Intestinal Autophagy Research Articles

MiR-34a/Notch1b mediated autophagy and apoptosis contributes to oxidative stress amelioration by emodin in the intestine of teleost Megalobrama amblycephala

Stress stimulated reactive oxygen species (ROS) excessive accumulation is the principal cause of oxidative damage in aquatic animals. Severe oxidative stress could induce cell fate dysfunction, affect physiologic derangement, growth, and even survival. This study aims to investigate the ameliorative function of emodin in resist to oxidative stress via histomorphology, transcriptome, miRNAs, and mRNA-miRNA integrate analysis in the intestine of Megalobrama amblycephala. Morphologically, emodin alleviated oxidative stress induced autophagy in intestine. Differentially expressed miRNAs induced by oxidative stress and emodin were mainly involved in energy metabolism, oxidative homeostasis, cell apoptosis and cancerization signaling. Specifically, target prediction of miRNAs reveals Notch and apoptosis signaling were dysregulated. mRNA-miRNA integrated analysis reveals miR-34a and Notch1b were actively co-related in the regulatory network, and miR-34a was solidly confirmed as the negative regulator of Notch1b by targeting to the 3’-UTR. Transcriptional expression of key genes reveal emodin alleviates intestinal oxidative stress by activating miR-34a, and subsequently inhibit the expression of Notch receptors (DllA, Jag1b, and Jag2) and down-stream regulators (Hes1, Hey1, and FBW7). Meanwhile, apoptosis-related gene expression (Bax, Casp3, Casp8, Casp9, AIF1, AIF3, CytoC, and CDK4) were activated by oxidative stress, while were alleviated to the control level when treated with emodin. In conclusion, these results suggest that miR-34a/Notch1b is the potential therapeutic target for emodin to ameliorate oxidative stress in M. amblycephala.

Impact of Different Durations of Fasting on Intestinal Autophagy and Serum Metabolome in Broiler Chicken.

Fasting-induced autophagy in the intestine is beneficial for body health. This study was designed to explore the relationship between the host metabolism and intestinal autophagy. Broilers were randomly assigned into 48 cages. At 0 (CT), 12 (FH12), 24 (FH24), 36 (FH36), 48(FH48), and 72 h (FH72) before 09:00 a.m. on day 25, eight cages of birds were randomly allotted to each fasting time point using completely random design, and their food was removed. At 09:00 a.m. on day 25, the blood and jejunum were sampled for serum metabolome and autophagy gene analyses, respectively. The results showed that the autophagy gene Atg7 has a good quadratic fit with fasting duration (R2 = 0.432, p < 0.001). Serum phosphatidylethanolamine (PE) and lyso-PE were decreased in the birds that were fasted for 24 h or longer. Conversely, the serum phosphatidylcholine (PC) and lyso-PC were increased in the birds that were fasted for 36 h or longer. Metabolism pathway analysis showed that the serum glycerophospholipid, phenylalanine, and GnRH signaling pathways were downregulated with the extended fasting duration. The serum metabolites involved in glycosylphosphatidylinositol anchor biosynthesis, autophagy, and ferroptosis were upregulated in all of the fasted groups. Correlation analysis showed that serum PE (18:3(9Z,12Z,15Z)/P-18:0) was a potential biomarker for intestinal autophagy. Our findings provide a potential biomarker related to intestinal autophagy.

Characterizing Autophagy in the Cold Ischemic Injury of Small Bowel Grafts: Evidence from Rat Jejunum.

