川陳皮素通過調節腸肝循環改善高脂飲食誘導非酒精性脂肪肝的作用

Abstract

肥胖會導致肝臟脂質堆積，而在沒有大量飲酒的情況下肝臟脂肪堆積程度大於肝臟面積的 5% 時，稱為非酒精性脂肪肝 (Non-alcoholic fatty liver disease, NAFLD)。目前 NAFLD 的潛在機制仍未完全明瞭，因為其致病機制是複雜和多因素的，而肥胖所造成的肝損傷、發炎反應、腸道菌相及膽酸平衡的失調，皆可能影響 NAFLD 的疾病進程，研究證明高脂飲食會影響腸道菌相和膽酸代謝，並影響腸肝循環中法尼醇 X 受體 (FXR) 的表現量，導致膽酸及脂質形成的失衡，使堆積於肝臟中的脂肪增加且血清中的膽固醇含量也有所提升，從而增加形成 NAFLD 之風險。因此有研究認為，串聯著腸道及肝臟之間的腸肝循環機制，可能為延緩 NAFLD 的重要討論方向；腸肝循環是指腸道及其微生物群和肝臟之間，藉由肝門靜脈所建立的雙向關係，肝門靜脈能夠將腸道的膽酸及成纖維細胞生長因子 15/19 (FGF15/19) 運輸到肝臟，並與肝臟中的成纖維細胞生長因子受體 4 (FGFR4) 結合，進而調節肝臟中膽固醇 7α 羥化酶 (CYP7A1) 和 FXR 蛋白質表現量的變化，從而影響肝臟中膽固醇的代謝及脂質的形成，因此腸肝循環中腸道菌群、膽酸及 FXR-FGF15 訊號影響著整個循環，也是本次實驗探討 NAFLD 疾病進程的研究目標。 川陳皮素 (Nobiletin) 為存在於柑橘類水果皮中的多甲氧基黃酮化合物，先前的研究顯示其在調節腸道菌相、抗肥胖等作用上具有一定功效；然而其能否藉由腸肝循環及膽酸代謝的變化下影響肝臟之狀態仍未知；因此本研究探討以60% 高脂飲食誘導小鼠非酒精性脂肪肝的模式下，同時以管餵的方式給予低劑量 (20 mg/kg BW) 及高劑量 (100 mg/kg BW) 之川陳皮素，評估其改善 NAFLD 的效果。實驗結果顯示，川陳皮素可以減緩高脂飲食誘導之小鼠體重上升、減少肝臟器官重量、以及減少血液中的膽固醇、三酸甘油酯、低密度脂蛋白以及血糖濃度。於肝臟組織切片染色結果顯示，川陳皮素具有抑制肝臟脂質蓄積的能力，使其脂肪的體積明顯較高脂飲食組小。西方墨點法結果顯示，川陳皮素可以通過調節 FXR-FGF15 路徑，提升肝臟 CYP7A1 蛋白質表現量，從而降低肝臟膽固醇的形成，此外，川陳皮素也可以降低肝臟中的 SREBP-1c 和 FASN 蛋白質表現量，進而減少脂質堆積於肝臟中。在腸道菌群與膽酸分析結果方面，高脂飲食使迴腸膽酸量有所增加，而隨著川陳皮素的介入後，迴腸中能夠活化 FXR 的膽酸含量皆有所下調，而能夠抑制 FXR 的膽酸 (如 αMCA) 含量則上升，其結果可能解釋了川陳皮素使迴腸 FXR 蛋白表現量下降的原因，從而影響腸肝循環並因此調節了肝臟膽酸的形成。16S rRNA 基因序列數據顯示，川陳皮素改變腸道菌群的組成，增加 Akkermansia、Oscillibacter 及 Romboutsia 菌屬的數量和降低 F/B ratio，顯示餵食川陳皮素後能夠調節高脂飲食造成之腸道菌相的失衡並增加益菌之生長。相關性結果顯示 高脂飲食的組別中 Lactobacillus johnsonii、Ligilactobacillus murinus、Anaerostipes hadrus 與膽酸 TCDCA 具正相關性，高劑量川陳皮素的組別中 Dubosiella newyorkensis 與膽酸 UDCA 和 LCA 具正相關；Kineothrix alysoides 與膽酸 DCA 具正相關性，顯示川陳皮素影響高脂飲食小鼠的腸道菌群與膽酸的變化中具潛在相關性的結果，而相關文獻中尚未有研究探討這些菌群和膽酸間的代謝變化關係，因此能藉由本實驗之結果，為潛在影響膽酸變化的腸道菌群提供新的研究方向，也能夠更全面的推論攝取川陳皮素後所影響的腸道菌群、膽酸的變化與腸肝循環機制間的關係，顯示川陳皮素可以調節因高脂飲食引起的腸道菌相失衡，並透過調節膽酸來影響腸肝循環中的下游蛋白質 FXR 表現，從而達到預防非酒精性脂肪肝的進展。 綜合上述結果川陳皮素可以通過改變腸道菌相進而影響膽酸代謝，且藉由腸肝循環使得肝臟 CYP7A1 蛋白質表現量的上升從而降低肝臟膽固醇的含量，此外也降低肝臟 SREBP-1c 和 FASN 的蛋白質表現量，從而減少肝臟的脂肪堆積，最終改善 NAFLD 的疾病進展。

