Chinese Journal of Animal Nutrition >

2023 , Vol. 35 >Issue 12: 8097 - 8110

DOI: https://doi.org/10.12418/CJAN2023.733

RESEARCH PAPER

Effects of Sparassis latifolia Polysaccharide on Oxidative Stress and Autophagy Injury of Kidney in Mice Induced by Lead

  • LU Xingru , 1 ,
  • WU Rui 1 ,
  • AI Honghu 1 ,
  • WU Shanshan 1 ,
  • CHENG Yanfen 1, 2 ,
  • YUN Shaojun 1, 2 ,
  • LIU Jingyu 1, 2 ,
  • CHENG Feier 1, 2 ,
  • FENG Cuiping , 1, 2, * ,
  • CAO Jinling , 1, 2, *
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  • 1 College of Food Science and Engineering, Shanxi Agricultural University, Taigu 030801, China
  • 2 Shanxi Key Laboratory of Edible Fungi for Loess Plateau, Taigu 030801, China
* FENG Cuiping, professor, E-mail: ;
CAO Jinling, professor, E-mail:

Received date: 2023-07-07

  Online published: 2023-12-11

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Abstract

To investigate the alleviating effects of Sparassis latifolia polysaccharide on the nephrotoxicity in lead (Pb) exposed mice and analyze the transmissibility of the alleviating effects under the co-housing mode, seventy-five Kunming mice were randomly divided into 5 groups, they were blank control group (NC group), Pb exposed group (Pb group), Sparassis latifolia polysaccharide group (SLP group), co-housing Pb exposed group (Co-Pb group) and the co-housing Sparassis latifolia polysaccharide group (Co-SLP group) (mice in the Co-Pb group and the Co-SLP group were put into the same cage), with 15 mice in each group. Drinking water of mice contained 1 g/L Pb2+ except NC group. In addition to this, mice in the SLP and Co-SLP groups were intragastric with Sparassis latifolia polysaccharide, and mice in the NC, Pb and Co-Pb groups were given the same volume of saline, once a day. After 8 weeks of feeding, the lipid metabolism related indicators in the plasma and kidney were detected by a fully automated biochemical analyzer; the Pb content in the blood and kidney was measured by a graphite furnace atomic absorption spectroscopy; the renal tissue structure was observed using optical microscope; the oxidative stress related indicators including the activities of total superoxide dismutase (T-SOD), glutathione peroxidase (GSH-Px) and catalase (CAT), and the glutathione (GSH) and malondialdehyde (MDA) contents in the kidney were determined by enzyme-linked immunosorbent assay (ELISA) kits; the mRNA relative expression levels of the inflammatory factor genes [interleukin-6 (IL-6), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), interleukin-4 (IL-4) and interleukin-10 (IL-10)] and autophagy genes [Beclin1, microtubule-associated protein light chain 3-Ⅰ (LC3-Ⅰ), microtubule-associated protein light chain 3-Ⅱ (LC3-Ⅱ), autophagy related gene 5 (Atg5), ubiquitin binding protein p62 (p62) and lysosomal associated membrane protein 2 (Lamp2)]were detected using real-time fluorescence quantitative PCR; the relative abundance of intestinal flora at phylum level was analyzed by gene sequencing. The results showed that compared with the NC group, Pb exposure significantly increased the total cholesterol (TC), triglyceride (TG) and free fatty acids (FFA) contents in the plasma, the TG and TC contents in the kidney, and the Pb content in the blood and kidney (P<0.05); Pb exposure significantly decreased the activities of T-SOD, GSH-Px, CAT and GSH content (P<0.05), but significantly increased the MDA content (P<0.05); the pathological changes and deposition of lipid droplets in the renal tissues were observed in Pb group; Pb exposure significantly elevated the mRNA relative expression levels of inflammatory factor genes, such as IL-6, IL-1β and TNF-α, and the autophagy genes including Beclin1, LC3-Ⅰ, LC3-Ⅱ, Atg5, p62 and Lamp2 (P<0.05), while significantly decreased the mRNA relative expression levels of inflammatory factor genes including TGF-β, IL-4 and IL-10 (P<0.05); Pb exposure significantly increased the relative abundance of Firmicutes in intestinal microbiota (P<0.05), while significantly decreased the relative abundance of Bacteroides (P<0.05). Compared with the Pb group, the TG, TG and FFA contents in the plasma, the TG and TC contents in the kidney, and the Pb content in the blood and kidney in the SLP and Co-SLP groups were significantly decreased (P<0.05); the activities of T-SOD, GSH-Px, CAT and GSH content in the SLP and Co-SLP groups were significantly decreased (P<0.05), and the MDA content was significantly decreased (P<0.05); the glomerular atrophy and lipid droplet deposition in the renal tissues were obviously alleviated in the SLP and Co-SLP groups; the mRNA relative expression levels of the inflammatory factor genes IL-6 and IL-1β and the autophagy genes Beclin1, LC3-Ⅰ, LC3-Ⅱ, Atg5, p62 and Lamp2 in the kidney were significantly decreased (P<0.05), while the mRNA relative expression levels of TGF-β, IL-4 and IL-10 in the kidney were significantly increased in the SLP and Co-SLP groups (P<0.05); the relative abundance of Firmicutes in intestinal microbiota was significantly decreased (P<0.05), while the relative abundance of Bacteroides in intestinal microbiota was significantly increased in the SLP and Co-SLP groups (P<0.05). Compared with the Pb group, the Co-Pb group showed a significant decrease in the plasma TC and TG contents, and the Pb content in the blood and kidney (P<0.05); the activities of T-SOD, CAT and GSH content in the kidney were significantly elevated in the Co-Pb group (P<0.05); treatment with co-housing and Pb exposure significantly alleviated the glomerular atrophy and lipid droplet deposition in the renal tissue; the mRNA relative expression levels of the inflammatory factor genes TGF-β, IL-4, IL-10 and the autophagy genes Beclin1 and p62 were significantly reduced in the Co-Pb group (P<0.05); the relative abundance of Firmicutes in intestinal microbiota was significantly decreased in the Co-Pb group (P<0.05). All above indicate that the Sparassis latifolia polysaccharide can alleviate the renal damage caused by Pb exposure by reducing the Pb content in the blood and kidney, regulating the lipid metabolism, alleviating the oxidative stress, modulating the inflammatory reaction and inhibiting the autophagy injury, and it can transmit this protective effect through co-housing.

