Intestinal Epithelial Injury Research Articles

The Effects of Prebiotic Supplementation on Markers of Exercise-Induced Gastrointestinal Syndrome in Response to Exertional Heat Stress.

Exercise perturbs various aspects of gastrointestinal integrity and function, which may lead to performance impeding gastrointestinal symptoms (GIS) and/or precipitate clinical issues warranting medical management. This study aimed to determine the impact of prebiotic supplementation on gastrointestinal integrity and functional status in response to exertional heat stress (EHS). Sixteen endurance athletes completed two trials of 3-hr running at 60% V˙O2max in 30°C at baseline (T1) and following an 8-week supplementation period (T2), with 16g/day prebiotic (PREBIOTIC) or matched placebo (PLACEBO). Blood samples were collected pre-EHS and post-EHS and in recovery for determination of stress response (cortisol), intestinal epithelial injury (intestinal fatty acid binding protein), bacterial endotoxemia (sCD14), and systemic inflammation (C-reactive protein). GIS and feeding tolerance variables were assessed throughout the EHS. Orocecal transit time was determined via a lactulose challenge given at 2.5hr into EHS. Plasma cortisol (combined mean: +252ng/ml), intestinal fatty acid binding protein (+800pg/ml), and sCD14 (+487ng/ml) concentrations increased in response to EHS in T1 (p ≤ .05), but not for C-reactive protein (+0.8μg/ml; p > .05), in both PREBIOTIC and PLACEBO. PREBIOTIC supplementation resulted in a blunted intestinal fatty acid binding protein response on T2 (+316pg/ml) compared with an increase (+1,001ng/ml) in PLACEBO (p = .005). Lower sCD14 was observed at T2 (2,799ng/ml) versus T1 (3,246ng/ml) in PREBIOTIC only (p = .039). No intervention effects were observed for C-reactive protein. No difference within or between PREBIOTIC and PLACEBO at T1 and T2 was observed for orocecal transit time, GIS, and feeding tolerance. In conclusion, 8 weeks of prebiotic supplementation modestly attenuates EHS associated perturbations to intestinal integrity, but does not further impair gastrointestinal transit and/or exacerbate EHS associated GIS or feeding tolerance.

Polyamines regulate mitochondrial metabolism essential for intestinal epithelial renewal and wound healing.

Homeostasis of the mammalian intestinal epithelium is tightly regulated by multiple factors, including cellular polyamines, but the exact mechanism underlying polyamines in this process remains largely unknown. Mitochondria are the powerhouse of cells and can also function as signaling organelles by releasing metabolic by-products. Here, we determined whether polyamines regulate intestinal epithelial renewal and wound healing by altering mitochondrial activity. Depletion of cellular polyamines by inhibiting ornithine decarboxylase with α-difluoromethylornithine (DFMO) resulted in mitochondrial dysfunction as evidenced by decreases in basal and maximal respiration levels, ATP production, and spare respiration capacity. Polyamine depletion by DFMO also decreased the levels of mitochondria-associated proteins prohibitin 1 and COX-IV. Mitochondrial dysfunction induced by DFMO was associated with an inhibition of intestinal organoid growth and epithelial repair after wounding, and this inhibition was ameliorated by administration of the mitochondrial activator Mito-Tempo or exogenous polyamine putrescine. These results indicate that polyamines are necessary for mitochondrial metabolism, in turn, controlling constant intestinal mucosal growth and epithelial repair after acute injury. NEW & NOTEWORTHY Our results indicate that polyamines are required for maintaining mitochondrial integrity in intestinal epithelial cells. Polyamine depletion led to mitochondrial dysfunction, along with an inhibition of intestinal epithelial renewal and delayed wound healing. Reinforcing mitochondrial activity by Mito-Tempo ameliorated reduced epithelial renewal and delayed healing in polyamine-deficient cells, demonstrating the importance of mitochondrial metabolism in polyamine-regulated mucosal growth and repair after injury.

Unraveling the role of Ctla-4 in intestinal immune homeostasis through a novel Zebrafish model of inflammatory bowel disease.