Obesity would lead to the accumulation of lipids in the liver, and when the degree of hepatic steatosis was greater than 5% of the liver area without heavy alcohol consumption, it was called non-alcoholic fatty liver disease (NAFLD). The pathogenesis of NAFLD has not been fully understood because the underlying mechanisms were complex and multifactorial. Studies have shown that high-fat diet may affect gut bacteria, thereby influencing bile acid metabolism and the protein expression of farnesoid X receptor (FXR) in the enterohepatic circulation. It would lead to serum triglyceride and cholesterol level increase and contribute to the progression of NAFLD. Enterohepatic circulation referred to the bidirectional relationship between liver, gut and its microbiota. This interaction was established by the hepatic portal vein which transported bile acids and Fibroblast growth factor 15/19 (FGF15/19) to the liver and bind to Fibroblast growth factor receptor 4 (FGFR4), thereby influencing Cholesterol 7 alpha-hydroxylase (CYP7A1) and FXR protein expression in liver which regulated the metabolism of cholesterol and the formation of lipids. Nobiletin was a non-polar polymethoxylated flavone found in the citrus peel. In previous studies, it had exhibited significant biological activity, including anti-inflammatory and anti-obesity properties. Besides, it was capable of regulating the ratio of Firmicutes and Bacteroidetes genera in the intestinal flora. However, it was still unknown whether it could affect the state of the liver through the regulation of enterohepatic circulation and bile acid. Therefore, our study aimed to explore the interaction between enterohepatic circulation and NAFLD progression with the nobiletin intake in the high-fat-diet animal model. The results showed that nobiletin could reduce body weight and the total triacylglyceride level in the liver in high-fat diet feeding mice. In addition, the section staining results showed that nobiletin had the ability to inhibit the accumulation of lipids in the liver so that the formation volume of fat droplets was significantly smaller than the high-fat diet group. Nobiletin also had the effect of lowering low density lipoprotein cholesterol (LDL), triacylglyceride and glucose levels in the blood, showing the potential effect of improving fatty liver. Data of immune-blotting further showed that nobiletin could improve bile acid homeostasis through FXR-FGF15 pathway and upregulated CYP7A1 protein levels. Nobiletin could also downregulate SREBP-1c and FASN protein expressions which were related to lipid synthesis in liver. In terms of bile acid analysis results, the high-fat diet would increase the content of bile acid with FXR agonist function, while nobiletin intake would reduce these bile acid, thereby downregulating FXR protein expression in ileum. The 16S rRNA gene sequence data indicated that nobiletin could increase the number of Dubosiella, Oscillibacte, Romboutsia and Akkermensia genera in the intestinal bacteria. In the correlation analysis chart, it showed that Lactobacillus johnsonii, Ligilactobacillus murinus, Anaerostipes hadrus, Dubosiella newyorkensis and Kineothrix alysoides were correlated with taurochenodeoxycholic acid (TCDCA), Ursodeoxycholic acid (UDCA), Lithocholic acid (LCA) and Deoxycholic acid (DCA). These results showed that these bacterial genera may have the ability to influence bile acid conversion. In conclusion, our study showed that nobiletin could affect gut microbiota composition which may potentially influence TCDCA, UDCA, LCA and DCA bile acid metabolism. Besides, nobiletin could also ameliorate NAFLD progression by reducing cholesterol and triglycerides level in liver through regulating CYP7A1, SREBP-1c and FASN protein expression in enterohepatic circulation.