动物营养学报    2019, Vol. 31 Issue (11): 5378-5390    PDF    
慢性铅胁迫对日本沼虾硫氧还蛋白和热应激蛋白系统基因表达及肠道菌群的影响
郑纯纯 , 李小雨 *, 聂欢 , 周厚杰 , 丁志丽 , 田钰滢 , 杜明川 , 刘翥     
浙江省水生生物资源养护与开发技术研究重点实验室, 中国水产科学研究院水生动物繁育与营养重点实验室, 湖州师范学院生命科学学院, 湖州 313000
摘要: 本试验旨在研究水体铅的慢性胁迫对日本沼虾硫氧还蛋白和热应激蛋白系统基因表达及肠道菌群的影响。将450尾均重为(0.10±0.02)g的日本沼虾随机分为3组,每组3个重复,每个重复50尾,进行为期60 d的慢性胁迫试验。水体铅的胁迫浓度分别为0(对照组)、13.13和26.26 μg/L。试验结束后,采用实时荧光定量PCR分析肝胰腺中硫氧还蛋白(Trx)、硫氧还蛋白还原酶(TrxR)、热应激蛋白60(HSP60)、热应激同源蛋白70(HSC70)和热应激蛋白90(HSP90)mRNA相对表达量,同时通过16S rRNA高通量测序对日本沼虾肠道菌群组成及多样性进行分析。结果发现:1)慢性铅胁迫抑制TrxRTrx mRNA表达,26.26 μg/L组TrxRTrx mRNA相对表达量显著低于对照组(P < 0.05)。2)随着铅浓度的增加,HSP60和HSC70 mRNA相对表达量逐渐降低,26.26 μg/L组HSP60和HSC70 mRNA相对表达量显著低于对照组(P < 0.05),而HSP90 mRNA相对表达量呈先增加后降低的趋势,13.13 μg/L组HSP90 mRNA相对表达量显著高于其余2组(P < 0.05)。3)从门水平上分析日本沼虾所有组的肠道菌群,其主要以3个优势菌门为主,分别为变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和软壁菌门(Tenericutes)。采用常规方差统计肠道菌群丰度发现,未分类气单胞菌种(Aeromonas_unclassified)的相对丰度在铅胁迫后显著下降(P < 0.05)。采用线性判别分析效应值(LEfSe)方法分析肠道宏基因组,发现对照组与26.26 μg/L组肠道菌群存在差异,对照组中肠杆菌属(Enterobacteriales)、肠杆菌科(Enterobacteriaceae)、肠杆菌目(Enterobacter)、柠檬杆菌属(Citrobacter)、疣微菌目(Verrucomicrobiae)、疣微菌纲(Verrucomicrobiales)、疣微菌科(Verrucomicrobiaceae)相对丰度较高,而26.26 μg/L组中变形菌纲(Deltaproteobacteria)、黄杆菌纲(Flavobacteriia)、黄杆菌目(Flavobacteriaceae)、黄杆菌科(Flavobacteriales)、黄杆菌属(Flavobacterium)、拟杆菌门(Bacteroidetes)和井杆菌属(Phreatobacter)相对丰度较高。由此可见,慢性铅胁迫降低日本沼虾肝胰腺TrxTrxR的转录水平,调控HSP60、HSC70和HSP90 mRNA相对表达量,且日本沼虾肠道内存在不受慢性铅胁迫干扰的核心微生物门类,但高浓度的铅胁迫会产生丰度有显著性差异的菌群,维持正常代谢的菌群丰度减少,与降解污染物、调节机体免疫和抗氧化应激相关的菌群丰度增加。
关键词: 热应激蛋白    硫氧还蛋白    肠道菌群        日本沼虾    
Effects of Chronic Lead Exposure on Expressions of Thioredoxin and Heat Shock Protein System Genes and Intestinal Microbiota in Macrobrachium nipponense
ZHENG Chunchun , LI Xiaoyu *, NIE Huan , ZHOU Houjie , DING Zhili , TIAN Yuying , DU Mingchuan , LIU Zhu     
Zhejiang Provincial Key Laboratory of Aquatic Resources Conservation and Development, Key Laboratory of Aquatic Animal Genetic Breeding and Nutrition of Chinese Academy of Fishery Sciences, College of Life Science, Huzhou University, Huzhou 313000, China
Abstract: The aim of this study was to investigate the effects of chronic lead exposure on expressions of thioredoxin and heat shock protein system genes and intestine microbiota in Macrobrachium nipponense. Juvenile M.nipponense with an average weight of (0.10±0.02) g were randomly divided into three groups in three replicates (50 tails/replicate) for chronic lead exposure trial. The concentration of lead in water were 0 (control group), 13.13 and 26.26 μg/L and exposure time lasted for 60 days. After the exposure trial, quantitative real-time polymerase chain reaction (qRT-PCR) was used to analyze the mRNA expressions of thioredoxin (Trx), thioredoxin reductase (TrxR), heat shock protein 60 (HSP60), heat shock cognate protein 70 (HSC70) and heat shock protein 90 (HSP90). Meanwhile, the composition and diversity of intestine microbiota were analyzed by 16s RNA high throughput sequencing. The results showed as follows 1) chronic lead exposure inhibited the mRNA expressions of TrxR and Trx. The mRNA relative expressions of TrxR and Trx in the 26.26 μg/L group were significantly lower than those in the control group (P < 0.05). 2) With the increase of lead concentration, the mRNA relative expressions of HSP60 and HSC70 decreased gradually, and the mRNA relative expressions of HSP60 and HSC70 in 26.26 μg/L group were significantly lower than those in control group (P < 0.05). But the mRNA relative expressions of HSP90 increased first and then decreased, and the HSP90 mRNA relative expressions in 13.13 μg/L lead group was significantly higher than that in the other two groups (P < 0.05). 3) Analysis of intestinal bacteria of all processing groups of M.nipponense at phyla level, three dominant phyla, Proteobacteria, Firmicutes and Tenericutes were observed in the three groups at the phylum level in M.nipponense. It was found that the relative abundance of Aeromonas_unclassified significantly decreased after lead stress by the conventional variance analysis approach. Intestinal metagenome was analyzed by LEfSe approach, and it was found that there were differences in intestinal bacteria between the control group and the 26.26 μg/L group. The results showed that the relative abundant of Enterobacteriales, Enterobacteriaceae, Enterobacter, Citrobacter, Verrucomicrobiae, Verrucomicrobiales, Verrucomicrobiaceae were higher in the control group, and the relative abundant of Deltaproteobacteria, Flavobacteriia, Flavobacteriales, Flavobacteriaceae, Flavobacterium, Bacteroidetes, Phreatobacter were higher in the 26.26 μg/L group. Thus it can be concluded that chronic lead exposure decreases the transcription level of Trx and TrxR genes and regulates the mRNA relative expressions of HSP60, HSC70 and HSP90 in the hepatopancreas of M.nipponense. Regardless of the concentration of the lead exposure, there are core microorganisms in the intestinal tract of M.nipponense, but high-concentration lead exposure will produce bacteria with significant difference in abundance, and the abundance of bacteria to maintain normal metabolism will be reduced, and the bacteria which is related to the degradation of pollutants and the regulation of immune and antioxidant stress will increase.
Key words: heat shock protein    thioredoxin    intestinal microbiota    lead    Macrobrachium nipponense    

