摘要
本研究通过二个动物饲养试验及一个动物细胞体外培养试验,系统地探讨了刺五加多糖(ASPS)对断奶仔猪免疫功能的调控作用及其作用机理。
(1)研究不同水平的ASPS对断奶仔猪生长性能和免疫功能的影响,筛选ASPS的适宜添加量。将96头断奶仔猪随机分为6个处理,分别饲喂添加0、150、300、500、800、1000mg/kg ASPS的饲粮,试验期21d。试验第14d,仔猪肌肉注射OVA：试验第14和21d,测定生长性能,采血测定血液指标。试验结果表明：随ASPS添加量增加,仔猪ADG、FCR、外周血白细胞数量、淋巴细胞数量、淋巴细胞转化率、CD4淋巴细胞业群含量、IFN-y和皮质醇水平均呈二次曲线变化(P<0.05),外周血IL-2水平呈线性改善(P＜0.05)；ASPS对血清IL-4、OVA抗体、IgG、IgA、IgM抗体水平无显著影响(P>0.05)；综合生长性能和免疫功能考虑,ASPS适宜添加量为800mg/kg.结果提示,ASPS能提高仔猪的细胞免疫功能和抗断奶应激能力,改善断奶仔猪的生长性能。
(2)注射LPS构建仔猪的免疫应激模型,研究ASPS缓解免疫应激仔猪的生长抑制及免疫调控的作用机理。试验采用2×2因素设计,即饲粮处理和免疫应激处理,试验期21d。将64头断奶仔猪随机分为2组(每组8个重复),分别饲喂添加0或800mg/kg ASPS的饲粮,试验第14和21d,给每组4个重复的仔猪腹腔注射LPS,另4个重复注射等量生理盐水。注射后3h和第2d,采血测定血液指标。结果表明：①ASPS对试验1-14d仔猪的生长性能无影响(P>0.05),但缓解了因注射LPS导致的试验14～21d仔猪ADG和ADFI的下降(P<0.05)；②ASPS提高了淋巴细胞转化率和试验第15d CD4"、CD8+淋巴细胞亚群含量(P＜0.05),缓解了因注射LPS导致的CD4+/CD8+比值的上升(P<0.05)；③ASPS缓解了免疫应激仔猪血浆IL-1β、IL-6、TNF-α、PGE2、皮质醇、葡萄糖、NEFA和α-AGP水平的升高(P＜0.05),提高了血浆IL-10、TP、GH和IGF-I水平(P＜0.05),降低了UN浓度(P＜0.05),对胰岛素水平无影响(P>0.05)。结果提示,ASPS缓解免疫应激仔猪的生长抑制与其减少前炎性细胞因子的分泌,从而间接影响机体的内分泌调节有关；ASPS调节应激仔猪的免疫功能是通过直接影响T淋巴细胞而起作用。
(3)研究ASPS对免疫应激仔猪脾脏IL-1β、ILF6和TNF-α mRNA相对表达量的影响。试验设计同(2)。试验第21d,仔猪注射LPS后3h,从每个重复中取1头仔猪屠宰取脾脏。试验结果表明：ASPS显著降低了脾脏IL-1β、IL-6和TNF-α mRNA表达量(P＜0.05),且能缓解免疫应激仔猪脾脏IL-6、IL-1β和TNF-α mRNA相对含量上升(P<0.05)。结果提示,ASPS缓解免疫应激仔猪的生长抑制是从分子水平上降低了前炎性细胞因子IL-6、IL-1β和TNF-α mRNA的相对含量而实现的。
(4)研究ASPS对体外培养的仔猪外周血淋巴细胞信号传导通路的影响。结果表明：①随ASPS浓度增加,B细胞增殖无变化(P＞0.05),T细胞增殖呈二次曲线变化(P<0.05),且较高剂量ASPS (80、160、320、μg/mL)对其影响显著(P<0.05)。②随ASPS浓度增加,IL-4水平无显著变化(P>0.05),IL-2水平呈二次曲线变化(P<0.05),且随培养时间和ASPS浓度变化表现出剂量和时间依赖关系。③无anti-TLR4处理时,N0、iNOS、NF-κB和TNF-α水平随ASPS浓度增加呈二次曲线变化(P＜0.05),anti-TLR4孵育淋巴细胞悬液后,ASPS对这些指标无影响(P＞0.05)。④ASPS对仔猪外周血淋巴细胞内cAMP、cGMP浓度和cAMP/cGMP匕值的影响具有时间和剂量依赖性,作用20min,80、160μg/mL ASPS显著提高了淋巴细胞内cAMP浓度和cAMP/cGMP比值(P<0.05),降低了cGMP浓度(P＜0.05)。作用60min,320μg/mL ASPS对上述指标出现显著影响(P<0.05)。结果提示,ASPS能增强体外培养的淋巴细胞的细胞免疫功能,ASPS可能通过与淋巴细胞表面TLR4受体结合,影响淋巴细胞内cAMP、cGMP等信号分子浓度,通过提高NF-KB活性从而影响TNF-α等细胞因子的浓度。
Two animal feeding experiments and one experiment in vitro were conducted to investigate the immunomodulation and its mechanism of Acanthopanax senticosus polysaccharide (ASPS) on immune function of weaned pigs.
