Compatibility mechanism of Chrysanthemi Indici Flos compound against hepatocellular carcinoma based on balance of DNMTs/TET2
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摘要:
目的：当前中药复方配伍研究中药效评价指标较为单一，本研究基于DNMTs/TET2的平衡关系探讨了野菊花复方抗肝癌的配伍机制。方法：以细胞活力和DNMTs/TET2平衡关系作为评价指标，采用CCK-8法、RT-PCR等检测方法，比较了单味药及不同配比的中药对HepG2和H22细胞的影响，筛选出复方最佳配伍。通过药效学实验进一步验证最佳配伍复方的抗肝癌作用。结果：不同配比药物的细胞增殖抑制率和DNMTs/TET2平衡关系的变化趋势始终一致，复方( 5 g / kg )的抑瘤率为62.97 %。野菊花复方通过减少MDA的积累，避免氧化应激，降低AST、ALT和AFP水平，避免肝功能损伤；通过增强CD3 + T细胞、CD4 + T细胞、CD8 + T细胞的表达，增强肿瘤免疫功能，发挥抗肝癌作用。结论：本研究证实了DNMTs/TET2平衡关系作为中药复方配伍机制研究评价指标的合理性及可行性。DNMTs/TET2平衡关系指导下筛选出的野菊花复方具有肝保护和肿瘤免疫功能，表现出良好的抗肝癌作用。
Abstract:
Objective: At present, traditional Chinese medicine (TCM) compounds is widely used in tumor therapy. However, due to the current single and simplistic efficacy evaluation indicators that are detached from the guidance of TCM theory, the elucidation of its mechanisms of efficacy is hindered. Therefore, the compatibility mechanism of Chrysanthemi Indici Flos compound (YJHFF) against hepatocellular carcinoma based on DNA methyltransferases/DNA demethylase ten-eleven translocation 2 (DNMTs/TET2) balanced relationship was explored according to the concept of the overall balance of TCM. Methods: This study screened the best compound compatibility ratio in the in vitro experiments, cell viability and DNMTs/TET2 balance were used as evaluation indicators to investigate the effects of single herb and different compatibility of compound on HepG2 and H22 cells. In vivo experiments were conduct on H22 liver orthotopic transplantation model mice. After treatment, the tumor tissues were observed by hematoxylin-eosin (HE), TUNEL and Ki67 immunohistochemical staining, respectively. The oxidative stress and liver function indicators in serum were detected by enzyme-linked immunosorbent assay. In addition, the proportion of tumor-infiltrating lymphocytes was detected by immunofluorescence staining. The mRNA expression levels of DNMTs and TET2 in tumor tissues were detected by RT-PCR. Results: The results of in vitro experiments showed that the cell proliferation inhibition rate was highly consistent with the change trend of DNMTs/TET2 balance, accordingly, this study obtained the optimal formula YJHFF. In the in vivo experiments, the tumor growth inhibition rate of YJHFF in 5 g/kg was 62.97%. Extensive necrosis, increased apoptosis and inhibited proliferation activity were observed in tumor tissue. Furthermore, the serum SOD activity was significantly increased, MDA, ALT, AST and AFP levels were significantly decreased, suggesting YJHFF could systematically alleviated oxidative stress and improved liver function. Besides, the infiltration of CD3+ , CD4+ and CD8+ T lymphocytes increased significantly, indicating that the body’s anti-tumor immune response was enhanced. Finally, RT-PCR analysis confirmed that antitumor effects were closely related to the core mechanism of DNMTs/TET2 expression regulation and reconstruction of epigenetic balance. Conclusion: This work confirmed the rationality and feasibility of the DNMTs/TET2 balance relationship as an evaluation indicator for the study of the compatibility mechanism of TCM compounds. The YJHFF screened under the guidance of DNMTs/TET2 balance has liver protection and tumor immune function, showing good anti-hepatocellular carcinoma effect.
关键词:
DNMTs/TET2平衡;野菊花复方;配伍机制;疗效评价指标;肝癌
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This work was supported by National Natural Science Foundation of China (No. 82304732), the Postdoctoral Fellowship Program of CPSF (No. GZC20232456), the Fellowship from the China Postdoctoral Science Foundation (No. 2024M752966), and the Key Project of the Nature Fund of Henan Province, China (No.232300421118).
