消瘤散调控AKT/mTOR-Rac1通路抑制肝癌生长机制研究

Xiaoliu Powder inhibits hepatocellular carcinoma by suppressing the AKT/mTOR-Rac1 pathway

关键词:  消瘤散  肝癌  Rac1  AKT/mTOR

Key words:  Xiaoliu Powder  hepatocellular carcinoma  Rac1  AKT/mTOR pathway

摘要:

[目的] 探讨具有健脾消癥、扶正祛邪作用的消瘤散（Xiaoliu San，XLS）对肝癌荷瘤裸鼠模型及肝癌细胞株HepG2生长的抑制作用及其机制。[方法] 动物实验构建HepG2荷瘤鼠模型，将24只裸鼠随机分为模型组（生理盐水）和消瘤散低（12.5 g/kg）、中（25 g/kg）、高（50 g/kg）剂量组，每组6只，以8×106个细胞注射进裸鼠皮下，成瘤后灌胃给药14 d。统计裸鼠肿瘤大小、肝脏及脾脏数值。HE染色检测裸鼠荷瘤病理状态；生化检测裸鼠血清丙氨酸氨基转移酶（ALT）、天门冬氨酸氨基转移酶（AST）、总胆红素（TBil）、谷氨酰转移酶（GGT）、碱性磷酸酶（ALP）；免疫组化检测裸鼠肿瘤组织增殖细胞核抗原（Ki67）、血小板-内皮细胞黏附分子（CD31）；免疫蛋白印迹法（Western blot）检测裸鼠荷瘤丝氨酸/苏氨酸激酶（AKT）、p-AKT、哺乳动物雷帕霉素靶蛋白（mTOR）、p-mTOR、磷脂酰肌醇3激酶（PI3K）、Ras相关的C3肉毒素底物1（Rac1）表达。细胞实验选取HepG2细胞株，并将其分为XLS-L（1.25 mg/mL）、XLS-M（1.5 mg/mL）、XLS-H（1.75 mg/mL）剂量组给药，细胞计数试剂盒-8（CCK-8）法检测消瘤散对HepG2细胞的半抑制浓度（IC₅₀）；Transwell实验检测HepG2细胞侵袭能力；流式细胞术测定HepG2细胞凋亡能力；构建过表达Rac1的HepG2细胞（HepG2-Rac1^OE），分为XLS组（1.5 mg/mL）、Rac1OE组、Rac1OE+XLS组和对照组，Western blot检测各组细胞AKT、p-AKT、mTOR、p-mTOR、PI3K的表达。[结果] 动物实验提示，模型组HE染色可见大量肿瘤细胞。与模型组比较，消瘤散中、高剂量组的裸鼠荷瘤体积减小（P<0.05），消瘤散高剂量组的脾脏质量较模型组减轻（P<0.05）；消瘤散给药组的裸鼠血清的AST、TBil、GGT降低（P<0.01）；消瘤散给药组苏木精-伊红（HE）染色可见肿瘤细胞坏死，细胞质疏松，核仁固缩；消瘤散高剂量组Ki-67表达降低（P<0.05）；消瘤散中剂量组的Rac1表达下降（P<0.01），消瘤散高剂量组的Rac1、p-AKT、p-mTOR、PI3K表达下降（P<0.05或P<0.01）。细胞实验提示，随着给药浓度的增加，细胞存活率下降，IC₅₀值约1.5 mg/mL，细胞侵袭能力明显减弱（P<0.01），细胞凋亡比例升高，1.5 mg/mL的消瘤散处理72 h的HepG2细胞凋亡率最高（P<0.01）；与对照组相比，消瘤散组的p-AKT、p-mTOR、表达下降（P<0.05）。与Rac1^OE组相比，Rac1^OE+消瘤散组的p-AKT、p-mTOR表达下降（P<0.05或P<0.01）。[结论] 1）消瘤散可以抑制肝癌荷瘤裸鼠的肿瘤生长、改善肝脏功能，降低AKT/mTOR-Rac1通路蛋白表达。2）消瘤散可抑制HepG2细胞株生长、降低其迁移及侵袭能力、促进其凋亡，其机制可能与抑制AKT/mTOR-Rac1通路有关。