Cold ischemic injury to the intestine during preservation remains an unresolved issue in transplantation medicine. Autophagy, a cytoplasmic protein degradation pathway, is essential for metabolic adaptation to starvation, hypoxia, and ischemia. It has been implicated in the cold ischemia (CI) of other transplantable organs. This study determines the changes in intestinal autophagy evoked by cold storage and explores the effects of autophagy on ischemic grafts. Cold preservation was simulated by placing the small intestines of Wistar rats in an IGL-1 (Institute George Lopez) solution at 4 °C for varying periods (3, 6, 9, and 12 h). The extent of graft preservation injury (mucosal and cellular injury) and changes in autophagy were measured after each CI time. Subsequently, we determined the differences in apoptosis and preservation injury after activating autophagy with rapamycin or inhibiting it with 3-methyladenine. The results revealed that ischemic injury and autophagy were induced by cold storage. Autophagy peaked at 3 h and subsequently declined. After 12 h of storage, autophagic expression was reduced significantly. Additionally, enhanced intestinal autophagy by rapamycin was associated with less tissue, cellular, and apoptotic damage during and after the 12-h long preservation. After reperfusion, grafts with enhanced autophagy still presented with less injury. Inhibiting autophagy exhibited the opposite trend. These findings demonstrate intestinal autophagy changes in cold preservation. Furthermore, enhanced autophagy was protective against cold ischemia–reperfusion damage of the small bowels.

A TORC1-histone axis regulates chromatin organisation and non-canonical induction of autophagy to ameliorate ageing.

Age-related changes to histone levels are seen in many species. However, it is unclear whether changes to histone expression could be exploited to ameliorate the effects of ageing in multicellular organisms. Here we show that inhibition of mTORC1 by the lifespan-extending drug rapamycin increases expression of histones H3 and H4 post-transcriptionally through eIF3-mediated translation. Elevated expression of H3/H4 in intestinal enterocytes in Drosophila alters chromatin organisation, induces intestinal autophagy through transcriptional regulation, and prevents age-related decline in the intestine. Importantly, it also mediates rapamycin-induced longevity and intestinal health. Histones H3/H4 regulate expression of an autophagy cargo adaptor Bchs (WDFY3 in mammals), increased expression of which in enterocytes mediates increased H3/H4-dependent healthy longevity. In mice, rapamycin treatment increases expression of histone proteins and Wdfy3 transcription, and alters chromatin organisation in the small intestine, suggesting that the mTORC1-histone axis is at least partially conserved in mammals and may offer new targets for anti-ageing interventions.

N-Acetylcysteine improves intestinal function and attenuates intestinal autophagy in piglets challenged with \u03b2-conglycinin

β-Conglycinin (β-CG), an anti-nutritional factor, is a major allergen in soybeans to induce intestinal dysfunction and diarrhea in neonatal animals, including piglets and human infants. This study with a piglet model determined the effects of N-acetylcysteine (NAC) on intestinal function and autophagy in response to β-CG challenge. Twenty-four 12-day-old piglets (3.44 ± 0.28 kg), which had been weaned at 7 days of age and adapted for 5 days after weaning, were randomly allocated to the control, β-CG, and β-CG + NAC groups. Piglets in the control group were fed a liquid diet containing 10% casein, whereas those in the β-CG and β-CG + NAC groups were fed the basal liquid diets containing 9.5% casein and 0.5% β-CG for 2 days. Thereafter, pigs in the β-CG + NAC group were orally administrated with 50 mg (kg BW)−1 NAC for 3 days, while pigs in the other two groups were orally administrated with the same volume of sterile saline. NAC numerically reduced diarrhea incidence (− 46.2%) and the concentrations of hydrogen peroxide and malondialdehyde, but increased claudin-1 and intestinal fatty-acid binding protein (iFABP) protein abundances and activities of catalase and glutathione peroxidase in the jejunum of β-CG-challenged piglets. Although β-CG challenge decreased the villus height, villus height/crypt depth ratio, and mRNA levels of claudin-1 and occludin, no significant differences were observed in these indices between the control and β-CG + NAC groups, suggesting the positive effects of NAC supplementation on intestinal mucosal barrier function. Moreover, NAC increased the concentrations of citrulline and D-xylose in the plasma, as well as the expression of genes for aquaporin (AQP) 3, AQP4, peptide transporter 1 (PepT1), sodium/glucose co-transporter-1 (SGLT-1), potassium inwardly-rectifying channel, subfamily J, member 13 (KCNJ13), and solute carrier family 1 member 1 (SLC1A1) in the jejunum, demonstrating that NAC augmented intestinal metabolic activity and absorptive function. Remarkably, NAC decreased Atg5 protein abundance and the LC3II/LC3I ratio (an indicator of autophagy) in the jejunum of β-CG-challenged piglets. Taken together, NAC supplementation improved intestinal function and attenuated intestinal autophagy in β-CG-challenged piglets.