Key words： Sparassis latifolia polysaccharide; lead; kidney; oxidative stress; autophagy injury

Cite this article

LU Xingru , WU Rui , AI Honghu , WU Shanshan , CHENG Yanfen , YUN Shaojun , LIU Jingyu , CHENG Feier , FENG Cuiping , CAO Jinling . Effects of Sparassis latifolia Polysaccharide on Oxidative Stress and Autophagy Injury of Kidney in Mice Induced by Lead[J]. Chinese Journal of Animal Nutrition, 2023 , 35(12) : 8097 -8110 . DOI: 10.12418/CJAN2023.733

铅是自然界中广泛存在的一种具有蓄积毒性的重金属,由于其具有延展性高、熔点低和耐腐蚀等诸多独特的性能,被广泛地应用于汽车、颜料、陶瓷制造业等领域,从而造成严重的环境污染和健康问题[1-2]。铅可经过呼吸、饮食及皮肤黏膜等途径进入人和动物体内对机体组织产生毒性作用[3-4]。研究表明,氧化应激与铅对机体的损伤有关,如张迪[5]发现铅暴露可以抑制大鼠血清和脏器中抗氧化酶活性,加快自由基的产生,造成机体组织的氧化损伤;王颖等[6]发现铅蓄积于机体后,可以显著破坏机体抗氧化酶系,增加脂质过氧化物的生成及存在而对小鼠肾脏组织造成损伤。当机体处于氧化应激状态时,严重的脂质过氧化会破坏细胞,而细胞自噬通过清除胞内错误折叠的蛋白质、受损的细胞器等来抵御这种应激损伤[7],如周蓉等[8]研究发现血红素氧合酶-1可通过上调自噬减轻铅诱导的细胞氧化应激损伤。
多糖是一类由单糖构成的天然大分子化合物,并广泛地存在动物、植物、微生物等有机体中。研究发现多种多糖具有缓解铅中毒的作用,如樊永恒[9]研究发现板蓝根多糖可改善铅对小鼠肝脏、十二指肠、心脏的损伤,降低铅对肝脏的氧化应激;高文静等[10]研究发现岩藻多糖对铅中毒大鼠体内蓄积的铅有较好的促排作用,同时还可有效缓解铅毒性造成的体内矿物元素稳态失衡;谢志明等[11]研究发现柴胡多糖可减轻铅诱导小鼠引起的脂质过氧化和炎症反应,从而缓解铅对小鼠肝脏、肾脏的损伤作用。绣球菌(Sparassis latifolia)是一种珍稀名贵的食药用菌,不但多糖含量十分丰富,而且具有调节免疫、降血脂、抗氧化等多种生理作用,已被开发用于功能性食品、化妆品等领域[12⇓-14]。但是,在绣球菌多糖对于铅暴露肾脏毒性效应和自噬损伤影响方面的研究还很少。
因此,本试验以染铅小鼠为模型,来探讨绣球菌多糖通过抑制氧化应激、调控炎症因子和自噬基因对铅致小鼠肾脏损伤的缓解效应,以期为绣球菌多糖作为一种绿色有效铅拮抗剂的开发利用提供理论依据。