Inflammatory bowel disease (IBD) is a chronic and relapsing immune-mediated disorder characterized by intestinal inflammation and epithelial injury. The underlying causes of IBD are not fully understood, but genetic factors have been implicated in genome-wide association studies, including CTLA-4, an essential negative regulator of T cell activation. However, establishing a direct link between CTLA-4 and IBD has been challenging due to the early lethality of CTLA-4 knockout mice. In this study, we identified zebrafish Ctla-4 homolog and investigated its role in maintaining intestinal immune homeostasis by generating a Ctla-4-deficient (ctla-4-/-) zebrafish line. These mutant zebrafish exhibited reduced weight, along with impaired epithelial barrier integrity and lymphocytic infiltration in their intestines. Transcriptomics analysis revealed upregulation of inflammation-related genes, disturbing immune system homeostasis. Moreover, single-cell RNA-sequencing analysis indicated increased Th2 cells and interleukin 13 expression, along with decreased innate lymphoid cells and upregulated proinflammatory cytokines. Additionally, Ctla-4-deficient zebrafish exhibited reduced diversity and an altered composition of the intestinal microbiota. All these phenotypes closely resemble those found in mammalian IBD. Lastly, supplementation with Ctla-4-Ig successfully alleviated intestinal inflammation in these mutants. Altogether, our findings demonstrate the pivotal role of Ctla-4 in maintaining intestinal homeostasis. Additionally, they offer substantial evidence linking CTLA-4 to IBD and establish a novel zebrafish model for investigating both the pathogenesis and potential treatments.

Unraveling the role of Ctla-4 in intestinal immune homeostasis through a novel Zebrafish model of inflammatory bowel disease

Inflammatory bowel disease (IBD) is a chronic and relapsing immune-mediated disorder characterized by intestinal inflammation and epithelial injury. The underlying causes of IBD are not fully understood, but genetic factors have been implicated in genome-wide association studies, including CTLA-4, an essential negative regulator of T cell activation. However, establishing a direct link between CTLA-4 and IBD has been challenging due to the early lethality of CTLA-4 knockout mice. In this study, we identified zebrafish Ctla-4 homolog and investigated its role in maintaining intestinal immune homeostasis by generating a Ctla-4-deficient (ctla-4-/-) zebrafish line. These mutant zebrafish exhibited reduced weight, along with impaired epithelial barrier integrity and lymphocytic infiltration in their intestines. Transcriptomics analysis revealed upregulation of inflammation-related genes, disturbing immune system homeostasis. Moreover, single-cell RNA-sequencing analysis indicated increased Th2 cells and interleukin 13 expression, along with decreased innate lymphoid cells and upregulated proinflammatory cytokines. Additionally, Ctla-4-deficient zebrafish exhibited reduced diversity and an altered composition of the intestinal microbiota. All these phenotypes closely resemble those found in mammalian IBD. Lastly, supplementation with Ctla-4-Ig successfully alleviated intestinal inflammation in these mutants. Altogether, our findings demonstrate the pivotal role of Ctla-4 in maintaining intestinal homeostasis. Additionally, they offer substantial evidence linking CTLA-4 to IBD and establish a novel zebrafish model for investigating both the pathogenesis and potential treatments.

Nicotinamide riboside alleviates heat stress-induced intestinal barrier dysfunction in mice.

