| 引用本文: |
黄超文,刘艳娟,王璐,包晴,蒋宇,杨磊.基于转录组学探讨何首乌不同炮制品的肝细胞毒性作用机制[J].湖南中医药大学学报,2023,43(6):1028-1034[点击复制] |
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| 基于转录组学探讨何首乌不同炮制品的肝细胞毒性作用机制 |
| 黄超文,刘艳娟,王璐,包晴,蒋宇,杨磊 |
| (湖南中医药大学第一附属医院, 湖南 长沙 410007;湖南省人民医院, 湖南 长沙 410005) |
| 摘要: |
| 目的 探讨何首乌（Polygonum Multiflorum, PM）炮制后降低肝毒性的作用机制。方法 通过使用生何首乌（raw Polygonum Multiflorum, RPM）与制何首乌（processed Polygonum Multiflorum, PPM）处理L02肝细胞，采用CCK-8与EdU试剂检测L02肝细胞的细胞活力与增殖能力；运用转录组学技术分析正常组、RPM、PPM组之间的差异基因，并对差异基因进行富集分析，在此基础上采用Western blot和免疫荧光验证相关通路上关键蛋白的表达水平。结果 CCK-8和EdU实验中发现，与正常组相比，RPM组能够显著抑制肝细胞的细胞活力与增殖能力（P<0.01），与RPM组相比，PPM组的肝细胞活力与增殖能力明显升高（P<0.01）。RNA测序结果发现，与正常组相比，RPM组有3449个上调基因、3793个下调基因。与RPM组相比，PPM组有3788个基因上调、3515个基因下调。将正常组与RPM组的差异基因和RPM组与PPM组的差异基因进一步取交集，得到6854个共同差异基因；GO分析类结果显示主要参与细胞杀伤、细胞聚集、新陈代谢、生长等生物过程；KEGG富集分析结果显示，这些差异基因主要富集在内吞作用、溶酶体、自噬作用等细胞过程。Western blot与免疫荧光结果显示，与正常组相比，RPM组中自噬相关基因LC3蛋白水平显著上调（P<0.01），P62蛋白水平显著降低（P<0.01）；与RPM组相比，PPM组中LC3蛋白水平显著降低（P<0.01），P62蛋白水平显著升高（P<0.01）。结论 何首乌炮制后能够降低肝细胞毒性，其机制可能与减少细胞自噬有关，为降低何首乌毒性提供了新的干预措施。 |
| 关键词: 何首乌|炮制|自噬|肝毒性|转录组学 |
| DOI：10.3969/j.issn.1674－070X.２０23.06.010 |
| 投稿时间：2022-11-22 |
| 基金项目:国家自然科学基金资助项目（81804075）；张志国“全国名老中医药专家传承工作室建设项目”（国中药人教发〔2016〕42）。 |
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| Hepatocytotoxic mechanism of different processing products of Heshouwu (Polygonum Multiflorum) based on transcriptomics |
| HUANG Chaowen,LIU Yanjuan,WANG Lu,BAO Qing,JIANG Yu,YANG Lei |
| (The First Hospital of Hunan University of Chinese Medicine, Changsha, Hunan 410007, China;Hunan Provincial People's Hospital, Changsha, Hunan 410005, China) |
| Abstract: |
| Objective To investigate the mechanism of action of the processed Heshouwu (Polygoni Multiflori) (PM) in reducing hepatocytotoxicity. Methods By treating L02 hepatocytes with raw Heshouwu (Polygonum Multiflora) (RPM) and processed Heshouwu (Polygoni Multiflori) (PPM), cell viability and proliferative capacity of L02 hepatocytes were detected using CCK-8 with EdU reagent; the gene differences among the normal, RPM and PPM groups were analyzed and the gene set enrichment analysis was performed by transcriptomics. Finally, Western blot and immunofluorescence were used to verify the expression levels of key proteins on the relevant pathways. Results CCK-8 and EdU experiments showed that RPM significantly inhibited cell viability and proliferative capacity of hepatocyte compared to the normal group (P<0.01), hepatocyte viability and proliferative capacity in the PPM group were significantly highercompared to the RPM group(P<0.01). The RNA sequencing revealed: 3449 up-regulated and 3793 down-regulated genes in RPM group compared with normal group; 3788 up-regulated and 3515 down-regulated genes in PPM group compared with RPM group. GO classification showed that the genes were mainly involved in cell killing, cell aggregation, metabolism, growth and other biological processes; KEGG classification suggested that these genes were mainly enriched in endocytosis, lysosome, autophagy and other cellular processes. The Western blot and immunofluorescence analyses showed the significantly higher protein level of LC3 (P<0.01) and lower protein level of P62 (P<0.01) in RPM group than in normal group; PPM group had the significantly lower protein level of LC3 (P<0.01) and higher level of P62 (P<0.01) than RPM group. Conclusion PPM is able to alleviate hepatocytotoxicity, whose mechanism may be reducing autophagy. The study may provide a new intervention to alleviate the toxicity of PM. |
| Key words: Heshouwu (Polygonum Multiflorum)|processing|autophagy|hepatotoxicity|transcriptomics |
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