目的 通过体内外实验及转录组测序技术探讨养肝软坚丸（Yanggan Ruanjian Wan，YGRJW）对肝纤维化（hepatic fibrosis，HF）的干预作用及其作用机制。方法 采用转化生长因子-β1（transforming growth factor-β1，TGF-β1）诱导LX-2细胞活化模型，通过CCK-8、细胞划痕、qRT-PCR、Western blotting实验考察YGRJW含药血清对细胞活力、增殖、炎症因子[白细胞介素-1β（interleukin-1β，IL-1β）、IL-6、肿瘤坏死因子-α（tumor necrosis factor-α，TNF-α）]及纤维化标志物[α-平滑肌肌动蛋白（α-smooth muscle actin，α-SMA）、Ⅰ型胶原蛋白α1链（collagen type Ⅰ alpha 1 chain，COL1A1）、Ⅲ型胶原蛋白α1链（collagen type Ⅲ alpha 1 chain，COL3A1）]表达的影响；通过转录组测序技术筛选YGRJW含药血清干预活化LX-2细胞的差异表达基因及关键通路，并使用qRT-PCR和免疫荧光实验验证测序分析结果。小鼠ip含10%四氯化碳（carbon tetrachloride，CCl₄）的玉米油溶液诱导HF模型，给予药物干预后，通过检测体质量、脏器指数、肝肾功能、肝组织病理学改变、肝组织炎症因子及纤维化标志物的表达、血清中炎症因子及肝纤四项的水平，综合评价YGRJW对HF小鼠的治疗作用，并验证YGRJW对潜在药效靶点及关键通路指标表达的影响。结果 体外实验结果显示，10% YGRJW含药血清干预24 h可显著抑制LX-2细胞活化（P＜0.01），并下调炎症因子和纤维化标志物的mRNA表达水平（P＜0.05、0.01、0.001），抑制效果优于10%水飞蓟宾含药血清。转录组学分析及验证结果显示，YGRJW可能通过集落刺激因子3受体（colony stimulating factor 3 receptor，CSF3R）/Janus激酶2（Janus kinase 2，JAK2）/信号传导及转录激活因子3（signal transducer and activator of transcription 3，STAT3）途径发挥抗HF作用；与模型组比较，YGRJW含药血清干预能显著下调CSF3R、JAK2、STAT3的mRNA和蛋白表达（P＜0.05、0.01）。体内实验结果显示，YGRJW能显著改善HF小鼠的体质量增长缓慢（P＜0.05），降低肝脏指数、脾脏指数及肝肾功能指标（P＜0.05、0.01、0.001），减轻肝组织炎症浸润和胶原沉积，降低肝组织中TGF-β、TNF-α、IL-6、α-SMA、COL1A1、COL3A1、金属蛋白酶组织抑制因子-1（tissue inhibitor of metalloproteinase-1，TIMP-1）mRNA表达水平（P＜0.05、0.01、0.001），升高成纤维细胞生长因子7（fibroblast growth factor 7，FGF7）mRNA表达水平（P＜0.05、0.01），降低血清中炎症因子及肝纤四项水平（P＜0.05、0.01、0.001），并下调肝组织CSF3R、JAK2、STAT3的mRNA和蛋白表达（P＜0.05、0.01）。结论 YGRJW通过调控CSF3R/JAK2/STAT3信号通路，抑制肝星状细胞（hepatic stellate cell，HSC）活化并减少细胞外基质（extracellular matrix，ECM）沉积，进而发挥抗HF作用。

Objective To explore the effect and mechanism of Yanggan Ruanjian Wan (养肝软坚丸, YGRJW) in prevention and treatment of hepatic fibrosis (HF) based on in vivo and in vivo experiments and transcriptome sequencing technology. Methods LX-2 cell activation model was induced by transforming growth factor-β1 (TGF-β1), CCK-8 method, cell scratch assay, qRT-PCR and Western blotting were used to detect the effects of YGRJW-medicated serum on cell viability, proliferation, expressions of inflammatory cytokines [interleukin-1β (IL-1β), IL-6, tumor necrosis factor-α (TNF-α)] and fibrosis markers [α-smooth muscle actin (α-SMA), collagen type I alpha 1 chain (COL1A1), collagen type III alpha 1 chain (COL3A1)]. Transcriptome sequencing was employed to identify differentially expressed genes and key pathways in activated LX-2 cells treated with YGRJW-containing serum, followed by validation through qRT-PCR and immunofluorescence experiments. A HF model was induced in mice via intraperitoneal injection of a 10% carbon tetrachloride (CCl₄)-containing corn oil solution. After drug intervention, the therapeutic effects of YGRJW on HF mice were comprehensively evaluated by measuring body weight, organ indices, liver and kidney function, pathological changes in liver tissue, expressions of inflammatory factors and fibrosis markers in liver tissue, as well as levels of inflammatory factors and four liver fibrosis indicators in serum, while also verifying the effect of YGRJW on potential pharmacodynamic targets and key pathway indicators. Results In vitro experiments demonstrated that 10% YGRJW-containing serum significantly inhibited LX-2 cell activation after 24 h (P < 0.01) and downregulated mRNA expression levels of inflammatory factors and fibrosis markers (P < 0.05, 0.01, 0.001), with superior inhibitory effects compared to 10% silybinin-containing serum. Transcriptomic analysis and validation results showed that YGRJW may exert anti-HF effects through the colony stimulating factor 3 receptor (CSF3R)/Janus kinase 2 (JAK2)/signal transducer and activator of transcription 3 (STAT3) pathway. Compared with model group, YGRJW drug containing serum intervention could significantly downregulate the mRNA and protein expressions of CSF3R, JAK2 and STAT3 (P < 0.05, 0.01). The in vivo experimental results showed that YGRJW could significantly improve the slow growth of body weight in HF mice (P < 0.05), reduce liver index, spleen index, liver and kidney function indicators (P < 0.05, 0.01, 0.001), alleviate liver tissue inflammation infiltration and collagen deposition, reduce the mRNA expression levels of TGF-β, TNF-α, IL-6, α-SMA, COL1A1, COL3A1 and tissue inhibitor of metalloproteinases-1 (TIMP-1) in liver tissue (P < 0.05, 0.01, 0.001), increase the mRNA expression level of fibroblast growth factor 7 (FGF7) (P < 0.05, 0.01), and reduce levels of inflammatory factors and four indicators of liver fibrosis in serum (P < 0.05, 0.01, 0.001), and downregulated mRNA and protein expressions of CSF3R, JAK2, STAT3 in liver tissue (P < 0.05, 0.01). Conclusion YGRJW exerts anti-HF effects by regulating CSF3R/JAK2/STAT3 signaling pathway, to inhibit hepatic stellate cell (HSC) activation and reducing extracellular matrix (ECM) deposition.

R285.5

广州中医药大学“固本”工程一级学科能力提升项目（广中医校办[2026]105号）；广东省中医药局科研项目（20261121）；国家中医药传承创新中心科研专项重点项目（2023ZD07）；邝卫红省名中医工作室（粤府函[2023]16号）