蜂胶对细菌脂多糖刺激下奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接蛋白的影响

本试验旨在研究中国蜂胶乙醇提取物（ethanol extract of Chinese propolis，EECP）对细菌脂多糖（lipopolysaccharide，LPS）刺激下体外培养奶牛乳腺上皮细胞炎症相关基因mRNA转录水平和紧密连接渗透性的影响。EECP中总酚酸和总黄酮含量测定采用福林酚法和硝酸铝法，并建立LPS诱导奶牛乳腺上皮细胞（bovine mammary epithelial cells，MAC-T）炎症模型，采用CCK-8法测定EECP对MAC-T相对增殖率的影响，利用实时荧光定量PCR（RT-qPCR）评估EECP对LPS诱导的MAC-T细胞炎症相关因子（IL-6、IL-8、TNF-α和IL-1β）相对mRNA转录水平；以及对紧密连接蛋白（occludin、ZO-1）相对mRNA转录水平进行检测，并进一步利用免疫荧光技术对紧密连接膜蛋白进行定位，确定EECP对LPS诱导MAC-T细胞炎症紧密连接渗透性的影响。结果显示：EECP中总酚酸含量为106.35 mg没食子酸当量（GAE）·g^-1、总黄酮含量为320.85 mg芦丁当量（RE）·g^-1；CCK-8结果显示EECP的安全浓度为0~15 μg·mL^-1，并可有效提高LPS刺激下MAC-T的活力；LPS刺激显著增加了细胞炎症相关因子IL-6、IL-8、TNF-α和IL-1β mRNA的转录量（P<0.001）；但2.5~15.0 μg·mL^-1 EECP预处理显著降低了IL-6、IL-8、TNF-α和IL-1β mRNA的转录量；与此类似，LPS刺激显著抑制了紧密连接蛋白基因（occludin、ZO-1）mRNA的转录量（P<0.01），而EECP预处理后紧密连接蛋白基因（occludin和ZO-1）mRNA的转录量显著增加（P<0.05）；免疫荧光染色试验也证实EECP能通过上调紧密连接蛋白（occludin、ZO-1）的表达，缓解LPS诱导的乳腺上皮细胞屏障功能紊乱。该结果证实，EECP对细菌脂多糖诱导奶牛乳腺上皮细胞炎症具有良好的保护作用，这为利用中国蜂胶预防奶牛乳腺炎提供了试验基础。     

Alteration of the haemocyte parameters,expression of immune‐related and neurohormone bursicon genes of giant river prawn Macrobrachium rosenbergii (de Man) in the response to salinity stress and ammonia stress

We examined the effects of salinity stress and ammonia stress on alteration of the haemocyte count, phenoloxidase activity, expressions of immune‐related genes including prophenoloxidase (proPO), crustin, heat shock protein 70 (HSP70), and expressions of stress‐responsive neurohormone (Bur‐α and Bur‐β) in the thoracic and abdominal ganglia of giant river prawn Macrobrachium. These parameters of prawn that subjected to salinity stress (transferred from 0‰ to 5‰ and 10‰), and subjected to ammonia‐nitrogen (ammonia‐N) stress (transferred from 0 to 0.262 mg/L and 0.786 mg/L) were examined after 0, 3, 6, 24, 72 and 168 hr respectively. During the initial period of 3, 6 and 24 hr, granulocyte haemocyte (granular and semi‐granular hemocyte) count and PO activity significantly decreased, while expressions of Bur‐α and Bur‐β significantly increased. After 24 hr, granulocyte haemocyte count and PO activity significantly increased, whereas expressions of Bur‐α and Bur‐β significantly decreased. The expressions of proPO, crustin and HSP70 were significantly downregulated in the prawn that subjected to salinity stress and ammonia‐N stress at all time periods of 3–168 hr. In conclusion, changes in the granulocyte haemocyte count of M. rosenbergii following salinity stress and ammonia‐N stress are closely associated with the changes of Bur‐α and Bur‐β expressions.     

Dietary ginger administration attenuates oxidative stress and immunosuppression caused by oxytetracycline in rainbow trout (Oncorhynchus mykiss)

In the present study, potential ameliorative effects of dietary ginger (GN) were investigated on antioxidant and immune responses of rainbow trout (Oncorhynchus mykiss) during oxytetracycline (OX) administration. As a 2 × 3 factorial design, the fish were orally treated with OX (a daily dose of 100 mg/kg) and GN (either 10 or 20 g/kg diet) for 10 days. Then, blood samples were taken from each treatment to monitor plasma lysozyme, complement (ACH50), superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx), glutathione‐S‐transferase (GST) activities, and reduced glutathione (GSH), malondialdehyde (MDA), total immunoglobulin (Ig) and globulin levels. OX treatment significantly decreased SOD (30%), GPx, (10%) and lysozyme (23%) activities, and GSH (19%) levels; however, it increased GST (16%) activity and MDA (28%) levels. Ten grams GN per kg levels significantly decreased SOD (35%), CAT (13%), GST (20%) and MDA (30%), but increased GSH (30%), lysozyme (48%) and globulin (16%). Twenty grams GN per kg diet significantly decreased SOD (26%) and MDA (17%), but increased lysozyme (31%) levels. Interaction effects of dietary GN and OX were observed on plasma MDA and GPx levels, as 10 g GN per kg diet prevented the OTC‐induced changes in these parameters. Moreover, 20 g GN per kg diet prevented the OX‐induced change in GPx activity and mitigated the MDA elevation by 20%. It is concluded that GN administration at 10 g/kg diet is beneficial in mitigating oxidative stress and immunosuppression of rainbow trout during OX administration.     

