BACKGROUND:The phosphoinositide 3-kinase/protein kinase-B/mechanistic target of rapamycin (PI3K/Akt/mTOR) signalling pathway is crucial for cell survival, differentiation, apoptosis and metabolism. Xihuang pills (XHP) are a traditional Chinese preparation with antitumour properties. They inhibit the growth of breast cancer, glioma, and other tumours by regulating the PI3K/Akt/mTOR signalling pathway. However, the effects and mechanisms of action of XHP in hepatocellular carcinoma (HCC) remain unclear. Regulation of the PI3K/Akt/mTOR signalling pathway effectively inhibits the progression of HCC. However, no study has focused on the XHP-associated PI3K/Akt/mTOR signalling pathway. Therefore, we hypothesized that XHP might play a role in inhibiting HCC through the PI3K/Akt/mTOR signalling pathway. AIM:To confirm the effect of XHP on HCC and the possible mechanisms involved. METHODS:The chemical constituents and active components of XHP were analysed using ultra-performance liquid chromatography-quadrupole time of flight mass spectrometry (UPLC-Q-TOF-MS). Cell-based experiments and in vivo xenograft tumour experiments were utilized to evaluate the effect of XHP on HCC tumorigenesis. First, SMMC-7721 cells were incubated with different concentrations of XHP (0, 0.3125, 0.625, 1.25, and 2.5 mg/mL) for 12 h, 24 h and 48 h. Cell viability was assessed using the CCK-8 assay, followed by an assessment of cell migration using a wound healing assay. Second, the effect of XHP on the apoptosis of SMMC-7721 cells was evaluated. SMMC-7721 cells were stained with fluorescein isothiocyanate and annexin V/propidium iodide. The number of apoptotic cells and cell cycle distribution were measured using flow cytometry. The cleaved protein and mRNA expression levels of caspase-3 and caspase-9 were detected using Western blotting and quantitative reverse-transcription polymerase chain reaction (RT-qPCR), respectively. Third, Western blotting and RT-qPCR were performed to confirm the effects of XHP on the protein and mRNA expression of components of the PI3K/Akt/mTOR signalling pathway. Finally, the effects of XHP on the tumorigenesis of subcutaneous hepatocellular tumours in nude mice were assessed. RESULTS:The following 12 compounds were identified in XHP using high-resolution mass spectrometry: Valine, 4-gingerol, myrrhone, ricinoleic acid, glycocholic acid, curzerenone, 11-keto-β-boswellic acid, oleic acid, germacrone, 3-acetyl-9,11-dehydro-β-boswellic acid, 5β-androstane-3,17-dione, and 3-acetyl-11-keto-β-boswellic acid. The cell viability assay results showed that treatment with 0.625 mg/mL XHP extract decreased HCC cell viability after 12 h, and the effects were dose- and time-dependent. The results of the cell scratch assay showed that the migration of HCC cells was significantly inhibited in a time-dependent manner by the administration of XHP extract (0.625 mg/mL). Moreover, XHP significantly inhibited cell migration and resulted in cell cycle arrest and apoptosis. Furthermore, XHP downregulated the PI3K/Akt/mTOR signalling pathway, which activated apoptosis executioner proteins (e.g., caspase-9 and caspase-3). The inhibitory effects of XHP on HCC cell growth were determined in vivo by analysing the tumour xenograft volumes and weights. CONCLUSION:XHP inhibited HCC cell growth and migration by stimulating apoptosis via the downregulation of the PI3K/Akt/mTOR signalling pathway, followed by the activation of caspase-9 and caspase-3. Our findings clarified that the antitumour effects of XHP on HCC cells are mediated by the PI3K/Akt/mTOR signalling pathway, revealing that XHP may be a potential complementary therapy for HCC.

