評估蟬花及北蟲草對於酒精性脂肪肝治療與預防之效用

Abstract

酒精性脂肪肝（alcoholic fatty liver disease）是酒精性肝病（alcoholic liver disease，ALD）的早期階段，會進一步惡化成脂肪性肝炎、肝硬化、纖維化和肝癌。酒精透過多種機制造成酒精性脂肪肝，包括增加脂肪生成、脂質過氧化、氧化壓力和抑制脂肪酸氧化。有許多研究指出蟬花（Cordyceps cicadae）以及北蟲草（Cordyceps militaris）之多醣、腺苷，以及蟲草素等成分具有抗發炎、抗氧化、免疫調節和改善脂質代謝等多種功效。而大多研究旨在非酒精性脂肪肝病（nonalcoholic fatty liver disease, NAFLD），對於酒精性脂肪肝的研究相對較少。故本試驗將分為治療與預防之策略，並透過酒精性肝病的二次打擊模型探討蟬花菌絲（Cordyceps cicadae liquid mycelia，CCM）、蟬花米基（Cordyceps cicadae mycelia and rice medium，CCRM）及北蟲草米基（Cordyceps militaris fruiting bodies and rice medium，CMRM）之水萃物，以及蟬花發酵液（Cordyceps cicadae fermentation broth，CCB）在該模型中的抗氧化、發炎反應，以及脂質代謝之效用。 本試驗透過0.25 mM的棕櫚酸（palmitate acid，PA）以及400 mM的乙醇處理AML-12小鼠肝臟細胞誘導肝臟細胞死亡以及產生氧化壓力、發炎反應和脂質堆積的效果。初步以不同濃度的CCM、CCB、CCRM、CMRM水萃物與PA/EtOH共同處理作為治療組，以及在PA/EtOH處理前先給予CCM、CCB、CCRM、CMRM水萃物作為預防組，分析水萃物能否提升細胞活力。脂質堆積方面，則是利用油紅染色和測定三酸甘油酯的含量，探討水萃物是否擁有減少脂質堆積的效果，並透過分析與脂質生合成相關基因之表現量，以了解相關機制。氧化壓力之評估，則透過細胞ROS的含量測定，以及利用MDA作為脂質過氧化的指標，並分析與發炎反應和纖維化相關基因的表現。 試驗結果為CCM和CCB作為治療組能夠提升細胞存活率，而CCM在預防組也具有相同之效果。在脂質堆積方面，預防組之CCM與CCRM能夠減少三酸甘油酯之含量，進一步分析基因的表現顯示，預防組之CCM和CCRM能夠顯著降低脂質生合成相關基因sterol regulatory element binding protein 1c（SREBP1c）和stearoyl-coenzyme A desaturase 1（SCD-1）之表現。另外CCM、CCB和CCRM之治療組能減少MDA之含量，降低脂質過氧化。另外CMRM雖然無法減少脂質，但在PPAR-α、PPAR-γ、CPT-1之基因表達中具有顯著差異，說明在調控脂質合成代謝相關基因方面，CMRM具有一定潛力。 綜上所述，蟬花菌絲水萃物效用較為全面，能夠減緩肝細胞受到PA/EtOH所造成的死亡，以及減少三酸甘油酯和MDA的含量。然而，許多詳細相關機制仍需進一步研究。

Alcoholic fatty liver disease (AFLD) is the early stage of alcoholic liver disease (ALD), which can further deteriorate into steatohepatitis, cirrhosis, fibrosis, and liver cancer. Alcohol causes AFLD through various mechanisms, including increased lipogenesis, lipid peroxidation, oxidative stress, and inhibition of fatty acid oxidation. Numerous studies have indicated that the polysaccharides, adenosine, and cordycepin in Cordyceps cicadae and Cordyceps militaris possess anti-inflammatory, antioxidant, immunomodulatory, and lipid metabolism-improving effects. Most of these studies focus on nonalcoholic fatty liver disease (NAFLD), with relatively few addressing AFLD. Therefore, this study aims to explore the therapeutic and preventive strategies of Cordyceps cicadae liquid mycelia (CCM), Cordyceps cicadae mycelia and rice medium (CCRM), Cordyceps militaris fruiting bodies and rice medium (CMRM) extracts, and Cordyceps cicadae fermentation broth (CCB) in an AFLD model induced by a two-hit process. This study induces liver cell death, oxidative stress, inflammatory response, and lipid accumulation in AML-12 mouse hepatocytes by treating them with 0.25 mM palmitic acid (PA) and 400 mM ethanol. Initial experiments involve co-treatment of different concentrations of CCM, CCB, CCRM, and CMRM extracts with PA/EtOH as the treatment model, and pre-treatment with these extracts before PA/EtOH exposure as the prevention model. The MTT assay evaluates the cell viability-enhancing potential of these extracts. Oil Red O staining and triglyceride content measurement assess lipid accumulation reduction, while gene expression analysis of lipogenesis-related genes elucidates the underlying mechanisms. Oxidative stress is measured by DCFDA for cellular ROS levels and MDA as a lipid peroxidation marker, followed by the analysis of inflammation and fibrosis-related gene expression. Results indicate that CCM and CCB in the treatment model improve cell viability, with CCM also effective in the prevention model. CCM and CCRM in the prevention model reduce triglyceride levels. Gene expression analysis shows that CCM and CCRM in the prevention model significantly decrease the expression of lipogenesis-related genes sterol regulatory element binding protein 1c (SREBP1c) and stearoyl-Coenzyme A desaturase 1 (SCD-1). Additionally, CCM, CCB, and CCRM in the treatment model reduce MDA levels, mitigating lipid peroxidation. Although CMRM shows no significant effects in most assays, it exhibits notable differences in the expression of PPAR-α, PPAR-γ, and CPT-1 genes. In summary, the water extract of Cordyceps cicadae mycelium demonstrates comprehensive efficacy in mitigating PA/EtOH-induced hepatocyte death, reducing triglyceride and MDA levels. However, further research is required to elucidate the detailed mechanisms involved.