随着现代工业化的快速发展,许多含有重金属离子的污染物进入包括水生系统在内的生态环境中[1]。由于铅具有持久性积累和高毒性,其已成为对人类和其他生物最危险的金属之一[2]。对哺乳动物的研究发现,铅对中枢神经和外周神经系统、造血系统、心血管系统、肾脏、生殖器官和抗氧化防御系统都有不良影响[3-6]

在水生系统中,急性或慢性铅胁迫可干扰水生生物的生理和生化反应,改变血液酶活性等生化指标[7-8],降低抗氧化能力[9],产生活性氧(ROS)并诱导氧化应激[10]。体内氧化还原系统如硫氧还蛋白(thioredoxin,Trx)系统是参与去除细胞内ROS毒害的重要功能蛋白,在抗细胞氧化应激、维持细胞内蛋白质还原状态并发挥正常功能等方面起着重要的作用[11]。事实上,机体为了应对氧化应激,除了可通过抗氧化防御系统来消除ROS之外,分子伴侣在这个过程中也起到重要的作用。热应激蛋白(heat shock protein,HSP)作为分子伴侣,不仅在亚致死应激条件下维持蛋白质稳态方面发挥着关键作用[12],而且可作为水生系统中重金属污染监测的敏感分子生物标志物[13]

