Anemonin improves intestinal barrier restoration and influences TGF-ß1 and EGFR signaling pathways in LPS-challenged piglets.

The present study was aimed at investigating whether dietary anemonin could alleviate LPS-induced intestinal injury and improve intestinal barrier restoration in a piglet model. Eighteen 35-d-old pigs were randomly assigned to three treatment groups (control, LPS and LPS+anemonin). The control and LPS groups were fed a basal diet, and the LPS + anemonin group received the basal diet + 100 mg anemonin/kg diet. After 21 d of feeding, the LPS- and anemonin-treated piglets received i.p. administration of LPS; the control group received saline. At 4 h post-injection, jejunum samples were collected. The results showed that supplemental anemonin increased villus height and transepithelial electrical resistance, and decreased crypt depth and paracellular flux of dextran (4 kDa) compared with the LPS group. Moreover, anemonin increased tight junction claudin-1, occludin and ZO-1 expression in the jejunal mucosa, compared with LPS group. Anemonin also decreased TNF-α, IL-6, IL-8 and IL-1ß mRNA expression. Supplementation with anemonin also increased TGF-ß1 mRNA and protein expression, Smad4 and Smad7 mRNA expressions, and epidermal growth factor and epidermal growth factor receptor (EGFR) mRNA expression in the jejunal mucosa. These findings suggest that dietary anemonin attenuates LPS-induced intestinal injury by improving mucosa restoration, alleviating intestinal inflammation and influencing TGF-ß1 canonical Smads and EGFR signaling pathways.

L-cysteine protects intestinal integrity, attenuates intestinal inflammation and oxidant stress, and modulates NF-κB and Nrf2 pathways in weaned piglets after LPS challenge.

In this study we investigated whetherL-cysteine (L-cys) could alleviate LPS-induced intestinal disruption and its underlying mechanism. Piglets fed with anL-cys-supplemented diet had higher average daily gain.L-cys alleviated LPS-induced structural and functional disruption of intestine in weanling piglets, as demonstrated by higher villus height, villus height (VH) to crypt depth (CD) ratio, and transepithelial electrical resistance (TER) and lower FITC-dextran 4 (FD4) kDa flux in jejunum and ileum. Supplementation withL-cys up-regulated occludin and claudin-1 expression, reduced caspase-3 activity and enhanced proliferating cell nuclear antigen expression of jejunum and ileum relative to LPS group. Additionally,L-cys suppressed the LPS-induced intestinal inflammation and oxidative stress, as demonstrated by down-regulated TNF-α, IL-6 and IL-8 mRNA levels, increased catalase, superoxide dismutase, glutathione peroxidase activity, glutathione (GSH) contents and the ratio of GSH and oxidized glutathione in jejunum and ileum. Finally, a diet supplemented withL-cys inhibited NF-κB(p65) nuclear translocation and elevated NF erythroid 2-related factor 2 (Nrf2) translocation compared with the LPS group. Collectively, our results indicated the protective function ofL-cys on intestinal mucosa barrier may closely associated with its anti-inflammation, antioxidant and regulating effect on the NF-κB and Nrf2 signaling pathways.

Zinc oxide influences intestinal integrity, the expressions of genes associated with inflammation and TLR4-myeloid differentiation factor 88 signaling pathways in weanling pigs.

This study explored whether zinc oxide (ZnO) supplementation could alleviate weanling-induced intestinal injury through TLR and NOD-like receptor signaling pathways. Twelve early-weanling piglets were allotted to two dietary treatments (control vs 2200 mg Zn/kg from ZnO) for 1 wk. The results showed that supplemental ZnO improved daily gain and feed intake, decreased post weaning scour scores, increased villus height and villus height:crypt depth ratio at the jejunal mucosa, and decreased diamine oxidase activity and endotoxin concentration in plasma. The intestinal mRNA levels of TLR4 and its downstream signals, including MyD88, IL-1 receptor-associated kinase 1 and TNF-α receptor-associated factor 6, were decreased, and the expressions of intestinal pro-inflammatory cytokines and chemokines were decreased simultaneously in the ZnO-supplemented piglets. Although NF-κB p65 mRNA abundance was not affected by ZnO supplementation, NF-κB p65 protein expression was down-regulated by ZnO. However, ZnO supplementation had no effect on intestinal expressions of NOD1 and NOD2, and their adaptor molecule receptor-interacting serine/threonine-protein kinase 2, as well as protein expressions of caspase-3 and heat shock protein 70. The results indicated that the protective effects of ZnO on intestinal integrity were closely related to decreasing the expressions of genes associated with inflammation through inhibiting the TLR4-MyD88 signaling pathways.