1 材料与方法

1.1 试验动物

无特定病原体(SPF)级雄性昆明(KM)小鼠,3周龄,购于斯贝福(北京)生物技术有限公司[许可证号:SCXK(京)2019-0010]。

1.2 主要试剂

绣球菌由山西省清徐县太和食用菌栽培基地提供,乙酸铅购自天津市大茂化学试剂厂,苏木素、伊红购自天津市赛瑞达生物工程有限公司,考马斯亮兰(G-250)、总超氧化物歧化酶(total superoxide dismutase,T-SOD)、过氧化氢酶(catalase,CAT)、丙二醛(malondialdehyde,MDA)、谷胱甘肽(glutataione,GSH)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)测定试剂盒均购自南京建成生物工程研究所,RNAiso Plus、PrimeScriptTM RT Master Mix、SYBR® Premix Ex TaqTM Ⅱ购自北京索莱宝科技有限公司,其余试剂均为国产分析纯。

1.3 主要仪器

电感耦合等离子发射光谱仪(Optima 5300DV型,美国PE公司)、石墨消解仪(YXMD-Ⅰ型,天津云祥仪器有限公司)、冰冻切片机(Leica CM3050,德国Leica公司)、实时荧光定量PCR仪(Mx3000P,美国Agilent公司)、手持式匀浆器(PRO 200,美国Pro Scientific公司)、冷冻离心机(Allegra 25R,美国Beckman公司)、酶标仪(iMark,美国BioRad公司)。

1.4 试验方法

1.4.1 绣球菌多糖的制备

参照本课题组前期方法[14]提取绣球菌多糖。经高效凝胶渗透色谱和离子色谱分析,所提取的绣球菌多糖分子质量为1.04×104 u,由葡萄糖、甘露柚、半乳糖、木糖、果糖组成,摩尔比为13∶4∶1∶2∶3;根据1D和2D核磁共振(NMR)谱分析,确定该多糖结构主链是β-(1→3)-D-葡聚糖,支链是β-(1→6)-D-葡聚糖,分支频率约为3个主链单位1个分支的β-葡聚糖。

1.4.2 动物分组及样品采集

小鼠适应性饲养7 d后随机分为5组,每组15只,包括空白对照组(NC组)、染铅模型组(Pb组)、绣球菌多糖组(SLP组)、同笼染铅组(Co-Pb组)和同笼绣球菌多糖组(Co-SLP组),同笼即是把Co-Pb组和Co-SLP组小鼠标志好后饲养在同一笼子里面。除NC组外,其余小鼠均给予含1 g/L二价铅离子(Pb2+)的水溶液作为饮用水。SLP组、Co-SLP组小鼠每天灌胃400 mg/kg BW的绣球菌多糖(溶于0.5 mL生理盐水),NC组、Pb组和Co-Pb组灌胃相同体积的生理盐水,每天1次。试验时间为8周。
试验结束后将小鼠禁食12 h,尾静脉采血,血液置于抗凝管中(一部分用于血液中铅含量的测定,一部分3 000 r/min离心,取血浆,用于脂质代谢相关指标的测定),接着颈椎脱臼法处死小鼠进行肾脏组织和肠道内容物的采集。

