三氯生诱导小鼠肝纤维化的细胞和分子事件的单细胞转录组学分析,Military Medical Research

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三氯生诱导小鼠肝纤维化的细胞和分子事件的单细胞转录组学分析
Military Medical Research ( IF 16.7 ) Pub Date : 2023-02-22 , DOI: 10.1186/s40779-023-00441-3
Yun-Meng Bai ₁ , Fan Yang _{2,

3} , Piao Luo ₂ , Lu-Lin Xie ₂ , Jun-Hui Chen ₁ , Yu-Dong Guan ₂ , Hong-Chao Zhou ₂ , Teng-Fei Xu ₂ , Hui-Wen Hao ₂ , Bing Chen _{2,

3} , Jia-Hui Zhao _{2,

3} , Cai-Ling Liang ₂ , Ling-Yun Dai _{1,

2} , Qing-Shan Geng _{1,

2} , Ji-Gang Wang _{1,

2,

4,

5,

6}

Affiliation

三氯生 [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS] 是许多个人护理和保健产品中常见的抗菌添加剂，经常在人体血液和尿液中检测到。因此，近年来它被认为是一种新兴的具有潜在毒性的污染物。长期暴露于 TCS 已被建议发挥内分泌干扰作用，并促进肝纤维化和肿瘤发生。本研究旨在阐明 TCS 在起始阶段肝毒性作用的潜在细胞和分子机制。C57BL/6 小鼠暴露于不同剂量的 TCS 2 周，并通过各种测量评估器官毒性，包括全血细胞计数、组织学分析和 TCS 定量。然后对 TCS 或模拟处理的小鼠肝脏进行单细胞 RNA 测序 (scRNA-seq)，以描述 TCS 诱导的肝毒性。从差异基因表达、转录因子 (TF) 调控网络、伪时间轨迹和细胞通讯等不同方面分析获得的单细胞转录组数据，以系统地剖析 TCS 暴露后的分子和细胞事件。为了验证 TCS 诱导的肝纤维化，通过蛋白质印迹、免疫荧光、马松三色和天狼星红染色检查了关键纤维化蛋白的表达水平。此外，以正常肝细胞MIHA和肝星状细胞LX-2为体外细胞模型，通过免疫学、蛋白质组学和代谢组学技术，通过实验验证TCS的作用。我们建立了一个相对短期的 TCS 暴露小鼠模型，发现 TCS 主要积累在肝脏中。在 TCS 处理组和对照组的肝脏上进行的 scRNA-seq 分析了属于 13 种主要细胞类型的 > 76,000 个细胞的基因表达。在这些类型中，肝细胞和肝星状细胞 (HSC) 在 TCS 治疗组中显着增加。我们发现 TCS 促进了肝细胞的纤维化相关增殖，其中 Gata2 和 Mef2c 是驱动转录因子的关键。我们的数据还表明 TCS 诱导了 HSC 的增殖和活化，这在肝组织和细胞模型中都得到了实验验证。此外，其他变化包括内皮细胞的功能障碍和毛细血管化，B浆细胞纤维化特征的增加，和 M2 表型偏斜的巨噬细胞，也从 scRNA-seq 分析中推断出来，这些变化可能有助于肝纤维化的进展。最后，确定了参与细胞通讯的关键差异配体-受体对，并证实了 GAS6_AXL 相互作用介导的细胞通讯在促进肝纤维化中的作用。TCS调节肝脏中几种特定细胞类型（包括肝细胞、HSC、内皮细胞、B细胞、Kupffer细胞和肝包膜巨噬细胞）的细胞活动和命运，并调节这些细胞之间的配体-受体相互作用，从而促进增殖和 HSC 的激活，导致肝纤维化。全面的，

"点击查看英文标题和摘要"

Single-cell transcriptomic dissection of the cellular and molecular events underlying the triclosan-induced liver fibrosis in mice

Triclosan [5-chloro-2-(2,4-dichlorophenoxy) phenol, TCS], a common antimicrobial additive in many personal care and health care products, is frequently detected in human blood and urine. Therefore, it has been considered an emerging and potentially toxic pollutant in recent years. Long-term exposure to TCS has been suggested to exert endocrine disruption effects, and promote liver fibrogenesis and tumorigenesis. This study was aimed at clarifying the underlying cellular and molecular mechanisms of hepatotoxicity effect of TCS at the initiation stage. C57BL/6 mice were exposed to different dosages of TCS for 2 weeks and the organ toxicity was evaluated by various measurements including complete blood count, histological analysis and TCS quantification. Single cell RNA sequencing (scRNA-seq) was then carried out on TCS- or mock-treated mouse livers to delineate the TCS-induced hepatotoxicity. The acquired single-cell transcriptomic data were analyzed from different aspects including differential gene expression, transcription factor (TF) regulatory network, pseudotime trajectory, and cellular communication, to systematically dissect the molecular and cellular events after TCS exposure. To verify the TCS-induced liver fibrosis, the expression levels of key fibrogenic proteins were examined by Western blotting, immunofluorescence, Masson’s trichrome and Sirius red staining. In addition, normal hepatocyte cell MIHA and hepatic stellate cell LX-2 were used as in vitro cell models to experimentally validate the effects of TCS by immunological, proteomic and metabolomic technologies. We established a relatively short term TCS exposure murine model and found the TCS mainly accumulated in the liver. The scRNA-seq performed on the livers of the TCS-treated and control group profiled the gene expressions of > 76,000 cells belonging to 13 major cell types. Among these types, hepatocytes and hepatic stellate cells (HSCs) were significantly increased in TCS-treated group. We found that TCS promoted fibrosis-associated proliferation of hepatocytes, in which Gata2 and Mef2c are the key driving TFs. Our data also suggested that TCS induced the proliferation and activation of HSCs, which was experimentally verified in both liver tissue and cell model. In addition, other changes including the dysfunction and capillarization of endothelial cells, an increase of fibrotic characteristics in B plasma cells, and M2 phenotype-skewing of macrophage cells, were also deduced from the scRNA-seq analysis, and these changes are likely to contribute to the progression of liver fibrosis. Lastly, the key differential ligand-receptor pairs involved in cellular communications were identified and we confirmed the role of GAS6_AXL interaction-mediated cellular communication in promoting liver fibrosis. TCS modulates the cellular activities and fates of several specific cell types (including hepatocytes, HSCs, endothelial cells, B cells, Kupffer cells and liver capsular macrophages) in the liver, and regulates the ligand-receptor interactions between these cells, thereby promoting the proliferation and activation of HSCs, leading to liver fibrosis. Overall, we provide the first comprehensive single-cell atlas of mouse livers in response to TCS and delineate the key cellular and molecular processes involved in TCS-induced hepatotoxicity and fibrosis.

更新日期：2023-02-22

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