College of Pharmaceutical Sciences, Zhejiang University of Technology, Hangzhou, 310014, People's Republic of China.
Collaborative Innovation Center of Yangtze River Delta Region Green Pharmaceuticals, Zhejiang University of Technology, Hangzhou, Zhejiang, 310014, People's Republic of China.
J Ethnopharmacol. 2022 Mar 1;285:114873. doi: 10.1016/j.jep.2021.114873. Epub 2021 Nov 27.
Traditional Chinese medicine considers that the etiology and pathogenesis of non-alcoholic fatty liver disease (NAFLD) are related to liver depression and qi stagnation. Saffron and its active ingredient, crocetin (CCT), are used for the treatment of metabolic diseases owing to their "Liver deobstruent" and "Liver tonic" effects. However, the effect of CCT on NAFLD has not been fully elucidated. In the present study, the effect and potential molecular mechanism of CCT were explored in both in vivo and in vitro models of NAFLD.
CCT was isolated from saffron and purity and structure characterization were performed using HPLC, MS, H-NMR, and C-NMR. The effect of CCT on the viability of L02 cells and its maximum tolerable concentration (MTC) in zebrafish were investigated. Free fatty acids (FFA) and thioacetamide (TAA) were used to induce lipid accumulation in L02 cells and steatosis in zebrafish, respectively. The effects of CCT on indexes related to lipid metabolism, oxidative stress, and mitochondrial function in NAFLD models were explored using biochemical assay kits, Western blot analysis, Reverse Transcription-Polymerase Chain Reaction (RT-PCR), histopathology analysis, and determination of mitochondrial membrane potential (ΔΨm). Morphological analysis of mitochondria was performed using transmission electron microscopy (TEM).
The levels of triglyceride (TG), total cholesterol (TC), malondialdehyde (MDA), and alanine/aspartate aminotransferases (ALT/AST) activities in FFA treated L02 cells were significantly reduced after CCT treatment. CCT treatment significantly increased ATP concentration, ΔΨm, and activities of superoxide dismutase (SOD), catalase (CAT), and cytochrome c oxidase (COX IV) in FFA treated L02 cells. TEM images showed restoration of mitochondrial morphology. CCT decreased ATP concentration and upregulated expression of B-cell lymphoma-2 (Bcl-2) and COX IV, whereas, CCT downregulated expression of BCL2-Associated X (Bax) and cleaved caspase-3 in TAA treated zebrafish. These findings indicated that mitochondrial dysfunction was alleviated after CCT treatment. Oil Red O staining of L02 cells and zebrafish showed that CCT treatment reversed the accumulation of lipid droplets.
In summary, CCT treatment effectively alleviated the symptoms of NAFLD and restored mitochondrial function in L02 cells and zebrafish NAFLD model.
ETHNOPHARMACOLOGICAL 相关性：传统中医认为非酒精性脂肪性肝病 (NAFLD) 的病因和发病机制与肝郁气滞有关。藏红花及其活性成分西红花酸 (CCT) 因其“疏肝”和“养肝”作用而用于治疗代谢性疾病。然而，西红花酸对 NAFLD 的作用尚未得到充分阐明。在本研究中，西红花酸在 NAFLD 的体内和体外模型中进行了作用和潜在分子机制的探索。
西红花酸从藏红花中分离得到，采用高效液相色谱 (HPLC)、质谱 (MS)、氢核磁共振 (H-NMR) 和碳核磁共振 (C-NMR) 进行纯度和结构表征。考察了 CCT 对 L02 细胞活力的影响及其在斑马鱼中的最大耐受浓度 (MTC)。游离脂肪酸 (FFA) 和硫代乙酰胺 (TAA) 分别用于诱导 L02 细胞内脂质堆积和斑马鱼脂肪变性。采用生化试剂盒、Western blot 分析、逆转录聚合酶链反应 (RT-PCR)、组织病理学分析和测定线粒体膜电位 (ΔΨm) 等方法探讨 CCT 对 NAFLD 模型中与脂质代谢、氧化应激和线粒体功能相关指标的影响。采用透射电子显微镜 (TEM) 进行线粒体形态分析。
FFA 处理的 L02 细胞中甘油三酯 (TG)、总胆固醇 (TC)、丙二醛 (MDA) 和丙氨酸/天冬氨酸转氨酶 (ALT/AST) 活性水平显著降低，经 CCT 处理后均显著降低。CCT 处理可显著增加 FFA 处理的 L02 细胞中 ATP 浓度、ΔΨm 以及超氧化物歧化酶 (SOD)、过氧化氢酶 (CAT) 和细胞色素 c 氧化酶 (COX IV) 的活性。TEM 图像显示线粒体形态得到恢复。CCT 降低了 ATP 浓度，上调了 B 细胞淋巴瘤-2 (Bcl-2) 和 COX IV 的表达，而下调了 TAA 处理的斑马鱼中 BCL2 相关 X (Bax) 和 cleaved caspase-3 的表达。这些发现表明，CCT 处理后线粒体功能障碍得到缓解。L02 细胞和斑马鱼的油红 O 染色表明，CCT 处理逆转了脂质滴的积累。
综上所述，CCT 治疗可有效缓解 NAFLD 症状，并恢复 L02 细胞和斑马鱼 NAFLD 模型中的线粒体功能。