Heat stress can damage the intestinal epithelial barrier, leading to harmful substances entering the body. Although oxidative stress and inflammation are implicated in heat-induced intestinal barrier dysfunction, the protective effect of anti-inflammatory and antioxidant agents on the intestinal epithelial barrier against heat insult remains inconsistent. Evidence suggests that nicotinamide adenine nucleotide (NAD+) is a central signaling molecule in the regulation of redox and inflammatory reactions. In this study, we examined the effects of the NAD+ precursor nicotinamide riboside (NR) on heat-induced intestinal epithelial barrier injury. Male C57BL/6 J mice were orally administered vehicle or NR for 10 days and subsequently were subjected to a single heat or sham exposure test. NR significantly increased intestinal NAD+ and NADH levels but did not change the NAD+/NADH ratio in both sham and heat-exposed mice. Heat-exposed mice reduced intestinal NAD+/NADH ratio, caused intestinal barrier impairment and dysfunction, and increased intestinal IL-6, TNFα and TBARS levels. NR reduced these effects of heat exposure. Heat also reduced mtDNA copy number and ATP content in the intestinal tissue, but NR did not impact these changes. Heat stress disrupts intestinal NAD+/NADH homeostasis and NR pretreatment prevents this effect. The protective effect of NR on mouse intestinal barrier against heat is associated with reduced inflammation and oxidative stress. However, NR has no effect on heat-induced intestinal mitochondrial dysfunction.

LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis.

Long non-coding RNA(LncRNA) has been reported to be associated with intestinal barrier damage. The aim of this study was to explore the mechanism of lncRNA Nuclear enriched abundant transcript 1 (NEAT1) in uremic toxin-induced intestinal epithelial barrier injury. Human colon cancer cells (Caco-2) were used to establish intestinal epithelial injury models with the urea treatment in different conditions. Cell Counting Kit-8 (CCK-8) and Western blot screening the best concentration and time. The expressions of lncRNA NEAT1 and miR-122-5p were measured by quantitative real-time polymerase chain reaction (qRT-PCR). Western blot and immunofluorescence were used to detect the expression of tight junction proteins Occludin, ZO-1 and Claudin-1. Sodium fluorescein was used to detect the paracellular permeability of intestinal epithelial injury models. The binding of miR-122-5p to lncRNA NEAT1 and Occludin was determined by bioinformatics analysis and dual luciferase reporter assay. The best condition for the injury model was urea treatment in 144 mg/dl for 48 hours. With the increase of urea intervention time and concentration, the damage degree of intestinal epithelial cells is aggravated. Based on the qRT-PCR results, lncRNA NEAT1 was significantly down-regulated in the model group. Meanwhile, the tight junction proteins Occludin, ZO-1 and Claudin-1 were significantly reduced. The permeability of sodium fluorescein was significantly increased in the model group. Overexpression of lncRNA NEAT1 can alleviate the above performances. As the target gene of lncRNA NEAT1, miR-122-5p is significantly up-regulated in the model group. The dual luciferase reporter assay proved that miR-122-5p was targets to Occludin. The protective effect of overexpression lncRNA NEAT1 on intestinal epithelial barrier function is reversed by miR-122-5p mimics. LncRNA NEAT1 protects uremic toxin-induced intestinal epithelial barrier injury by regulating miR-122-5p/Occludin axis.

NUDT1 aggravates intestinal epithelial barrier injury through oxidative stress in ulcerative colitis.

NUDT1 aggravates intestinal epithelial barrier injury through oxidative stress in ulcerative colitis.

A novel scheme for non-invasive drug delivery with a magnetically controlled drug delivering capsule endoscope.

A novel scheme for non-invasive drug delivery with a magnetically controlled drug delivering capsule endoscope.

Kaempferol ameliorated central nervous injury induced by alcohol uptake through improving intestinal barrier function.

Excessive neuroinflammation resulting from chronic alcohol intake is an important risk factor for central nervous system injury. The aim of this study was to investigate the effect of kaempferol (KAE) on alcohol-induced neural injury and its underlying mechanism. C57BL/6 N mice were employed to develop a binge-on-chronic alcohol exposure model, with different doses of KAE as an interventional drug for 6 weeks. Neuronal damage and microglial activation in the brain, as well as colonic tissue damage and serum lipopolysaccharide (LPS) concentrations, were systematically assessed. Additionally, Caco-2 cells were exposed to alcohol to induce intestinal epithelial injury in vitro. Chronic alcohol exposure let to significant neuronal damage in the cortex and hippocampus of mice. KAE treatment effectively attenuated microglial activation and reduced neuronal damage in the brains of alcohol-exposed mice. Analysis of colonic tissues revealed that KAE administration inhibited miRNA-122a expression, alleviated pathological damage, and enhanced occludin expression, thereby significantly lowing serum LPS concentrations in alcohol-fed mice. In vitro, KAE markedly decreased miRNA-122a expression and enhanced occludin levels in Caco-2 cells treated with alcohol. Furthermore, overexpression of miRNA-122a was found to diminish occludin protein production in Caco-2 cells, which was significantly counteracted by KAE treatment. KAE treatment enhanced intestinal barrier function to alleviate neuronal damage caused by microglial activation mediated by gut-derived LPS under alcohol expose. This effect of KAE was involved in the enhance of intestinal occludin expression by inhibiting the expression of miRNA-122a. This suggested that KAE had the potential to prevent alcohol-induced neurological damage.