近年来,关于肠道菌群与环境重金属污染密切相关的研究也有初步报道[1, 14-15]。众所周知,动物的肠道不仅是吸收营养的场所,也是重要的免疫器官[16]。稳定的肠道菌群可以对宿主的健康起到有益的作用[17-19],然而,有关鱼和虾类等水生动物的肠道菌群对重金属污染胁迫的反应鲜有研究报道。因此,为了进一步揭示重金属污染的毒理机制,加强对肠道菌群分析与水生动物应激生理关系的认识显得至关重要。

日本沼虾是一种在经济和营养上重要的水产养殖品种,属甲壳纲,十足目,长臂虾科,沼虾属,广泛分布于中国、日本和东南亚国家[20],我国的主要产区集中在河北的白洋淀、江浙的太湖等地区[21]。已有的研究发现,日本沼虾对养殖环境如饲养密度[22]、水体亚硝酸盐含量[23]和硫化物含量[24]等应激胁迫感知灵敏,而合理的营养是应对各种应激胁迫简单有效的措施[6, 25]。本研究拟从肝胰腺中TrxHSP系统基因表达以及肠道菌群着手,揭示日本沼虾应对铅胁迫的可能机制。

1 材料与方法 1.1 试验动物及饲料

试验用日本沼虾购于当地1个养殖场,在慢性胁迫试验之前,暂养1周以适应试验条件。日本沼虾摄食饲料为浙江一星实业公司提供的商业饲料,该饲料含39.2%粗蛋白质、6.3%粗脂肪、9.6%粗纤维、4.3%粗灰分以及40.6%无氮浸出物。

1.2 慢性胁迫试验

根据先前的急性胁迫试验,得到日本沼虾96 h铅半致死浓度(LC50)为131.31 μg/L[26]。将1.83 g乙酸铅(CAS6080-56-4;纯度为99.5%)溶解在1 L ddH2O中,制备浓度为1 g/L母液,根据试验胁迫浓度进行稀释使用。

选择450尾健康状况良好、个体大小基本一致[(0.10±0.02) g]的日本沼虾,随机分为3组,水体铅的胁迫浓度分别为0(对照组)、13.13和26.26 μg/L,每组3个重复,每个重复50尾虾,随机放入白水槽内,每个水槽内放置一定量的网片作为虾的躲避物,以减少虾为争抢饲料而进行互相残杀。

采用静水养殖系统,养殖期间,每天投喂饲料2次,时间为08:00和16:00,投喂量为体重的6%,光照为自然光照,养殖用水为曝气的自来水。为了保持养殖水质,每天吸出虾的排泄物并换1/3的养殖水量。在整个慢性胁迫试验过程中,水质保持稳定[溶氧含量>6.5 mg/L,氨氮和硝酸盐含量 < 0.1 mg/L,水温(26±1) ℃],养殖时间为60 d。

1.3 样本采集

慢性胁迫试验结束后,饥饿处理1 d,采集各组虾肝胰腺,保存于-80 ℃超低温冰箱,同时每个水槽取10尾虾,在无菌条件下,采集虾的肠道用于16S rRNA高通量菌群测定分析。

1.4 细菌基因组DNA提取

使用微量样品基因组DNA提取试剂盒[天根生化科技(北京)有限公司]提取10尾虾肠道内菌群DNA,琼脂糖电泳检测提取效果,分光光度计(Thermo Fisher Scientific,美国)测量DNA产量。随后,将DNA样本送至元莘生物医药科技有限公司进行高通量测序分析。

1.5 Illumina高通量测序和处理

用原核生物通用引物F341/R806(F341:5′-CCTAYGGGRBGCASCAG-3′和R806:5′-GGACTACHVGGGTWTCTAAT-3′)扩增细菌16S rRNA基因V3~V4区域,每个引物添加独特的八碱基条形码以区分PCR产物。PCR反应体积为20 μL,包含5×FastPfu缓冲液4 μL、2.5 mmol/L dNTPs 2 μL、引物各0.8 μL(5 mmol/L)、FastPfu聚合酶0.4 μL、模板DNA 10 μL(1 ng/μL),补ddH2O至20 μL。PCR反应条件如下:95 ℃预变性5 min;95 ℃变性30 s,55 ℃退火30 s,72 ℃延伸45 s,循环27次;最后72 ℃延伸10 min。纯化后的PCR产物进行高通量测序,结果已上传至NCBI,SRA登录号为SRP158859。

