图 1(Figure 1)
表 1(Table 1)
抗菌肽Sublancin与黄芪多糖对免疫抑制小鼠免疫功能调节作用的比较分析
引用本文
尚丽君, 杨天任, 于海涛, 黄烁, 曾祥芳, 谯仕彦. 抗菌肽Sublancin与黄芪多糖对免疫抑制小鼠免疫功能调节作用的比较分析[J]. 畜牧兽医学报, 2019, 50(2): 406-414.
SHANG Lijun,
YANG Tianren,
YU Haitao,
HUANG Shuo,
ZENG Xiangfang,
QIAO Shiyan.
Immunomodulation Effect of Antimicrobial Peptide Sublancin and Astragalus Polysaccharides on Immunosuppressed Mice: A Comparative Study[J]. Acta Veterinaria et Zootechnica Sinica, 2019, 50(2): 406-414.
抗菌肽Sublancin与黄芪多糖对免疫抑制小鼠免疫功能调节作用的比较分析
1. 中国农业大学动物科技学院, 北京 100193;
2. 生物饲料添加剂北京市重点实验室, 北京 100193
2. 生物饲料添加剂北京市重点实验室, 北京 100193
摘要：旨在比较研究抗菌肽sublancin与黄芪多糖对环磷酰胺（cyclophosphamide，CTX）诱导的免疫抑制小鼠的免疫功能的调节作用。试验选取60只4~6周龄健康雌性BALB/c小鼠，随机分为6个处理组。正常对照组：第1-3天，腹腔注射生理盐水；第4-10天，灌胃生理盐水。其余五个处理组：第1-3天，腹腔注射CTX（80 mg·kg-1）；第4-10天，阴性对照组：灌胃生理盐水；低浓度抗菌肽组：灌胃4.0 mg·kg-1抗菌肽sublancin；高浓度抗菌肽组：灌胃8.0 mg·kg-1抗菌肽sublancin；黄芪多糖组：灌胃200.0 mg·kg-1黄芪多糖；阳性对照组：灌胃10.0 mg·kg-1左旋咪唑。所有处理均为每天一次，每次0.2 mL。于试验第1天小鼠腹腔注射CTX前、第4天灌胃前和第11天对小鼠进行称重。第11天称重结束后，采集全部小鼠的外周血及脾，检测外周血生理生化指标、CD4+与CD8+数量和脾细胞的细胞因子mRNA表达等指标。结果表明，与正常对照组相比，阴性对照组中小鼠体重、外周血的红细胞、血红蛋白和白细胞含量显著降低（P < 0.05），脾IL-2、IL-4和IL-6的基因表达显著降低（P < 0.05）。灌胃后，与阴性对照组相比，黄芪多糖组和高浓度sublancin组体重无差异但有升高趋势，说明高浓度sublancin与黄芪多糖对小鼠体重有积极影响；低、高浓度sublancin、黄芪多糖组和阳性对照组中小鼠外周血的白细胞含量显著升高（P < 0.05）；高剂量sublancin组中小鼠外周血的红细胞和血红蛋白显著升高；低、高浓度sublancin和黄芪多糖组中小鼠外周血的CD4+显著升高（P < 0.05）；低、高浓度sublancin、黄芪多糖组和阳性对照组中小鼠外周血的CD8+显著降低（P < 0.05）；低、高浓度sublancin组中小鼠外周血的CD4+/CD8+细胞显著升高（P < 0.05）；低、高浓度sublancin、黄芪多糖组和阳性对照组中小鼠脾IL-4的表达均显著升高（P < 0.05）；高浓度sublancin、黄芪多糖组和阳性对照组中小鼠脾IL-2、IL-6的表达显著升高（P < 0.05）。综合上述结果，适宜剂量的抗菌肽sublancin和黄芪多糖均可缓解环磷酰胺造成的免疫抑制。与200.0 mg·kg-1黄芪多糖相比，8.0 mg·kg-1抗菌肽sublancin对缓解环磷酰胺免疫抑制造成的细胞因子降低的效果更好。
关键词：抗菌肽 sublancin 免疫调节 环磷酰胺 黄芪多糖
Immunomodulation Effect of Antimicrobial Peptide Sublancin and Astragalus Polysaccharides on Immunosuppressed Mice: A Comparative Study
1. College of Animal Science and Technology, China Agricultural University, Beijing 100193, China;
2. Beijing Key Laboratory of Biological Feed Additive, Beijing 100193, China
2. Beijing Key Laboratory of Biological Feed Additive, Beijing 100193, China
