大蒜精油及其活性主成份抗非酒精性脂肪肝病程發展之代謝體學研究

Abstract

非酒精性脂肪肝疾病（nonalcoholic fatty liver disease, NAFLD）為代謝性疾病在肝臟中的表徵。肥胖、血脂異常、胰島素阻抗、氧化壓力與發炎為其病程進展的關鍵危險因子。目前在相似於臨床病因及發病機制的 NAFLD 動物模式下尚未有完整之代謝體學研究，且大蒜精油（garlic essential oil, GEO）對 NAFLD 保護功效與代謝機制亦仍未被探討。故本研究以代謝體學探討正常或高脂飲食（high fat diet, HFD）分別餵食 C57BL/6J 小鼠 4、8、12 與 16 週後 NAFLD 病程發展下病理與生理代謝之變化。並應用所建立動物模式評估 GEO 及其有機含硫主成份 diallyl disulfide（DADS）對 NAFLD 保護之功效及代謝機制。 代謝體學分析方面採用超高效能液相層析結合四極-飛行式質譜儀及氣相層析-質譜儀分析血清代謝物，數據以主成份分析（principal component analysis, PCA）進行評估。結果顯示各誘導週期正常與 HFD 組間依據 NAFLD 嚴重程度而具有明顯之差異代謝物，其病程發展下共有 30 個相關之潛力性指標代謝物。其中在具肥胖、高血清葡萄醣、總膽固醇與肝臟三酸甘油脂特徵之第 4 週早期 NAFLD 發病下有 11 個代謝物可能主要影響醣類代謝、肝臟生物轉化作用、膠原合成作用及腸道微生物代謝；第 8 週誘導中期 NAFLD 進展下，5 個額外代謝物可能主要影響脂質代謝及胰島素分泌作用，而引起高血脂、高胰島素血症及肝臟脂質堆積；而第 12 與 16 誘導末期，14 個額外代謝物可能主要引發膽汁酸合成作用、氧化與發炎反應異常變化，而使 NAFLD 發展下產生肝臟發炎浸潤情形。此結果顯示這些潛力性指標代謝物可能作為 NAFLD 病程發展前期或進展下的風險評估指標。 GEO 功效評估方面，C57BL/6J 小鼠餵食正常或 HFD，並管餵 GEO（25、50與100 mg/kg）或 DADS（10與20 mg/kg）共12週。結果顯示 GEO 及 DADS 可劑量依賴性減緩小鼠 HFD 誘導體重、脂肪組織與血清生化指標增加，具延緩肥胖及降血脂之作用。給予小鼠 50、100 mg/kg GEO 與 20 mg/kg DADS 顯著降低 NAFLD 病程發展所引起肝臟促發炎細胞因子釋放，抑制 CYP2E1 蛋白表現提升抗氧化酵素能力。GEO 及 DADS 可通過抑制 SREBP-1c、ACC、FAS 與 HMGCR 蛋白表現量，促進 PPARα 與 CPT-1 蛋白表現而減緩脂質堆積於肝臟所引發的 NAFLD。代謝體學分析結果亦顯示，GEO 與 DADS 可調控部份 HFD 誘導 NAFLD 病程發展下參與醣類代謝、膠原合成作用、腸道微生物代謝、胰島素分泌、脂質代謝及氧化與發炎代謝的關鍵指標性代謝物。上述證實 GEO 及 DADS 可有效延緩長期 HFD 下肥胖小鼠誘導 NAFLD 病程發展，而其中 20 mg/kg DADS 劑量與含有等量主成份的 50 mg/kg GEO 有相似改善 NAFLD 之作用，顯示 GEO 保護功效可能主要來自 DADS 之活性主成份。 綜合以上本研究發現數種代謝物為參與 NAFLD 病程發展關鍵代謝途徑或與臨床 NAFLD 相似的關鍵代謝物，這些具潛力的指標性代謝物可能作為病程發展前期的風險指標。所建立之小鼠 NAFLD 誘導模式可提供 NAFLD 病程發展下病理生理改變及相關分子機制的探討。此外，本研究亦證實 GEO 與 DADS 具有效延緩小鼠 HFD 所誘導 NAFLD 病程的發展。