使用QIIME(1.17版)对原始读取数据进行了分析和质量筛选,并用Usearch(7.1版)对有效的数据进行归类,设定97%以上相似度的序列为一个操作分类单元(OTU)。用70%的置信度阈值的RDP分类器与Silva 16S rRNA数据库对比,分析了每个16S rRNA基因序列的亲缘分类关系。用Mothur(v.1.30.1版)确定分类的丰富度估计数和群落多样性,包括Chao指数在内的α多样性指数来反映群落的丰富度,Shannon指数和Simpson指数来反映群落的多样性,覆盖率来检测该群体中鉴定的16S rRNA基因序列是否能代表本研究样本中的大多数细菌。使用未加权的Unifrac距离评价细菌群落之间的β多样性,对非加权距离矩阵的Unifrac距离进行主坐标分析(principal co-ordinates analysis,PCoA),以研究菌群组成和结构上的差异。用线性判别分析(linear discriminant analysis,LDA)效应值(LEfSe)法(http://huttenhower.sph.harvard.edu/lefse/)从统计学意义和生物学相关性确定铅胁迫后的肠道菌群特征,用于发现生物标志物。

1.6 基因表达的实时荧光定量PCR(qRT-PCR)分析

采用组织/细胞RNA快速提取试剂盒(艾德莱,中国)提取各组肝胰腺总RNA,用1%琼脂糖凝胶电泳检测RNA的完整性,与此同时,RNA纯度和浓度用Thermo NanoDrop 2000核酸蛋白测定仪检测。用反转录试剂盒(TaKaRa,日本)将总RNA反转录合成cDNA,并于-20 ℃保存备用。

用qRT-PCR对胁迫后各组虾肝胰腺中Trx(登录号KY465768)、硫氧还蛋白还原酶(thioredoxin reductase,TrxR)(登录号KY465769)、热应激蛋白60(heat shock protein 60,HSP60)(登录号KF028596)、热应激同源蛋白70(heat shock cognate protein 70,HSC70)(登录号DQ660140)和热应激蛋白90(heat shock protein 90,HSP90)(登录号GU319963)基因mRNA表达进行定量分析,以β-肌动蛋白(β-actin)(登录号FL589653)作为qRT-PCR反应中的内参基因,所有基因的扩增效率都大于97%。所用基因的引物序列见表 1。qRT-PCR反应体积为20 μL,包括10 μL 2×SYBR Green Premix Ex Taq、引物(10 μmmol/L)各0.2 μL、2 μL cDNA和7.6 μL ddH2O。qRT-PCR反应条件为:95 ℃预变性10 min;94 ℃变性15 s、58 ℃退火20 s、72 ℃延伸20 s,共40个循环。PCR反应后绘制熔解曲线以判断扩增产物的正确性,温度以0.5 ℃/5 s的速度从60 ℃上升到95 ℃,所有基因的扩增效率基本相同,使用2-ΔΔCt方法[26]对基因mRNA相对表达量进行分析。

表 1 qRT-PCR所用引物序列 Table 1 Primer sequences used for qRT-PCR
1.7 统计分析

试验结果用平均值±标准差表示,使用SPSS 17.0对数据进行单因素方差分析(one-way ANOVA)后,若差异达到显著水平,则进行Tukey氏法多重比较,显著水平为P < 0.05。LEfSe分析使用非参数因子的Kruskal-Wallis(KW)和秩检验来检测具有显著丰度差异的特征,并使用LDA检测每个组分丰度对差异效果影响的大小。默认LDA值大于2,P < 0.05,该物种为差异物种。

2 结果 2.1 不同铅浓度对Trx系统基因表达的影响

图 1可知,随着铅浓度的增加,TrxTrxR mRNA相对表达量逐渐降低,其中,26.26 μg/L组Trx mRNA相对表达量显著低于其余2组(P<0.05),13.13和26.26 μg/L组TrxR mRNA相对表达量显著低于对照组(0 μg/L)(P<0.05)。