[Biological characteristics of hematopoietic progenitor cells at different stages of hematopoietic development].

The aim of the present paper is to better understand the mechanism of hematopoietic development through studying the biological characteristics of hematopoietic progenitor cells at different stages of development. Firstly, the c-kit expression levels of the mononuclear cells from murine embryonic aorta-gonad-mesonephros (AGM) region at embryonic day (E)10.5 and E11.5, fetal liver (FL) at E12.5, E14.5, E16.5, E18 and bone marrow (BM) were assayed with fluorescence activated cell sorting (FACS). Secondly, hematopoietic progenitor cells derived from AGM at E10.5, FL at E14.5 and BM were isolated by using c-kit microbeads. Isolated c-kit(+) population cells from AGM, FL and BM were then co-cultured with E14.5 FL-derived stromal cells in transwell co-culture system in vitro. After 3, 7, 10 days of co-culture, numerous floating cells were generated. The floating cells generated in transwell inserts were collected for FACS cell count, migration activity detection and colony forming unit (CFU) formation assay. The results showed that the c-kit was highly expressed in E10.5 AGM, with the percentage of c-kit(+) cells declining during AGM development. c-kit expression was highly expressed again in E12.5 FL, declining along with the progressive development of the FL region. Co-cultured with FL-derived stromal cells, E10.5 AGM-derived c-kit(+) cells produced the highest number of hematopoietic cells, while BM-derived c-kit(+) cells produced the lowest number of hematopoietic cells. Compared with E10.5 AGM-derived c-kit(+) cells, E14.5 FL- and BM- derived c-kit(+) cells inclined to differentiate after 7 to 10 days of culture in vitro. E10.5 AGM and E14.5 FL-derived c-kit(+) cells exhibited a higher migration activity than BM-derived c-kit(+) cells. Moreover, E10.5 AGM-derived c-kit(+) cells showed a higher ability to form mixed colony-forming unit (CFU-Mix) colony. In conclusion, compared with FL- and BM-derived c-kit(+) cells, E10.5 AGM-derived c-kit(+) hematopoietic progenitor cells exhibit better proliferation, migration potential, and have a higher ability to maintain the undifferentiation state in vitro, providing an insight into their clinical manipulation.

[Study on Wnt and Notch signalling involves in regulation of hematopoietic microenvironment.].

OBJECTIVE: To explore the mechanism of Wnt and Notch pathway involved modulating time and spatial restricted hematopoiesis. METHODS: Murine hematopoietic stem and progenitor cells (HSPCs) were isolated from bone marrow (BM) by using c-kit microbeads. E10.5 aorta-gonad-mesonephros (AGM), E12.5, E14.5, E16.5 fetal liver (FL) and adult BM derived stromal cells (StroCs) were isolated and co-cultured with c-kit(+)HSPCs. The floating cells in co-culture system were sorted and counted by FACS. Gene expressions of Wnt and Notch pathway were detected by quantitative PCR and protein expressions by immunostaining. RESULTS: Co-culturing HSPCs with AGM and FL-derived StroCs resulted in an expansion of c-kit(+)population from 0.4 x 10(5)/well to (19.2 +/- 3.2) x 10(5)/well and (26.8 +/- 5.4) x 10(5)/well, respectively, being greater than that with BM-derived StroCs (P < 0.05). The percentage of c-kit(+)cells detected in AGM- and BM- derived StroCs culture system was (75.2 +/- 7.1)%, (74.1 +/- 6.2)% respectively, being higher than FL- derived StroCs culture system (63.4 +/- 5.3)% (P < 0.05). Wnt and Notch pathway genes expression varied at different phases of hematopoiesis. Wnt was highly expressed in AGM and FL derived StroCs, and, Notch did in AGM and BM derived StroCs. CONCLUSION: Wnt and Notch pathway are important modulators in regulating time and spatial restricted hematopoiesis.