Nonalcoholic fatty liver disease (NAFLD) is regarded as the hepatic manifestation of the metabolic syndrome. Obesity, dyslipidemia, insulin resistance, oxidative stress, and inflammation are key clinical risk factors for the progression of NAFLD. Currently there is no comprehensive metabolic profile in a well-established animal model that effectively mimics the etiology and pathogenesis of NAFLD in humans. Also the protective effects and underlying mechanisms of garlic essential oil (GEO) on the development of NAFLD have not been explored. Here, we report the pathophysiological and metabolomic changes associated with NAFLD development in a C57BL/6J mouse model induced by a high-fat diet (HFD) for 4, 8, 12, and 16 weeks. Then, the established mouse model of NAFLD was used to investigate the protective properties of GEO and its major organosulfur component, diallyl disulfide (DADS), against the development of NAFLD. For the metabolomic approach, ultra-high performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UHPLC-QTOF-MS) and gas chromatography-quadrupole mass spectrometry (GC/MS) was employed to assess the metabolites in serum samples of mice. The datasets were statistically analyzed using principal component analysis (PCA). These results revealed marked differences in metabolites between the control and HFD group depending on NAFLD severity. Thirty potential biomarkers were strongly associated with the development of NAFLD. Among them, 11 metabolites were mainly related to the impact of carbohydrate metabolism, hepatic biotransformation, collagen synthesis, and gut microbial metabolism, which are characteristics of obesity, as well as significantly increased serum glucose, total cholesterol, and hepatic triglycerides levels during the onset of NAFLD (4 weeks). At 8 weeks, 5 additional metabolites that are chiefly involved in perturbation of lipid metabolism and insulin secretion were associated with the hyperinsulinemia, hyperlipidemia, and hepatic steatosis in the mid-term of NAFLD progression. At the end of 12 and 16 weeks, 14 additional metabolites were predominantly correlated to abnormal bile acid synthesis, oxidative stress, and inflammation, representing hepatic inflammatory infiltration in the development of NAFLD. These results provide potential biomarkers for early risk assessment of NAFLD and further insight into NAFLD development. For efficacy assessment of GEO, C57BL/6J mice were fed a normal or HFD with/without GEO (25, 50, and 100 mg/kg) or DADS (10 and 20 mg/kg) for 12 weeks. GEO and DADS dose-dependently exerted antiobesity and antihyperlipidemic effects by reducing HFD-induced body weight gain, adipose tissue weight, and serum biochemical parameters. Administration of 50 and 100 mg/kg GEO and 20 mg/kg DADS significantly decreased the release of pro-inflammatory cytokines in liver, accompanied by elevated antioxidant capacity via inhibition of CYP2E1 expression during NAFLD development. The anti-NAFLD effects of GEO and DADS were mediated through down-regulation of SREBP-1c, ACC, FAS, and HMGCR, as well as stimulation of PPARα and CPT-1. The results of metabolomics analysis also revealed that GEO and DADS could particaully regulate the potential biomarkers involved in carbohydrate metabolism, collagen synthesis, gut microbial metabolism, insulin secretion, lipid metabolism, oxidative stress, and inflammation during NAFLD development. These results demonstrate that GEO and DADS dose-dependently protected obese mice with long-term HFD-induced NAFLD. The dose of 20 mg/kg DADS was equally as effective in preventing NAFLD as 50 mg/kg GEO containing the same amount of DADS, which demonstrates that DADS may be the main bioactive component in GEO. In conclusion, the present study reports several key metabolites and potential biomarkers in the metabolic pathways involved in the pathogenesis of NAFLD, similar to those observed in clinical cases of NAFLD. The potential biomarkers for early risk assessment of NAFLD were also discovered. The established mouse model of NAFLD could be used to elucidate the pathophysiologic alterations and molecular mechanisms underlying the development of NAFLD. In addition, this study demonstrated the protective attributes of GEO and DADS against the progression of HFD-induced NAFLD in mice.