数据柱上标不同字母表示差异显著(P < 0.05)。图 2同。 Value columns with different letters mean significant difference (P < 0.05). The same as Fig. 2. 图 1 不同铅浓度下日本沼虾肝胰腺TrxTrxR mRNA相对表达量 Fig. 1 mRNA relative expression of Trx and TrxR in Macrobrachium nipponense under different lead concentrations exposure
图 2 不同铅浓度下日本沼虾肝胰腺HSP60、HSC70和HSP90 mRNA相对表达量 Fig. 2 mRNA relative expression of HSP60, HSP90 and HSC70 in Macrobrachium nipponense under different lead concentrations exposure
2.2 不同铅浓度对HSP系统基因表达的影响

图 2可知,在26.26 μg/L铅的胁迫下,HSP60 mRNA相对表达量降低,显著低于对照组和13.13 μg/L组(P<0.05);HSC70 mRNA相对表达量也随着铅浓度的增加而降低,26.26 μg/L组HSC70 mRNA相对表达量显著低于对照组(P<0.05);HSP90 mRNA相对表达量也随着铅浓度的增加呈先增加后降低的趋势,其中26.26 μg/L组和对照组HSP90 mRNA相对表达量显著低于13.13 μg/L组(P < 0.05)。

2.3 肠道菌群分析

日本沼虾在0、13.13和26.26 μg/L铅浓度胁迫下的肠道菌群的生态特征的评估使用基于OTU水平的各种指数。见表 2。3个组的高质量序列总数为385 987条,平均每个样本有42 866条序列。每组的覆盖率超过99%,表明这些群体中的16S rRNA基因序列代表了本研究样本中存在的大多数细菌。各组OTU的数量、菌群丰度(Chao指数)和多样性(Shannon指数和Simpson指数)均没有显著差异(P>0.05)。从这些指标可知,不同铅浓度的胁迫对日本沼虾肠道菌群总的丰度和多样性没有显著影响。

表 2 不同铅浓度对日本沼虾肠道菌群的α多样性的影响 Table 2 Alpha diversity of intestinal microbiota in Macrobrachium nipponense under different lead concentrations exposure

根据UniFrac距离,用PCoA图显示在不同铅浓度胁迫下日本沼虾肠道菌群组成。从图 3可知,对照组和13.13 μg/L组菌群没有因为铅浓度而分离。与对照组和13.13 μg/L组相比,26.26 μg/L组在PC2水平上的菌群组成模式有一定的差异。

Pb1-1、Pb1-2和Pb1-3为对照组;Pb2-1、Pb2-2和Pb2-3为13.13 μg/L组;Pb3-1、Pb3-2和Pb3-3为26.26 μg/L组。图 4同。 Pb1-1, Pb1-2 and Pb1-3 were control group, Pb2-1, Pb2-2 and Pb2-3 were 13.13 μg/L group, and Pb3-1, Pb3-2 and Pb3-3 were 26.26 μg/L group. The same as Fig. 4. 图 3 在不同铅水平下的日本沼虾肠道菌群的主坐标分析 Fig. 3 Principal co-ordinates analysis of intestinal bacteria in Macrobrachium nipponense under different lead concentrations exposure
Proteobacteria:变形菌门;Firmicutes:厚壁菌门;Tenericutes:软壁菌门;Others:其他。 图 4 不同铅浓度下日本沼虾肠道微生物群在门水平上组成的研究 Fig. 4 Gut microbiota composition at phylum level in Macrobrachium nipponense under different lead concentrations exposure

图 4显示了3组日本沼虾肠道菌群在门分类水平上相对丰度。在这3组中,观察到3个优势菌门,即变形菌门(Proteobacteria)、厚壁菌门(Firmicutes)和软壁菌门(Tenericutes)。在对照组中,这3个菌门的相对丰度分别为93.99%、5.43%和0.22%。在13.13 μg/L组中,这3个菌门的相对丰度分别为92.24%、2.15%和5.46%。在26.26 μg/L组中,这3个菌门的相对丰度分别为93.61%、3.55%和2.23%。