Curcumin Alleviates DON-Induced Intestinal Epithelial Barrier Disruption by Improving Ribotoxic Stress-Associated p38 Pathway-Mediated TJ Injury, Apoptosis, and Cell Cycle Arrest.

Deoxynivalenol (DON) is a pervasive ribotoxic stressor that induces intestinal epithelial barrier disruption by impairing tight junctions (TJs) and causing cellular damage. Curcumin (CUR), known for its enteroprotective properties and low toxicity, has been shown to attenuate DON-induced intestinal epithelial barrier injury. However, the underlying mechanisms are still unclear. In this study, we established in vivo and in vitro models using 30 male Kunming mice and IPEC-J2 cells to investigate the mechanisms by which CUR alleviates DON-induced intestinal epithelial barrier injury. The results showed that CUR markedly reduced DON-induced increases in intestinal permeability by restoring TJ protein expression (Claudin-4 and occludin) and preventing fiber-shaped actin (F-actin) contraction. CUR also attenuated DON-induced apoptosis by downregulating p53 and caspase activation and alleviated the G1 cell cycle arrest by reducing p21 expression. Mechanistically, CUR inhibited the activation of the ribosomal stress response (RSR)-associated p38 pathway, evidenced by decreased phosphorylation of p38, GSK3β, and ATF-2. The p38 activator dehydrocorydaline reversed CUR's protective effects. In conclusion, CUR alleviates DON-induced intestinal epithelial barrier disruption by improving RSR-associated p38 pathway-mediated TJ injury, apoptosis, and cell cycle arrest. These findings highlight the potential of CUR as a therapeutic agent for mitigating mycotoxin-induced intestinal dysfunction and suggest new avenues for drug target discovery.

Intestinal epithelial injury and inflammation after physical work in temperate and hot environments in older men with hypertension or type 2 diabetes.

We tested whether older adults with well-controlled type 2 diabetes or hypertension, compared with age-matched adults without chronic disease, exhibit greater intestinal damage, microbial translocation and inflammation during exertional heat stress. Twelve healthy men (age 59 years, SD 4 years), nine with type 2 diabetes (age 60 years, SD 5 years) and nine with hypertension (age 60 years, SD 4 years) walked for 180min at 200W/m2 in temperate conditions (wet-bulb globe temperature 16°C) and high-heat stress conditions (wet-bulb globe temperature 32°C). Serum intestinal fatty acid binding protein (IFABP), plasma soluble cluster of differentiation 14, lipopolysaccharide-binding protein (LBP), interleukin-6 and tumour necrosis factor-alpha were measured pre- and postexercise and after 60min recovery. Total exercise duration was lower in men with hypertension and diabetes (p≤0.049), but core temperature did not differ. All markers increased more in heat versus temperate conditions (p<0.002). In the heat, individuals with type 2 diabetes had greater postexercise increases in IFABP [+545pg/mL (95% confidence interval: 222, 869)] and LBP [+3.64µg/mL (1.73, 5.56)] relative to healthy control subjects (p<0.048), but these resolved after recovery. Despite reduced exercise duration, hypertensive individuals showed similar increases in IFABP and LBP to control subjects. Our findings suggest that older workers with well-controlled type 2 diabetes or hypertension might have greater vulnerability to heat-induced gastrointestinal barrier disturbance and downstream inflammatory responses when compared with otherwise healthy, age-matched adults during prolonged exercise in the heat.