Escalating dose-multiple binge methamphetamine treatment elicits neurotoxicity, altering gut microbiota and fecal metabolites in mice

Methamphetamine (METH) is an addictive and illegal psychostimulant drug that can cause multiple organ dysfunction, especially in the central nervous system (CNS). Gut microbiota have been implicated in development of various CNS-related diseases, via the gut-brain axis (GBA). However, effect of METH in the alteration of gut microbiota and fecal metabolites is unclear, whereas the relationship with METH-induced neurotoxicity remains unknown. In the current study, we investigated effect of METH on neurotoxicity in striatum and colonic damage by exposing BALB/c mice to an escalating dose-multiple binge regimen, and then analyzed protein expression using Western blot analysis. We further detected and sequenced the 16 S rRNA gene in fecal samples, and performed ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS)-based metabolomics to analyze gut microbes and fecal metabolites. Exposure to METH significantly downregulated tyrosine hydroxylase (TH) proteins, but upregulated MAOA, Beclin1, Atg5, and LC3-Ⅱ. METH up-regulated inflammation-related factors, such as caspase1, TNF-α and IL-18, by activating the toll-like receptors 4 (TLR4)/myeloid differentiation factor 88 (Myd88)/nuclear factor κB (NF-κB) pathway and reduced occludin protein expression. In addition, METH exposure changed α and β diversities of gut microbiota. Specifically, METH exposure elevated relative abundances of pathogenic bacteria, but reduced those of probiotics. Metabolomics, combined with enrichment analyses revealed that METH exposure altered fecal metabolites. Our findings suggest that METH exposure induced autophagy in the CNS, elevated intestinal autophagy flora, leading to accumulation of fecal metabolites in the autophagy pathway, and causing enteritis. Moreover, METH promoted intestinal inflammation by increasing the relative abundance of the pathogenic bacteria in the intestinal tract, and reduced intestinal TJ protein expression.

Chloroquine Improves Deoxynivalenol-Induced Inflammatory Response and Intestinal Mucosal Damage in Piglets.

We investigated the effects of rapamycin (RAPA) and chloroquine (CQ) in supporting growth performance and the intestinal mucosal barrier in response to deoxynivalenol (DON) in piglets. A total of 32 healthy weaned piglets (bodyweight 7.10 ± 0.58 kg) were divided into four groups and treated daily with RAPA (1 mg/kg BW), CQ (10 mg/kg BW), or a control volume of normal saline (two groups) until the end of the experiment. After feeding a basal diet for seven days, three groups were then switched to mildewed feed containing 1 mg kg/DON for a further seven days. In contrast to the control group, DON-treated piglets showed decreased average daily gain (ADG) and daily feed intake (ADFI), as well as negatively affected intestinal morphology as indicated by villus height, crypt depth, and tight junction protein expression. A group treated with RAPA and DON showed increased intestinal autophagy, aggravated inflammatory responses, and damage to the intestinal mucosa and permeability, leading to reduced growth performance. Meanwhile, a group treated with CQ and DON showed indices comparable to the non-DON control group, with alleviated inflammatory cytokines and healthy intestinal morphology and structure. They also showed better growth performance compared to DON treatment alone. These findings have important implications for mediating autophagy against DON in vivo, as well as the potential for CQ in improving growth performance and maintaining intestinal barrier integrity in weanling piglets.

Epidermal growth factor regulation by autophagy-mediated lncRNA H19 in murine intestinal tract after severe burn.

