Paneth cell-derived iNOS is required to maintain homeostasis in the intestinal stem cell niche.

BACKGROUND: Mammalian intestinal epithelium constantly undergoes rapid self-renewal and regeneration sustained by intestinal stem cells (ISCs) within crypts. Inducible nitric oxide synthase (iNOS) is an important regulator in tissue homeostasis and inflammation. However, the functions of iNOS on ISCs have not been clarified. Here, we aimed to investigate the expression pattern of inducible nitric oxide synthase (iNOS) within crypts and explore its function in the homeostatic maintenance of the ISC niche. METHODS: Expression of iNOS was determined by tissue staining and qPCR. iNOS-/- and Lgr5 transgenic mice were used to explore the influence of iNOS ablation on ISC proliferation and differentiation. Enteroids were cultured to study the effect of iNOS on ISCs in vitro. Ileum samples from wild-type and iNOS-/- mice were collected for RNA-Seq to explore the molecular mechanisms by which iNOS regulates ISCs. RESULTS: iNOS was physiologically expressed in Paneth cells. Knockout of iNOS led to apparent morphological changes in the intestine, including a decrease in the small intestine length and in the heights of both villi and crypts. Knockout of iNOS decreased the number of Ki67+ or BrdU+ proliferative cells in crypts. Loss of iNOS increased the number of Olfm4+ ISCs but inhibited the differentiation and migration of Lgr5+ ISCs in vivo. iNOS depletion also inhibited enteroid formation and the budding efficiency of crypts in vitro. Moreover, iNOS deficiency altered gluconeogenesis and the adaptive immune response in the ileum transcriptome. CONCLUSION: Paneth cell-derived iNOS is required to maintain a healthy ISC niche, and Knockout of iNOS hinders ISC function in mice. Therefore, iNOS represents a potential target for the development of new drugs and other therapeutic interventions for intestinal disorders.

Aged gut microbiota contribute to different changes in antioxidant defense in the heart and liver after transfer to germ-free mice.

Age-associated impairment in antioxidant defense is an important cause of oxidative stress, and elderly individuals are usually associated with gut microbiota (GM) changes. Studies have suggested a potential relationship between the GM and changes in antioxidant defense in aging animals. Direct evidence regarding the impact of aging-associated shifts in GM on the antioxidant defense is lacking. The heart is a kind of postmitotic tissue, which is more prone to oxidative stress than the liver (mitotic tissue). To test and compare the influence of an aged GM on antioxidant defense changes in the heart and liver of the host, in this study, GM from young adolescent (5 weeks) or aged (20 months) mice was transferred to young adolescent (5 weeks) germ-free (GF) mice (N = 5 per group) by fecal microbiota transplantation (FMT). Four weeks after the first FMT was performed, fecal samples were collected for 16S rRNA sequencing. Blood, heart and liver samples were harvested for oxidative stress marker and antioxidant defense analysis. The results showed that mice that received young or aged microbiota showed clear differences in GM composition and diversity. Mice that received aged microbiota had a lower ratio of Bacteroidetes/Firmicutes in GM at the phylum level and an increased relative abundance of four GM genera: Akkermansia, Dubosiella, Alistipes and Rikenellaceae_RC9_gut_group. In addition, GM α-diversity scores based on the Shannon index and Simpson index were significantly higher in aged GM-treated mice. Oxidative stress marker and antioxidant defense tests showed that FMT from aged donors did not have a significant influence on malondialdehyde content in serum, heart and liver. However, the capacity of anti-hydroxyl radicals in the heart and liver, as well as the capacity of anti-superoxide anions in the liver, were significantly increased in mice with aged microbiota. FMT from aged donors increased the activities of Cu/Zn superoxide SOD (Cu/Zn-SOD), catalase (CAT) and glutathione-S-transferase in the heart, as well as the activity of Cu/Zn-SOD in the liver. Positive correlations were found between Cu/Zn-SOD activity and radical scavenging capacities. On the other hand, glutathione reductase activity and glutathione content in the liver were decreased in mice that received aged GM. These findings suggest that aged GM transplantation from hosts is sufficient to influence the antioxidant defense system of young adolescent recipients in an organ-dependent manner, which highlights the importance of the GM in the aging process of the host.

Astragalus polysaccharide (APS) supplement in beagle dogs after castration: Effects on the haematology and serum chemistry profiles, immune response, and oxidative stress status.

