本人自攻读博士学位以来，主要从事肠道健康方面的相关研究，致力于研究抗生素替代品对缓解肠道炎症的作用。这不仅具有重要的科学研究价值，也为降低养殖经济损失具有重要意义，有利于我国畜牧业的健康发展。

肉鸡坏死性肠炎是由产气荚膜梭菌感染造成的肠道疾病。欧盟禁止在饲料中添加抗生素或者促生长物质后，坏死性肠炎的发生率持续增加，受该病严重影响的鸡群利润比受轻度影响的鸡群利润降低了33%。在2015年，该病给全球肉鸡生产造成的经济损失高达60亿美元。对于产气荚膜梭菌造成坏死性肠炎发生的致病机理目前尚不完全清楚。我们采用体外培养鸡胚肠道上皮细胞的方法，发现了产气荚膜梭菌及其产生的α-毒素是通过激活NOD1/TLR2受体和NF-κB信号通路诱导炎症因子的表达，从而引起强烈的炎症反应。该发现补充了产气荚膜梭菌致病机理的研究，具有重要的科学价值。

在深入了解产气荚膜梭菌致病机理的基础上，我们研究了木聚糖酶和益生菌干扰产气荚膜梭菌致病途径，缓解肉鸡坏死性肠炎的作用。研究发现，小麦基础日粮中添加木聚糖酶可以通过减轻气荚膜梭菌感染引起的肠道组织损伤，提高肠道的屏障功能，调节肠道消化酶的活性和养分转运载体的表达，优化肠道菌群组成，从而改善了养分的利用率，缓解了感染组肉仔鸡生产性能的下降。采用体外研究的方法，我们发现嗜酸乳杆菌和发酵乳杆菌通过竞争营养物质、降低pH值和分泌抑菌物质等方式抑制了产气荚膜梭菌的生长及其生物膜的形成。这两种乳杆菌对α-毒素分泌的抑制作用可能与降低培养基的pH值有关。它们还通过下调细胞受体(NOD1/TLR2)和NF-κB信号通路，缓解了产气荚膜梭菌感染细胞引起的炎症反应，从而对细胞起到保护作用。木聚糖酶和益生菌缓解肉仔鸡坏死性肠炎的研究，一方面具有理论创新性，另一方面对养殖生产具有指导作用，带来一定的经济效益。

坏死性小肠结肠炎是发生在体重极低的早产儿上的一种肠道疾病。基于早产儿服用益生菌可能诱发败血症的危险，我们研究了益生菌(婴儿双歧杆菌和嗜酸乳杆菌)的代谢产物对缓解坏死性小肠结肠炎的作用及其机理。益生菌代谢产物主要通过调控NF-κB信号通路和细胞外基质重构相关基因的表达来发挥抗炎作用，而且婴儿双歧杆菌比嗜酸乳杆菌的分泌物具有更显著的调控作用。两种益生菌的代谢产物还能缓解IL-1β刺激造成的肠道上皮屏障损伤，这主要是通过校正occludin和claudin-1的蛋白表达以及阻止NF-κB的激活来实现的。该研究不仅具有科学价值，还为防治坏死性小肠结肠炎提供指导方向。

1. Inflammatory responses to Clostridium perfringens type A strains and α-toxin in primary intestinal epithelial cells of chicken embryos.

摘要：The causative pathogen of necrotic enteritis is Gram-positive bacterium Clostridium perfringens. Its main cell wall component, peptidoglycan (PGN), can be recognized by Toll-like receptor 2 and nucleotide-binding oligomerization domain (NOD). Consequently, immune response is initiated via activation of nuclear factor kappa B (NF-κB) signaling pathway. An in vitro study was conducted to investigate chicken intestinal inflammatory responses to C. perfringens type A and one of its virulence factors, α-toxin. In primary intestinal epithelial cells,C. perfringens as well as commercially availablePGN and α-toxin challenge up-regulated mRNA expression of interleukin (IL)-6, IL-8 and inducible nitric oxide synthase (iNOS) with a dosage-dependent manner at 3 h post infection (p.i.) (P ≤ 0.001). Time-course effects of three stimulators at high concentration were further examined. C. perfringens infection elevated IL-6, IL-8 and iNOS levels from 1 h to 9 h p.i., while PGN treatment increased IL-6 and IL-8 expression at 1 h and 3 h p.i. (P< 0.05). Bacterial and PGN treatments induced NOD1 expression at 6 h p.i. and only bacterial infection boosted NF-κB p65 expression at 6 h and 9 h p.i. (P< 0.05).α-Toxin treatment up-regulated IL-6 and IL-8 expression along infection, as well as iNOS, TNF-α and NF-κB p65 expression at later hours p.i.(P< 0.05).In conclusion, both C. perfringens and α-toxin challenge induced intense cytokine expression associated with NF-κB activation in chicken intestinal epithelial cells. The receptors for the recognition of PGN component of C. perfringens need further investigation.