A195 MALNUTRITION IMPAIRS WOUND HEALING DURING DSS COLITIS AND AN IN-VITRO EPITHELIAL MIGRATION ASSAY

Abstract Background Malnutrition is a major public health burden. Caused by insufficient intake of food/nutrients, malnutrition often stems from poverty and lack of access to food. Malnutrition is also a common complication of many gastrointestinal diseases, including inflammatory bowel disease (IBD). Reportedly, more than half of all IBD patients suffer from micronutrient or macronutrient malnutrition, resulting from malabsorption, low nutrient intake, and excessive bleeding or diarrhea. Nutritional deficiencies can alter the course of IBD, prolong hospitalizations, alter treatment response, and delay wound healing. Notably, epithelial wound healing is critical in IBD, but efficient re-epithelization rely on nutrients such as zinc and vitamin A. I thus hypothesize that malnutrition will impair intestinal epithelial wound repair, in vivo and in vitro. Aims We tested how malnutrition affects wound healing following epithelial damage caused by dextran sulphate sodium (DSS)-induced colitis and in a 2D monolayer gap closure assay. Methods We used our previously published mouse model of multiple micronutrient deficiencies (MND) and induced colitis using dextran sodium sulfate (DSS). Weanling three-week-old male C57BL/6N mice were fed a diet deficient in (zinc, vitamin A, B12, folate and iron) or a control diet for 4 weeks. At 3 weeks, we induced colitis by administering 2.5% DSS in drinking water for 4 days, plus 3 days of water recovery. We investigated epithelial damage, wound repair and molecular responses through histological and gene expression profiling. We also generated 3D colonoids from non-colitic malnourished mice, converted them to a 2D monolayer and subjected them to a wound migration assay cultured using an established malnourished media containing standard nutrients, and 50% of normal levels. Results We found that while DSS induced tissue damage both in our control and MND mice, those on the MND diet exhibited impaired tissue repair with decreased re-epithelization over the wounded area compared to controls. Organoids cultured in the 50% malnourished media showed diminished growth, being smaller and less dense compared to standard media. Notably, epithelial migration towards gap closure in the 2D monolayer was delayed by 75% compared to standard media. Conclusions Our work demonstrates that malnutrition impairs intestinal wound repair mechanisms. We also demonstrate an effective organoid model for examining the effects of malnutrition in vitro. We propose that efforts to address underlying malnutrition may improve wound healing leading to better outcomes for patients with IBD. Funding Agencies CIHRTRIANGLE/Crohn’s and Colitis Canada, BC Children’s Hospital Research Foundation

Intermittent Fasting Reduces Intestinal Inflammation in Dextran Sulfate Sodium-Induced Colitis of Mice.

Inflammatory bowel disease (IBD), comprising ulcerative colitis (UC) and Crohn's disease (CD), is a chronic condition impacting both the gastrointestinal tract and the immune system. Intestinal inflammation and epithelial injury are the pathological features of IBD. Recent studies have reported that some strategies of dietary restriction (DR) can regulate immune system, correct the immune disorders, and improve some immune-associated diseases such as IBD. However, as a form of DR, the effect of intermittent fasting (IF) on the IBD remains unknown. In this study, we investigated the therapeutic efficacy of two cycles of IF on the IBD mouse model induced by dextran sulfate sodium (DSS). It was found that two cycles of IF significantly decreased the score of the disease activity index (DAI) and alleviated the IBD-related symptoms. In addition, IF reversed the shortening of colon length mediated by DSS, significantly increased the number of colonic crypts, and decreased the colonic histological score. Furthermore, the proportion of CD4+ T cells in both the spleen and mesenteric lymph node was reduced by IF treatment. The expression of serum pro-inflammatory cytokines IL-1β, TNF-α, and IL-6 was restrained by IF intervention. Moreover, IF administration significantly reduced the number of leukocytes and macrophages infiltrating around the crypt base in the colon. In conclusion, these results demonstrated that IF administration can alleviate the symptoms and pathology of IBD in the DSS-induced IBD mouse model by reducing the intestinal inflammation.

