the colocalization of RIP1 and RIP3, resulting in the phosphorylation and translocation of MLKL to the plasma membrane in IEC-6 cells. LPS also facilitates ROS production in IEC-6 cells, but this effect is further reversed by Nec-1, si-RIP1 and si-RIP3. Furthermore, LPS-induced necrosis in IEC-6 cells is counteracted by NAC. Thus, we conclude that RIP1/RIP3-dependent programmed cell necrosis participates in intestinal injury in sepsis and may be associated with RIP1/RIP3-mediated ROS.

Inhibition of circulating exosomes release with GW4869 mitigates severe acute pancreatitis-stimulated intestinal barrier damage through suppressing NLRP3 inflammasome-mediated pyroptosis

Intestinal barrier dysfunction frequently occurs as a complication in cases of severe acute pancreatitis (SAP); however, no effective therapeutic methods are available because the precise mechanism remains obscure. Recent research has elucidated the role of circulating exosomes in the progression of SAP. Therefore, the present study explored whether inhibiting circulating exosomes release would improve intestinal barrier injury triggered via SAP and investigated the possible underlying mechanism. In vivo, we found that circulating exosomes release exhibited a considerable increase in SAP rats than in SO rats, and GW4869, a suppressor of exosomes release, significantly decreased exosomes release in SAP rats. We also observed that GW4869 suppressed NLRP3 inflammasome-mediated pyroptosis within the intestine and alleviated intestinal barrier injury within SAP. Moreover, the inflammatory response and remote organ (kidney and lung) injury associated with SAP improved after GW4869 treatment. In vitro, we confirmed that depletion of exosomes with GW4869 could partially abolish the destructive effects of SAP rat plasma on the viability and barrier function of IEC-6 cells. In summary, our findings show that the suppression of the release of circulating exosomes effectively inhibits the process of pyroptosis mediated by the NOD-like receptor protein 3 (NLRP3) inflammasome and, therefore, mitigates intestinal barrier dysfunction in SAP, suggesting that circulating exosomes may be a potential target for treating SAP.

The brominated flame retardant tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) as well as hexabromocyclododecane lead to lipid disorders in mice

The brominated flame retardant tetrabromobisphenol A-bis(2,3-dibromo-2-methylpropyl ether) (TBBPA-DBMPE) is a recommended substitute for hexabromocyclododecane (HBCD), a banned persistent organic pollutant, yet its potential toxicities remains largely unexplored. Here, we investigated the effects of a long-term exposure to TBBPA-DBMPE at nominal doses of 50 and 1000 μg/kg/d on lipid homeostasis in CD-1 mice, in comparison with 50 μg/kg/d HBCD as a positive control. Male pups received chemical treatments through maternal administration via drinking water from postnatal day 0–21, followed by direct administration through drinking water after weaning. On the 23rd week after treatment, the oral lipid tolerance test revealed that low-dose TBBPA-DBMPE as well as HBCD affected lipid tolerance, although the fasting serum triglyceride (TG) levels were not altered. When chemical treatment was extended to the 32nd week, TBBPA-DBMPE-treated animals displayed adipocyte hypertrophy in both white adipose tissue (eWAT) and brown adipose tissue (BAT) and hepatic steatosis, which was largely consistent with the effects of HBCD. These findings indicate that like HBCD, TBBPA-DBMPE led to increased lipid load in mice. Interestingly, we also observed intestinal histological changes, coupled with increased expression of lipid absorption-related genes in both HBCD and TBBPA-DBMPE treatments, suggesting increased lipid absorption. This was supported by in vitro findings that both HBCD and TBBPA-DBMPE promoted lipid accumulation in IEC-6 cells under the stress of oleic acid for 6 h, implying that altered lipid absorption by the intestine may partly contributed to increased lipid load in mice. Overall, the effects of 50 μg/kg/d TBBPA-DBMPE in terms of some parameters were comparable with 50 μg/kg/d HBCD, suggesting that TBBPA-DBMPE may not be an ideal substitute of HBCD.