1.4.3 血浆和肾脏中脂质代谢相关指标检测

血浆中总胆固醇(TC)、甘油三酯(TG)、游离脂肪酸(FFA)和肾脏中TC、TG含量采用日本奥林巴斯AU2700全自动生化分析仪测定。

1.4.4 血液和肾脏中铅含量测定

取适量血液、肾脏组织样品置于消化管中,湿法消解样品,利用石墨炉原子吸收光谱法测定样品中铅含量。

1.4.5 肾脏组织切片的制备与观察

将肾脏组织置于20%蔗糖溶液沉底2 d,然后取出用预冷的磷酸盐缓冲液(PBS)清洗,适量OCT包埋剂包埋,液氮迅速冰结成块,冰冻切片机上切片,厚度5 μm。切片一部分用苏木精-伊红(HE)染色,另一部分用油红O染色,梯度酒精脱水,二甲苯透明,中性树胶封片,显微成像系统拍照观察。

1.4.6 肾脏中氧化应激相关指标检测

取小鼠肾脏组织,按照试剂盒说明书将其制备成组织匀浆液后,测定T-SOD、GSH-Px、CAT活性及GSH、MDA含量。

1.4.7 肠道菌群的测定

取适量小鼠肠道内容物,送至诺禾致源科技股份有限公司进行16S rDNA基因测序,分析肠道菌群门水平相对丰度。

1.4.8 肾脏中炎症因子基因和自噬基因mRNA表达的检测

由RNAiso Plus提取肾脏组织中的总RNA,按照PrimeScriptTM RT Master Mix说明书将RNA逆转录合成cDNA,利用实时荧光定量PCR技术对肾脏组织炎症因子基因白细胞介素-6(IL-6)、白细胞介素-1β(IL-1β)、肿瘤坏死因子-α(TNF-α)、转化生长因子-β(TGF-β)、白细胞介素-4(IL-4)、白细胞介素-10(IL-10)及自噬基因Beclin1、微管相关蛋白轻链3-Ⅰ(LC3-Ⅰ)、微管相关蛋白轻链3-Ⅱ(LC3-Ⅱ)、自噬相关蛋白5(Atg5)、泛素结合蛋白p62(p62)、溶酶体关联膜蛋白2(Lamp2)的mRNA表达水平进行检测,具体操作过程参照试剂盒的说明书进行,以β-肌动蛋白作为内参基因,采用2-ΔΔCt法分析目的基因mRNA相对表达量。应用在NCBI数据库查询的基因序列,采用Primer Premier 6软件设计引物,引物信息见表1。
表1 引物信息

Table 1 Primer information

基因名称
Gene names		 引物序列
Primer sequence (5'—3')		 片段长度
Fragment length/bp		 GenBank登录号
GenBank accession number
白细胞介素-6 IL-6 F:TATGGCTCAGGGTCCAACTC
R:GCTCCAGTGAATTCGGAAAG		 140 NM_013693.2
白细胞介素-1β IL-1β F:GAGCCCATCCTCTGTGTCTC
R:AGCTCATATGGGTCCGACAG		 131 NM_008361.3
肿瘤坏死因子-α TNF-α F:TATGGCTCAGGGTCCAACTC
R:GCTCCAGTGAATTCGGAAAG		 140 NM_013693.2
转化生长因子-β TGF-β F:CTGGAGTTGTACGGCAGTGG
R:GTTCATGTCATGGATGGTGCC		 161 NM_011577.2
白细胞介素-4 IL-4 F:CAGCTAGTTGTCATCCTGCTCT
R:CCTTCTCCTGTGACCTCGTTC		 116 NM_021283.2
白细胞介素-10 IL-10 F:GAGCCCATCCTCTGTGTCTC
R:AGCTCATATGGGTCCGACAG		 131 NM_008361.3
Beclin1 F:ATGCACAGACACTCTTTTAGACC
R:ACTGTCATCCTCATTCATCTGCT		 115 NM_019584.4
微管相关蛋白轻链3-Ⅰ LC3-Ⅰ F:GACCAGCACCCCAGTAAGATCC
R:GCCGGATGATCTTGACCAACT		 133 NM_025735.3
微管相关蛋白轻链3-Ⅱ LC3-Ⅱ F:CGAGAAGACCTTCAAGCAGCG
R:GCTCCTATGGGGTTAGGGTC		 132 NM_001364358.1
自噬相关基因5 Atg5 F:ACAAAGATGTGCTTCGAGATGT
R:CACTTTGTCAGTTACCAACGTCA		 146 NM_053069.6
泛素结合蛋白p62 p62 F:GGAGCTGGACTTTATCCTGTCG
R:GTAGGTCTTCTCACGCTCCTC		 130 NM_053074.2
溶酶体关联膜蛋白2 Lamp2 F:TCTCTCCGGTTAAAGGCGCAAA
R:CTGCATAAAGGCAAGTACCCT		 114 NM_001017959.2
β-肌动蛋白β-actin F:AGCCATGTACGTAGCCATCC
R:CTCTCAGCTGTGGTGGTGAA		 83 NM_007393.3

