A traditional Chinese medicine decoction developed based on the theory of “Yang transforming into Qi with Yin constituting Form” suppresses hepatic tumorigenesis in mouse models

Background Also the traditional Chinese medicine (TCM) theory of "Yang transforming into Qi with Yin constituting Form" was recognized as a potential mechanism underlying the tumorigenesis. The decoction based on this theory and the underlying molecular mechanisms remained exploration. Methods A TCM decoction was prepared based on "Yang transforming into Qi with Yin constituting Form". Its anti-tumorigenesis effect was evaluated on mouse models of hepatocellular carcinoma (HCC) (H-rasG12V transgenic mice (Ras-Tg) and Diethylnitrosamine (DEN)-induced HCC mice) by liver/body weight, number and size of tumors, histopathological changes and serum biochemistry. RNA sequencing was utilized to check differentially expressed genes (DEGs) and gene ontology (GO) and KEGG were used for bioinformatics analysis. The concerned biological processes were further confirmed by RT-qPCR and metabolite detection kits. Results The TCM decoction significantly suppressed both Ras oncogene- and DEN-induced hepatic tumorigenesis evidenced by reduced serum ALT and AST levels, liver weight, liver/body weight ratio, hepatic tumor numbers and sizes. RNA sequencing showed 659 up-regulated genes and 222 down-regulated genes in comparison of liver tissues between TCM-treated and untreated Rag-Tg (n=5). GO entries and KEGG analysis revealed multiple biological processes and pathways involving the hepatic tumorigenesis suppressing effective of the TCM. Further molecular examination confirmed that activation of retinal metabolism, reduction of ROS and electrophilic levels by inhibiting the H2O2 production and promoting GSH production, and inhibition of expression of inflammation factors play pivotal roles in the anti-tumorigenesis effects of TCM. Conclusion A TCM decoction based on "Yang transforming into Qi with Yin constituting Form" showed effective anti-hepatic tumorigenesis functions and its regulation functions on retinal, ROS and inflammation processes play important roles. The present study provides valuable clue for clinical therapy on tumorigenesis. Firstly, develop and prepare traditional Chinese medicine decoctions, and confirm the anti liver tumor occurrence or development effect of TCM by feeding Ras oncogene and DEN induced HCC mouse models. Perform bioinformatics analysis to investigate potential mechanisms by sequencing RNA from peri-tumor and tumor tissues.

Background：传统中医学（Traditional Chinese Medicine, TCM）“阳化气，阴成形”理论被认为是肿瘤发生的潜在机制，基于该理论构建的中药汤剂及其潜在分子机制仍有待探索。

Methods：本研究基于“阳化气，阴成形”理论制备中药汤剂，并以肝细胞癌（Hepatocellular carcinoma, HCC）小鼠模型为研究对象，涵盖H-rasG12V转基因小鼠（Ras-Tg）及二乙基亚硝胺（Diethylnitrosamine, DEN）诱导的HCC小鼠，通过肝重/体重比值、肿瘤数量与大小、组织病理学变化及血清生化指标，评估该汤剂的抗肿瘤发生作用。采用转录组测序（RNA sequencing）筛选差异表达基因（Differentially Expressed Genes, DEGs），并通过基因本体论（Gene Ontology, GO）与京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes, KEGG）开展生物信息学分析；进一步借助实时定量聚合酶链反应（RT-qPCR）及代谢物检测试剂盒，验证相关生物学过程。

Results：该中药汤剂可显著抑制Ras癌基因及DEN诱导的肝脏肿瘤发生，具体表现为血清谷丙转氨酶（Alanine Aminotransferase, ALT）、谷草转氨酶（Aspartate Aminotransferase, AST）水平降低，肝重、肝重/体重比值、肝脏肿瘤数量及体积均显著下降。对中药给药组与未给药组Ras-Tg小鼠（n=5）的肝组织进行转录组测序，结果显示共筛选得到659个上调基因与222个下调基因。GO富集分析与KEGG通路分析表明，该中药汤剂的抗肿瘤发生作用涉及多种生物学过程与信号通路。进一步的分子验证实验证实，其通过抑制过氧化氢（H₂O₂）生成、促进谷胱甘肽（Glutathione, GSH）合成，激活视黄醛代谢通路、降低活性氧（Reactive Oxygen Species, ROS）及亲电子物质水平，同时抑制炎症因子表达，上述过程在其抗肿瘤发生作用中发挥关键调控作用。

Conclusion：基于“阳化气，阴成形”理论制备的中药汤剂具有显著的抗肝脏肿瘤发生功效，其对视黄醛代谢、活性氧水平及炎症反应的调控作用是核心机制之一。本研究为肿瘤发生的临床治疗提供了宝贵线索：其一，开发并制备中药汤剂，通过饲喂Ras癌基因及DEN诱导的HCC小鼠模型，验证其抗肝脏肿瘤发生与进展的作用；其二，对肿瘤旁组织及肿瘤组织进行RNA测序，结合生物信息学分析探究潜在作用机制。