To investigate the regulation of epidermal growth factor (EGF) by autophagy‐mediated long non‐coding RNA (lncRNA) H19 in the intestinal tracts of severely burned mice. C57BL/6J mice received third‐degree burns to 30% of the total body surface area. Rapamycin and 3‐methyladenine (3‐MA) were used to activate and inhibit autophagy, and the changes in LC3 and Beclin1 levels were assessed by Western blotting. The effect of autophagy on lncRNA H19 was detected by qRT‐PCR. Adenovirus‐mediated overexpression of lncRNA H19 in IEC‐6 cells was used to assess the effects of lncRNA H19 on EGF and let‐7g via bioinformatics analysis, Western blotting and qRT‐PCR. let‐7g mimic/inhibitor was used to overexpress/inhibit let‐7g, and qRT‐PCR and Western blotting were used to detect the effects of let‐7g on EGF. The expression levels of LC3‐II, Beclin1 and lncRNA H19 were increased in intestinal tissues and IEC‐6 cells after rapamycin treatment but were reversed after 3‐MA treatment. LC3‐II, Beclin1 and lncRNA H19 levels increased in intestinal tissues after the burn, and these increases were more significant after rapamycin treatment but decreased after 3‐MA treatment. The lncRNA H19 overexpression in IEC‐6 cells resulted in increased and decreased expression levels of EGF and let‐7g, respectively. Furthermore, overexpression and inhibition of let‐7g resulted in decreased and increased expression of EGF, respectively. Taken together, intestinal autophagy is activated after a serious burn, which can increase the transcription level of lncRNA H19. lncRNA H19 may regulate the repair of EGF via let‐7g following intestinal mucosa injury after a burn.

Probiotic Bacillus Attenuates Oxidative Stress- Induced Intestinal Injury via p38-Mediated Autophagy.

Probiotics have been widely used in maintaining intestinal health and one of their benefits is to enhance host antioxidant capacity. However, the involved molecular mechanisms require further investigated. Autophagy is a self-protection process in response to diverse stresses. We hypothesized that probiotics could modulate intestinal autophagy to alleviate oxidative stress. Sprague-Dawley (SD) rats were orally administered Bacillus SC06 or SC08 daily for 24 days and thereafter received an intraperitoneal injection of diquat (DQ) to induce oxidative stress. We found that rats administered Bacillus SC06 showed more significant intestinal tissue repair and antioxidant properties than those administered SC08, which suggests a strain-specific effect of probiotics. Moreover, SC06 alleviated apoptosis by regulating the expression of Bcl2, Bax and cleaved caspase-3. Further investigations revealed that SC06 triggered autophagy, indicated by the upregulation of LC3 and Beclin1 and the degradation of p62 in rat jejunum and IEC-6 cells. Preincubation with autophagy inhibitor 3-methyladenine (3-MA) significantly aggravated reactive oxygen species (ROS) production and apoptotic cell formation. Furthermore, we demonstrated that p38 MAPK (mitogen-activated protein kinase), not AKT (alpha serine/threonine kinase)/mTOR (mammalian target of rapamycin), was involved in SC06-induced autophagy. Taken together, Bacillus SC06 can alleviate oxidative stress-induced disorders and apoptosis via p38-mediated autophagy. The above findings highlight a novel mechanism underlying the beneficial effects of probiotics as functional food and provide a new perspective on the prevention and treatment of oxidative damages.

Zebrafish modeling of intestinal injury, bacterial exposures and medications defines epithelial in vivo responses relevant to human inflammatory bowel disease.

ABSTRACTGenome-wide association studies have identified over 200 genomic loci associated with inflammatory bowel disease (IBD). High-effect risk alleles define key roles for genes involved in bacterial response and innate defense. More high-throughput in vivo systems are required to rapidly evaluate therapeutic agents. We visualize, in zebrafish, the effects on epithelial barrier function and intestinal autophagy of one-course and repetitive injury. Repetitive injury induces increased mortality, impaired recovery of intestinal barrier function, failure to contain bacteria within the intestine and impaired autophagy. Prostaglandin E2 (PGE2) administration protected against injury by enhancing epithelial barrier function and limiting systemic infection. Effects of IBD therapeutic agents were defined: mesalamine showed protective features during injury, whereas 6-mercaptopurine displayed marked induction of autophagy during recovery. Given the highly conserved nature of innate defense in zebrafish, it represents an ideal model system with which to test established and new IBD therapies targeted to the epithelial barrier.This article has an associated First Person interview with the first author of the paper.