1.5 数据处理及分析

采用SPSS 20.0对数据进行单因素方差分析,并采用Duncan氏法进行多重比较,数据用平均值±标准差的方式表示,采用GraphPad Prism 8.0进行作图,显著水平为P<0.05。

2 结果与分析

2.1 绣球菌多糖对铅暴露小鼠血浆和肾脏中脂质代谢相关指标的影响

由图1可知,Pb组血浆中TC、TG、FFA及肾脏中TG、TC含量较NC组显著升高(P<0.05);SLP组血浆中TC、TG及肾脏中TG、TC含量较Pb组显著下降(P<0.05);Co-Pb组血浆中TC、TG含量较Pb组显著下降(P<0.05);Co-SLP组血浆中TC、TG及肾脏中TG、TC含量较Co-Pb组显著下降(P<0.05)。
图1 绣球菌多糖对铅暴露小鼠血浆和肾脏中脂质代谢相关指标的影响

数据柱形标注不同小写字母表示差异显著(P<0.05),相同小写字母表示差异不显著(P>0.05)。下图同。

Fig.1 Effects of SLP on lipid metabolism related indicators in plasma and kidney of lead exposed mice

Value columns with different small letters mean significant difference (P<0.05), while with the same small letter superscripts mean no significant difference (P>0.05). The same as below.

2.2 绣球菌多糖对铅暴露小鼠血液和肾脏中铅含量的影响

由图2可知,Pb组血液和肾脏中铅含量较NC组显著增加(P<0.05);SLP组、Co-Pb组和Co-SLP组血液和肾脏中铅含量均较Pb组显著下降(P<0.05);Co-SLP组血液和肾脏中铅含量较Co-Pb组显著下降(P<0.05)。
图2 绣球菌多糖对铅暴露小鼠血液和肾脏中铅含量的影响

Fig.2 Effects of SLP on lead content in blood and kidney of lead exposed mice

2.3 绣球菌多糖对铅暴露小鼠肾脏组织结构的影响

由图3可知,NC组肾脏组织结构完整,肾小球形态规则,肾小管的结构完整清晰,界限明显排列有序;Pb组肾小球萎缩,肾小管上皮细胞肿胀,基底膜有断裂现象,肾小球毛细血管腔内和部分肾小管上皮细胞胞质内可见深染红色的脂滴;Co-Pb组肾小球轻微萎缩,肾小管上皮细胞轻微肿胀,肾小球毛细血管腔内和部分肾小管上皮细胞胞质内可见深染红色的脂滴;SLP组肾组织结构完整,肾小球形态规则,肾小管的结构完整清晰,上皮细胞轻微肿胀,部分肾小管上皮细胞胞质内可见少量深染红色的脂滴;Co-SLP组肾小球轻微萎缩,肾小管上皮细胞可见轻微肿胀,部分肾小管上皮细胞胞质内可见少量深染红色的脂滴。
图3 绣球菌多糖对铅暴露小鼠肾脏组织结构的影响

Fig.3 Effects of SLP on renal tissue structure of lead exposed mice

2.4 绣球菌多糖对铅暴露小鼠肠道菌群门水平相对丰度的影响

由图4可知,与NC组相比,Pb组肠道菌群中厚壁菌门的相对丰度显著升高(P<0.05),拟杆菌门的相对丰度显著降低(P<0.05);与Pb组比较,SLP组、Co-SLP组和Co-Pb组肠道菌群中厚壁菌门的相对丰度显著下降(P<0.05),拟杆菌门的相对丰度显著升高(P<0.05)。
图4 绣球菌多糖对铅暴露小鼠肠道菌群门水平相对丰度的影响