Abstract: The purpose of this study was to evaluate the immunomodulatory effects of antimicrobial peptide sublancin and astragalus polysaccharides (APS) on immunosuppressive mice induced by cyclophosphamide (CTX). Sixty healthy female BALB/c mice aged 4-6 weeks were randomly divided into 6 groups of 10 each. Normal control group:d 1 to d 3, intraperitoneally injected with 0.9% physiological saline, d 4 to d 10, gavage 0.9% physiological saline. The other 5 groups:d 1 to d 3, injected with CTX (80 mg·kg-1). On the d 4 to d 10, the negative control group:gavage 0.9% physiological saline; low or high concentration sublancin treatment group:sublancin solution was administered at a dose of 4.0 and 8.0 mg·kg-1; the APS-treated group:gavage APS (200 mg·kg-1); positive control group:gavage levamisole (10 mg·kg-1, BW). All treatments were performed once a day and 0.2 mL each time. The mice were weighed before the mice were intraperitoneally injected with CTX on the first day of the experiment, on the d 4 before gavage and on the d 11. After weighing the body weight on the d 11, peripheral blood and spleen were collected. Compared with the normal control group, the peripheral blood red blood cell, hemoglobin, and white blood cell counts in the negative control group were significantly reduced (P < 0.05); the gene expression of IL-2, IL-4, and IL-6 in the spleen was significantly reduced (P < 0.05). Compared with the negative control group, there was no difference in body weight between the APS group and the high concentration Sublancin group after intragastric administration but there is an upward trend, indicating that high concentration Sublancin and APS had a positive effect on body weight of mice; the leukocyte content in peripheral blood of low and high concentration Sublancin, APS, and positive control groups were significantly increased (P < 0.05); in the high concentration Sublancin-treated group, red blood cells and hemoglobin were significantly increased (P < 0.05); CD4+ levels in peripheral blood of mice treated with low or high concentration Sublancin and APS were significantly increased (P < 0.05); in both low and high concentration Sublancin, APS and positive control groups, CD8+ was significantly decreased (P < 0.05); in low and high concentration sublancin groups, CD4+/CD8+ was significantly increased (P < 0.05); in low