Intestinal autophagy links psychosocial stress with gut microbiota to promote inflammatory bowel disease

Psychosocial stress is a critical inducing factor of inflammatory bowel diseases (IBD), while autophagy is a novel central issue of IBD development. The present study investigated the potential role of autophagy in stress-related IBD in patients and animal model. The correlation between psychosocial stress and intestinal autophagy was determined in 23 patients with IBD. Corticotropin-releasing hormone (CRH), a well-established inducer of psychosocial stress, was administrated in dextran sulfate sodium (DSS)-induced IBD mice and lipopolysaccharide (LPS)-stimulated bone marrow-derived macrophages (BMDM). In IBD patients, the autophagy markers beclin-1, LC3-II/I ratio, Atg16L1, and Atg4B were significantly enhanced. The psychosocial stress score was positively associated with the levels of beclin-1 and the LC3II/I ratio in intestinal biopsy specimens. In IBD mouse model, CRH significantly aggravated intestinal inflammation, increased Paneth cell metaplasia, and enhanced intestinal autophagy (beclin-1, Atg16L1, PIK3R4, and Atg4B upregulation; GAA, CTSD, and PPKAA1 downregulation). Additionally, the CRH-induced gut microbial dysbiosis was evidenced by a marked increase in the number of detrimental bacteria. In LPS-stimulated BMDM, CRH substantially increased M1/M2 polarization and thus promoted inflammation. In both IBD mice and LPS-treated BMDM, blockade of autophagy by chloroquine abrogated the unbeneficial effects of CRH, whereas autophagy inducer rapamycin resulted in a pronounced protective effect against IBD lesion. Our data demonstrate that psychosocial stress may link the enhanced intestinal autophagy by modulating gut microbiota and inflammation to aggravate IBD. These data indicate autophagy as a promising therapeutic target for psychosocial stress-related IBD.

Deciphering the ionic homeostasis, oxidative stress, apoptosis, and autophagy in chicken intestine under copper(II) stress.

As cofactors of several enzymatic, copper (Cu) participates in many essential metabolic processes. Also, as a heavy metal, it exhibits highly toxic to the organism if excessive. This study endeavored to detect the pathophysiological changes in the jejunum of chickens, which were insulted by CuSO4 (300mg/kg diet) for 90days. Results showed metabolic disorders of trace elements evidenced by their significant downregulations (Na, Al, Li, B, Cr, Ni, Sn, Sb, Ba) and upregulations (Cu, Si, As, Cd, Se, and Tl) in 90days. Simultaneously, increased TdT-mediated dUTP nick end labeling (TUNEL)-positive nuclei and distinct ultrastructural apoptotic features were observed. Meanwhile, in 30, 60, and 90days, indicators of oxidative stress, apoptosis, autophagy, and mitochondrial dynamic were detected to uncover the molecular mechanism behind these pathological changes. The results showed that suppressed antioxidant ability was companied by increased mRNA and protein levels of proapoptosis and mitochondrial fission activating genes in the Cu group compared with chickens in the control group (P < 0.05). Moreover, the markers of autophagy long-chain 3 (LC3-II/LC3-I), Bcl-2-interacting protein (beclin-1), and autophagy-related gene (ATG4B and ATG5) displayed a time-dependent increase during 30, 60, and 90days. We conjectured that subchronic copper poisoning, under the background of redistribution of trace elements, induced oxidative stress and cascaded apoptosis, autophagy, and mitochondrial disorder, which contributed to jejunotoxicity in chicken. Collectively, our study provides a basic assessment of subchronic Cu exposure on poultry, voicing concerns about copper pollution by anthropogenic activities.