鱼类组蛋白核苷酸的多态性及其在杀鱼爱德华氏菌感染中的作用

组蛋白是染色体核小体的重要组分,在调控染色质结构、基因转录、个体发育等不同生物学过程中起着重要作用。为了研究鱼类组蛋白基因是否存在核苷酸的多态性以及组蛋白核苷酸多态性是否会影响鱼类的抗病力,本实验以斑马鱼和草鱼为研究对象,通过PCR扩增克隆了组蛋白H2A的全长开放阅读框;利用过表达技术、菌落平板计数、感染存活分析以及荧光定量PCR技术,研究了斑马鱼和草鱼组蛋白H2A核苷酸多态性不同变异体在杀鱼爱德华氏菌感染中的作用。在本研究中,实验发现鱼类组蛋白H2A存在着丰富的核苷酸多态性。序列分析结果显示斑马鱼和草鱼组蛋白H2A核苷酸多态性的变异体核苷酸序列相似性为90%～100%;而两两H2A核苷酸多态性变异体氨基酸序列之间最多只有3个位点存在差异。通过体内和体外抗菌实验可知,斑马鱼和草鱼组蛋白H2A核苷酸的多态性显著影响H2A的抗菌活性。此外,筛选出的抗菌组蛋白H2A核苷酸多态性的变异体在斑马鱼体内的过表达,不仅具有免疫增强的作用,还能显著增强斑马鱼对杀鱼爱德华氏菌感染的抗病力。本研究为筛选具有抗病作用的组蛋白H2A免疫保护原奠定了重要基础。     

Effects of oral administration of Spirulina platensis and probiotics on serum immunity indexes,colonic immune factors,fecal odor,and fecal flora in mice

To evaluate the effects of Spirulina platensis and probiotics on growth, immunity indexes, fecal flora, and fecal odor in mice, 40 mice were randomly allotted to four groups, and each was administrated with nothing, S. platensis, probiotics, or both for 28 days, respectively. Then, many indexes were measured. The results showed that S. platensis was more effective (P < 0.001) than probiotics in improving mice's feed conversion ration (FCR). In immunity, probiotics administration increased (P < 0.042) serum IgE, IgM, IFN-γ, colonic AHR, TLR4, and NF-κB protein expression and decreased (P < 0.039) serum IL-1α, IL-21, IL-22, and colonic ARNT gene expression. However, the S. platensis showed weaker effect, which increased (P < 0.025) the serum IgE, IgM, TNF-α, and the colonic AHR and NF-κB protein expression, and decreased (P < 0.01) serum IL-21. Probiotics consumption decreased the fecal odor by decreasing (P < 0.02) fecal Escherichia coli, indole-3-acetic acid (IAA), and skatole contents, and the S. platensis decreased (P = 0.04) the IAA. These results indicated that oral administration of probiotics, S. platensis, or both of them in mice probably benefited body's immunity and reduced fecal odor. However, their mechanisms were still unclear and need further study.     

Outer membrane protein A of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in RAW264.7 cells

AIM:To analyze the effects of outer membrane protein A (OmpA) from Acinetobacter baumannii ATCC 19606 on the autophagy of RAW264.7 cells. METHODS:The RAW264.7 cell model stimulated by OmpA was established. The effects of OmpA on the autophagy of RAW264.7 cells were detected by immunofluorescence, Western blot and transmission electron microscopy. RESULTS:The OmpA increased the expression of LC3B-Ⅱ and reduced the phosphorylation levels of Akt, mTOR and p70S6K. Rapamycin further reduced the phosphorylation levels of mTOR and p-70S6K, and increased the expression of LC3B-Ⅱ induced by OmpA. CONCLUSION:The OmpA of Acinetobacter baumannii induces autophagy via Akt/mTOR/p70S6K signaling pathway in the RAW264.7 cells. This work provides a basis for further research on the molecular mechanism of autophagy induced by Acinetobacter baumannii to find a new method against the infection of Acinetobacter baumannii.     