and high concentration sublancin, APS and positive control groups, mouse spleen IL-4 were significantly increased (P < 0.05); in high concentration sublancin, APS and positive control groups, the expressions of IL-2 and IL-6 in the spleen of mice were significantly increased (P < 0.05). In summary, it can be concluded that appropriate doses of the antimicrobial peptides sublancin and APS can all alleviate the immunosuppression caused by cyclophosphamide. Compared with APS, the role of antimicrobial peptide sublancin in the effect on alleviating the immunosuppression of CTX is more comprehensive.
Key words:
antimicrobial peptide
sublancin
immunomodulation
cyclophosphamide
astragalus polysaccharide
用作补充或替代药物的免疫调节剂已经成为治疗免疫疾病的常用手段。抗菌肽是生物进化史上最古老的抗微生物感染多肽[1]，不仅具有广谱抗菌活性，而且具有调节炎症反应、免疫细胞趋化、促进细胞分化和激活先天性免疫等多种免疫调节功能[2-3]，因此具有作为新型免疫调节剂的潜力。抗菌肽subalncin的多种抗菌活性已被证实，对革兰阳性菌具有强力的广谱抗菌活性[4]。Sublancin是从枯草芽孢杆菌发酵产物中分离鉴定到一种抗菌肽[4]，可耐受高低pH[5]。王帅[6]的研究表明，抗菌肽sublancin可提高免疫抑制小鼠外周血红细胞、血小板和白细胞的含量以及显著上调脾中细胞因子的基因表达。黄芪多糖是中国农业农村部批准使用的免疫增强剂[7]，具有多重免疫调节功能，包括调节细胞因子的不平衡以及影响T细胞的生长等[8]，从而增强机体免疫能力。环磷酰胺(cyclophosphamide, CTX)可以对机体骨髓、胃肠道及免疫系统产生明显的抑制作用[9]，常用来制备免疫抑制模型[10-11]。为比较口服抗菌肽sublancin和黄芪多糖对机体的免疫调节能力，建立CTX诱导的小鼠免疫抑制模型，继而比较二者对模型动物免疫抑制的缓解作用，从而探讨sublancin在动物免疫抑制中的免疫增强作用。
1 材料与方法 1.1 试验材料黄芪多糖：由北京爱迪森生物科技有限公司生产提供，生产批号1703241，1 g粉末含黄芪多糖不少于450 mg；抗菌肽sublancin：由国家饲料工程技术研究中心提供，纯度为99.6%；左旋咪唑和CTX：美国Sigma公司。
4~6周龄健康雌性BALB/c小鼠购自北京华阜康生物科技股份有限公司。本试验在农业部饲料效价和安全评价监督检验测试中心(北京)鼠营养代谢室进行。电脑控制鼠房温度为18~22 ℃，相对湿度为35%~55%，昼夜光照交替时间为12 h: 12 h。小鼠自由采食和饮水，小鼠饲粮购自北京华阜康生物科技股份有限公司。
1.2 试验设计将60只小鼠随机分为6组，每组10只，试验期10天。正常对照组前3 d腹腔注射生理盐水，而后灌胃生理盐水7 d；其余各组先腹腔注射80.0 mg·kg-1的CTX连续3 d，而后分别灌胃7 d不同物质：阴性对照组灌胃生理盐水；低、高剂量抗菌肽组分别灌胃4.0和8.0 mg·kg-1的sublancin；黄芪多糖组灌胃200.0 mg·kg-1的黄芪多糖；阳性对照组选取传统的免疫调节剂左旋咪唑(10.0 mg·kg-1)灌胃。试验中各处理均为每天一次，每次0.2 mL。在试验第1天注射CTX前、试验第4、11天记录小鼠体重，最后一次记录体重后采集全部小鼠的外周血及脾，检测血清中CD4+、CD8+细胞数量及比例、血常规和脾细胞细胞因子mRNA表达情况等指标(如图 1)。
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图 1 试验设计 Figure 1 Experiment design |
小鼠眼球采血，注入EDTA-K2抗凝管。采血完成后，将小鼠脱臼处死，无菌取脾。
1.4 检测指标 1.4.1 体重于试验第1天小鼠腹腔注射环磷酰胺前、第4天灌胃前和最后一次灌胃结束后24 h对小鼠进行称重。
1.4.2 血液指标的测定小鼠眼球采血，注入EDTA-K2抗凝管中，采用Coulter LH 755全自动血细胞分析仪测定外周血血常规指标，包括白细胞(white blood cell，WBC)、红细胞(red blood cell，RBC)、血红蛋白(hemoglobin，HGB)和血小板(platelets，PLT)。