PSXIV-8 Comparison of the Effects of Hydroxy-Selenomethionine and Selenium-Enriched Yeast Against Hydrogen Peroxide-Induced Oxidative Stress in Rat Intestinal Epithelial Cells

Abstract Superior bioavailability of organic selenium (Se) has been extensively studied. However, limited research has been conducted to evaluate the effect of organic forms on intestinal redox potential to support gut health and animal well-being. Two experiments (Exp) were conducted to compare the effect of either hydroxy-selenomethionine (OH-SeMet) or Se-enriched yeast (Se-yeast) on ameliorating hydrogen peroxide (H2O2)-induced oxidative stress in a rat intestinal epithelial cell line (IEC-6). A cell viability test was initially performed to validate the Se level (0.15, 0.3, 0.6 ug/mL) used for the reactive oxygen species (ROS) assay. In both studies, OH-SeMet or Se-yeast promoted cell viability and attenuated cell apoptosis indicating no negative impact on cell growth. However, the Se-yeast sources showed considerably greater effectiveness than OH-SeMet. To quantify redox potential, confluent cells were treated with DCFDA, an indicator of ROS production. Briefly, IEC-6 cells were seeded into 96-well plates (4 ×104 cells per well) with 3 replicates per treatment (Trt) and incubated with a fresh solution of DCFDA. The ROS production, in response to 30µM H2O2, was quantified by fluorescence emission at 485 nm. Data were analyzed with GLIMMIX procedure of SAS. In both Exp, Se inclusion in all Se-Trts was 0.3 ppm. In Exp 1, Trts included: Non treated (cells only), H2O2, H2O2 + 2mM Trolox (Vit. E analog), H2O2 + sodium selenite, H2O2 + OH-SeMet, H2O2 + Se-yeast product A, H2O2 + Se-yeast product B. Cells treated with H2O2 had 4.6-fold increase (P &amp;lt; 0.05) in ROS production relative to non-H2O2 treated cells. Trolox reduced ROS 7.6-fold (P &amp;lt; 0.05) relative to H2O2 only cells, OH-SeMet and sodium selenite did not ameliorate (P &amp;gt; 0.35) the negative impact of H2O2 on ROS production. However, cells treated with H2O2 + Se-yeast product A or B reduced ROS production by 15% (P = 0.07) and 32% (P &amp;lt; 0.01), respectively, compared with cells treated with H2O2 only. To validate the redox potential of OH-SeMet, Exp 2 included a pure form of selenomethionine. Treatments in Exp 2. were similar to Exp 1 with the inclusion of H2O2 + seleno-L-methionine. Selenomethionine, either as a pure source or the hydroxy analog, did not prevent (P &amp;gt; 0.40) H2O2-induced oxidative stress. However, Exp 2 confirmed the effectiveness of Se-Yeast products on reducing ROS production. More specifically, Se-Yeast product B (52% reduction, P = 0.04) was superior to product A (32% reduction, P = 0.17) in comparison to cells treated with H2O2 only. These findings suggest that the type of organic Se source differs in their ability to regulate intestinal redox potential, with Se-yeast sources being more effective than hydroxy analogs. Selenomethionine content in Se-yeast does not appear to be the main driver in reducing ROS in intestinal cells, suggesting that livestock fed Se-yeast can support better intestinal health during oxidative stress.

Longan Polysaccharides with Covalent Selenylation Combat the Fumonisin B1-Induced Cell Toxicity and Barrier Disruption in Intestinal Epithelial (IEC-6) Cells.