草地贪夜蛾寄生蜂资源及其调控寄主免疫反应的研究

草地贪夜蛾Spodoptera frugiperda（J.E.Smith）原产于美洲，自2019年初入侵我国以来迅速向北蔓延，对农业造成了巨大损失。其寄生蜂作为重要的天敌资源之一，在害虫防治中具有不可替代的作用。本文对目前已鉴定到的9个科170种草地贪夜蛾寄生蜂天敌等进行了分类总结，而后讨论了其在害虫生物防治方面的应用前景，并围绕草地贪夜蛾寄生蜂寄生因子调控寄主免疫反应的分子机理展开详细论述。该研究旨在丰富草地贪夜蛾的生物防治资源素材，并为该害虫的绿色防控提供重要的理论与实践指导意义。     

Partial fishmeal replacement by mussel meal or meat and bone meal in low‐fishmeal diets for juvenile Ussuri catfish (Pseudobagrus ussuriensis): Growth,digestibility, antioxidant capacity and IGF‐I gene expression

This study evaluated the effects of replacing fishmeal with mussel meal or meat and bone meal in low‐FM diet on growth performance, body composition, digestibility, antioxidant capacity and IGF‐I gene expression in juvenile Ussuri catfish (Pseudobagrus ussuriensis). The results showed that no significant changes in SGR, FE and PER were observed between MM₄₀ and LFM groups, but significantly reduced result was found in MBM₄₀ group. MM₄₀ group showed the higher ADC of lipid (93.30%) and lipase activity (95.00 U/gprot) than LFM group (90.97%; 70.18 U/gprot). MM₄₀ or MBM₄₀ diets led to significant reduction of SOD and CAT activities. MM₄₀ group showed significantly higher MDA level (5.84 nmol/mg) than LFM group (4.73 nmol/mg). The activities of LZM decreased significantly in MM₄₀ and MBM₄₀ groups compared with LFM group. MM₄₀ and MBM₄₀ groups showed no significant difference in hepatic IGF‐I gene expression levels compared with LFM group. The findings demonstrated that MM could substitute 400 g/kg of FM in low‐FM diet for Ussuri catfish without influencing the growth, but to some extent, spontaneous oxidative stress and immune damage could occur; when 400 g/kg of FM was replaced by MBM, significantly negative effects were observed on growth, antioxidant capacity and non‐specific immune response of Ussuri catfish.     

Surplus spermine does not protect salmon cells against lipopolysaccharide (LPS)‐induced stress,but excess spermine is eliminated through spermidine/spermine N1‐acetyltransferase (SSAT)

The present trials tested the efficiency of surplus spermine to reduce inflammation and oxidative stress following LPS‐induced stress using an in vitro model of head kidney and liver cells isolated from Atlantic salmon. Spermine did not protect cells from LPS‐induced inflammatory response at either 0.3, 0.6 or 0.9 mM. However, as the gene expression of spermidine/spermine N1‐acetyltransferase (SSAT) increased with increasing spermine concentration, we addressed possible oxidative effects of the increased SSAT using its activator DENSPM or inhibitor of polyamine oxidation of the acetylated polyamines using MDL72527 at a spermine concentration of 0.6 mM. There was no significant effect of DENSPM, but MDL72527 decreased gene expression of GPX‐3 (p = .04), while gene expression of catalase and MnSOD was unaffected by treatment (p = .30 and p = .48, respectively). In conclusion, spermine did not protect cells from LPS‐provoked inflammation. The higher the spermine concentration, the more SSAT producing acetylated spermine occurred. Inhibiting the acetylated polyamine oxidases by MDL72527 improved oxidation status as expected due to a lower endogenous production of H₂O₂ by polyamine and acetylated polyamine oxidases. Probably care should be taken using polyamines or arginine as functional ingredients to avoid any increased oxidation within cells.     

Effects of alternative dietary lipid sources on growth performance,health status and fillet fatty acid composition of hybrid sturgeon (Acipenser baeri Brandt ♀ × Acipenser schrenckii Brandt ♂)

An 8‐week trial was conducted to determine the effects of total replacement of 12.9% fish oil (FO) with soybean oil (SBO), peanut oil (PNO), sunflower seed oil (SFSO), corn oil (CO) and canola oil (CNO) on growth performance, health status and fillet fatty acid composition of hybrid sturgeon (194.28 ± 0.14 g). Compared to the FO group, dietary SBO decreased growth performance (p < .05), increased serum glucose and hepatic lipid content (p < .05). No obvious adverse effects on growth performance and health status were observed in PNO, SFSO and CO groups (p > 0.05). The fish fed with CNO had increased growth performance (p < .05), reduced serum ALT, AST, LDL‐C (p < .05) and enhanced serum GSH‐Px, T‐AOC, and LZM, MPO, C4 (p < .05). The contents of C18:1n9, C18:2n6, and ∑n‐3 PUFA and ∑n‐6 PUFA in fillets showed a positive linear correlation with the diets (p < .05). In summary, PNO, SFSO and CO are probable alternative lipid sources to fully replace FO. Hybrid sturgeon prefers to use CNO as a lipid source with improved growth performance and health status. The fillet fatty acid composition mirrors the dietary fatty acid composition.