中文关键词:产气荚膜梭菌，α毒素，炎症反应，肠道上皮细胞，肉鸡

所属研究课题：中国农业研究体系(CARS-42-G13)

研究方向：家禽营养与免疫

2. Two Lactobacillus speciesinhibit the growth and α-toxin production of Clostridium perfringens and induced proinflammatory factors in chicken intestinal epithelial cells in vitro.

摘要：Clostridiumperfringens is the causative pathogen of avian necrotic enteritis. Lactobacillusspp. are well-characterized probiotics with anti-microbial and immune-modulatoryactivities. In the present study, we investigated the effects of L. acidophilus andL. fermentum on the growth, α-toxin production and inflammatory responses ofC. perfringens. In in vitro culture experiments, both lactobacilli inhibited the growthof C. perfringens ( P < 0.01), accompanied with a decrease in pH ( P < 0.01).Supernatants from lactobacilli cultures also suppressed the growth of C. perfringensduring 24 h of incubation ( P < 0.01), but this inhibitory effect disappeared after 48 h.Both lactobacilli decreased the α-toxin production of C. perfringens ( P < 0.01) withoutinfluencing its biomass, and even degraded the established a-toxin ( P < 0.01). Lowerenvironmental pH reduced the a-toxin production as well ( P< 0.01). Preincubation withL. acidophilus decreased the attachment of C. perfringens to cells ( P < 0.01) with the cellcytotoxicity being unaffected. Both lactobacilli pretreatment reduced the up-regulationof proinflammatory factors, peptidoglycan (PGN) receptors and nuclear factor kappa B

(NF-kB) p65 in C. perfringens-challenged chicken intestinal epithelial cells ( P < 0.05).In conclusion, L. acidophilus and L. fermentum inhibited the pathological effects ofC. perfringens in vitro conditions.

中文关键词：乳酸杆菌，产气荚膜梭菌，α毒素，肉鸡，肠道上皮细胞，炎症

所属研究课题：中国农业研究体系(CARS-42-G13)

研究方向：家禽营养与免疫

3. Xylanase supplementation of a wheat-based diet improved nutrient digestion and mRNA expression of intestinal nutrient transporters in broiler chickens infected with Clostridium perfringens.

摘要：Necrotic enteritis caused by Clostridium perfringens has been becoming prevalent in European Uniondue to withdrawal of antibiotics in poultry feed. In an experiment with 2 × 2 factorial arrangement,336 1-day-old male broiler chicks (Ross 308) were assigned to four groups with or without C. perfringenschallenge and fed wheat-based diets supplemented with or without xylanase 5,500 U/kg of diet. The study aimed toinvestigate effects of xylanase addition on growth performance as well as nutrient digestion and absorption ofC. perfringens-infected broilers. Before challenge (day 0 to 14), xylanase-supplemented birds had greater ADG and lower feed conversion ratio (FCR) (P < 0.05). During infection (day 14 to 21), challengetended to decreaseADG (P = 0.063) and significantly increased FCR (P < 0.05), whilexylanase addition greatly reduced FCR (P < 0.05).C. perfringens infection decreased AME values and apparent ileal digestibility of dry matter of diets (P < 0.05).Xylanase supplementation increased AME values regardless of infection and apparent ileal digestibility of CP in challenged birds (P < 0.05). Activities of duodenal α-amylase and chymotrypsin and pancreatic trypsin were decreased by C. perfringens infection (P < 0.05). Xylanase supplementation elevated pancreatic chymotrypsin activity and reduced duodenal α-amylase and trypsin activities(P < 0.05). It also decreasedjejunalα-amylase activityand increased pancreatic α-amylase as well as jejunalsucrase activities in uninfected birds (P < 0.05). The duodenal mRNA expression of sodium glucose co-transporter 1 (SGLT1), H+-dependent peptide transporter 1 (PepT1) and liver fatty acid-binding protein (L-FABP) were down-regulated (P < 0.05), but ileal SGLT1 gene expression was increased by infection (P < 0.05). Xylanase addition up-regulated expression of jejunal SGLT1, PepT1 and L-FABP genes as well as ileal PepT1 and L-FABP genes in challenged broilers (P < 0.05). In conclusion, xylanase supplementation of wheat-based dietsimprovedFCR and AMEin birdsirrespective of C. perfringensinfection and elevated apparent ileal digestibility of CP and mRNA expression of nutrient transporters in challenged birds.

