- 學位論文
18碳多元不飽和脂肪酸對棕櫚酸介導脂毒性肝細胞損傷的保護效應與相關機制
Protective effects and related mechanism of 18-carbon polyunsaturated fatty acids on palmitic acid mediated lipotoxic liver cell injury
摘要
肝臟中過量脂質堆積會導致脂質變性(Steatosis)，並進一步誘發肝臟氧化壓力和內質網壓力，造成細胞損傷與發炎，引起非酒精性脂肪肝病(Non-alcoholic fatty liver disease, NAFLD)。若肝臟細胞無法透過抗氧化與自噬作用改善脂質變性引起的細胞損傷，則會造成肝臟細胞纖維化及死亡。在本實驗室過去研究中已證實，亞麻油酸(Linoleic acid, C18:2, LA)、γ-次亞麻油酸(Gamma-Linolenic acid, 18:3, GLA)與α-次亞麻油酸(α-Linolenic acid, C18:3, LNA)等18碳多元不飽和脂肪酸(18-carbon chain polyunsaturated fatty acid, 18C-PUFAs)具有改善棕櫚酸(Palmitate, PA)誘發C2C12肌管細胞發炎與胰島素阻抗的效果。眾所周知，發炎反應是NAFLD進展的過程之一，因此本研究欲評估18C-PUFAs對改善PA誘導HepG2人類肝臟細胞產生脂毒性的細胞模式，評估其保護效果與相關機制。實驗結果顯示，PA處理HepG2細胞後，給予18C-PUFAs能夠增加單磷酸腺苷活化蛋白質激酶(Adenosine monophosphate-activated protein kinase, AMPK)與sirtuin-1 (SirT1)，並降低fatty acid synthase (FAS)蛋白質表達，且油紅染色後可以觀察到脂質油滴的堆積量降低。PA處理HepG2細胞後，給予18C-PUFAs能夠增加抗氧化酵素超氧化物歧化酶1(Superoxide dismutases 1, SOD1)、SOD2、麩胱甘肽過氧化物酶2(Glutathione peroxidase 2, GPx2)與其相關的轉錄因子核因子紅細胞2-相關因子2 (Nuclear factor erythroid 2-related factor 2, Nrf2)表達，且H2DCFDA與MitoSOX染色後分別能看到肝臟總細胞與粒線體ROS的降低。PA處理HepG2細胞後，給予18C-PUFAs亦能增加自噬相關蛋白Beclin1、microtubule-associated protein 1 light chain 3 (LC3)與sequestosome 1 (P62/SQSTM1)表達，並降低phospho-inositol-requiring enzyme-1α (p-IRE1α)表達，改善內質網壓力的影響。此外，給予18C-PUFAs可以降低PA誘導HepG2細胞凋亡相關蛋白Bax、caspase 9與caspase 3，並增加B淋巴细胞瘤-2 (B cell lymphoma-2, Bcl-2)表達，且TMRE染色可以觀察到肝臟細胞粒線體膜電位的增加，說明給予18C-PUFAs可以阻止肝臟細胞中粒線體介導的細胞凋亡。綜合以上結果得知，給予18C-PUFAs有助於提升肝臟細胞抗氧化與自噬能力，並改善脂質變性引起的氧化壓力與內質網壓力，避免粒線體介導的細胞凋亡發生，維持肝臟細胞活性。未來可以進一步探討18C-PUFAs是否能夠改善脂質毒性對肝臟或其他組織的影響，預防慢性代謝性疾病的發生。
並列摘要
Excessive lipid accumulation in the liver will lead to steatosis, and further induce liver oxidative stress and endoplasmic reticulum stress, resulting in cell damage and inflammation, leading to non-alcoholic fatty liver disease (NAFLD). If liver cells cannot improve the cell damage caused by lipid degeneration through anti-oxidation and autophagy, it will cause liver cell fibrosis and death. Past studies in our laboratory had confirmed that 18-carbon polyunsaturated fatty acids (18C-PUFAs) such as linolenic acid (LA), γ-linolenic acid (GLA) and α-linolenic acid (LNA) improved Palmitate (PA)-induced inflammation and insulin resistance in C2C12 myotube cells. Since inflammation is one of the processes of NAFLD progression, this study aims to evaluate the cellular model of 18C-PUFAs in improving PA-induced lipotoxicity in HepG2 human liver cells, and evaluate its protective effect and related mechanism. Experimental results showed that after PA treatment of HepG2 cells, administration of 18C-PUFAs could increase Adenosine monophosphate-activated protein kinase (AMPK) and sirtuin-1 (SirT1), and decrease fatty acid synthase (FAS) protein expression, and the accumulation of lipid oil droplets can be observed to decrease after oil red staining. After PA treatment of HepG2 cells, administration of 18C-PUFAs increase the expression of Superoxide dismutases 1 (SOD1), SOD2, Glutathione peroxidase 2 (GPx2) and its related transcription factor Nuclear factor erythroid 2-related factor 2 (Nrf2), and observed reduction of ROS in total liver cells and mitochondria after H2DCFDA and MitoSOX staining respectively.After PA treatment of HepG2 cells, administration of 18C-PUFAs can also increase the expression of autophagy-related proteins Beclin1, microtubule-associated protein 1 light chain 3 (LC3) and sequestosome 1 (P62/SQSTM1), and reduce the expression of phospho-inositol-requiring enzyme-1α (p-IRE1α) expression, ameliorating the effects of endoplasmic reticulum stress.In addition, administration of 18C-PUFAs can reduce PA-induced apoptosis-related proteins Bax, caspase 9 and caspase 3 in HepG2 cells, and increase the expression of B cell lymphoma-2 (Bcl-2), and observed increasion of mitochondrial membrane potential in liver cells after TMRE staining, indicated that administration of 18C-PUFAs could prevent mitochondria-mediated apoptosis in liver cells.In conclusion, the administration of 18C-PUFAs improve the antioxidant and autophagy capabilities of liver cells, improve the oxidative stress and endoplasmic reticulum pressure caused by lipid degeneration, and avoid the occurrence of mitochondria-mediated apoptosis. Maintain liver cell viability. In the future, we can further explore whether 18C-PUFAs can improve the effects of lipid toxicity on the liver or other tissues, and prevent the occurrence of chronic metabolic diseases.