目的 探究下瘀血汤的活性成分及其抗肝癌的作用机制.方法 应用超高效液相色谱-四极杆-飞行时间质谱(UPLC-Q-TOF-MS)分析鉴定下瘀血汤的活性成分,借助TCMSP、PubChem、SwissTargetPrediction、UniProt数据库收集活性成分对应靶点.通过GeneCards、OMIM数据库筛选原发性肝癌的相关靶点,采用Cytoscape3.8.2构建成分-靶点-疾病网络.借助STRING数据库构建蛋白相互作用网络,运用Metascape数据库对靶点进行GO功能及KEGG通路富集分析.利用分子对接验证下瘀血汤主要活性成分与核心靶点的相互作用.采用CCK8法检测下瘀血汤含药血清对肝癌细胞HepG2和Huh-7增殖的影响,并通过RT-qPCR验证其对关键靶点的作用.结果 鉴定下瘀血汤活性成分49个,其中山柰酚、儿茶素、苦杏仁苷、芦荟大黄素、大黄素、大黄酚葡萄糖苷等是其抗肝癌潜在活性成分.预测得到其抗肝癌潜在靶点112个,关键靶点涉及CASP3、ESR1、PPARG、MYC等.GO功能及KEGG通路富集分析结果显示,下瘀血汤抗肝癌作用机制主要与PI3K-Akt信号通路、炎症反应、激素水平调节、cGMP-PKG信号通路调控的抗血管生成、紧密连接调控的肠屏障、肿瘤细胞的缺氧、应激、激酶结合等途径相关.分子对接结果显示,下瘀血汤中大黄酚葡萄糖苷和儿茶素二聚体等活性成分与核心靶点具有较好的亲和能力.细胞实验证实,下瘀血汤含药血清浓度为20％时可有效抑制肝癌细胞增殖,并可降低核心靶点CASP3、ESR1、PPARG、MYC表达.结论 下瘀血汤中多种成分具有抗肝癌活性,通过CASP3、ESR1、PPARG、MYC等多靶点及多途径发挥抗肝癌作用.

BACKGROUND:In traditional Chinese medicine (TCM), frankincense and myrrh are the main components of the antitumor drug Xihuang Pill. These compounds show anticancer activity in other biological systems. However, whether frankincense and/or myrrh can inhibit the occurrence of hepatocellular carcinoma (HCC) is unknown, and the potential molecular mechanism(s) has not yet been determined.AIM:To predict and determine latent anti-HCC therapeutic targets and molecular mechanisms of frankincense and myrrh in vivo.METHODS:In the present study, which was based on the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (http://tcmspw.com/tcmsp.php), Universal Protein database (http://www.uniprot.org), GeneCards: The Human Gene Database (http://www.genecards.org/) and Comparative Toxicogenomics Database (http://www.ctdbase.org/), the efficacy of and mechanism by which frankincense and myrrh act as anti-HCC compounds were predicted. The core prediction targets were screened by molecular docking. In vivo, SMMC-7721 human liver cancer cells were transplanted as xenografts into nude mice to establish a subcutaneous tumor model, and two doses of frankincense plus myrrh or one dose of an EGFR inhibitor was administered to these mice continuously for 14 d. The tumors were collected and evaluated: the tumor volume and growth rate were gauged to evaluate tumor growth; hematoxylin-eosin staining was performed to estimate histopathological changes; immunofluorescence (IF) was performed to detect the expression of CD31, α-SMA and collagen IV; transmission electron microscopy (TEM) was conducted to observe the morphological structure of vascular cells; enzyme-linked immunosorbent assay (ELISA) was performed to measure the levels of secreted HIF-1α and TNF-α; reverse transcription-polymerase chain reaction (RT-qPCR) was performed to measure the mRNA expression of HIF-1α, TNF-α, VEGF and MMP-9; and Western blot (WB) was performed to determine the levels of proteins expressed in the EGFR-mediated PI3K/Akt and MAPK signaling pathways.RESULTS:The results of the network pharmacology analysis showed that there were 35 active components in the frankincense and myrrh extracts targeting 151 key targets. The molecular docking analysis showed that both boswellic acid and stigmasterol showed strong affinity for the targets, with the greatest affinity for EGFR. Frankincense and myrrh treatment may play a role in the treatment of HCC by regulating hypoxia responses and vascular system-related pathological processes, such as cytokine-receptor binding, and pathways, such as those involving serine/threonine protein kinase complexes and MAPK, HIF-1 and ErbB signaling cascades. The animal experiment results were verified. First, we found that, through frankincense and/or myrrh treatment, the volume of subcutaneously transplanted HCC tumors was significantly reduced, and the pathological morphology was attenuated. Then, IF and TEM showed that frankincense and/or myrrh treatment reduced CD31 and collagen IV expression, increased the coverage of perivascular cells, tightened the connection between cells, and improved the shape of blood vessels. In addition, ELISA, RT-qPCR and WB analyses showed that frankincense and/or myrrh treatment inhibited the levels of hypoxia-inducible factors, inflammatory factors and angiogenesis-related factors, namely, HIF-1α, TNF-α, VEGF and MMP-9. Furthermore, mechanistic experiments illustrated that the effect of frankincense plus myrrh treatment was similar to that of an EGFR inhibitor with regard to controlling EGFR activation, thereby inhibiting the phosphorylation activity of its downstream targets: the PI3K/Akt and MAPK (ERK, p38 and JNK) pathways.CONCLUSION:In summary, frankincense and myrrh treatment targets tumor blood vessels to exert anti-HCC effects via EGFR-activated PI3K/Akt and MAPK signaling pathways, highlighting the potential of this dual TCM compound as an anti-HCC candidate.