在门、纲、目等不同分类水平下不同组肠道主要菌群采用常规方差统计分析,结果见表 3。由表可知,各组间菌群相对比较稳定,但也存在组间差异较大导致标准差较大的现象。在纲水平,主要为甲型变形菌纲(Alphaproteobacteria)和丙型变形菌纲(Gammaproteobacteria),在目水平主要为立克次氏体目(Rickettsiales)和气单胞菌目(Aeromonadales),在科水平主要为立克次氏体科(Rickettsiales Incertae Sedis)、气单胞菌科(Aeromonadaceae)和毛螺菌科(Lachnospiraceae),在属水平主要为Candidatus Hepatincola、气单胞菌属(Aeromonas)和Tyzzerella 3,在种水平主要为Candidatus Hepatincola_unclassified、未分类气单胞菌种(Aeromonas_unclassified)和嗜水气单胞菌嗜水亚种(Aeromonas hydrophila subsp. hydrophila)。其中铅胁迫后未分类气单胞菌种相对丰度显著低于对照组(P < 0.05)。

表 3 不同铅浓度下日本沼虾肠道主要菌群相对丰度 Table 3 Relative abundance of main gut microbiota species in Macrobrachium nipponense under different lead concentrations exposure

采用宏基因组LEfSe分析方法分析对照组和13.13或26.26 μg/L组之间差异的关键菌群类型(图 5),结果发现,对照组与26.26 μg/L组某些肠道菌群相对丰度存在显著差异(P < 0.05),对照组中肠杆菌属(Enterobacteriales)、肠杆菌科(Enterobacteriaceae)、肠杆菌目(Enterobacter)、柠檬杆菌属(Citrobacter)、疣微菌目(Verrucomicrobiae)、疣微菌纲(Verrucomicrobiales)、疣微菌科(Verrucomicrobiaceae)相对丰度较高,而26.26 μg/L组中δ-变形菌纲(Deltaproteobacteria)、黄杆菌纲(Flavobacteriia)、黄杆菌科(Flavobacteriales)、黄杆菌目(Flavobacteriaceae)、黄杆菌属(Flavobacterium)、拟杆菌门(Bacteroidetes)和井杆菌属(Phreatobacter)相对丰度较高。这些菌群也是导致对照组与26.26 μg/L组肠道菌群差异的主要类型。

Deltaproteobacteria:δ-变形菌纲;Phreatobacter:井杆菌属;Bacteroidetes:拟杆菌门;Flavobacteriia:黄杆菌纲;Flavobacteriaceae:黄杆菌目;Flavobacteriales:黄杆菌科;Flavobacterium:黄杆菌属;Verrucomicrobiales:疣微菌纲;Verrucomicrobiae:疣微菌目;Verrucomicrobiaceae:疣微菌科;Enterobacteriaceae:肠杆菌目;Enterobacter:肠杆菌科;Enterobacteriales:肠杆菌属;Citrobacter:柠檬杆菌属。
26.26 μg/L组富集的分类群以正的LDA评分(绿色)表示,而在对照组(0 μg/L)中富集的分类群以负的LDA评分(红色)表示。只显示LDA显著阈值>2的分类群。
Taxa enriched in 26.26 μg/L group indicated with a positive LDA score (green), and taxa enriched in control group (0 μg/L) indicated with a negative score (red). Only taxa meeting a LDA significant threshold>2 were shown. 图 5 LEfSe鉴定对照组和26.26 μg/L组之间差异最大的类群结果 Fig. 5 Results of the greatest differnce between control group and 26.26 μg/L group identified by LEfSe
3 讨论

本研究主要从TrxHSP系统基因表达以及肠道菌群的角度探讨重金属铅对日本沼虾生理影响的机制。产生ROS而导致氧化应激、降低机体抗氧化防疫系统是重金属铅引起肝脏毒性的机制之一[27],氧化应激可引起关键细胞分子如脂质、蛋白质和DNA的损伤[28]。Trx系统作为机体内的氧化还原系统,在抗氧化方面发挥着至关重要的作用。Trx系统由还原型烟酰胺腺嘌呤二核苷酸磷酸(NADPH)、TrxR和Trx组成,是一种主要的二硫还原酶系统,可为多种酶提供电子,对DNA合成和抗氧化应激具有重要作用[29]。已有相关研究证明,环境胁迫可影响机体TrxRTrx的表达,如盐胁迫使加工番茄幼苗叶片TrxR活性降低[30];高浓度石英粉尘能显著降低人胚肺成纤维细胞的TrxTrxR基因和蛋白的表达[31];长时间饲喂高铜饲粮可导致肉鸡肝脏中Trx蛋白表达量降低[32]。本研究中,发现铅的胁迫浓度达到26.26 μg/L时,TrxTrxR mRNA相对表达量相对于对照组显著降低,说明高铅导致肝脏细胞内产生大量的ROS,引起了日本沼虾的氧化应激,破坏了机体氧化-抗氧化平衡;此外,高铅可能抑制了TrxTrxR mRNA相对表达,使得虾体的抗氧化能力降低。

