目的 运用网络药理学结合体外实验探讨姜黄素对血管紧张素Ⅱ （Ang Ⅱ）诱导的肝星状细胞系LX-2焦亡和活化的影响，为姜黄素治疗肝纤维化提供参考。方法 应用网络药理学筛选姜黄素对LX-2细胞活化作用的关键靶点和通路。体外培养LX-2细胞，Ang Ⅱ（10 μmol?L^-1）诱导活化24 h后加药，经CCK-8法筛选后，确定姜黄素加药浓度为5、10、20 μmol?L^-1，药物干预48 h；显微镜下观察细胞形态； Hoechst 33342/PI染色法观察细胞焦亡； DCFH-DA荧光探针检测细胞内活性氧（ROS）含量； ELISA法检测细胞上清液中白细胞介素（IL）-1β和IL-18水平；采用Western blotting检测细胞中焦亡相关蛋白[NOD样受体热蛋白结构域蛋白3（NLRP3）、半胱氨酸蛋白水解酶（Caspase）-1、gasdermin D-N（GSDMD-N）、IL-18、IL-1β]、肝纤维化相关蛋白[胶原蛋白Ⅰ （Collagen Ⅰ）、α-平滑肌肌动蛋白（α-SMA）]和小窝蛋白-1（Cav-1）蛋白表达。使用GV146-CAV1质粒过表达Cav-1，设置对照组、模型组、姜黄素（20 μmol?L^-1）组、CAV1组、姜黄素（20 μmol?L^-1） ＋CAV1组，ELISA法检测细胞上清液中IL-1β和IL-18水平； Western blotting检测焦亡相关蛋白、肝纤维化相关蛋白和Cav-1蛋白的表达。结果 共获得13个姜黄素和LX-2活化共有靶点。蛋白质-蛋白质互作网络（PPI）网络中的IL-1β为核心靶点。基因本体（GO）富集分析发现姜黄素干预LX-2活化与小窝（caveola）等多种生物功能相关。京都基因与基因组百科全书（KEGG）结果显示，细胞焦亡密切相关的NOD样受体信号通路与PPI网络中的核心靶点IL-1β紧密联系。体外实验表明，与模型组相比，姜黄素能够显著抑制活化状态下的LX-2细胞增殖，显著下调肝纤维化相关蛋白Collagen-I和α-SMA水平，显著改善LX-2细胞焦亡，显著降低LX-2细胞内ROS含量，显著降低Ang II诱导LX-2细胞上清液中IL-1β和IL-18水平，显著下调NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表达水平，显著上调Cav-1蛋白表达，差异均有统计学意义（P＜0.05、0.01、0.001）；此外，过表达Cav-1能够降低Ang II诱导LX-2细胞上清液中IL-1β和IL-18水平（P＜0.01），下调焦亡相关蛋白和Collagen-Ⅰ蛋白表达（P＜0.05、0.01）。结论 姜黄素抑制Ang II诱导的LX-2活化减轻肝纤维化，机制可能与调控Cav-1密切相关。

Objective To investigate the effects of curcumin on pyroptosis and activation of hepatic stellate cell line LX-2 induced by angiotensin II (Ang II) using network pharmacology combined with in vitro experiments, and to provide a reference for the treatment of liver fibrosis with curcumin. Methods Network pharmacology was used to screen the key targets and pathways of curcumin on the activation of LX-2 cells. LX-2 cells were cultured in vitro, and after 24 h of activation induced by Ang II (10 μmol?L^-1), curcumin was added. After screening by CCK-8 method, the curcumin concentration was determined to be 5, 10, and 20 μmol?L^-1, and the drug intervention lasted for 48 h. Cell morphology was observed under a microscope; pyroptosis was observed by Hoechst 33342/PI staining, intracellular reactive oxygen species (ROS) content was detected by DCFH-DA fluorescent probe, levels of interleukin (IL)-1β and IL-18 in the cell supernatant were detected by ELISA, expressions of pyroptosis-related proteins [NOD-like receptor pyrin domain-containing protein 3 (NLRP3), caspase-1, gasdermin D-N (GSDMD-N), IL-18, IL-1β], liver fibrosis-related proteins [collagen type I (Collagen-Ⅰ), α-smooth muscle actin (α-SMA)], and caveolin-1 (Cav-1) were detected by Western blotting. The GV146-CAV1 plasmid was used to overexpress Cav-1, and the control group, model group, curcumin (20 μmol?L^-1) group, CAV1 group, and curcumin (20 μmol?L^-1) + CAV1 group were set up. Levels of IL-1β and IL-18 in the cell supernatant were detected by ELISA, expressions of pyroptosis-related proteins, liver fibrosis-related proteins, and Cav-1 were detected by Western blotting. Results A total of 13 common targets of curcumin and LX-2 activation were obtained. IL-1β was the core target in the protein-protein interaction (PPI) network. Gene Ontology (GO) enrichment analysis revealed that curcumin intervention on LX-2 activation was related to multiple biological functions such as caveola. Kyoto Encyclopedia of Genes and Genomes (KEGG) results showed that the NOD-like receptor signaling pathway closely related to cell pyroptosis was closely linked to the core target IL-1β in the PPI network. In vitro experiments showed that compared with the model group, curcumin could significantly inhibit the proliferation of activated LX-2 cells, significantly downregulate the levels of liver fibrosis-related proteins Collagen-I and α-SMA, significantly improve LX-2 cell pyroptosis, significantly reduce intracellular ROS content, significantly reduce the levels of IL-1β and IL-18 in the LX-2 cell supernatant induced by Ang II, significantly down-regulate the expression levels of NLRP3, caspase-1, GSDMD-N, IL-18, and IL-1β proteins, and significantly upregulate the expression of Cav-1 protein, and the differences were statistically significant (P<0.05, 0.01, 0.001); In addition, overexpression of Cav-1 could reduce the levels of IL-1β and IL-18 in the LX-2 cell supernatant induced by Ang II (P<0.05, 0.01), and down-regulate the expressions of pyroptosis and liver fibrosis-related proteins (P<0.05, 0.01). Conclusion Curcumin inhibits Ang II-induced LX-2 activation and alleviates liver fibrosis, likely through the regulation of Cav-1, although the specific mechanisms require further investigation.

R285.5

河南省科技攻关计划项目（ 222102310408）； 河南省科技攻关项目（252102310135）