鳖甲是临床常用药物,现代对鳖甲的研究主要聚焦于实验研究及临床观察,鲜见文献研究.该文在系统查阅鳖甲古今文献的基础上,通过分析历代本草方书中的相关内容,对鳖甲的名称、基原、产地、品质评价、功效主治、炮制方法及用药禁忌进行全面考证.通过考证发现,在基原上,古籍文献所载的鳖甲当来源于中华鳖Trionyx sinensis的背甲,山瑞鳖T.steindachneri的背甲不宜作为中药鳖甲的来源.鳖甲的道地产区在今长江中下游的岳阳、荆州、安徽东南部及江苏西部.关于鳖甲的品质评价,本草古籍中常以鳖甲的肋数如七肋、九肋作为品质评价的标准,但通过文献研究及对药材市场的实地考察,发现以肋数作为品质的评价标准在现代并不可取.随着时代的更迭,鳖甲的功效主治在《神农本草经》的基础上逐渐扩展,后世将鳖甲广泛应用于内外妇儿各科.需要注意的是,鳖甲治劳热骨蒸当来源于《神农本草经》,而不是《本草衍义》等古籍所言的《药性论》.在炮制方面,鳖甲炮制方法多样,主要以醋制为主.在用药禁忌方面,鳖甲不能与矾石、理石配伍,孕妇禁用,脾虚胃弱、肝虚无热者慎用.该文的考证结果为鳖甲的正本清源及进一步资源开发利用提供了参考依据.

目的:分析、总结经方中黄连"角药"的配伍特点与临床应用,并深入剖析以黄连为角药的类方鉴别,为现代临床用药谴方提供理论依据.方法:谨遵经旨,对经方中黄连"角药"的配伍特点与临床应用进行详尽梳理,并从独立成方之"角药"、作为方剂主要部分之"角药"以及类方鉴别三方面进行论述.结果:经方中独立成方之黄连"角药"两对、作为方剂主要部分之"角药"7 对,黄连的角药配伍,寒热并用,表里同调,虚实相兼,使得其可用于下利、呕吐、肠鸣、烦躁、心下痞等多个方证.结论:经方中蕴含着丰富的黄连"角药"配伍知识,理解掌握经方中黄连"角药"的配伍特点与临床应用及其类方的准确运用,可扩大主治,减少药味,精准治疗.