C. elegans Eats Its Own Intestine to Make Yolk Leading to Multiple Senescent Pathologies

SummaryAging (senescence) is characterized by the development of numerous pathologies, some of which limit lifespan. Key to understanding aging is discovery of the mechanisms (etiologies) that cause senescent pathology. In C. elegans, a major senescent pathology of unknown etiology is atrophy of its principal metabolic organ, the intestine. Here we identify a cause of not only this pathology but also of yolky lipid accumulation and redistribution (a form of senescent obesity): autophagy-mediated conversion of intestinal biomass into yolk. Inhibiting intestinal autophagy or vitellogenesis rescues both visceral pathologies and can also extend lifespan. This defines a disease syndrome leading to multimorbidity and contributing to late-life mortality. Activation of gut-to-yolk biomass conversion by insulin/IGF-1 signaling (IIS) promotes reproduction and senescence. This illustrates how major, IIS-promoted senescent pathologies in C. elegans can originate not from damage accumulation but from direct effects of futile, continued action of a wild-type biological program (vitellogenesis).

&lt;i&gt;C. Elegans&lt;/i&gt; Eats Its Own Intestine to Make Yolk: A Cause of Senescent Polymorbidity

Aging (senescence) is characterized by the development of numerous pathologies, some of which limit lifespan. Key to understanding aging is discovery of the mechanisms (etiologies) that cause senescent pathology. In Caenorhabditis elegans a major senescent pathology of unknown etiology is atrophy of its principal metabolic organ, the intestine. Here we identify a cause of not only this pathology, but also of yolky lipid accumulation and redistribution (a form of senescent obesity): autophagymediated conversion of intestinal biomass into yolk. Inhibiting intestinal autophagy or vitellogenesis rescues both visceral pathologies, and can also extend lifespan. This defines a disease syndrome leading to polymorbidity and contributing to late-life mortality. Activation of gut-to-yolk biomass conversion by insulin/IGF-1 signaling (IIS) promotes reproduction and senescence. This illustrates how major, IIS-promoted senescent pathologies in C. elegans can originate not from damage accumulation, but from continued action of a wild-type function (vitellogenesis), consistent with the recently proposed hyperfunction theory of aging.

Role of autophagy and its molecular mechanisms in mice intestinal tract after severe burn.

Severe burn can lead to hypoxia/ischemia of intestinal mucosa. Autophagy is the process of intracellular degradation, which is essential for cell survival under stresses, such as hypoxia/ischemia and nutrient deprivation. The present study was designed to investigate whether there were changes in intestinal autophagy after severe burn in mice and further to explore the effect and molecular mechanisms of autophagy on intestinal injury. This study includes three experiments. Kunming species mice were subjected to 30% total body surface area third-degree burn. First, we determined protein of LC3 (light chain 3), beclin-1, and cleaved-caspase3 by Western blotting and immunohistochemical (paraffin) staining to investigate whether there were changes in intestinal autophagy after severe burn in mice. Then, changes of the status of enteric damage postburn were measured by observing intestinal mucosa morphology under a magnifier, hematoxylin and eosin staining, enzyme-linked immunosorbent assay, Western blotting under the condition that the intestinal autophagy was respectively activated by rapamycin and inhibited by 3-methyladenine. Finally, protein of the AMP-activated protein kinase (AMPK)/mammalian target of rapamycin (mTOR) pathway, LC3-II and beclin-1 were assayed, and mice were treated with compound C before burn. The protein of LC3 and beclin-1 were observed at 1 hour postburn and increased to peak-point at 24 hours, reaching the normal level at 96 hours. The cleaved caspase-3 expression increased at 1 hour postburn, but the peak point occurred at 12 hours and had dropped to normal level at 72 hours. In addition, rapamycin enhanced intestinal autophagy and alleviated burn-induced gut damage, while 3-methyladenine showed the against behavior. The AMPK/mTOR pathway which was inhibited decreased the expression of phosphorylated AMPK, LC3-II, and beclin-1, increasing the expression of phosphorylated mTOR. Intestinal autophagy is activated and response to intestinal apoptosis after serious burn, which alleviated burn-induced intestinal injury. The AMPK/mTOR pathway may involve in the activation of burn-induced autophagy. Therapeutic/care management, levels of evidence are not applicable to some studies, such as in vitro work, animal models, cadaver studies.