Tryptophan ameliorates soybean meal-induced enteritis via remission of oxidative stress, mitophagy hyperactivation, and apoptosis inhibition in hybrid yellow catfish gut (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂)

Tryptophan ameliorates soybean meal-induced enteritis via remission of oxidative stress, mitophagy hyperactivation, and apoptosis inhibition in hybrid yellow catfish gut (Pelteobagrus fulvidraco ♀ × Pelteobagrus vachelli ♂)

Rhamnogalacturonan promotes intestinal mucosal repair through increased cell migration.

Mucosal healing is the primary goal for inflammatory bowel disease (IBD) treatment. We previously showed the direct beneficial effects of rhamnogalacturonan (RGal) on intestinal epithelial barrier function. Here, we aimed to evaluate the effect of RGal in intestinal epithelial wound healing. Confluent cancer cell lines and colonoid monolayers were wounded, treated with RGal for 48 h, and assessed using a live cell imaging system. Proliferation and apoptosis of cells were evaluated using 5-ethynyl-2'-deoxyuridine (EdU) and TUNEL assays, respectively. Inhibitors were used to determine the receptor and signaling pathways involved. Female and male mice with DSS-induced colitis were treated orally with RGal for 7 days during the recovery phase. RGal enhanced wound healing in Caco-2, T84, and primary cells by increasing cell migration. Inhibition of pretranscriptional signaling pathways FAK, Src, PI3K, Rho family, and JNK reversed the RGal-induced wound healing. RNAseq data from Caco-2 and primary cells treated with RGal showed the upregulation of the NF-κB pathway at 12 h. Actinomycin D, Bay 11-7082 or JSH-23, and NS-398 treatment significantly reversed the effect of RGal on wound healing, confirming that the response was also transcriptionally dependent and involved NF-κB signaling and downstream COX-2 protein activity. RGal treatment of male mice enhanced recovery from DSS colitis. RGal promoted wound healing in cancer and primary cells by increasing cell migration and accelerated epithelial mucosal healing in male mice. Our findings show a novel mechanism of action of RGal in wound healing that could help in mucosal healing and the resolution of intestinal inflammation.NEW & NOTEWORTHY RGal increases wound healing in colon cancer cell lines and primary cells through increased cell migration and participation of important pretranscriptional signaling pathways and the transcription factor NF-κB. In addition, RGal also accelerates intestinal mucosal healing of male mice with DSS-induced colitis.

Protective effect of soluble protein hydrolysate against H2O2‑induced intestinal injury: An interventional study.

The present study aimed to investigate whether soluble protein hydrolysate (SPH) protects against intestinal oxidative stress injury. An in vitro lactate dehydrogenase assay was used to assess the cytotoxicity and protective effect of SPH. For in vivo assessment, friend virus B NIH Jackson mouse pups aged 21 days were administered with 5% w/v soluble protein hydrolysate (SPH) through drinking water for 14 days and then luminally injected with 0.3% or 0.6% H2O2. Thereafter, the fecal samples of mice were collected, and the mice were sacrificed. Intestinal epithelial injury was assessed, and the expressions of 84 oxidative stress‑related genes in intestinal tissues was determined. SPH prophylactically protected against H2O2‑induced oxidative stress injury in human intestinal epithelial cells. An animal model of oxidative stress‑induced intestinal injury was established using 0.3 and 0.6% H2O2. SPH treatment reduced oxidative stress (0.3% H2O2)‑induced gut injury in mice. As no accelerated body growth was observed in SPH‑treated mice, it was hypothesized that the underlying protective mechanism of SPH is not related to nutrient oversupply. Treatment with SPH upregulated five oxidative protective genes that were not consistent between the sexes. Some antioxidative genes, including ferritin heavy polypeptide‑1 (Fth1), heme oxygenase‑1 (Hmox1), NAD(P)H dehydrogenase quinone 1 (Nqo1) and superoxide dismutase 1 (Sod1), were commonly upregulated in both male and female mice. Overall, an antioxidative protective effect was observed following SPH treatment, which may be attributed to the upregulation of genes that protect against oxidative damage. The findings of the present study highlight the promising potential of SPH as a functional food for alleviating intestinal oxidative stress injury.