In this study, the soluble, but non-digestible, longan (Dimocarpus longan Lour.) polysaccharides (LP) were extracted from dried longan fruits and then chemically selenylated to produce two selenylated products, namely SeLP1 and SeLP2, with different selenylation extents. The aim was to investigate their protective effects on rat intestinal epithelial (IEC-6) cells exposed to the food toxin fumonisin B1 (FB1). LP only contained total Se content of less than 0.01 g/kg, while SeLP1 and SeLP2 were measured with respective total Se content of up to 1.46 and 4.79 g/kg. The cell viability results showed that these two selenylated products were more efficient than LP in the IEC-6 cells in alleviating FB1-induced cell toxicity, suppressing lactate dehydrogenase (LDH) release, and decreasing the generation of intracellular reactive oxygen species (ROS). These two selenylated products were also more effective than LP in combating FB1-induced barrier disruption via increasing the transepithelial electric resistance (TEER), reducing the paracellular permeability, decreasing the mitochondrial membrane potential (MMP) loss, and maintaining cell barrier integrity by upregulating the tight-junction-related genes and proteins. FB1 caused cell oxidative stress and barrier dysfunction by activating the MAPK and mitochondrial apoptosis signaling pathways, while SeLP1 and SeLP2 could regulate the tMAPK- and apoptosis-related proteins to suppress the FB1-mediated activation of the two pathways. Overall, SeLP2 was observed to be more active than SeLP1 in the IEC-6 cells. In conclusion, the chemical selenylation of LP caused an activity enhancement to ameliorate the FB1-induced cell cytotoxicity and intestinal barrier disruption. Meanwhile, the increased selenylation of LP would endow the selenylated product SeLP2 with more activity.

Favipiravir Protects Enterocytes From Cell Death After Inflammatory Storm.

Over the past years, inflammatory bowel disease (IBD) treatment has become more targeted, anticipating the use of immune-modifying therapies at an earlier stage. During the treatment process prevention and management of viral infections hold significant importance. The protective role of favipiravir on enterocytes which are affected by inflammation is still unknown. We aim to analyze the effects of favipiravir on enterocytes after an inflammatory condition. We conducted a 2,5-diphenyl-2H-tetrazolium bromide (MTT) assay to assess the cytotoxicity of favipiravir on intestinal epithelioid cells (IEC-6). To mimic the inflammation model in cell culture conditions, we exposed IEC-6 cells to tumor necrosis factor-α (TNF-α) and interferon-γ (IFN-γ). The cells were categorized into four groups: control, inflammation model, application of favipiravir before inflammation (prophylactic), and application of favipiravir after inflammation (treatment). We assessed the presence and distribution of caspase 1, caspase 3, interleukin6 (IL6), interleukin 8 (IL8), mixed lineage kinase domain-like protein (MLKL), receptor-interacting protein kinase 1 (RIPK1), and TNF-α using indirect immunoperoxidase staining. TNF-α and IL8 levels were analyzed with enzyme-linked immunosorbent assay (ELISA) in a culture medium. Caspase 1 was observed to be strong (+++) in the treatment group and weak (+) in the prophylactic group compared to the inflammation group. Caspase 3 was weak (+) in the inflammation group, and it was strong (+++) in the prophylactic and treatment group, the increase in the treatment group was significant. Therefore administering favipiravir before inducing inflammation appears to control the inflammatory caspase pathway in intestinal enterocytes, protecting them from inflammatory responses, while the caspase 3-dependent apoptotic pathway may not be active in enterocytes during inflammation. IL6 and IL8 were negative (-) in control, IL6 was weak (+) in inflammation and favipiravir treated groups; IL8 increased significantly in favipiravir groups compared to control and inflammation groups. Consequently, favipiravir may trigger IL6 release, initiating the inflammatory pathway and potentially enhancing IL8 interactions with other cytokines. TNF-α immunoreactivity was strong (+++) in the inflammation group, while it was moderate (++) in favipiravir-administered groups. MLKL immunoreactivity was strong (+++) in all groups, RIPK1 was weak (+) in control, strong (+++) in the inflammation and treatment group, moderate (++) in the prophylactic group, and the increase in inflammation and treatment group was significant compared to control. Our findings suggest that in the treatment group, necroptosis was triggered by increased MLKL and RIPK1, key players in inflammation and cell death. After immunocytochemical evaluation, our findings suggest that, after the onset of inflammation, favipiravir may play a role in cell death by increasing necroptosis rather than apoptosis.

