目的 基于肝星狀細(xì)胞（hepatic stellate cells，HSCs）鐵代謝研究沙苑子總黃酮（total flavonoids from Astragali Complanati Semen，TFACS）對(duì)肝纖維化（hepatic fibrosis，HF）小鼠的影響。方法 小鼠連續(xù)8周ip 10%四氯化碳（carbon tetrachloride，CCl₄）建立HF小鼠模型，HF小鼠隨機(jī)分為對(duì)照組、模型組、水飛薊賓（100 mg/kg）組和TFACS低、高劑量（8、20 mg/kg）組，連續(xù)給藥6周，末次給藥16 h后收集血清，計(jì)算肝臟和脾臟指數(shù)；采用全自動(dòng)血清生化儀測定血清肝功能指標(biāo)；采用ELISA試劑盒檢測小鼠血清中Ⅳ型膠原蛋白（collagen type IV，Col-IV）、透明質(zhì)酸（hyaluronic acid，HA）、層黏蛋白（laminin，LN）和Ⅲ型前膠原（procollagen Ⅲ，PCⅢ）水平；采用蘇木素-伊紅（HE）和Masson染色觀察肝組織病變和膠原沉積；采用試劑盒測定肝組織中鐵調(diào)素、鐵以及丙二醛（malondialdehyde，MDA）含量；采用普魯士藍(lán)染色觀察肝組織中鐵的分布；采用Western blotting檢測肝組織中Ⅰ型膠原蛋白（CollagenⅠ）、Ⅳ型膠原蛋白（Collagen Ⅳ）、α-平滑肌肌動(dòng)蛋白（α-smooth muscle actin，α-SMA）和轉(zhuǎn)鐵蛋白受體1（transferrin receptor 1，TFRC）的表達(dá)。采用轉(zhuǎn)化生長因子-β1（transforming growth factor-β1，TGF-β1）誘導(dǎo)HSC-T6細(xì)胞活化，CCK-8法檢測TFACS對(duì)細(xì)胞增殖的影響，試劑盒測定細(xì)胞中MDA和鐵含量，采用免疫熒光法檢測細(xì)胞中活性氧（reactive oxygen species，ROS）水平，采用Western blotting檢測細(xì)胞中α-SMA和TFRC蛋白的表達(dá)。結(jié)果 TFACS顯著降低HF小鼠的脾臟指數(shù)（P＜0.05、0.01），降低血清中丙氨酸氨基轉(zhuǎn)移酶（alanine aminotransferase，ALT）、天冬氨酸氨基轉(zhuǎn)移酶（aspartate aminotransferase，AST）、堿性磷酸酶（alkaline phosphatase，ALP）活性和Col-Ⅳ、HA、LN、PCⅢ水平（P＜0.05、0.01、0.001），改善HF小鼠肝細(xì)胞損傷、炎癥浸潤和膠原沉積，減少肝組織鐵染色面積（P＜0.05），降低肝組織中鐵調(diào)素、鐵和MDA水平（P＜0.05、0.01），并下調(diào)肝組織Collagen Ⅰ、Collagen Ⅳ、TFRC和α-SMA蛋白表達(dá)（P＜0.05）。此外，TFACS可明顯抑制TGF-β1誘導(dǎo)的HSC-T6細(xì)胞增殖（P＜0.05、0.01），下調(diào)TFRC和α-SMA蛋白表達(dá)（P＜0.05、0.01），并降低MDA、ROS和鐵水平（P＜0.001）。結(jié)論 TFACS對(duì)HF具有保護(hù)作用，其作用機(jī)制可能與調(diào)節(jié)HSCs鐵代謝有關(guān)。

Objective To study the effect of total flavonoids from Shayuanzi (Astragali Complanati Semen, TFACS) on hepatic fibrosis (HF) mice based on iron metabolism in hepatic stellate cells (HSCs). Methods HF mice model was established by ip 10% carbon tetrachloride (CCl₄) for eight consecutive weeks. HF mice were randomly divided into control group, model group, silibinin (100 mg/kg) group, and TFACS low-, high-dose (8, 20 mg/kg) groups. The mice were administered continuously for six weeks, and serum was collected 16 h after the last administration. Liver and spleen indexes were calculated; Serum liver function index was measured by automatic serum biochemical instrument; ELISA was used to measure the levels of collagen type IV (Col-IV), hyaluronic acid (HA), laminin (LN) and procollagen Ⅲ (PCⅢ) in serum; The liver lesions and collagen deposition were observed by hematoxylin-eosin (HE) and Masson staining; The contents of hepcidin, iron and MDA in liver tissue were determined by kit; The distribution of iron in liver tissue was observed by prussian blue staining; Western blotting was used to detect the expressions of Collagen Ⅰ, Collagen Ⅳ, α-smooth muscle actin (α-SMA) and transferrin receptor (TFRC) in liver tissue. Transforming growth factor-β1 (TGF-β1) was used to induce the activation of HSC-T6 cells, and the effect of TFACS on the proliferation of cells was evaluated by CCK-8 method, the contents of MDA and iron in cells were determined by kits, the level of reactive oxygen species (ROS) in cells was detected by immunofluorescence method, the expressions of α-SMA and TFRC proteins in cells were detected by Western blotting. Results TFACS significantly reduced the spleen index of HF mice (P < 0.05, 0.01), reduced the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP) and levels of Col-IV, HA, LN and PCIII in serum (P < 0.05, 0.01, 0.001), improved liver cell injury, inflammatory infiltration and collagen deposition in HF mice, reduced the iron staining area of liver tissue (P < 0.05), decreased the levels of hepcidin, iron and MDA in liver tissue (P < 0.05, 0.01), and down-regulate the expressions of Collagen I, Collagen IV, TFRC and α-SMA proteins in liver tissue (P < 0.05). In addition, TFACS could significantly inhibit the proliferation of HSC-T6 cells induced by TGF-β1 (P < 0.05, 0.01), down-regulate the expressions of TFRC and α-SMA proteins (P < 0.05, 0.01), and reduce MDA, ROS and iron levels (P < 0.001). Conclusion TFACS has a protective effect on HF, and its mechanism may be related to regulating iron metabolism in HSCs.

R285.5

陜西省科技廳重點(diǎn)研發(fā)計(jì)劃項(xiàng)目（2024SF-YBXM-458，2024NC-YBXM-263）；陜西省教育廳重點(diǎn)科學(xué)研究計(jì)劃項(xiàng)目（22JY019）；陜西省科技創(chuàng)新團(tuán)隊(duì)（2022TD-56）；陜西高校青年創(chuàng)新團(tuán)隊(duì)建設(shè)項(xiàng)目（2023）；秦創(chuàng)原中醫(yī)藥產(chǎn)業(yè)創(chuàng)新聚集區(qū)項(xiàng)目（L2024-QCY-ZYYJJQ-X201）；陜西省重點(diǎn)研發(fā)計(jì)劃-重點(diǎn)產(chǎn)業(yè)創(chuàng)新鏈（群）（社會(huì)發(fā)展領(lǐng)域）（2023-ZDLSF-58）