1.4.3 细胞因子的测定RNA提取按美基生物通用型RNA小量提取试剂盒(上海伯易生物科技有限公司)操作说明进行，并使用NanoDrop 2000分光光度计(Termo Fisher Scientifc，MA，USA)测定RNA量和质量。随后反转录为cDNA。使用Primer 3.0设计引物，引物序列见表 1。采用SYBR Green Ⅰ染料法在ABI StepOnePlusTM实时荧光定量PCR扩增仪上进行扩增。
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表 1 引物序列 Table 1 Primers for gene expression test by using real-time PCR |
将采集的外周血用EDTA-K2抗凝后通过流式细胞术(FCM)检测CD4+、CD8+和CD4+/CD8+细胞水平。PE Anti-mouse CD8与PE-Cy5 Anti-mouse CD4由北京四正柏生物科技有限公司提供，流式细胞仪采用BD(Becton, Dickinson and Company)公司FACS CantoⅡ型流式细胞仪。
1.5 统计方法用SAS 9.4中的一般线性模型(GLM)程序对数据进行单因素方差分析，方差分析差异显著者，以Duncan法比较平均数间的差异显著性。统计结果以“x±s”表示，P＜0.05表示差异显著。
2 结果 2.1 体重各组小鼠的体重变化见表 2。在注射CTX前，各组间小鼠体重无显著差异(P＞0.05)；连续注射CTX 3 d构建免疫抑制模型后，与正常对照组相比，腹腔注射环磷酰胺的小鼠体重显著下降(P＜0.05)。试验结束后，除对照组外，各试验组之间无显著差异，但是治疗组小鼠体重比模型组高，尤其是黄芪多糖组和高浓度sublancin组，说明对小鼠体重有积极影响。
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表 2 各组小鼠体重变化(x±s) Table 2 Effects of body weight in CTX treated mice (x±s) |
与正常对照组比较，阴性对照组小鼠外周血中RBC、HGB和WBC含量显著下降(P＜0.05)。高浓度抗菌肽组小鼠外周血中RBC、HGB含量均显著高于模型组(P＜0.05)；低、高浓度抗菌肽、黄芪多糖和阳性对照组中，小鼠外周血中WBC含量显著高于阴性对照组(P＜0.05)；各组间血小板无明显差异(表 3)。
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表 3 对小鼠血液指标的影响(x±s) Table 3 Effects on blood routine index in CTX treated mice (x±s) |
对CTX诱导免疫抑制小鼠的脾细胞因子基因转录的影响见表 4。阴性对照组小鼠脾IL-2、IL-4和IL-6相应基因转录显著低于正常对照组(P＜0.05)。高浓度抗菌肽、黄芪多糖和阳性对照组中小鼠脾IL-2和IL-6相应基因转录显著高于阴性对照组(P＜0.05)，并且与正常对照组差异不显著性(P＞0.05)；此外，低、高浓度抗菌肽、黄芪多糖和阳性对照组中小鼠脾IL-4的转录均显著高于阴性对照组(P＜0.05)。
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表 4 抗菌肽sublancin对环磷酰胺免疫抑制小鼠脾细胞因子基因转录的影响(x±s) Table 4 Effects of sublancin on cytokine transcrption in spleen in CTX treated mice(x±s) |
如表 5所示，与阴性对照组相比，低、高浓度的抗菌肽sublancin灌胃7 d后，均使小鼠CD4+水平显著升高(P＜0.05)，CD8+水平显著降低(P＜0.05)。两个抗菌肽组CD4+/CD8+显著上升(P＜0.05)，低浓度组与正常对照组相比无显著差异(P＞0.05)，高浓度组显著高于正常对照组(P＜0.05)。黄芪多糖仅使CD4+显著提升(P＜0.01)。
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表 5 对小鼠T淋巴细胞亚群的影响(x±s) Table 5 The effect on T cell subsets of peripheral blood (x±s) |
免疫系统通过多层防御机制来保护机体免受疾病感染[12]，它在抵御疾病感染和治疗疾病中起着不可替代的作用[13]。因此免疫调节剂与传统治疗法相比具有明显的优势，成为当前研究热点之一[14]。抗菌肽的免疫调节作用已被大家熟知[15]。之前也有文献报道了sublancin在耐甲氧西林金黄色葡萄球菌(MRSA)感染期间，通过平衡免疫应答并通过抑制NF-κB活化来缓解肠道炎症。由于其具有抗菌和免疫调节活性，因此sublancin可能具有治疗抗药性感染和败血症的潜力[16]。中草药同样具有免疫调节作用，从中药中分离出的多糖因其低毒性和强免疫增强作用而引起了人们的关注[17]。