Gegen-Sangshen oral liquid and its active fractions mitigate alcoholic liver disease in mice through repairing intestinal epithelial injury and regulating gut microbiota

Abstract Background Liuweizhiji Gegen-Sangshen oral liquid (LGS), as a Chinese medicinal preparation, is developed from a Traditional Chinese medicinal formula consisting of six Chinese medicinal herbs, including Puerariae lobatae radix, Hoveniae semen, Imperatae rhizoma, Crataegi fructus, Mori fructus and Canarli fructus, and has been extensively utilized in the prevention and treatment of alcoholic liver disease (ALD) clinically. Previous study has demonstrated that LGS dose-dependently mitigated ALD in rat models. However, whether and how the main characteristic constituents of LGS (the flavonoid and polysaccharide fractions, LGSF and LGSP) contribute to the anti-ALD effect remains unclear. This study aimed to assess the anti-ALD effect of LGS and its main fractions (LGSF and LGSP) in a murine model of ALD and to explore the underlying mechanisms. Methods ALD mouse model was constructed using the chronic and binge ethanol feeding method. Biochemical determinations of AST, ALT, TC, TG, ADH, ALDH, HDL, LDL, IL-1β, IL-6, and TNF-α were performed using corresponding kits. Histopathological examination of liver and intestinal sections was conducted based on the H&amp;E staining. Lipid accumulation in hepatocytes was evaluated by oil red O staining. Ethanol metabolism was assessed by determining the activity of ADH and ALDH enzymes. Intestinal barrier function was analyzed based on immunohistochemistry analysis of ZO-1 and occludin and immunofluorescence analysis of epithelial markers, Lgr5, Muc2, and Lyz1. Intestinal epithelial apoptosis was detected by TUNEL staining. Mouse fecal microbiota alterations were analyzed by 16S rRNA sequencing. An in vitro epithelial injury model was established by developing TNF-α-induced 3D-cultured intestinal organoids. In vitro culture of specific bacterial strains was performed. Results The results showed that LGS and its flavonoid and polysaccharide fractions (LGSF and LGSP) significantly alleviated ALD in mice through attenuating hepatic injury and inflammation, improving liver steatosis and promoting ethanol metabolism. Notably, LGS, LGSP, and LGSF mitigated intestinal damage and maintained barrier function in ALD mice. The intestinal barrier protection function of LGS, LGSP, and LGSF was generally more obvious than that of the positive drug meltadosine. Further study demonstrated that LGS, LGSP, and LGSF promoted intestinal epithelial repair via promoting Lgr5+ stem cell mediated regeneration in TNF-α-induced intestinal organoids. LGS and LGSF, other than LGSP, had a better effect on repair of epithelial injury in vitro. Moreover, LGS, LGSP, and LGSF remarkably alleviated gut dysbiosis in ALD mice via at least partially recovery of alcohol-induced microbial changes and induction of specific bacterial groups. In vitro culture of bacterial strains indicated that LGS, LGSP, and LGSF had a specific impact on bacterial growth. LGS and LGSP, but not the LGSF, significantly promoted the growth of Lactobacillus. Similarly, LGS and LGSP significantly increased the proliferation of Bacteroides sartorii, and LGSF had a minimal effect. LGS, LGSP and LGSF all promoted the growth of Bacillus coagulans, Bifidobacterium adolescentis, and Bifidobacterium bifidum. LGS and LGSP promoted the growth of Dubosiella newyorkensis, but the LGSF had no effect. Conclusions LGS exerts its anti-ALD effect in mice through regulating gut-liver axis, and its flavonoid and polysaccharide fractions, LGSF and LGSP, are responsible for its protective effect.

