目的 運用網(wǎng)絡(luò)藥理學(xué)結(jié)合體外實驗探討姜黃素對血管緊張素Ⅱ （Ang Ⅱ）誘導(dǎo)的肝星狀細(xì)胞系LX-2焦亡和活化的影響，為姜黃素治療肝纖維化提供參考。方法 應(yīng)用網(wǎng)絡(luò)藥理學(xué)篩選姜黃素對LX-2細(xì)胞活化作用的關(guān)鍵靶點和通路。體外培養(yǎng)LX-2細(xì)胞，Ang Ⅱ（10 μmol?L^-1）誘導(dǎo)活化24 h后加藥，經(jīng)CCK-8法篩選后，確定姜黃素加藥濃度為5、10、20 μmol?L^-1，藥物干預(yù)48 h；顯微鏡下觀察細(xì)胞形態(tài)； Hoechst 33342/PI染色法觀察細(xì)胞焦亡； DCFH-DA熒光探針檢測細(xì)胞內(nèi)活性氧（ROS）含量； ELISA法檢測細(xì)胞上清液中白細(xì)胞介素（IL）-1β和IL-18水平；采用Western blotting檢測細(xì)胞中焦亡相關(guān)蛋白[NOD樣受體熱蛋白結(jié)構(gòu)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]、肝纖維化相關(guān)蛋白[膠原蛋白Ⅰ （Collagen Ⅰ）、α-平滑肌肌動蛋白（α-SMA）]和小窩蛋白-1（Cav-1）蛋白表達(dá)。使用GV146-CAV1質(zhì)粒過表達(dá)Cav-1，設(shè)置對照組、模型組、姜黃素（20 μmol?L^-1）組、CAV1組、姜黃素（20 μmol?L^-1） ＋CAV1組，ELISA法檢測細(xì)胞上清液中IL-1β和IL-18水平； Western blotting檢測焦亡相關(guān)蛋白、肝纖維化相關(guān)蛋白和Cav-1蛋白的表達(dá)。結(jié)果 共獲得13個姜黃素和LX-2活化共有靶點。蛋白質(zhì)-蛋白質(zhì)互作網(wǎng)絡(luò)（PPI）網(wǎng)絡(luò)中的IL-1β為核心靶點。基因本體（GO）富集分析發(fā)現(xiàn)姜黃素干預(yù)LX-2活化與小窩（caveola）等多種生物功能相關(guān)。京都基因與基因組百科全書（KEGG）結(jié)果顯示，細(xì)胞焦亡密切相關(guān)的NOD樣受體信號通路與PPI網(wǎng)絡(luò)中的核心靶點IL-1β緊密聯(lián)系。體外實驗表明，與模型組相比，姜黃素能夠顯著抑制活化狀態(tài)下的LX-2細(xì)胞增殖，顯著下調(diào)肝纖維化相關(guān)蛋白Collagen-I和α-SMA水平，顯著改善LX-2細(xì)胞焦亡，顯著降低LX-2細(xì)胞內(nèi)ROS含量，顯著降低Ang II誘導(dǎo)LX-2細(xì)胞上清液中IL-1β和IL-18水平，顯著下調(diào)NLRP3、Caspase-1、GSDMD-N、IL-18和IL-1β蛋白表達(dá)水平，顯著上調(diào)Cav-1蛋白表達(dá)，差異均有統(tǒng)計學(xué)意義（P＜0.05、0.01、0.001）；此外，過表達(dá)Cav-1能夠降低Ang II誘導(dǎo)LX-2細(xì)胞上清液中IL-1β和IL-18水平（P＜0.01），下調(diào)焦亡相關(guān)蛋白和Collagen-Ⅰ蛋白表達(dá)（P＜0.05、0.01）。結(jié)論 姜黃素抑制Ang II誘導(dǎo)的LX-2活化減輕肝纖維化，機制可能與調(diào)控Cav-1密切相關(guān)。

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

河南省科技攻關(guān)計劃項目（ 222102310408）； 河南省科技攻關(guān)項目（252102310135）