为了对比评估口服抗菌肽sublancin和黄芪多糖对免疫抑制小鼠的免疫调节作用，笔者使用CTX建立了小鼠的免疫抑制模型。CTX是一种重要的化疗药物，主要用于治疗癌症，但也可引起机体免疫功能紊乱[18]，损伤机体DNA，杀死免疫细胞，干扰巨噬细胞、T细胞和B细胞的增殖和分化[19-21]。因此，CTX常用于建立免疫缺陷或免疫抑制模型。之前的研究表明，CTX对小鼠造成免疫抑制会表现为骨髓抑制以及各细胞因子的变化[22-23]。在该试验中，小鼠腹腔内注射80 mg·kg-1的CTX后，各项指标的检测结果表明成功建立免疫抑制模型。然而与之前文献中不一致的是，模型组中血小板的量高于正常对照组。笔者推测可能是由于骨髓造血干细胞中含有高度表达的酶(醛脱氢酶)，可以分解CTX从而保护自身免受损伤[24]。
部分抗菌肽作为趋化因子的作用以及诱导趋化因子的产生，导致在感染部位招募白细胞和淋巴细胞，促进伤口愈合和调节适应性免疫。免疫系统受细胞因子网络调节。IL-2是由T细胞产生的一种多效性细胞因子，可驱动T细胞生长，增强NK细胞溶解活性，诱导调节性T细胞分化并介导活化诱导的细胞死亡，在免疫系统中发挥关键作用[12, 25]。IL-4可促进Th2细胞的发育。Th2参与体液免疫，并产生抗炎细胞因子IL-4。而IL-4又可以拮抗IFN-γ并通过转录因子GATA-3的作用使T细胞极化为2型表型[26]。IL-6是由多种免疫细胞产生的急性炎症的重要介质[27]，通过刺激骨髓来源的抑制细胞(myeloid-derived suppressor cells，MDSCs)、嗜中性粒细胞、巨噬细胞和C-反应蛋白的产生在先天免疫应答中起重要作用[28-30]。腹腔注射环磷酰胺显著抑制了脾中IL-2、IL-4和IL-6的表达。Hou等[31]发现提高脾中IL-2、IL-4和IL-6的表达，可以缓解CTX引起的免疫抑制。高浓度sublancin组和黄芪多糖组均能使以上细胞因子的mRNA表达量显著上升，而且与空白对照组没有显著差别。这表明抗菌肽sublancin和黄芪多糖都可以通过增加上述脾中细胞因子mRNA的表达而在免疫抑制小鼠中发挥免疫调节功能，从而缓解免疫抑制。
骨髓抑制是指骨髓中血细胞前体活性的降低，是癌症患者接受化疗时遇到的主要挑战之一。一般来说，CTX可通过影响造血干细胞的增殖、分化和凋亡来引起骨髓抑制[32]。造血干细胞是能够自我更新并转化为成熟血细胞的多能细胞，如RBC、WBC和HGB。之前有研究表明，提升外周血中WBC计数可以缓解骨髓抑制[33]。之前有研究表明，黄芪多糖可改善CTX致小鼠外周血红细胞、白细胞等被抑制的现象[34]，提高骨髓细胞增殖[35]。此外，在本实验室前期的研究中，sublancin可以改善环磷酰胺诱导免疫抑制小鼠外周血中红细胞、白细胞、血红蛋白和血小板的数量，从而缓解环磷酰胺对小鼠造成的骨髓抑制[6]。本试验中，sublancin与黄芪多糖均改善了环磷酰胺致免疫抑制小鼠外周血中红细胞、白细胞以及血红蛋白的数量，说明两种药物均对小鼠的骨髓抑制有缓解作用。高浓度抗菌肽sublancin效果优于黄芪多糖，使RBC、WBC和HGB均显著增加。这说明sublancin可以有效缓解CTX对造血系统和免疫细胞的损伤，调节机体免疫功能，使CTX诱导的骨髓抑制被抑制或逆转，并且sublancin的免疫调节效果比黄芪多糖更加全面。
正常的免疫反应取决于免疫细胞，特别是T细胞亚群之间的相互制约。免疫功能紊乱通常是由T细胞亚群之间的不平衡引起的[36-37]。根据成熟的T淋巴细胞表面分化表达受体CD4+和CD8+分为辅助性T细胞(CD4+)和细胞毒性T细胞(CD8+)两大亚群。调节性T细胞(Treg)占外周血CD4+T细胞群的5%~10%，许多文献一致地报道了在啮齿动物模型中CTX给药后Treg细胞的下降以及Treg细胞比例的改变，这可能反映了细胞数量以及不同淋巴细胞亚群间平衡的改变[12, 22, 38-43]。笔者的试验显示，sublancin和黄芪多糖均能使CD4+水平升高。说明二者对于环磷酰胺引起的T细胞失衡均有缓解作用。此外，现代免疫学认为，外周血中T细胞亚群的数量和CD4+/CD8+比值是评估免疫平衡最重要的参数之一，也是疾病严重程度和预后的重要标志[36]。CD4+/CD8+的高比例表明免疫系统被高度激活，反之亦然。Sublancin(4.0或8.0 mg·kg-1)能使CD4+/CD8+的比值显著增加，而黄芪多糖未能发挥这种作用。由此可见，抗菌肽sublancin还具有激活机体免疫的能力，在调节T细胞亚群平衡、调动机体免疫方面更优于黄芪多糖。
4 结论通过对比空白对照组和模型组小鼠体重、血常规、脾细胞因子mRNA的表达情况等可知，本试验成功构建了环磷酰胺致小鼠免疫抑制模型。适宜剂量的sublancin与黄芪多糖及左旋咪唑相似，可增强小鼠先天性免疫功能，还可缓解环磷酰胺造成的免疫抑制；在缓解骨髓抑制和调节T细胞亚群平衡方面效果优于黄芪多糖，为其作为新一代免疫调节剂提供了理论依据。
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