BACKGROUND: Castration is one of the most common surgical procedures performed in dogs. However, based on increasing evidence, male animals experience significant pain after castration. Astragalus polysaccharide (APS), one of the main bioactive components in A. membranaceus bunge, has been widely used as part of Fu-Zheng therapy to enhance natural defense mechanisms. INTRODUCTION: This study was carried out to determine the effects of supplementing different doses of Astragalus polysaccharide (APS; control, 0 mg/kg; APSL, 400 mg/kg; and APSH, 800 mg/kg) for 8 weeks on the haematology and serum chemistry profiles, immune response, and oxidative stress status in weanling beagle dogs. METHODS: After adapting to the experimental environment for 1 week, 18 male beagle dogs (Sichuan Institute of Musk Deer Breeding, China; average initial weight, 3.80 ± 0.43 g; age, 3-month-old) were randomly allotted to diets supplemented with three doses of APS (Control, 0 mg/kg; low, 400 mg/kg; and high, 800 mg/kg), referred to as control, APSL, and APSH, respectively; six dogs were assigned to each treatment. The dogs were fed the respective diets twice daily at 08:30 and 16:30 h in sufficient quantity to supply the metabolizable energy requirements for 8 weeks. On day 43 (19 weeks old), the dogs were castrated. On days 42 (prior to castration, 19 weeks old), 50 (day 7 after castration, 20 weeks old), and 57 (day 14 after castration, 21 weeks old) to measure the haematology, blood chemistry, immune response, and oxidative stress status parameters. RESULTS: Based on our findings, the APSH diet decreased weight gain and increased the feed to gain ratio in dogs (P < 0.05). At 14 days after castration, the wound was almost closed, slightly swollen, dry, and clean in the groups supplemented with APS. In addition, optimal APS supplementation was found to decrease erythrocyte count (RBC), haematocrit (HCT), alkaline phosphatase (ALP), alanine aminotransferase (ALT), C-reactive protein (CRP), interleukin 1ß (IL-1ß), and tumor necrosis factor-α (TNF-α) levels, and cortisol and protein carbonyl (PC) concentrations (P < 0.05). Moreover, the mean corpuscular haemoglobin (MCH) and platelet (PLT) levels, interleukin 10 (IL-10) and glutathione (GSH) content, and Cu/Zn superoxide dismutase (SOD1), catalase (CAT), and glutathione peroxidase (Se-GPx) activities were increased in the APS supplemented groups (P < 0.05) CONCLUSION: This study demonstrated that supplementing weanling beagle dogs with optimum APS could positively affect wound healing by improving their haematological profile (decreased RBC and HCT content, increased MCH and PLT levels), serum biochemical parameters (decreased ALP and ALT content), immune status (decreased CRP, IL-1ß, and TNF-α levels; increased IL-10 content), and antioxidant defense (decreased cortisol and PC content; increased GSH content, and SOD1, CAT, and Se-GPx activities). However, the detailed mechanism whereby APS regulates these changes requires further investigation. In addition, the results of this study suggest that 400 mg/kg diet is the optimum APS dose for beagle dogs.

Enhancing immune response, antioxidant capacity, and gut health in growing beagles through a chitooligosaccharide diet.

Chitooligosaccharides (COS) have attracted significant attention due to their unique biological activities, water solubility, and absorbable properties. The objective of the present study was to investigate the impact of COS-supplemented diets on the immune response, antioxidative capacity, hematology, serum biochemistry, and modulation of intestinal microbiota in growing beagles. Twelve weaning male beagles (6 weeks old; weighing 3.6 ± 0.6 kg) were fed either a control diet (food without COS, n = 6) or a COS-supplemented diet (n = 6) twice daily for 7 weeks. Blood samples collected at weeks 4 and 7 indicated that hematology and serum biochemistry remained unaffected by COS supplementation. Compared with the control group, the test group showed higher levels of serum antibodies against the canine distemper virus and parvovirus, higher levels of immunoglobulin A, G, and M, and increased activities of superoxide dismutase, glutathione peroxidase, and catalase. In addition, COS was observed to modulate the intestinal flora by enhancing the presence of probiotics, such as Muribaculaceae, Prevotellaceae_Ga6A1_group, Lactobacillus, Collinsella, Blautia, and Lachnospiraceae_NK4A136_group. In summary, a COS-supplemented diet could effectively improve dog health by regulating immune function and antioxidant responses and modulating intestinal microbiota. This study highlights the potentiality of using COS as a valuable nutraceutical for growing dogs.