Corilagin attenuates intestinal ischemia/reperfusion injury in mice by inhibiting ferritinophagy-mediated ferroptosis through disrupting NCOA4-ferritin interaction

AimsIntestinal ischemia reperfusion (II/R) is a common clinical emergency. Ferroptosis is reported to play a role in II/R injury. Our previous studies revealed that corilagin significantly attenuates intestinal ischemia/reperfusion injuries. However, the underlying molecular mechanism is unclear and requires further study. Materials and methodsDAO, GSSG/T-GSH, MDA, and Fe2+ were measured by assay kits, 4-HNE was assessed by IHC, and 15-LOX was measured by ELISA. Mitochondrial damage was observed by TEM and cellular oxidation levels were detected by C11-BODIPY 581/591 and DHE probes. LC3, p62, Beclin1, ACSL4, GPX4, NCOA4, and ferritin expression were examined by WB in vivo and in vitro. IF, co-IF, q-PCR, and constructed NCOA4-knock-down IEC-6 cells were used to evaluate the role of NCOA4 in the effect of corilagin against II/R injury. Temporal and nucleoplasmic variations with or without corilagin were observed by WB. Co-IP and molecular docking were used to investigate the NCOA4-ferritin interaction. Key findingsCorilagin attenuated II/R-induced ferroptosis both in vitro and in vivo. Further study revealed that the anti-ferroptosis bioactivity of corilagin might be due to the modulation of iron homeostasis via inhibition of ferritinophagy in an NCOA4-dependent manner. SignificanceCorilagin might be a potential therapeutic agent for II/R-induced tissue injury.

Inhibition of IEC-6 Cell Proliferation and the Mechanism of Ulcerative Colitis in C57BL/6 Mice by Dandelion Root Polysaccharides.

An exploration was conducted on the potential therapeutic properties of dandelion polysaccharide (DP) in addressing 3% dextran sodium sulfate (DSS)-induced ulcerative colitis (UC) in murine models. Subsequent assessments focused on DP's influence on inflammation, oxidative stress, and ferroptosis in IEC-6 cells damaged by H2O2. Results highlighted the efficacy of DP in mitigating weight loss, improving disease activity index scores, normalizing colon length, and alleviating histological abnormalities in the affected mice. DP repaired colonic mitochondrial damage by enhancing iron transport and inhibited iron death in colonic cells. Moreover, DP played a pivotal role in enhancing the antioxidant potential. This was evident from the increased expression levels of Nrf2, HO-1, NQO-1, and GSH, coupled with a decrease in MDA and 4-HNE markers in the UC-afflicted mice. Concurrently, DP manifested inhibitory effects on MPO activation and transcription levels of inflammatory mediators such as IL-1β, IL-6, TNF-α, and iNOS. An upsurge in the expression of occludin and ZO-1 was also observed. Restoration of intestinal tightness resulted in decreased serum LPS and LDH levels. Thereafter, administration of DP by gavage increased fecal flora diversity and relative abundance of probiotics in UC mice. Analysis of metabolites indicated that DP counteracted metabolic disturbances and augmented the levels of short-chain fatty acids in ulcerative colitis-affected mice. In vitro studies underscored the role of DP in triggering Nrf2 activation, which in turn exhibited anti-inflammatory, antioxidant, and anti-ferroptotic properties. Summarily, DP's capacity to activate Nrf2 contributes to the suppression of ferroptotic processes in intestinal epithelial cells of UC-affected mice, enhancing the intestinal barrier's integrity. Beyond that, DP possesses the ability to modulate the gut microbiome, rectify metabolic imbalances, rejuvenate short-chain fatty acid levels, and bolster the intestinal barrier as a therapeutic approach to UC.