Fig.4 Effects of SLP on relative abundance of intestinal microbiota at phylum level of lead exposed mice

2.5 绣球菌多糖对铅暴露小鼠肾脏中氧化应激相关指标的影响

由图5可知,与NC组比较,Pb组肾脏中T-SOD、CAT、GSH-Px活性与GSH含量显著降低(P<0.05),MDA含量显著升高(P<0.05);Co-Pb组肾脏中T-SOD、CAT活性与GSH含量较Pb组显著升高(P<0.05),而GSH-Px活性和MDA含量均无显著变化(P>0.05);SLP组肾脏中T-SOD、CAT、GSH-Px活性与GSH含量较Pb组显著升高(P<0.05),而MDA含量则显著下降(P<0.05);Co-SLP组肾脏中T-SOD、CAT、GSH-Px活性与GSH含量较Co-Pb组显著升高(P<0.05),而MDA含量则显著下降(P<0.05)。
图5 绣球菌多糖对铅暴露小鼠肾脏中氧化应激相关指标的影响

Fig.5 Effects of SLP on oxidative stress related indicators in kidney of lead exposed mice

2.6 绣球菌多糖对铅暴露小鼠肾脏中炎症因子基因mRNA表达的影响

由图6可知,与NC组相比,Pb组肾脏中IL-6、IL-1β、TNF-α mRNA相对表达量均显著升高(P<0.05),而TGF-β、IL-4、IL-10 mRNA相对表达量均显著下降(P<0.05);Co-Pb组肾脏中TGF-β、IL-4、IL-10 mRNA相对表达量较Pb组显著升高(P<0.05),而IL-6、IL-1β、TNF-α mRNA相对表达量均无显著变化(P>0.05);SLP组肾脏中IL-6、IL-1β、TNF-α mRNA相对表达量较Pb组均显著下降(P<0.05),而TGF-β、IL-4、IL-10 mRNA相对表达量均显著升高(P<0.05);Co-SLP组肾脏中IL-6、IL-1β和TGF-β mRNA相对表达量较Co-Pb组显著下降(P<0.05),而TNF-α、IL-4和IL-10 mRNA相对表达量均无显著变化(P>0.05)。
图6 绣球菌多糖对铅暴露小鼠肾脏中炎症因子基因mRNA表达的影响

Fig.6 Effects of SLP on mRNA expression of inflammatory factor genes in kidney of lead exposed mice

2.7 绣球菌多糖对铅暴露小鼠肾脏中自噬基因mRNA表达的影响

由图7可知,与NC组相比,Pb组肾脏中Beclin1、LC3-Ⅰ、LC3-Ⅱ、Atg5、p62、Lamp2 mRNA相对表达量均显著升高(P<0.05);Co-Pb组肾脏中Beclin1、Atg5 mRNA相对表达量较Pb组显著下降(P<0.05);SLP组肾脏中Beclin1、LC3-Ⅰ、LC3-Ⅱ、Atg5、p62、Lamp2 mRNA相对表达量较Pb组显著下降(P<0.05);Co-SLP组肾脏中Beclin1、LC3-Ⅰ、LC3-Ⅱ、Atg5、p62、Lamp2 mRNA相对表达量较Co-Pb组显著下降(P<0.05)。
图7 绣球菌多糖对铅暴露小鼠肾脏自噬基因mRNA表达的影响

Fig.7 Effects of SLP on mRNA expression of autophagy genes in kidney of lead exposed mice

