LRRK2蛋白表現之調降參與非酒精性脂肪肝與靈芝水萃物抑制肝臟細胞發炎之機制研究

Down-regulation of LRRK2 contributes to the nonalcoholic fatty liver disease (NAFLD) and the mechanism by which Ganoderma lucidum water extract suppresses the inflammation in hepatocyte

林巧威(Chiao-Wei Lin)

指導教授 ： 丁詩同

國立臺灣大學/生物資源暨農學院/生物科技研究所/博士(2021年)

https://doi.org/10.6342/NTU202100471

摘要

目前的研究指出，肝臟發炎為非酒精性脂肪肝病程推展至肝纖維化或肝癌中扮演重要的角色。然而，於非酒精性脂肪肝中，肝臟如何進展至發炎之機制，尚未被完全了解。近來研究指出Leucine-rich repeat kinase 2 (LRRK2) 蛋白參與脂肪代謝，但其所扮演的功能，以及是否參與非酒精性脂肪肝的疾病發展，目前尚不清楚。利用高脂飼料誘導的非酒精性脂肪肝小鼠模式中發現，LRRK2基因在肝臟中的表現量，相較於健康小鼠明顯降低。在人類肝臟細胞株(HepG2) 中，處理棕櫚酸後，LRRK2蛋白的表現也被抑制。為了進一步探討LRRK2於脂肪代謝中所伴演的角色，在HepG2細胞中過表現 (overexpression) 及基因靜默 (knockdown) LRRK2基因。結果顯示過表現LRRK2蛋白可促進脂肪β氧化，反之，抑制LRRK2基因的表現則抑制脂肪β氧化。進一步探究LRRK2如何調控脂肪的β氧化後發現，操控LRRK2蛋白表現對調控脂肪β氧化反應的重要酵素CPT1A的表現量。且可能是透過活化AMPK或PPARα蛋白，而調控CPT1A的表現有一致性結果。此外，過表現LRRK2抑制由棕櫚酸所誘導的促發炎細胞素TNFα表現。前人的研究顯示，促進細胞的脂肪β氧化可抑制由棕櫚酸所誘導的細胞發炎。本研究指出，LRRK2促進脂肪β氧化並抑制肝細胞的發炎反應。因此，非酒精性脂肪肝中被下調的LRRK2基因，可能為肝臟發炎的原因之一。目前的研究支持藉由抑制肝臟發炎可抑制非酒精性脂肪肝發展成肝纖維化或肝癌的機會。靈芝 (Ganoderma lucidum) 為一傳統中草藥，具有免疫調節、抗發炎、抗氧化、抗菌以及改善腸道菌相的功效。細胞及動物實驗顯示靈芝萃取物及從靈芝分離之活化化合物具有改善非酒精性脂肪肝的效果；然而，其分子機制尚未清楚。從雞初代肝臟細胞及人類肝臟細胞株HepG2的實驗結果顯示，靈芝水萃物可抑制由棕櫚酸誘導的促發炎因子TNFα的表現，可增加抗氧化酵素catalase於經棕櫚酸處理HepG2細胞中的表現量。並且也可有效抑制因H2O2處理所產生的活性氧化物(ROS)。此外，於經棕櫚酸處理HepG2細胞中，靈芝水萃物增加FOXO1的表現量以及促進AMPK的活化。因此，AMPK及FOXO1分子路徑可能參與了靈芝水萃物的抗氧化效果。靈芝未來或許可應用於治療或預防與氧化壓力所導致的相關疾病，例如非酒精性脂肪肝。