Spatiotemporal regulation of autophagy during Caenorhabditis elegans aging.

Autophagy has been linked to longevity in many species, but the underlying mechanisms are unclear. Using a GFP-tagged and a new tandem-tagged Atg8/LGG-1 reporter, we quantified autophagic vesicles and performed autophagic flux assays in multiple tissues of wild-type Caenorhabditis elegans and long-lived daf-2/insulin/IGF-1 and glp-1/Notch mutants throughout adulthood. Our data are consistent with an age-related decline in autophagic activity in the intestine, body-wall muscle, pharynx, and neurons of wild-type animals. In contrast, daf-2 and glp-1 mutants displayed unique age- and tissue-specific changes in autophagic activity, indicating that the two longevity paradigms have distinct effects on autophagy during aging. Although autophagy appeared active in the intestine of both long-lived mutants, inhibition of intestinal autophagy significantly abrogated lifespan extension only in glp-1 mutants. Collectively, our data suggest that autophagic activity normally decreases with age in C. elegans, whereas daf-2 and glp-1 long-lived mutants regulate autophagy in distinct spatiotemporal-specific manners to extend lifespan.

Autophagy can alleviate severe burn-induced damage to the intestinal tract in mice

The present study was designed to examine the effect of autophagy and apoptosis on intestinal injury in mice after severe burns. Kunming mice were subjected to third degree burns over 30% of the total body surface area. Damage to the intestine was assessed by examining changes in intestinal mucosal morphology, enzyme-linked immunosorbent assay of serum d-lactate, diamine oxidase, lipopolysaccharide, interleukin-6, and tumor necrosis factor α (marker of intestinal damage), hematoxylin and eosin staining, and Western blotting under 4 experimental conditions: control group, burn only (burn group), burn and administration of rapamycin to stimulate intestinal autophagy (rapamycin group), or burn and administration of 3-methyladenine to inhibit intestinal autophagy (3-methyladenine group). At day 1 postburn, the expression levels of light chain 3 II, beclin-1, and cleaved caspase-3 were significantly greater in all 3 groups of mice subjected to the burn injury than in the control group 1day postburn; while the levels of light chain 3 II and beclin-1 were significantly greater and those of cleaved caspase-3 were significantly less in the rapamycin group than in the burn group. In contrast, light chain 3 II and beclin-1 levels were significantly less and those of cleaved caspase-3 significantly greater in the 3-methyladenine group. All 3 groups subjected to burn injury showed significantly increased levels of d-lactate, diamine oxidase, lipopolysaccharide, interleukin-6, and tumor necrosis factor α. Of the 3 groups, the rapamycin group exhibited the least observed levels, the 3-methyladenine group the greatest, and the burn group intermediate. Pathologic sections of the intestinal tissue showed that all 3 burn groups exhibited severe intestinal mucosal damage at 1day postburn. The condition of the 3-methyladenine treatment group was worse than that of the rapamycin treatment group, but better than that of the burn group. Intestinal autophagy is activated in response to intestinal apoptosis after severe burns and may alleviate burn-induced intestinal injury.

Effects of probiotic Bacillus as a substitute for antibiotics on antioxidant capacity and intestinal autophagy of piglets

