目的 探讨砂生槐子总生物碱（TA-SM）抗肝细胞癌（HCC）的潜在作用靶点和作用机制。方法 采用网络药理学方法筛选TA-SM抗HCC的候选靶点与富集通路，构建相关网络，利用分子对接技术揭示砂生槐子中6种主要生物碱成分与潜在靶点的结合活性。体外以Huh-7、HepG2细胞为模型，CCK-8法检测TA-SM对2种细胞增殖的抑制效应，并借助Western blotting及实时荧光定量PCR（qRT-PCR）实验测定磷脂酰肌醇-3-激酶-蛋白激酶B（PI3K-Akt）信号通路中相关蛋白与基因的表达。体内构建HepG2裸鼠异种移植瘤模型，评价TA-SM对小鼠体质量与肿瘤体积的影响；通过Western blotting、qRT-PCR、免疫组化与免疫荧光评估PI3K-Akt通路变化； TUNEL检测肿瘤细胞凋亡；苏木精-伊红（HE）染色评估主要脏器毒性。结果 网络药理学分析显示TA-SM治疗HCC的核心靶点38个；基因本体（GO）富集654条，京都基因与基因组百科全书（KEGG）富集162条通路，涉及癌症通路、PI3K-Akt信号通路及内分泌抵抗等；分子对接显示6种生物碱与PI3K-Akt通路关键靶点结合能均＜-20.93 kJ·mol-1。体外实验显示TA-SM在1～64 μg·mL^-1范围内对Huh-7与HepG2细胞呈浓度相关性增殖抑制，并下调PI3K、Akt1、糖原合成酶激酶-3β（GSK3β）、p-Akt、p-GSK3β、p-Caspase-9的蛋白及mRNA表达，上调Caspase-9的蛋白及mRNA表达。体内实验显示TA-SM抑制移植瘤生长且对主要脏器无明显毒性；能显著下调PI3K、Akt1、GSK3β、p-Akt、p-GSK3β、p-Caspase-9的蛋白及基因表达（P＜0.05），升高Caspase-9表达（P＜0.05）； TUNEL染色结果显示索拉非尼组、TA-SM组肿瘤细胞凋亡率均较对照组显著升高（P＜0.001），TA-SM组诱导肿瘤细胞凋亡的能力显著优于索拉非尼。结论 基于网络药理学筛选提示TA-SM可能通过多靶点、多通路发挥抗HCC作用，其中PI3K-Akt信号通路为关键机制之一；体外与体内实验结果共同支持TA-SM通过抑制PI3K-Akt通路、促进Caspase-9相关凋亡从而抑制HCC生长的作用机制。

Objective To explore the potential target and mechanism of total alkaloids of Sophora moorcroftiana (TA-SM) against hepatocellular carcinoma. Methods Firstly, the network pharmacology method was used to screen the candidate targets and enrichment pathways of TA-SM against HCC, and the related network was constructed. The molecular docking technique was used to suggest the binding activity of six main alkaloid components of Sophora moorcroftiana with potential targets. In vitro, Huh-7 and HepG2 cells were used as models. CCK-8 method was used to detect the inhibitory effect of TA-SM on the proliferation of Huh-7 and HepG2 cells. Western Blotting and qRT-PCR experiments were used to determine the expression of related proteins and genes in the PI3K-Akt signaling pathway. The effect of TA-SM on the weight and tumor volume of mice was evaluated by constructing HepG2 xenograft tumor model in nude mice. The changes of PI3K-Akt pathway were evaluated by Western blotting, qRT-PCR, immunohistochemistry and immunofluorescence. Apoptosis of tumor cells was detected by TUNEL. The main organ toxicity was evaluated by HE staining. Results Network pharmacology analysis showed that there were 38 core targets of TA-SM in the treatment of HCC. There were 654 GO enrichment and 162 KEGG enrichment pathways, involving cancer pathway, PI3K-Akt signaling pathway and endocrine resistance. Molecular docking showed that the binding energy of the six alkaloids to the key targets of the PI3K-Akt pathway was less than -20.93 kJ·mol-1. In vitro experiments showed that TA-SM inhibited the proliferation of Huh-7 and HepG2 cells in a concentration-dependent manner in the range of 1-64 μg·mL^-1, and down-regulated the protein and mRNA expression of PI3K, Akt1, GSK3β, p-Akt, p-GSK3β and p-Caspase-9, and up-regulated the protein and mRNA expression of Caspase-9. In vivo experiments showed that TA-SM inhibited the growth of transplanted tumors and had no obvious toxicity to major organs. PI3K, Akt1, GSK3β, pAkt, p-GSK3β, p-Caspase-9 protein and gene expression were significantly down-regulated (P < 0.05), and Caspase-9 expression was increased (P < 0.05). TUNEL staining results showed that the apoptosis rate of tumor cells in sorafenib group and TA-SM group was significantly higher than that in Saline group (P < 0.001), and the ability of TA-SM group to induce apoptosis of tumor cells was significantly better than that of sorafenib group. Conclusion Based on network pharmacology screening, it is suggested that TA-SM may play an anti-HCC role through multiple targets and multiple pathways, among which PI3K-Akt signaling pathway is one of the key mechanisms. The results of in vitro and in vivo experiments support that TA-SM inhibits the growth of hepatocellular carcinoma by inhibiting the PI3K-Akt pathway and promoting Caspase-9-related apoptosis.

R285.5

青海省科技厅科技援青合作专项（2024-QY-201）