中文关键词：木聚糖酶，产气荚膜梭菌，营养物质消化率，消化酶活性，营养物质转运载体

所属研究课题：中国农业研究体系(CARS-42-G13)

研究方向：家禽营养与免疫

4. Secreted metabolites of Bifidobacteriuminfantis and Lactobacillus acidophilus protect immature human enterocytes from IL-1β-induced inflammation: A transcription profiling analysis.

摘要：Combination regimens of Bifidobacteriuminfantis and Lactobacillus acidophilus have beendemonstrated to prevent necrotizing enterocolitis (NEC) in clinical trials. However, the molecularmechanisms responsible for this protective effect are not well understood. Additionally,conditioned media from individual cultures of these two probiotics show strain specificmodulation of inflammation using in vitro human intestinal NEC models. Here we report atranscription profiling analysis of gene expression in immature human fetal intestinal epithelialcells (H4 cells) pretreated with conditioned media from B. infantis (BCM) or L. acidophilus(LCM) prior to IL-1β stimulation. Compared with control media, the two probioticconditionedmedia (PCM) treatments altered the expression of hundreds of genes involved in the immune response, apoptosis and cell survival, cell adhesion, the cell cycle, developmentand angiogenesis. In IL-1β-stimulated cells, PCM treatment decreased the upregulationof genes in the NF-κB activation pathway and downregulated genes associated withextracellular matrix (ECM) remodeling. Compared with LCM, BCM showed more significantmodulatory effects on ECM remodeling, reflected by a lower p value. IL-6 and IL-8 productionwas significantly reduced in IL-1β-stimulated cells pretreated with PCM (p<0.05), whichwas consistent with their altered gene expression. Western blot analysis showed that comparedwith IL-1β stimulation alone, PCM treatment attenuated the decrease of cytoplasmicIκBα and NF-κB p65 levels as well as the increase of nuclear NF-κB p65 levels in the stimulatedcells (p<0.05). In conclusion, PCM treatment exerted anti-inflammatory effects in immaturehuman fetal enterocytes primarily by modulating genes in the NF-κB signaling pathways, particularly the ECM remodeling pathway, were more profoundly affected byBCM than LCM.

中文关键词：婴儿双歧杆菌，嗜酸乳杆菌，分泌物，坏死性小肠结肠炎

所属研究课题：美国NIH

研究方向：营养与肠道健康

5. Secretions of Bifidobacteriuminfantis and Lactobacillus acidophilus protect intestinal epithelial barrier function.

摘要：Objectives: The secreted metabolites of probiotics are cytoprotective to intestinal epithelium and have been shown to attenuate inflammation and reduce gut permeability. The present study was designed to determine the protective effects of probiotic conditioned media (PCM) from Bifidobacteriuminfantis (BCM) and Lactobacillus acidophilus (LCM) on interleukin (IL)-1β–induced intestinal barrier compromise.

Methods: The epithelial barrier was determined by measuring the transepithelial electrical resistance (TER) across a Caco-2 cell monolayer using a Transwell model. The paracellular permeability was determined by fluorescein isothiocyanate–labeled dextran flux. The expression of tight junction (TJ) proteins and nuclear factor-kappa B (NF-κB) p65 were determined using Western blot and the distribution of NF-κB p65 was determined by immunofluorescence staining.

Results: BCM and LCM induced a dose-dependent increase in Caco-2 TER after 4 and 24 hours of incubation (P<0.05). The maximal increase of Caco-2 TER occurred at 4 hours of treatment with a PCM concentration of 15%. Preincubation with BCM and LCM for 4 hours significantly prevented the decrease of Caco-2 TER induced by 24 hours of stimulation with 10 ng/mL IL-1β. BCM and LCM decreased paracellular permeability in both stimulated and unstimulated Caco-2 monolayers (P<0.05). IL-1β stimulation decreased occludin expression and increased claudin-1 expression in Caco-2 cells (P<0.05), which was prevented in cells treated with BCM or LCM. The changes of claudin-1 expression in H4 cells were similar to Caco-2 cells in response to PCM treatment and IL-1β stimulation; however, a similar response in occludin was not demonstrated. The IL-1β–induced nuclear translocation of NF-κB p65 in Caco-2 cells was prevented by pretreatment with both PCMs.

Conclusions: BCM and LCM protected the intestinal barrier against IL-1β stimulation by normalizing the protein expression of occludin and claudin-1 and preventing IL-1β –induced NF-κB activation in Caco-2 cells, which may be partly responsible for the preservation of intestinal permeability.

中文关键词：Caco-2单层细胞，IL-1β，NF-κB p65，紧密连接

所属研究课题：美国NIH

研究方向：营养与肠道健康