Department of Biology, Hong Kong Baptist University (HKBU), Kowloon Tong, Kowloon, Hong Kong, China; Golden Meditech Center for NeuroRegeneration Sciences (GMCNS), HKBU, Kowloon Tong, Hong Kong, China; HKBU Institute of Research and Continuing Education, Shenzhen, China.
Department of Biology, Hong Kong Baptist University (HKBU), Kowloon Tong, Kowloon, Hong Kong, China; Golden Meditech Center for NeuroRegeneration Sciences (GMCNS), HKBU, Kowloon Tong, Hong Kong, China.
J Ethnopharmacol. 2023 Aug 10;312:116548. doi: 10.1016/j.jep.2023.116548. Epub 2023 Apr 24.
Hepatocellular carcinoma (HCC) poses a growing challenge to global health efforts. The 5-year survival rate of HCC patients is still dismal. A traditional prescription Qi-Wei-Wan (QWW) comprising Astragali Radix and Schisandra chinensis Fructus has traditionally been used for HCC treatment according to traditional Chinese medicine theory, but the pharmacological basis is not clear.
This study aims to investigate the anti-HCC effects of an ethanolic extract of QWW (hereafter, QWWE) and the mechanism of action.
An UPLC-Q-TOF-MS/MS method was developed to control the quality of QWWE. Two human HCC cell lines (HCCLM3 and HepG2) and a HCCLM3 xenograft mouse model were employed to investigate the anti-HCC effects of QWWE. The anti-proliferative effect of QWWE in vitro was determined by MTT, colony formation and EdU staining assays. Apoptosis and protein levels were examined by flow cytometry and Western blotting, respectively. Nuclear presence of signal transducer and activator of transcription 3 (STAT3) was examined by immunostaining. Transient transfection of pEGFP-LC3 and STAT3C plasmids was performed to assess autophagy and determine the involvement of STAT3 signaling in QWWE's anti-HCC effects, respectively.
We found that QWWE inhibited the proliferation of and triggered apoptosis in HCC cells. Mechanistically, QWWE inhibited the activation of SRC and STAT3 at Tyr416 and Tyr705, respectively; inhibited the nuclear translocation of STAT3; lowered Bcl-2 protein levels, while increased Bax protein levels in HCC cells. Over-activating STAT3 attenuated the cytotoxic and apoptotic effects of QWWE in HCC cells. Moreover, QWWE induced autophagy in HCC cells by inhibiting mTOR signaling. Blocking autophagy with autophagy inhibitors (3-methyladenine and chloroquine) enhanced the cytotoxicity, apoptotic effect and the inhibitory effect on STAT3 activation of QWWE. Intragastric administration of QWWE at 10 mg/kg and 20 mg/kg potently repressed tumor growth and inhibited STAT3 and mTOR signaling in tumor tissues, but did not significantly affect mouse body weight.
QWWE exhibited potent anti-HCC effects. Inhibiting the STAT3 signaling pathway is involved in QWWE-mediated apoptosis, while blocking mTOR signaling contributes to QWWE-mediated autophagy induction. Blockade of autophagy enhanced the anti-HCC effects of QWWE, indicating that the combination of an autophagy inhibitor and QWWE might be a promising therapeutic strategy for HCC management. Our findings provide pharmacological justifications for the traditional use of QWW in treating HCC.
肝细胞癌（HCC）对全球健康努力构成了日益严峻的挑战。HCC 患者的 5 年生存率仍然不容乐观。根据中医理论，一种由黄芪和五味子组成的传统方剂——七味丸（QWW）一直被用于 HCC 的治疗，但药理基础尚不清楚。
本研究旨在探讨 QWW 的醇提物（QWWE）的抗 HCC 作用及其作用机制。
采用 UPLC-Q-TOF-MS/MS 方法控制 QWWE 的质量。采用两种人 HCC 细胞系（HCCLM3 和 HepG2）和 HCCLM3 异种移植小鼠模型来研究 QWWE 的抗 HCC 作用。通过 MTT、集落形成和 EdU 染色实验测定 QWWE 在体外的抗增殖作用。通过流式细胞术和 Western blot 分别检测细胞凋亡和蛋白水平。通过免疫染色检测信号转导和转录激活因子 3（STAT3）的核定位。通过瞬时转染 pEGFP-LC3 和 STAT3C 质粒分别评估自噬和确定 STAT3 信号通路在 QWWE 抗 HCC 作用中的参与情况。
我们发现 QWWE 抑制 HCC 细胞的增殖并触发其凋亡。在机制上，QWWE 分别抑制 SRC 和 STAT3 的 Tyr416 和 Tyr705 的激活；抑制 STAT3 的核转位；降低 HCC 细胞中的 Bcl-2 蛋白水平，同时增加 Bax 蛋白水平。过表达 STAT3 减弱了 QWWE 对 HCC 细胞的细胞毒性和凋亡作用。此外，QWWE 通过抑制 mTOR 信号通路诱导 HCC 细胞自噬。用自噬抑制剂（3-甲基腺嘌呤和氯喹）阻断自噬增强了 QWWE 的细胞毒性、凋亡作用和对 STAT3 激活的抑制作用。QWWE 以 10mg/kg 和 20mg/kg 的剂量灌胃给药可有效抑制肿瘤生长并抑制肿瘤组织中的 STAT3 和 mTOR 信号，但对小鼠体重无明显影响。
QWWE 表现出强大的抗 HCC 作用。抑制 STAT3 信号通路参与 QWWE 介导的细胞凋亡，而阻断 mTOR 信号通路有助于 QWWE 介导的自噬诱导。阻断自噬增强了 QWWE 的抗 HCC 作用，表明自噬抑制剂与 QWWE 的联合应用可能是 HCC 管理的一种有前途的治疗策略。我们的研究结果为 QWW 治疗 HCC 的传统应用提供了药理学依据。