Abstract:

[Objective] Exploring the inhibitory effect and mechanism of the Xiaoliu Powder（XLS），which has the functions of strengthening the spleen，eliminating masses，reinforcing healthy qi，and dispelling pathogenic factors， on the growth of a hepatocellular carcinoma（HCC）bearing nude mouse model and the hepatocellular carcinoma cell line HepG2. [Methods] Animal experiment：HepG2 xenograft mouse model was established，24 nude mice were randomly divided into 4 groups（n=6 per group）：model group（normal saline），XLS low-dose（12.5 g/kg），medium- dose（25 g/kg），and high-dose（50 g/kg）groups. Subcutaneous injection of 8×10⁶ HepG2 cells was performed. After tumor formation，drugs were administered via gastric gavage for 14 days. Tumor size，liver，and spleen indices were recorded. HE staining assessed tumor pathology. Serum levels of alanine aminotransferase（ALT），aspartate aminotransferase（AST），total bilirubin（TBil），gamma-glutamyl transferase（GGT），and alkaline phosphatase（ALP） were measured biochemically. Immunohistochemistry（IHC）detected Ki67 and CD31 expression in tumor tissues. Western blot（WB）analyzed the expression of AKT，p-AKT，mTOR，p-mTOR，PI3K，and Ras-related C3 botulinum toxin substrate 1（Rac1）in tumors. Cell experiment：HepG2 cells were treated with XLS-L（1.25 mg/mL），XLS-M （1.5 mg/mL），and XLS-H（1.75 mg/mL）. The half-maximal inhibitory concentration（IC₅₀）was determined by the CCK-8 assay. The transwell assay assessed cell invasion. Flow cytometry measured apoptosis. Rac1-overexpressing HepG2 cells（HepG2 -Rac1^OE）were constructed and divided into XLS（1.5 mg/mL），Rac1^OE，Rac1^OE + XLS，and control groups. WB detected AKT，p-AKT，mTOR，p-mTOR，and PI3K expression. [Results] Animal experiment： HE staining revealed numerous tumor cells in the model group. Compared to the model group，tumor volume significantly decreased in the XLS medium and high-dose groups（P<0.05），and spleen weight decreased in the XLS high-dose group（P<0.05）. Serum AST，TBil，and GGT levels decreased in all XLS-treated groups（P<0.01）. HE staining of XLS-treated groups showed tumor cell necrosis，cytoplasmic vacuolation，and nuclear pyknosis. Ki67 expression decreased in the XLS high-dose group（P<0.05）. Rac1 expression decreased in the XLS medium-dose group（P<0.01）. Rac1，p-AKT，p-mTOR，and PI3K expression decreased in the XLS high-dose group（P<0.05，P<0.01）.Cell experiment：Cell viability decreased with increasing XLS concentration；the IC₅₀ was approximately 1.5 mg/mL. Cell invasion ability was significantly weakened（P<0.01），and the apoptosis rate increased. The highest apoptosis rate occurred after 72h treatment with 1.5 mg/mL XLS（P<0.01）. Compared to the control group，p-AKT and p-mTOR expression decreased in the XLS group（P<0.05）. Compared to the Rac1^OE group，p-AKT and p- mTOR expression decreased in the Rac1^OE + XLS group（P<0.05，P<0.01）. [Conclusion] 1）Xiaoliu Powder can inhibit tumor growth in HCC-bearing nude mice，improve liver function，and reduce the expression of proteins in the AKT/mTOR-Rac1 signaling pathway.2）Xiaoliu Powder can inhibit HepG2 cell proliferation，reduce migration and invasion，and promote apoptosis，potentially through suppressing the AKT/mTOR-Rac1 pathway.