机体受到应激之后,HSP可通过维持蛋白质构象、抗凋亡、细胞保护等作用以维持机体稳态,抵抗一系列应激[33]。HSP60是一种主要存在于线粒体和质体的蛋白质,其主要功能是折叠、组装蛋白质,形成最终结构[34]HSP90在真核细胞的细胞质中表达量较高,不仅具有分子伴侣的功能,在胞内运输、蛋白质降解、细胞信号传导等过程中也发挥着重要作用[35]。HSC70作为组成型热应激蛋白的一种,能够降解和清除受损、变性的蛋白,起着保护和修复细胞的作用[36-37]。本研究中,发现铅的胁迫浓度为13.13 μg/L时,HSP60和HSC70 mRNA相对表达量相对于对照组没有显著变化,而当铅的胁迫浓度达到26.26 μg/L时,HSP60和HSC70 mRNA相对表达量相对于对照组显著降低,说明在此浓度下,发生了HSP介导的细胞损伤。然而,在铅胁迫浓度为13.13 μg/L时,HSP90 mRNA相对表达量显著增加,说明HSP90可成为铅胁迫的潜在指示分子生物标记。当铅的浓度继续增加时,HSP90 mRNA相对表达量又显著降低,说明高浓度的铅引起的机体损伤可能不能通过HSP90得以修复。不同物种在不同重金属应激下,HSP调控灵敏度存在一定的差异。对剑水蚤(Tigriopus japonicus)的研究发现,HSP20和HSC70对5种重金属的胁迫都有较强的敏感性,而HSP10在100 μg/L的砷、镉和锌胁迫时,转录水平降低,HSP60在100 μg/L银和砷胁迫时,转录水平也同样降低[13]。对大西洋牡蛎(Crassostrea gigas)的研究发现,镉处理后增加HSP90的表达,而且这种表达具有浓度和时间依赖性[38]。有研究报道,镉胁迫(50、500、2 000 μmol/L)4 h增加了HSP60和HSC70基因的表达,但不影响HSP90基因的表达[39]