SAT031 The Effect Of Metformin On Increasing Glucose Metabolism And Secretion In The Distal Intestine

Abstract Disclosure: J. Nam: None. C. Kang: None. J. Oh: None. E. Wang: None. S. Lee: None. J. Hong: None. E. Lee: None. C. Ku: None. Object: Although the effect of metformin is widely known, little research has been performed on clinical significance and potential mechanism of increased intestinal glucose uptake which is commonly found in [18F]-fluoro-D-glucose (FDG) positron emission tomography. The aim of this study is to assess the altered glucose metabolism in the combined colon and terminal small intestine after metformin treatment. Material and Method: Radioactivity of FDG was measured in metformin treated mouse to evaluate the glucose uptake in the various tissue. qPCR and immunoblot were performed to examine the glucose metabolism in small intestine and colon. Autoradiography and gamma counts were used for measuring FDG uptake in small intestine and colon. IEC6 and Caco-2 cell lines were used for small intestine and colon respectively for in vitro experiment. Result: 150mg/kg metformin treatment for 7 days improved glucose homeostasis in c57BL6 mice (AUC 0.8- fold, p&amp;lt;0.05). Gradual increase in FDG absorption was observed along the intestine in an autoradiography and gamma counter. Especially distal ileum (9-fold, p &amp;lt;0.0001) and colon (15-fold, &amp;lt;0.0001) showed increased glucose uptake than proximal intestine (Duodenum no significance, Jejunum 3-fold, P&amp;lt;0.01). Interestingly, glucose content in the fecal flush was elevated in metformin treated mice (13.5-fold, p&amp;lt;0.01). In line with the in vivo result, metformin enhanced glucose uptake in IEC6 (3-fold, p&amp;lt;0.01) cell compared to the non-treat control cells (NT). qPCR and Immunoblot results showed that metformin treatment enhanced GLUT1 expression (1.6-fold, p&amp;lt;0.01) compared to the NT. Hexokinase2 (HK2), known as glycolysis marker, shown to be increased (1.4-fold, p&amp;lt;0.01) by the metformin treatment in IEC6 and Caco-2 cells compared to the NT. GLUT1 expression was also increased in the metformin treated mice small intestine and colon than in vehicle treated mice. Conclusion: Taken together, our data suggest that metformin increases the glucose metabolism of distal small intestine and colon by the upregulation of GLUT1 expression. Enhanced glucose metabolism induced by metformin was correlated with the improved glucose homeostasis. Our data suggests the novel mechanism of metformin in the G.I tract that involved to some extent in the disposal of excess glucose in in vitro and in vivo system. Presentation: Saturday, June 17, 2023

Lung protection of Chimonanthus nitens Oliv. essential oil driven by the control of intestinal disorders and dysbiosis through gut-lung crosstalk

This work aimed to investigate whether Chimonanthus nitens Oliv. essential oil (CEO)-mediated lung protection was implicated in gut-lung crosstalk. Results showed that CEO attenuated lung and intestinal impairment by improving histopathological changes and inhibiting TLR4/NF-κB signaling pathway in LPS-stimulated rats, suggesting that there might be a mechanism for its lung protection involved in gut-lung interaction through manipulating the overlap in pathological changes via the similar inflammatory response. Furthermore, CEO-triggered intestinal protection was in parallel with the mitigation of ROS production, apoptosis, Ca2+ transport and mitochondrial membrane potential loss in vivo, and its intestinal protection was confirmed in vitro through IEC-6 cells. Importantly, a combination with CEO and LPS significantly remodeled gut microbiota composition compared with LPS alone in rats, while no significant impact on lung microbiota. Therefore, CEO-exerted lung protection was linked to gut and lung interactions involvement with the control of intestinal disorders and dysbiosis.

Forsythiae Fructus attenuates cisplatin-induced cytotoxicity in IEC-6 ​cells and J774A.1 macrophages by inhibiting NLRP3/caspase-1/GSDMD mediated pyroptosis

