Table 1_Effects of combined exposure to heavy metals on lower respiratory flora and its role of lung injury in rats.docx

IntroductionExposure to heavy metals is a growing environmental concern. Although exposure to individual metals is associated with pulmonary damage, real-world exposure typically involves multiple metals simultaneously. This study hypothesizes that combined exposure to nickel, copper, and arsenic induces lung injury through disruption of the bronchoalveolar lavage fluid (BALF) microbial ecosystem in rats. The primary objective of this study was to verify the hypothesis.
MethodsThirty-two male Sprague–Dawley (SD) rats were randomly assigned to four groups: one control group and three exposure groups (low, medium, and high doses). The exposed groups received mixed heavy metal aerosols containing nickel (Ni), copper (Cu), and arsenic (As) at low (Ni: 210.9 ng/m3, Cu: 108.4 ng/m3, As: 104.6 ng/m3), medium (5 × low), and high (10 × low) concentrations. Exposure occurred via inhalation twice daily for 1 h over 90 days. Lung function was assessed non-invasively, and histological examinations, 16S ribosomal DNA (16S rDNA) sequencing, and microbial functional predictions were performed to evaluate exposure effects. We measured heavy metal concentrations in lung tissues and assessed the associations with microbial changes. Microbial community structure and function were further analyzed using LEfSe, PICRUSt2, and ecological network analysis.
ResultsCompared exposure to Ni, Cu, and As induced dose-dependent lung damage, including inflammation, alveolar deformation, and bronchial thickening, accompanied by significant declines in lung function, including a 21.2% reduction in tidal volume and a 34.5% increase in airway resistance in the high-dose group (P < 0.05). Microbial diversity and phylogenetic richness were significantly reduced (Chao1, Richness, ACE, P < 0.05), with taxonomic shifts characterized by the enrichment of metal-resistant genera (Pseudomonas, Burkholderia) and depletion of sensitive taxa (Ralstonia, Achromobacter). Functional prediction suggested impairments in xenobiotic metabolism and amino acid biosynthesis. Ecological network complexity declined with increasing exposure dose. Microbiota dysbiosis is strongly associated with lung function impairments.
ConclusionsCombined exposure to Ni, Cu, and As disrupts respiratory microbiota and impairs lung function in rats. These findings highlight a critical link between environmental heavy metal exposure and respiratory health, underscoring the need for stricter regulation of metal pollutants and further research into microbiota-related lung injury mechanisms.

引言
重金属暴露日益成为备受关注的环境问题。尽管单一金属暴露已被证实与肺脏损伤相关，但现实环境中的暴露通常是多种金属同时存在的复合暴露。本研究提出假说：镍、铜、砷联合暴露可通过破坏大鼠支气管肺泡灌洗液（bronchoalveolar lavage fluid，以下简称BALF）微生物生态系统，进而诱发肺损伤，本研究的主要目的即为验证该假说。

方法
将32只雄性Sprague-Dawley（SD）大鼠随机分为4组：1个对照组与3个暴露组（低、中、高剂量组）。各暴露组分别暴露于含镍（Ni）、铜（Cu）、砷（As）的混合重金属气溶胶，其浓度分别为低剂量组（Ni：210.9 ng/m³，Cu：108.4 ng/m³，As：104.6 ng/m³）、中剂量组（低剂量的5倍）与高剂量组（低剂量的10倍）。暴露方式为每日两次经呼吸道吸入，每次1小时，持续90天。采用无创方式评估肺功能，并通过组织病理学检查、16S核糖体DNA（16S ribosomal DNA，简称16S rDNA）测序及微生物功能预测，对暴露效应进行评价。检测肺组织内重金属浓度，并分析其与微生物群落变化的关联。此外，采用LEfSe、PICRUSt2及生态网络分析，进一步解析微生物群落的结构与功能特征。

结果
镍、铜、砷联合暴露可诱导剂量依赖性肺损伤，具体表现为炎症反应、肺泡变形及支气管壁增厚，同时伴随肺功能显著下降：高剂量组潮气量降低21.2%，气道阻力升高34.5%（P < 0.05）。微生物多样性及系统发育丰富度显著降低（Chao1、Richness、ACE指数均P < 0.05），群落分类学组成发生显著偏移：金属抗性菌属假单胞菌属（Pseudomonas）、伯克霍尔德菌属（Burkholderia）丰度升高，而敏感类群罗尔斯通氏菌属（Ralstonia）、无色杆菌属（Achromobacter）丰度降低。功能预测显示，外源物质代谢与氨基酸生物合成通路功能受损。随着暴露剂量升高，微生物生态网络的复杂度逐渐降低。微生物群落失调与肺功能损伤显著相关。

结论
镍、铜、砷联合暴露可破坏大鼠呼吸道微生物群落，并损伤肺功能。本研究结果揭示了环境重金属暴露与呼吸道健康之间的关键关联，强调了对金属污染物实施更严格监管的必要性，同时也为深入研究微生物群落失调相关的肺损伤机制指明了方向。