Rhamnocitrin 和 Ombuin降低肝臟細胞脂肪堆積下之抗氧化抗發炎的作用機制

Abstract

非酒精性脂肪肝病 (nonalcoholic fatty liver disease, NAFLD) 為全球最常見的肝病之一，從單純的脂肪變性到發炎的非酒精性脂肪肝炎 (non-alcoholic steatosis hepatitis, NASH)，甚至再演變成肝硬化或肝癌。有許多天然的黃酮類化合物能透過改善發炎、氧化壓力和脂質代謝來達到護肝的效果，rhamnocitrin和ombuin為植物中常見的兩種黃酮類化合物，近年來，有許多研究指出它們具有出色的抗發炎和抗氧化的生物活性，但其在 NAFLD 作用的研究相對少。因此，本試驗旨在探討 rhamnocitrin 和 ombuin 對模擬NAFLD的脂肪肝細胞的影響，尤其是其對降低肝臟細胞脂肪堆積和抗氧化抗發炎的機制。 本試驗分別探討 rhamnocitrin 和 ombuin 對細胞脂質代謝及發炎反應的影響，利用 500 µM 的油酸來誘導人肝癌細胞株SK-HEP-1和HepG2 以及雞隻初代肝臟細胞的脂質堆積，細胞經過不同濃度的rhamnocitrin和ombuin處理後，進行紅油染色和測定三酸甘油酯的含量，以探討rhamnocitrin和ombuin是否具有減少肝細胞脂質堆積的效果，並進一步分析脂質合成和脂質分解相關基因的表現，以了解rhamnocitrin和ombuin 影響細胞脂質堆積的相關機制。另一方面，利用 100 µM 的棕櫚酸來誘導人肝癌細胞株SK-HEP-1和HepG2 以及雞隻初代肝臟細胞的氧化壓力和發炎反應，細胞經過不同濃度的rhamnocitrin和ombuin處理後，進行 ROS 含量的測定，並進一步分析發炎反應相關基因的表現，以了解rhamnocitrin和ombuin 影響細胞發炎反應的相關機制。 試驗結果顯示，rhamnocitrin和ombuin 的處理能顯著的降低紅油染色的染色面積 (P<0.05)，同時也能顯著降低油酸所誘導的總三酸甘油酯含量 (P<0.05)，進一步分子機制的研究顯示 rhamnocitrin和ombuin 能夠透過增加 AMP activated protein kinase (AMPK) 的活性，顯著增加脂質氧化相關基因 peroxisome proliferator activated receptor α (PPARα) 和 carnitine palmitoyl transferase 1a (CPT1a) 的 mRNA 及蛋白質的表現量 (P<0.05)，顯示會因此等處理增加細胞脂肪酸的分解；此二處理也同時降低脂質生合成相關基因 sterol regulatory element binding protein 1c (SREBP1c)、acetyl CoA carboxylase (ACC) 和 fatty acid synthase (FAS) 的mRNA 及蛋白質的表現量 (P<0.05)，因而會減少細胞脂質生合成的作用，最終達到減少細胞脂質堆積的效果。另一方面，rhamnocitrin和ombuin 的處理能夠顯著的降低棕櫚酸所誘導的細胞 ROS含量 (P<0.05)，減少細胞的氧化壓力。此外，rhamnocitrin和ombuin 能夠透過增加 AMPK 的活性，增加nuclear factor kappa-B (NF-κB) 的抑制因子IκB的表現，顯著抑制 NF-κB 活性，因而降低其下游促發炎因子tumor necrosis factor-α (TNFα)、interleukin 6 (IL-6) 和 interleukin-1β (IL-1β) 的表現量 (p<0.05)，顯示此等處理也可降低細胞的發炎反應。 綜上所述，rhamnocitrin和ombuin 透過活化 AMPK 的活性，調節脂質代謝和發炎反應相關基因的表現，促進脂質分解和抑制脂質生合成的作用，最終達到減少細胞脂質堆積和發炎的效果。

Nonalcoholic fatty liver disease (NAFLD) is one of the most common liver diseases worldwide, ranging from simple steatosis to inflammatory non-alcoholic steatohepatitis (NASH), and even progresses into cirrhosis or liver cancer. Natural flavonoids can achieve hepatoprotective effects by improving inflammation, oxidative stress, and lipid metabolism. Rhamnocitrin and ombuin are two common flavonoids found in plants. Recent studies have indicated their outstanding effects as anti-inflammatory and antioxidant bioactivities, but their effects on NAFLD are relatively understudied. Therefore, this study aims to investigate the effects of rhamnocitrin and ombuin on simulated NAFLD fatty liver cells, particularly for their mechanisms in reducing hepatic lipid accumulation and exerting antioxidant and anti-inflammatory effects. This study explored the effects of rhamnocitrin and ombuin on cellular lipid metabolism and inflammatory responses. Human liver cancer cell lines SK-HEP-1 and HepG2, as well as primary chicken hepatocytes, were induced with 500 µM oleic acid for lipid accumulation. After treatment with various concentrations of rhamnocitrin and ombuin, the cells were subjected to Oil red O staining and triglyceride content measurement. Further analysis of lipid metabolism-related genes expression were conducted to understand the mechanisms by which rhamnocitrin and ombuin affect cellular lipid accumulation. In another aspect, cells were induced with 100 µM palmitic acid to trigger oxidative stress and inflammatory responses. After treatment with different concentrations of rhamnocitrin and ombuin, ROS production was measured, and the expressions of inflammation-related genes were analyzed to understand how rhamnocitrin and ombuin affect cellular inflammatory responses. The results showed that treatment with rhamnocitrin and ombuin significantly reduced the stained area in Oil red O staining (P<0.05) and significantly decreased the total triglyceride content induced by oleic acid (P<0.05). Further molecular mechanism studies revealed that rhamnocitrin and ombuin significantly increased the expression of β-oxidation related genes peroxisome proliferator-activated receptor α (PPARα) and carnitine palmitoyltransferase 1a (CPT1a) at mRNA and protein levels (P<0.05) by activating AMP-activated protein kinase (AMPK), indicating an increase in cellular fatty acid oxidation. These treatments also reduced the mRNA and protein levels of de novo lipogenesis genes, sterol regulatory element-binding protein 1c (SREBP1c), acetyl-CoA carboxylase (ACC), and fatty acid synthase (FAS) (P<0.05), thereby decreasing lipid biosynthesis and ultimately reducing cellular lipid accumulation. Moreover, treatment with rhamnocitrin and ombuin significantly reduced palmitic acid-induced cellular ROS generation (P<0.05) to decrease cellular oxidative stress. Additionally, rhamnocitrin and ombuin increased the expression of the inhibitor of NF-κB (IκB) by activating AMPK, to inhibit nuclear factor kappa-B (NF-κB) activity and thus reduce the expression of downstream pro-inflammatory cytokines, tumor necrosis factor-α (TNFα), interleukin-6 (IL-6), and interleukin-1β (IL-1β) (P<0.05), indicating a reduction in cellular inflammatory responses. In conclusion, rhamnocitrin and ombuin activated AMPK signaling, regulated the expression of genes related to lipid metabolism and inflammatory responses, promoted β-oxidation, and inhibited de novo lipogenesis, ultimately reducing cellular lipid accumulation and inflammation.