ObjectiveForsythiae Fructus (lian qiao in Chinese), the dried fruit of Forsythia suspensa (Thunb.) Vahl, is a commonly used traditional Chinese medicine known for its diverse biological activities, including antiemetic, anti-inflammatory, antioxidant, antiviral, and neuroprotective properties. This study investigated the protective effects of Forsythiae Fructus and its primary components, phillyrin and forsythoside A, against cisplatin-induced cytotoxicity in vitro, specifically focusing on the intestinal epithelial cells (IEC-6) and the J774A.1 macrophage cell line. MethodsCisplatin and tert-butyl hydroperoxide (tBHP) were used to induce stress in IEC-6 ​cells, while cisplatin and lipopolysaccharides (LPS)/adenosine triphosphate (ATP) were employed for J774A.1 macrophages. The protective effects of Forsythiae Fructus aqueous extract (FAE), phillyrin, and forsythoside A against cytotoxicity in these cultured cells were evaluated. Cell viability was assessed using the Cell Counting Kit-8 assay, while cell membrane permeability was determined through Hoechst 33342 and propidium iodide staining. Intracellular reactive oxygen species (ROS) levels were investigated using DCFH-DA, and the expression of mRNA and protein related to the NLRP3 inflammasome and GSDMD-induced pyroptosis was quantified through qRT-PCR and western blotting. ResultsIn IEC-6 ​cells, combining FAE, phillyrin, or forsythrin A with a subthreshold dose of the antioxidant N-acetyl-L-cysteine (NAC) significantly mitigated cisplatin- or tBHP-induced cell necrosis and restored impaired cell viability. Additionally, the upregulation of NF-κB, ASC, NLRP3, caspase-1, GSDMD, and HMGB1 at both mRNA and protein levels induced by cisplatin or tBHP was markedly reversed with the joint intervention of FAE, phillyrin, or forsythrin A with NAC. Similarly, in cisplatin- or LPS/ATP-treated J774A.1 macrophages, the effects on cell necrosis, cell viability, and the NLRP3/caspase-1/GSDMD pathway mirrored our previous findings in IEC-6 ​cells. ConclusionThe study suggests that the alleviating effect of Forsythiae Fructus and its primary components, phillyrin and forsythoside A, against cisplatin-induced cytotoxicity may be attributed to inhibiting oxidative stress, downregulating the NLRP3/caspase-1/GSDMD pathway, and inhibiting pyroptosis.

Astragaloside IV inhibits AOM/DSS-induced colitis-associated tumorigenesis via activation of PPARγ signaling in mice

BackgroundColitis-associated colorectal cancer (CAC) is a severe complication of inflammatory bowel disease (IBD), resulting from long-term inflammation in the intestines. The primary cause of CAC is the imbalance of oxidative metabolism in intestinal cells, triggered by excessive reactive oxygen (ROS) and nitrogen (NO) species production due to prolonged intestinal inflammation. This imbalance leads to genomic instability caused by DNA damage, eventually resulting in the development of intestinal cancer. Previous studies have demonstrated that astragaloside IV is effective in treating dextran sulfate sodium salt (DSS)-induced colitis, but there is currently no relevant research on its efficacy in treating CAC. MethodsTo investigate the effect of astragaloside IV against CAC and the underlying mechanism, C57 mice were treated with (20, 40, 80 mg/kg) astragaloside IV while CAC was induced by intraperitoneal injection of 10 mg/kg azoxymethane (AOM) and ad libitum consumption of 2% dextran sulfate sodium salt (DSS). We re-verified the activating effects of astragaloside IV on PPARγ signaling in IEC-6 cells, which were reversed by GW9662 (the PPARγ inhibitor). ResultsOur results showed that astragaloside IV significantly improved AOM/DSS-induced CAC mice by inhibiting colonic shortening, preventing intestinal mucosal damage, reducing the number of tumors and, the expression of Ki67 protein. In addition, astragaloside IV could activate PPARγ signaling, which not only promoted the expression of Nrf2 and HO-1, restored the level of SOD, CAT and GSH, but also inhibited the expression of iNOS and reduced the production of NO in the intestine and IEC-6 cells. And this effect could be reversed by GW9662 in vitro. Astragaloside IV thus decreased the level of ROS and NO in the intestinal tract of mice, as well as reduced the damage of DNA, and therefore inhibited the occurrence of CAC. ConclusionAstragaloside IV can activate PPARγ signaling in intestinal epithelial cells and reduces DNA damage caused by intestinal inflammation, thereby inhibiting colon tumourigenesis. The novelty of this study is to use PPARγ as the target to inhibit DNA damage to prevent the occurrence of CAC.