目的 运用网络药理学方法及分子对接技术对雷公藤治疗肝癌的主要活性成分及其潜在作用机制进行探讨.方法 通过中药系统药理学数据库与分析平台(TCMSP)筛选雷公藤主要活性成分及其预测靶点;在DrugBank、GeneCards、OMIM、TTD数据库中筛选肝癌相关靶点;通过R语言软件映射得到雷公藤治疗肝癌靶点,并绘制韦恩图;在STRING网站构建治疗靶点PPI网络图;应用Cytoscape软件构建"雷公藤-活性成分-靶点-肝癌"互作网络图;通过R语言软件对治疗靶点进行GO功能分析和KEGG通路富集分析;应用PubChem、RCSB PDB数据库,AutoDock、PyMOL软件对药物潜在活性成分与关键靶点进行分子对接.结果 共筛选出活性成分51个和潜在治疗靶点120个,靶点主要涉及酰胺结合、肽结合、DNA结合转录因子结合等生物学过程,并主要富集于卡波西肉瘤相关疱疹病毒感染、乙型肝炎、流体剪切应力和动脉粥样硬化等信号通路中.分子对接验证显示对接得分大于-5kcal/mol占100％,即所有靶点与成分的结合活性较好.结论 通过网络药理学方法及分子对接技术证实了雷公藤多成分、多靶点、多途径的作用特点,预测了雷公藤治疗肝癌的潜在作用机制,为后续进一步开发和应用提供理论依据.

肿瘤目前已成为危害人类生命健康的主要疾病之一.研究表明,龟鹿二仙胶可通过增强机体对肿瘤细胞的免疫、降低肿瘤细胞耐药性、改善骨髓抑制,从而发挥抗肿瘤作用,而成方中单药抗肿瘤作用亦可通过诱导细胞凋亡、抑制新生血管形成等多种途径来实现.虽然龟鹿二仙胶抗肿瘤机制研究已取得一定进展,但主要聚焦于免疫系统,今后需进一步研究其相关作用机制,为肿瘤临床治疗拓宽思路和方法.

目的 基于方证对应理论探讨中医药治疗肝郁脾虚型肝癌临床及用药规律.方法 根据服用中药时间将患者分成中药弱暴露(less-CM)组及中药强暴露(CM)组,评价2组患者生存时间(OS)及无进展生存时间(PFS);利用中医传承辅助平台,对CM组患者处方进行分析.结果 CM组患者在OS及PFS优于Less-CM组(P<0.05);单味药物频次使用频率最高的中药是甘草,其次为黄芪,白术等,药物模式以补脾益气、疏肝行气、清热解毒等为主.获得新方3个,分别为柴胡-白芍-枸杞-白术-茵陈-郁金;黄精-补骨脂-透骨草-地龙-鸡血藤-骨碎补;田基黄-苏梗-海螵蛸-莪术-土鳖虫.结论 服用中药汤剂超过6个月可提高肝郁脾虚型肝癌患者PFS及OS.肝癌肝郁脾虚证患者在疾病过程中会出现热证、瘀证、痰湿证等兼证.

壁虎和石龙子药用历史悠久,但长期以来两者常混为一谈,而现代对两者的研究主要集中在临床观察、有效成分及抗癌机制等方面,鲜见文献研究.该文在系统查阅壁虎和石龙子古今文献的基础上,通过分析历代本草方书中的相关内容,对两者的名称、基原、功效主治、炮制方法及用药禁忌进行全面考证.通过考证发现,在先秦,壁虎与石龙子被当作同一物,至汉代则知两者为不同的动物,到三国时期两者又被混淆,直至明代《本草纲目》才明确两者的关系.在基原上,石龙子的基原包括蜥蜴科丽斑麻蜥Eremias argus,山地麻蜥E.brenchleyi及石龙子科中国石龙子Eumeses chinensis.壁虎的基原包括多疣壁虎Gekko japonicus,铅山壁虎G.hokouensis,无蹼壁虎G.swinhonis及蹼趾壁虎G.subpalmatus.功效主治方面,历代本草记载石龙子的主要功效为利水,壁虎的主要功效为祛风、化瘀.在炮制上壁虎与石龙子有多种炮制方法,但主要以传统"火制法"为主.关于用药禁忌,古籍文献记载石龙子恶硫黄、芜荑、斑蝥,孕妇忌用;壁虎慎用于血虚气弱之人.该文的考证结果厘清了壁虎和石龙子从名称、功效主治到炮制等多方面的历史沿革,为壁虎及石龙子的正本清源及进一步开发利用提供了理论依据.