目的 观察加减三甲散含药血清对人肝星状细胞LX-2自噬的影响。方法 将人肝星状细胞LX-2作为研究对象，采用转化生长因子-β1（TGF-β1，10 ng·mL^-1）诱导LX-2细胞活化构建肝纤维化细胞模型。通过CCK-8法检测不同体积分数（2.5%、5.0%、10.0%、15.0%、20.0%）加减三甲散含药血清对LX-2细胞活性的影响，筛选最佳给药浓度。将LX-2细胞分为对照组（20%空白血清）、模型（10 ng·mL^-1 TGF-β1）组、加减三甲散（2.5%加减三甲散含药血清+10 ng·mL^-1 TGF-β1）组、3-甲基腺嘌呤（3-MA）组（0.5 mg·mL^-1 3-MA+10 ng·mL^-1 TGF-β1）、3-MA+加减三甲散含药血清组（0.5 mg·mL^-1 3-MA+2.5%加减三甲散含药血清+10 ng·mL^-1 TGF-β1），培养48 h后，采用CCK-8法检测LX-2细胞存活率；采用实时荧光定量PCR（qRT-PCR）法检测α-平滑肌肌动蛋白（α-SMA）、TGF-β1、纤维连接蛋白（FN） mRNA表达情况；采用Westernblotting法检测自噬标志物LC3 Ⅱ/LC3 I、p62、Beclin-1、Atg5蛋白表达情况；采用免疫荧光法检测自噬蛋白LC3B、α-SMA、FN荧光表达情况； MDC染色检测各组细胞自噬活性情况；透射电镜观察各组细胞的自噬小体形成情况。结果 加减三甲散含药血清对LX2细胞活性有抑制作用，且浓度越高，抑制效果越强，选择2.5%含药血清作为最佳干预浓度进行后续实验。与对照组比较，模型组细胞活性显著增强（P＜0.01）； LX-2活化标记物α-SMA、TGF-β1、FN mRNA水平显著升高（P＜0.01）；自噬蛋白LC3 Ⅱ/LC3 I、Beclin-1、Atg5表达显著增加，p62表达显著降低（P＜0.05、0.01）；自噬活性增强，自噬小体增多； LC3B、α-SMA、FN蛋白荧光表达强度显著增强（P＜0.01）。与模型组比较，加减三甲散组、3-MA组、3-MA+加减三甲散组细胞活性均显著降低（P＜0.01），LX-2活化标记物α-SMA、TGF-β1、FN mRNA显著降低（P＜0.05、0.01）；自噬蛋白LC3 II/LC3 I、Beclin-1、Atg5显著降低，p62显著增高（P＜0.05、0.01）；自噬活性减弱，自噬小体较少； LC3B、α-SMA、FN蛋白荧光表达强度显著降低（P＜0.01）；其中3-MA+加减三甲散组干预效果最为明显。结论 加减三甲散可通过抑制自噬降低LX-2细胞活化发挥抗肝纤维化的效应。

Objective To investigate the effect of modified Sanjia San-containing serum on autophagy in human hepatic stellate cells (LX-2). Methods Human LX-2 cells were used as the research model, and a liver fibrosis cell model was established by activating LX-2 cells with transforming growth factor-β1 (TGF-β1) at 10 ng·mL^-1. The effects of different concentrations (2.5%, 5.0%, 10.0%, 15.0%, 20.0%) of modified Sanjia San-containing serum on LX-2 cell viability were assessed using the CCK-8 assay to determine the optimal concentration. Cells were divided into five groups: Control group (20% blank serum), model group (10 ng·mL^-1 TGF-β1), modified Sanjia San group (2.5% modified Sanjia San-containing serum + 10 ng·mL^-1 TGF-β1), 3-MA group (0.5 mg·mL^-1 3-MA + 10 ng·mL^-1 TGF-β1), and 3-MA + modified Sanjia San group (0.5 mg·mL^-1 3-MA + 2.5% modified Sanjia San-containing serum + 10 ng·mL^-1 TGF-β1). After 48 h of culture, cell viability was measured by CCK-8 assay; mRNA expression levels of α-smooth muscle actin (α-SMA), TGF-β1, and fibronectin (FN) were detected by real-time quantitative PCR (qRT-PCR); Protein expression of autophagy markers LC3 II/LC3 I, p62, Beclin-1, and Atg5 was analyzed by Western blotting; fluorescence intensity of autophagy-related proteins LC3B, α-SMA, and FN was evaluated by immunofluorescence; autophagic activity was assessed by MDC staining; and formation of autophagosomes was observed under transmission electron microscopy. Results Modified Sanjia San inhibited LX-2 cell viability, with higher concentrations showing stronger inhibition; Thus, 2.5% drug-containing serum was selected as the optimal intervention concentration for subsequent experiments. Compared with the control group, the model group showed significantly enhanced cell viability (P < 0.01); mRNA levels of activation markers α-SMA, TGF-β1, and FN were significantly upregulated (P < 0.01); Expression of autophagy-related proteins LC3 II/LC3 I, Beclin-1, and Atg5 increased significantly, while p62 expression decreased significantly (P < 0.05, 0.01); Autophagic activity was enhanced, and more autophagosomes were observed; fluorescence intensities of LC3B, α-SMA, and FN proteins were significantly increased (P < 0.01). Compared with the model group, cell viability was significantly reduced in the modified Sanjia San group, 3-MA group, and 3-MA + modified Sanjia San group (P < 0.01); mRNA levels of α-SMA, TGF-β1, and FN were significantly downregulated (P < 0.05, 0.01); Expression of LC3 II/LC3 I, Beclin-1, and Atg5 decreased significantly, while p62 expression increased significantly (P < 0.05, 0.01); Autophagic activity was weakened, and fewer autophagosomes were observed; Fluorescence intensities of LC3B, α-SMA, and FN proteins were significantly reduced (P < 0.01); Among these, the 3-MA + modified Sanjia San group exhibited the most pronounced inhibitory effect. Conclusion Modified Sanjia San can exert anti liver fibrosis effects by inhibiting autophagy and reducing LX-2 cell activation.

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宁夏自然科学基金资助项目（2023AAC05040）；宁夏医科大学校级科研资助项目（XT2022015）；国家中医药管理局高水平中医药重点学科建设项目资助（zyyzdxk-2023209）；宁夏自然科学基金资助项目（2023AAC03210）；国家自然科学基金资助项目（82305085）