In Vitro Protective Effects of a Standardized Extract of Opuntia ficus-indica (L.) Mill. Cladodes and Olea europaea L. Leaves Against Indomethacin-Induced Intestinal Epithelial Cell Injury.

Nonsteroidal anti-inflammatory drugs (NSAIDs) can induce serious adverse effects in gastrointestinal (GI) mucosa, increasing intestinal permeability and leading to mitochondrial dysfunction, oxidative stress, apoptosis and inflammation. As proton pump inhibitors are effective in protecting against NSAID-induced gastropathy but not NSAID-induced enteropathy, current research is focused on natural products as protective substances for therapy and prevention of intestinal injury. Herein, through the use of an in vitro model based on intestinal epithelial cell (Caco-2) damage caused by indomethacin (INDO), we examined the protective activity of a commercially available standardized extract (OFI+OE) from Opuntia ficus-indica (L.) Mill. cladodes and Olea europaea L. leaves. Pre-treatment with OFI+OE prevented INDO-induced intestinal epithelial barrier damage, as demonstrated by TEER measurement, fluorescein permeability, and tight junction protein expression. The extract showed positive effects against INDO-induced oxidative stress and correlated activation of apoptosis, decreasing pro-apoptotic markers BAX and Caspase-3 and increasing anti-apoptotic factor Bcl-2. Moreover, the extract inhibited the NF-κB pathway and pro-inflammatory cascade. In conclusion, these data support the use of OFI+OE extract as a natural strategy for therapy and prevention of intestinal mucosal damage, demonstrating its beneficial effects against INDO-induced intestinal damage, through modulation of oxidative, apoptotic, and inflammatory pathways.

Elucidating novel mechanism of action of spiperone for drug repurposing to prevent and treat murine colitis and sepsis

Elucidating novel mechanism of action of spiperone for drug repurposing to prevent and treat murine colitis and sepsis

Pig Milk Exosome Packaging ssc-miR-22-3p Alleviates Pig Intestinal Epithelial Cell Injury and Inflammatory Response by Targeting MAPK14

Inflammatory diseases of the intestinal tract in piglets severely impair the economic performance of pig farms. Pig milk exosomes can encapsulate miRNAs which can then enter the piglet intestine to play an immunomodulatory role. Previously, we comparatively analyzed and identified exosomal miRNAs in the colostrum and mature milk of Bamei and Landrace pigs, and we screened for ssc-miR-22-3p, which is associated with inflammation and immune response; however, the role played by ssc-miR-22-3p in the immune response in IPEC-J2 cells is not yet clear. In this study, we first constructed a pig intestinal inflammatory response model using Lipopolysaccharide (LPS) and Polyinosinic-polycytidylic acid (Poly (I:C)), and we investigated the role of ssc-miR-22-3p targeting MAPK14 in the regulation of LPS and Poly (I:C)-induced inflammatory injury in IPEC-J2 cells by RT-qPCR, cell counting kit-8 (CCK-8), EdU staining, lactate dehydrogenase (LDH) activity assay, and dual luciferase reporter gene assay. We successfully established LPS and Poly (I:C)-induced cell damage models in IPEC-J2 cells. The immune response of IPEC-J2 cells was stimulated by induction of IPEC-J2 cells at 10 μg/mL LPS and 20 μg/mL Poly (I:C) for 24 h. Overexpression of ssc-miR-22-3p decreased cytokine expression and promoted cell viability and proliferation. The functional enrichment analysis revealed that ssc-miR-22-3p targets genes enriched in the pathways of negative regulation of inflammatory response and bacterial invasion of epithelial cells. The validity of the binding site of ssc-miR-22-3p to MAPK14 was tested by a dual luciferase reporter gene. Pig milk exosome ssc-miR-22-3p promotes cell viability and proliferation by targeting MAPK14, and it alleviates LPS and Poly (I:C)-induced inflammatory responses in IPEC-J2 cells.