關鍵字

非酒精性脂肪肝 ； LRRK2 ； 脂肪β氧化 ； 脂肪代謝 ； 靈芝

並列摘要

Hepatic inflammation in non-alcoholic fatty liver disease (NAFLD), a metabolic hepatic disease, contributes to progress to cirrhosis or hepatocellular carcinoma. However, etiology of the hepatic inflammation has not been fully understood. In the current study, we identified that leucine-rich repeat kinase 2 (LRRK2) was significantly down-regulated in the liver of NAFLD mouse model induced by a high-fat diet. In HepG2 cells, exposure to palmitic acid (PA) also down-regulated LRRK2. Overexpression and genetic knockdown of LRRK2 were performed to further investigate the role of LRRK2 in lipid metabolism. Results of HepG2 cells showed that β-oxidation was promoted by LRRK2 overexpression and consistently inhibited by LRRK2 knockdown. The expression of critical enzyme in β-oxidation, carnitine palmitoyltransferase 1A (CPT1A), was positively regulated by LRRK2 expression. The results suggested that the regulation of CPT1A by LRRK2 is likely to involve the activation of AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor α (PPARα). Overexpression of LRRK2 reduced the production of a pro-inflammatory cytokine, tumor necrosis factor α (TNFα), induced by PA, suggesting that increase in β-oxidation may promote lipid catabolism to suppress inflammation induced by PA. These results indicated that LRRK2 participated in the regulation of β-oxidation and to suppress inflammation in the liver. Inhibiting hepatic inflammation may suppress the development of cirrhosis or hepatocellular carcinoma in NAFLD has been proposed. Ganoderma lucidum is a traditional herb having the activities of immunomodulation, anti-inflammation, anti-oxidation, antibacterial property, and improvement of microbiota. Moreover, supplementations of extracts or compounds isolated from G. lucidum have been shown to ameliorate NAFLD have been shown in cellular and animal models; however, despite that the molecular mechanisms have not been fully understood. In the current study, the water extracts of G. lucidum suppressed the increase of LITAF by PA in chicken primary hepatocytes and HepG2 cells. Treatment of G. lucidum water extract also increased the levels of catalase, an anti-oxidative enzyme, in PA-treated HepG2 cells. The treatment of G. lucidum water extract also increased total FOXO1 protein expression, the transcription factor that directly turns on catalase gene expression. Besides, activation of AMPK by the treatment of G. lucidum water extracts suggested the increase of catalase may be mediated through AMPK/FOXO1 pathway. Moreover, the treatment of G. lucidum water extracts suppressed the generation of reactive oxygen species (ROS) induced by H2O2. In conclusion, activation of AMPK/FOXO1 pathway may contribute to the anti-oxidant activity of G. lucidum water extracts. G. lucidum has the potential to treat the diseases which can be attributed to oxidative stress, such as NAFLD.

並列關鍵字

nonalcoholic fatty liver disease ； LRRK2 ； β-oxidation ； lipid metabolism ； Ganoderma lucidum

參考文獻

Ai, L., Xu, Q., Wu, C., Wang, X., Chen, Z., Su, D., Jiang, X., Xu, A., Lin, Q., and Fan, Z. (2015). A20 Attenuates FFAs-induced lipid accumulation in nonalcoholic steatohepatitis. Int J Biol Sci 11, 1436-1446.

Ampong, I., Watkins, A., Gutierrez-Merino, J., Ikwuobe, J., and Griffiths, H.R. (2020). Dietary protein insufficiency: an important consideration in fatty liver disease? Br J Nutr 123, 601-609.

Apostolopoulou, M., Gordillo, R., Koliaki, C., Gancheva, S., Jelenik, T., De Filippo, E., Herder, C., Markgraf, D., Jankowiak, F., Esposito, I., et al. (2018). Specific hepatic sphingolipids relate to insulin resistance, oxidative stress, and inflammation in nonalcoholic steatohepatitis. Diabetes Care 41, 1235-1243.

Arab, J.P., Arrese, M., and Trauner, M. (2018). Recent insights into the pathogenesis of nonalcoholic fatty liver disease. Annu Rev Pathol 13, 321-350.

Asgharpour, A., Cazanave, S.C., Pacana, T., Seneshaw, M., Vincent, R., Banini, B.A., Kumar, D.P., Daita, K., Min, H.K., Mirshahi, F., et al. (2016). A diet-induced animal model of non-alcoholic fatty liver disease and hepatocellular cancer. J Hepatol 65, 579-588.