3 讨论

铅是一种在自然环境中普遍存在且不易被降解的重金属,可通过呼吸、饮食及皮肤黏膜进入体内干扰机体多方面的正常生化和生理活动[15]。血液中的铅随血液循环分布于血液丰富的器官,且血液中铅含量升高还会导致血脂异常[16]。肾脏是机体供血量最丰富的器官,进入血液中的铅可经血液循环抵达肾脏,使其成为铅中毒的一个重要靶器官。已有研究表明铅暴露能够诱导肾脏功能损害[11]。本试验发现,铅暴露可以导致小鼠血液和肾脏中铅含量显著升高,而SLP组、Co-SLP组血液和肾脏中铅含量显著降低,可见灌胃绣球菌多糖的小鼠单独或与染铅小鼠同笼饲养均可以加快铅排出体外,减少铅在血液和肾脏的蓄积,从而减轻对肾脏的损伤;同时,Co-Pb组血液和肾脏中铅含量显著低于Pb组,肾脏组织切片HE染色后的观察结果也印证了这一点。另外,本研究还发现,铅暴露使小鼠血浆和肾脏中TC、TG含量显著升高,表明铅中毒使小鼠脂质代谢发生了紊乱,而绣球菌多糖可以明显改善这种脂质代谢的紊乱,且同笼饲养的染铅小鼠的脂质代谢也得到明显的改善,肾脏组织切片油红O染色观察到的肾脏中脂滴的变化也佐证了这一点,推测Co-SLP组与Co-Pb组小鼠同笼饲养可能通过某种方式来减轻铅对机体的毒性效应。小鼠具有食粪性,容易通过食用粪便接受其他个体的肠道菌群[17],本研究也发现小鼠能够通过同笼饲养来改变其肠道菌群门水平相对丰度,提示绣球菌多糖对于铅暴露小鼠的保护作用可能是由其重塑肠道菌群的能力介导的,且可以通过同笼饲养将保护作用传递,其机理还需进一步研究。
铅具有氧化性,可诱导组织发生氧化应激,产生自由基,损伤细胞膜结构[18]。MDA含量高低可反映脂质过氧化程度,是反映细胞膜氧化损伤的主要指标之一[19]。超氧化物歧化酶(SOD)、CAT和GSH-Px是机体内重要的抗氧化防御性功能酶,起平衡细胞氧化还原的作用,能够缓解细胞的氧化损伤,这些酶已经被广泛作为机体的氧化应激标记物[20-21]。GSH是机体内一种重要的抗氧化剂,能够清除体内的自由基。张迪[5]研究发现,急性铅损伤小鼠肝脏中SOD、GSH-Px的活性与GSH的含量下降,MDA的含量上升。本研究也发现,铅暴露会导致小鼠肾脏中CAT、T-SOD、GSH-Px活性与GSH含量显著下降,MDA含量显著升高;而SLP、Co-SLP组小鼠肾脏中CAT、T-SOD、GSH-Px活性与GSH、MDA含量均较Pb组得到了有效的改善,表明灌胃绣球菌多糖小鼠单独或与染铅小鼠同笼饲养均可以诱导小鼠肾脏组织抗氧化能力的增强和脂质过氧化能力的减弱,从而消除过多的自由基来缓解氧化应激,减轻肾脏的氧化损伤;此外,Co-Pb组小鼠氧化应激水平明显低于Pb组,提示染铅小鼠与灌胃绣球菌多糖小鼠同笼饲养在一定程度上抑制了染铅小鼠的氧化应激,从而缓解了氧化损伤,且组织切片观察发现SLP组、Co-Pb组和Co-SLP组小鼠肾脏组织的病理变化得到缓解的结果也佐证了这一点。
在机体损伤过程中,炎症细胞被激活,炎症因子被释放,导致其表达增加[11]。炎症因子TNF-α、IL-6和IL-1β在炎症反应中起重要的作用,可以诱导T细胞活化增殖、分化,促进炎症的发生;IL-10、IL-4是重要的抗炎细胞因子,能广泛抑制促炎细胞因子及炎症介质的表达;TGF-β是一种通过影响免疫细胞分化、发育、平衡和耐受性来调节免疫应答的细胞因子,其可以抑制T、B细胞增殖,减轻局部炎症反应[22]。已有研究表明,重金属会影响组织炎症因子的表达,如锰可以上调鸡睾丸中TNF-α mRNA的表达,铅可以诱导鸡外软骨组织中TNF-α mRNA的表达[23],抑制鸡肾脏中IL-4 mRNA的表达[24]。