The objective of this study was to evaluate effects of probiotic Bacillus amyloliquefaciens (Ba) as a substitute for antibiotics on growth performance, antioxidant ability and intestinal autophagy of piglets. Ninety piglets were divided into three groups: G1 (containing 150 mg/Kg aureomycin in the diet); G2 (containing 75 mg/Kg aureomycin and 1 × 108 cfu/Kg Ba in the diet); G3 (containing 2 × 108 cfu/Kg Ba in the diet without any antibiotics). Each treatment had three replications of ten pigs per pen. Results showed that Ba replacement significantly increased the daily weight gain of piglets. Moreover, improved antioxidant status in serum and jejunum was noted in Ba-fed groups as compared with aureomycin group. Increased gene expression of antioxidant enzymes and elevated nuclear factor erythroid 2 related factor 2 (Nrf2) in jejunum was also observed in Ba-fed groups. Besides, Ba replacement significantly decreased jejunal c-Jun N-terminal kinase (JNK) phosphorylation compared with antibiotic group. Western blotting results also revealed that replacing all antibiotics with Ba initiated autophagy in the jejunum as evidenced by increased microtubule-associated protein 1 light chain 3 II (LC3-II) abundance. Taken together, these results indicate that replacing aureomycin with Ba can improve growth performance and antioxidant status of piglets via increasing antioxidant capacity and intestinal autophagy, suggesting a good potential for Ba as an alternative to antibiotics in feed.

Intrauterine growth retardation promotes fetal intestinal autophagy in rats via the mechanistic target of rapamycin pathway.

Intrauterine growth retardation (IUGR) impairs fetal intestinal development, and is associated with high perinatal morbidity and mortality. However, the mechanism underlying this intestinal injury is largely unknown. We aimed to investigate this mechanism through analysis of intestinal autophagy and related signaling pathways in a rat model of IUGR. Normal weight (NW) and IUGR fetuses were obtained from primiparous rats via ad libitum food intake and 50% food restriction, respectively. Maternal serum parameters, fetal body weight, organ weights, and fetal blood glucose were determined. Intestinal apoptosis, autophagy, and the mechanistic target of rapamycin (mTOR) signaling pathway were analyzed. The results indicated that maternal 50% food restriction reduced maternal serum glucose, bilirubin, and total cholesterol and produced IUGR fetuses, which had decreased body weight; blood glucose; and weights of the small intestine, stomach, spleen, pancreas, and kidney. Decreased Bcl-2 and increased Casp9 mRNA expression was observed in IUGR fetal intestines. Analysis of intestinal autophagy showed that the mRNA expression of WIPI1, MAP1LC3B, Atg5, and Atg14 was also increased, while the protein levels of p62 were decreased in IUGR fetuses. Compared to NW fetuses, IUGR fetuses showed decreased mTOR protein levels and enhanced mRNA expression of ULK1 and Beclin1 in the small intestine. In summary, the results indicated that maternal 50% food restriction on gestational days 10–21 reduced maternal serum glucose, bilirubin, and total cholesterol contents, and produced IUGR fetuses that had low blood glucose and reduced small intestine weight. Intestinal injury of IUGR fetuses caused by maternal food restriction might be due to enhanced apoptosis and autophagy via the mTOR signaling pathway.

Intestinal Autophagy Improves Healthspan and Longevity in C. elegans during Dietary Restriction.

Dietary restriction (DR) is a dietary regimen that extends lifespan in many organisms. One mechanism contributing to the conserved effect of DR on longevity is the cellular recycling process autophagy, which is induced in response to nutrient scarcity and increases sequestration of cytosolic material into double-membrane autophagosomes for degradation in the lysosome. Although autophagy plays a direct role in DR-mediated lifespan extension in the nematode Caenorhabditis elegans, the contribution of autophagy in individual tissues remains unclear. In this study, we show a critical role for autophagy in the intestine, a major metabolic tissue, to ensure lifespan extension of dietary-restricted eat-2 mutants. The intestine of eat-2 mutants has an enlarged lysosomal compartment and flux assays indicate increased turnover of autophagosomes, consistent with an induction of autophagy in this tissue. This increase in intestinal autophagy may underlie the improved intestinal integrity we observe in eat-2 mutants, since whole-body and intestinal-specific inhibition of autophagy in eat-2 mutants greatly impairs the intestinal barrier function. Interestingly, intestinal-specific inhibition of autophagy in eat-2 mutants leads to a decrease in motility with age, alluding to a potential cell non-autonomous role for autophagy in the intestine. Collectively, these results highlight important functions for autophagy in the intestine of dietary-restricted C. elegans.