目的 运用网络药理学及动物实验研究肝积方（GJR）抗肝纤维化（LF）的机制。方法 构建LF模型，通过苏木素-伊红（HE）、Masson染色观察肝脏病理情况；蛋白免疫印迹法（Western blotting）检测肝组织中α-平滑肌肌动蛋白（α-SMA）、I型胶原蛋白alpha 1链（Col1a1）、转化生长因子-β(TGF-β)的蛋白表达水平；采用碱水解法检测肝组织中羟脯氨酸（HYP）水平；采用微板法检测小鼠血清中丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）的活性水平。基于中药系统药理学数据库与分析平台（TCMSP）和Swiss Target Prediction网站筛选GJR活性成分作用靶点，结合GeneCards数据库LF靶点，两者取交集获得共同靶点，构建蛋白质-蛋白质相互作用（PPI）网络筛选核心靶点，进行基因本体（GO）和京都基因与基因组百科全书（KEGG）富集分析，并通过分子对接预测关键靶点与GJR活性成分结合情况。肝组织原位末端标记法（TUNEL）染色检测肝细胞凋亡情况；实时荧光定量PCR(qRT-PCR)检测肝组织中半胱天冬氨酸蛋白酶3(Caspase3)、B细胞淋巴瘤/白血病-2(BCL2)、促凋亡蛋白（Bax）表达水平。体外实验通过TGF-β诱导LX-2细胞活化，加入GJR含药血清（10%、15%、20%）干预后进行qRT-PCR、Western blotting实验观察α-SMA、Col1a1、组织金属蛋白酶抑制因子-1(TIMP1)的m RNA和蛋白表达变化，通过流式细胞术观察LX-2细胞凋亡情况。结果 体内实验显示，GJR可减轻肝损伤及胶原沉积，降低HYP、ALT、AST水平，下调α-SMA、Col1a1、TGF-β表达，减少肝细胞凋亡并调控Caspase3、BCL2、Bax表达；网络药理学筛选出625个共同靶点，核心靶点与凋亡相关，富集于磷脂酰肌醇3?激酶/蛋白激酶B(PI3K-Akt)等通路，关键成分与核心靶点结合良好；体外实验显示，GJR含药血清可下调α-SMA、Col1a1、Timp mRNA及蛋白表达水平，促进LX-2细胞凋亡。结论 网络药理学及实验验证GJR具有抗LF作用，其机制可能是通过Caspase3/Bcl-2/Bax信号通路抑制肝细胞凋亡并促进LX-2细胞凋亡有关。

Objective To investigate the mechanism of Ganji Recipe(GJR) antiliver fibrosis(LF) using network pharmacology and experimental validation. Methods The LF model was established. Liver pathology was observed by hematoxylin-eosin(HE) and Masson staining. Protein expression levels of α smooth muscle actin(α-SMA), collagen type I alpha 1 chain(Col1a1), and TGF-β in liver tissue were detected by Western blotting. Hydroxyproline(HYP) content in liver tissue was measured by alkaline hydrolysis. Serum activities of alanine aminotransferase(ALT) and aspartate aminotransferase(AST) were determined using microplate assays. Potential targets of active components in GJR were screened from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform(TCMSP) and SwissTargetPrediction. LF-related targets were obtained from the GeneCards database. The intersection of these targets was used to identify common targets. A protein-protein interaction(PPI) network was constructed to screen core targets, followed by Gene Ontology(GO) and Kyoto Encyclopedia of Genes and Genomes(KEGG) pathway enrichment analysis. Molecular docking was performed to predict the binding activity between key targets and major active components of GJR. Hepatocyte apoptosis was detected by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling(TUNEL) staining. mRNA levels of Caspase3, BCL2, and Bax in liver tissue were measured by quantitative real time polymerase chain reaction(qRT-PCR). For in vitro experiments, LX-2 cells were activated by transforming growth factor-β(TGF-β) and then treated with GJR containing serum(10%, 15%, 20%). mRNA and protein expression ofα-SMA, Col1a1, and tissue inhibitor of metalloproteinase 1(TIMP1) were examined by qRT-PCR and Western blotting. Apoptosis of LX-2 cells was analyzed by flow cytometry. Results In vivo experiments showed that GJR can alleviate liver injury and collagen deposition, reduce levels of HYP, ALT, and AST, downregulate the expression of α-SMA, Col1a1, and TGF-β, decrease hepatocyte apoptosis, and regulate the expression of Caspase3, BCL2, and Bax. Network pharmacology identified 625 common targets, with core targets related to apoptosis enriched in pathways such as PI3K-Akt. The key components bound well with the core targets. In vitro experiments showed that GJR-containing serum downregulated the mRNA and protein expression of α-SMA, Col1a1, and TIMP1, promoting the apoptosis of LX-2 cell. Conclusion Network pharmacology and experimental verification demonstrate that GJR exerts anti-LF effects, which may be related to promoting a HSC apoptosis and inhibiting hepatocyte apoptosis via the Caspase3/Bcl-2/Bax signaling pathway.

R285.5

国家自然科学基金资助项目(82474591,82274605); 重庆市科卫联合中医药青年项目(2026ZYQN011); 重庆市中医药创新团队(渝卫办白头[2024]570)