本研究也发现,铅暴露导致小鼠肾脏中促炎因子TNF-α、IL-6和IL-1β mRNA的表达上调,而抗炎因子IL-10、IL-4和TGF-β mRNA的表达下调,这与前人的研究结果一致,说明铅暴露可以促进小鼠肾脏炎症反应,对肾脏造成损伤。因此,适当降低炎症反应水平有助于减轻铅引起的肾脏损伤。本研究证实,SLP组、Co-SLP组和Co-Pb组小鼠肾脏中TNF-α、IL-6和IL-1β的表达受到抑制,而IL-10、IL-4和TGF-β的表达受到促进,提示绣球菌多糖可以在一定程度上对炎症因子基因的表达起到调控作用,对缓减小鼠肾脏损伤具有积极作用。结合氧化应激指标的变化趋势,绣球菌多糖调控炎症因子基因表达的作用机制可能与其抗氧化作用有关。
自噬作为细胞中广泛存在的一种保护和防御机制,参与机体内多种生理和病理过程。已有研究表明,细胞自噬参与了铅暴露对机体组织毒性损伤的过程,如小鼠脑组织以及大鼠海马脑区、肝脏、肾脏等[25-26],可见细胞自噬在铅致病机制中具有关键的作用。自噬的活化过程关键步骤之一是自噬体的形成,自噬体的形成是一个动态过程,分为起始、延长和成熟与降解3个阶段,其形成受多种自噬相关基因的严格调控[26]。在起始阶段,Beclin1招募自噬相关蛋白形成复合物,启动自噬蛋白的级联反应,调控细胞自噬水平[27]。本研究发现,铅暴露促进了小鼠肾脏中Beclin1 mRNA的表达,提示肾脏中细胞自噬已经启动。在延长阶段,Atg5促进了自噬体膜的伸展扩张,诱导自噬体的形成。微管相关轻链蛋白3(LC3)作为自噬体膜标志蛋白参与了自噬体的形成,是自噬小体生成的标志因子之一,其有LC3-Ⅰ和LC3-Ⅱ 2种亚型,LC3-Ⅰ可经过一系列的泛素化反应形成LC3-Ⅱ,LC3-Ⅱ基因的相对表达量可反映自噬的水平[27-28]。Lamp2在自噬过程中介导自噬体与溶酶体的融合,在自噬体成熟中发挥重要作用[29]。p62是一种多结构域的自噬底物连接蛋白,它与泛素化修饰的待降解蛋白结合,并将其运送至自噬泡中降解,Lamp2、p62基因的相对表达量升高预示着自噬流末端阻遏现象的发生[30]。本研究结果发现,铅暴露小鼠肾脏中Atg5、LC3-Ⅰ和LC3-Ⅱ mRNA相对表达量升高,且自噬下游调控基因p62和Lamp2 mRNA相对表达量也在升高。已有研究也表明铅暴露可诱导Beclin1、LC3-Ⅰ、LC3-Ⅱ、Atg5、p62和Lamp2表达上升,促进细胞自噬的发生[22,30⇓⇓-33],与本研究结果相一致。然而,过度的自噬反应会加重肾脏损伤,我们在铅暴露小鼠上观察到的肾脏组织切片病理变化和氧化应激损伤也对其起到了印证作用。本研究中,灌胃绣球菌多糖的SLP组、Co-SLP组小鼠肾脏中Beclin1、LC3-Ⅰ、LC3-Ⅱ、Atg5、p62和Lamp2 mRNA相对表达量较铅暴露小鼠明显下降,提示绣球菌多糖可使铅暴露导致的小鼠肾脏细胞的过度自噬有所抑制,减缓其损伤;此外,Co-Pb组小鼠肾脏中自噬相关基因p62的表达也下降明显,提示染铅小鼠与灌胃绣球菌多糖的小鼠同笼饲养可以在一定程度上起到抑制自噬相关基因表达的作用。

4 结论

综上所述,绣球菌多糖可以降低铅暴露小鼠血液和肾脏中铅含量、调控脂质代谢、减轻铅对肾脏的病理损伤,对缓解铅诱导的肾脏毒性效应具有一定的调节作用,其作用机制可能与其降低氧化应激、调节炎症反应和抑制自噬损伤有关,且可以通过同笼饲养将这种缓解效应传递。因此,绣球菌多糖是一种具有一定应用潜力和研究价值的绿色铅拮抗剂。

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