动物肠道内生活着大量的微生物,包括病毒、细菌和真核微生物等[40]。在肠道微生态系统中,肠道菌群与宿主经过长期的共同进化,在抵御病害菌侵袭、消化吸收营养物质和调节宿主免疫功能等方面具有重要作用[41-43]。肠道菌群发生紊乱会引起宿主的各种疾病,因此保持肠道菌群的稳定是维系动物肠道健康的重要因素[44-45]。现如今,使用传统的形态学和生物化学标准很难确定肠道微生物的代表[46]。本研究采用16S rRNA Illumina高通量测序数据显示,不同的铅胁迫浓度不影响日本沼虾肠道细菌的丰富性和多样性,但会改变其菌群组成。研究还发现,不论铅胁迫浓度大小,日本沼虾中的主要门类是变形菌门,其次是厚壁菌门和软壁菌门,主要细菌是变形菌门,这与之前对鱼类[41, 47-48]、虾[49-50]或螃蟹[51]的研究结果一致,说明日本沼虾肠道存在不受慢性铅胁迫干扰的核心微生物群。在微生物种水平上,本研究发现未分类气单胞菌种的相对丰度在铅胁迫后显著下降。尽管一些气单胞菌是潜在的病原菌,但Gibson等[52]研究发现,气单胞菌在鱼类生物学中参与宿主的营养、黏膜防御和宿主免疫等方面的生理活动,扮演着比致病菌更为重要的角色,这一结果得到了其他学者[52-54]的支持。本研究中,正常虾肠道黏膜中具有相对较高丰度的未分类气单胞菌种与在其他鱼类中的研究结果[48, 55-56]一致。但应激条件下,该气未分类单胞菌种的减少机理还需进一步研究。除此之外,本研究还鉴定出了与铅处理相关的其他细菌表型,可作为生物标志物,如对照组肠杆菌属、柠檬杆菌属、疣微菌目、疣微菌科等相对丰度较高,而高铅组中δ-变形菌纲、黄杆菌属、拟杆菌门和井杆菌属相对丰度较高。肠杆菌属分泌产生的乳酸是促进餐后饱腹感的重要神经递质,能够调控机体的饥饿信号[57];柠檬杆菌属能协助多形拟杆菌清除肠腔病原菌,发挥免疫防御功能[58-59];疣微菌科中的Akkermansia muciniphila是一种具有益生菌特性的新型功能性微生物,能增强肠黏膜伤口的修复和诱导表达紧密连接蛋白,从而保护肠道屏障并降低肠道通透性[60-62]。因此,本研究对照组中上述菌群相对丰度较高,对于机体的正常代谢和免疫增强可能具有重要意义,而高铅胁迫组上述菌群相对丰度的降低说明铅的胁迫影响了这些菌群的富集。δ-变形菌纲是变形菌门的一个分支,变形菌门广泛存在于海洋环境,可参与复杂有机物的分解[63],但该菌门在肠道相对丰度过高会导致肠道微生物菌群结构失衡,引发炎症反应[64]。另外,有研究发现,黄杆菌属含有质粒,是分解有机物的功能菌,可降解复杂的有机污染物[65],同时黄杆菌属的某些菌株及其胞外代谢产物能显著增强吞噬细胞的吞噬活性,以及提高补体3(C3)的蛋白水平[66]。其中,吞噬活性是吞噬细胞作为机体第1道防线的重要部分,通过提高吞噬活性,机体可以通过吞噬细胞的吞噬作用进行自我保护;补体蛋白中C3浓度最高,其分解产物C3a和C3b能在病原体表面附着,吸引吞噬细胞进行吞噬作用[67-68]。但是,黄杆菌属也是潜在条件致病菌,它可分布于健康鱼类的肠道中[56],因此铅胁迫后黄杆菌属相对丰度的增加可能是一把双刃剑。拟杆菌门的拟杆菌属是短链脂肪酸(SCFAs)的产生菌株,SCFAs主要包括乙酸、丙酸和丁酸。乙酸能促进外周组织的发育,丙酸有助于肝细胞的生长,丁酸能促进肠上皮细胞的生长、成熟和适当分化,为肠道细胞提供营养[69-70]。严康等[71]研究发现,短链脂肪酸可通过诱导短链脂肪酸受体41(GPR41)的表达,显著提高炎性细胞因子[白细胞介素-1β(IL-1β)和肿瘤坏死因子-α(TNF-α)]的表达以及趋化因子(CCL20、CXCL2、CXCL3和CXCL8)的表达,从而介导瘤胃上皮细胞的免疫反应。其中,IL-1β基因可以通过接触吞噬细胞和淋巴细胞,诱导其释放细胞因子来刺激免疫应答[72]TNF-α基因的功能和IL-1β基因相似,在炎症反应中发挥重要的作用[66]。因此,本研究中高铅组与降解污染物和调节机体免疫性能相关的δ-变形菌纲、黄杆菌属、拟杆菌属的相对丰度增加,而维持正常代谢的部分菌群相对丰度减少。生存在重金属污染环境中的细菌往往通过基因的平行转移来抵抗环境的污染[73]。基因的转移不仅可为菌群的组成提供一定的保护作用,而且可增加菌群对重金属的脱毒或耐受能力[15, 74]。因此,本研究中这些肠道菌群相对丰度的变化可能是应对铅胁迫的一种适应性改变。

4 结论

慢性铅胁迫会降低日本沼虾肝胰腺TrxTrxR基因的转录水平,调控HSP60、HSC70和HSP90基因的表达,日本沼虾肠道内存在不受慢性铅胁迫干扰的核心微生物门类,但高浓度的铅胁迫会产生丰度显著性差异的菌群,维持正常代谢的菌群丰度减少,与降解污染物、调节机体免疫和抗氧化应激相关的菌群丰度增加。

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