(1) Experiment1was evaluated to explore the effects of ASPS on growth and immune response in weaning piglets and the optimal dose of dietary ASPS. A total of96crossbred pigs were randomly allotted to six diets supplemented with ASPS at0,150,300,500,800, and1000mg/kg. Pigs were fed ad libitum for21days and were intramuscularly injected with OVA on d14to evaluate humoral immune response. Growth performance, blood parameters were measured on d14and21. The results showed that ADG, FCR, the number of WBC and lymphocyte, lymphocyte proliferation, concentration of CD4+lymphocyte subset, the scrum concentration of IFN-y and cortisol were changed quadratically (P<0.05) with the increased dose of dietary ASPS, IL-2was changed linearly (P<0.05). However, concentration of specific OVA antibody, IgG, IgA, IgM, IL-4, CD8+lymphocyte subset, ADFI, CD4+to CD8+were not affected (P>0.05) by ASPS. For improving growth and enhancing immune response, optimal supplementation level might be800mg/kg in weaned pigs.The result indicates that ASPS supplementation can improve growth performance and regulate cellular immunity as well as relieve stress response in weaned pigs.
(2) Experiment2was conducted to further reseach the effects of ASPS on growth depression and its immunoregulatory role in pigs challenged with LPS. A2×2factorial design experiment including main factors such as dietary treatment and immunological challenge was conducted with64piglets which were assigned to two dietary treatments (each treatment was replicated in8pens). Piglets in4pens per dietary treatment were injected with LPS or sterile saline on day14and21. Blood samples were obtained3h and the next day after injection for analysis of blood parameters. The results showed that ASPS did not affect growth performance of piglets prior to LPS injection (P>0.05). From day15to21, ASPS improved ADG and ADFI (P<0.05) and has interaction relationship with LPS (P<0.05). Lymphocyte proliferation, CD4+, CD8+lymphocyte subset on day15were increased, and CD4+/CD8+was relieved to avoid increasing by ASPS(P<0.05). ASPS relieved the increased plasma concentration of IL-1β, IL-6, TNF-a, PGE2, cortisol, glucose, NEFA and a-AGP(P<0.05). GH, IGF-I, TP and IL-10were significantly increased by dietay ASPS (P<0.05). However, IL-4and insulin were not affected (P>0.05) by ASPS. This study suggests that dietary ASPS relieve growth depression through decreasing concentration of IL-1β, IL-6, and TNF-a but not controlling the activity of immune system. ASPS improve cellular immunity of stress pigs not only by direct effect on lymphocyte, but also by the indirect role through regulating the nerve-endocrine-immune system.
(3) Experiment3possessed the same experimental design with (2) was used to further evaluate the immnoregulatory mechanism of ASPS on growth depression and immune function in stress pigs. Spleen was taken out from the slaughtering pigs after3h of LPS-injection on21d, using real-time fluorescent quantitative PCR to study the effect of ASPS on IL-1β, IL-6, and TNF-α mRNA gene expression. The results showed that dietary ASPS could decrease IL-1β、IL-6and TNF-α mRNA transcription in spleen(P<0.05) and showed the interaction relationship with LPS(P<0.05). namely, ASPS relieved the increasion of transcription levels of IL-6、IL-1β and TNF-α mRNA induced by LPS (P<0.05). The results suggest that ASPS play the role of immunoregulation by reducing expression level of IL-6、 IL-1β and TNF-α mRNA of spleen in stress pigs.
(4) Experiment4researched the effects of ASPS in different level on signal transduction pathway of cultured peripheral blood lymphoeyte of weaned piglets. The results showed that lymphoeyte proliferation of T cell was changed quadratically (P<0.05) with the increased dose of ASPS.80、160、320μg/mL ASPS in higher dose affacted significantly(P<0.05). ASPS did not affect lymphoeyte proliferation of cultured B cell and IL-4(P>0.05). IL-2was changed quadratically (P<0.05) with the increased dose of ASPS at24h、48h and72h after culture, The concentration of NO、iNOS and NF-κB and TNF-α changed quadratically (P<0.05)with ASPS in no anti-TLR4group and they did not change in anti-TLR4group(P>0.05). ASPS improved the level of cAMP(P<0.05) and cAMP/cGMP(P<0.05), decreased the concentration of cGMP(P<0.05) at20min and60min after cultivation. The results suggest that ASPS can improve cellular immune function of cultured lymphoeyte in vitro, and it works through combinating with TLR4receptor of lymphoeyte surface and affecting the concentration of sigal molecules such as NO. cAMP、cGMP, influencing the cytokine of TNF-α by increasing the activity of NF-κB.
引文
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