探討大蟬花子實體水萃物保護 Propacetamol誘導小鼠肝損傷的效果與分子機制

大蟬花(Cordyceps cicadae)含有多醣、腺苷、蟲草素等等活性成分，與冬蟲夏草(Ophiocordyceps sinensis)相似，由於大蟬花可以人工培養，因此具有替代冬蟲夏草的潛力。乙醯胺酚(Acetaminophen, APAP)廣泛運用於鎮痛與解熱，但 APAP 過量是最常見的藥物引起急性肝損傷(Acute liver injury, ALI)。本研究使用過量 APAP 誘導小鼠產生 ALI，研究人工養殖大蟬花子實體(F)水萃取物護肝效果與可能作用機制。首先我們將人工養殖大蟬花子實體水萃物(FW)與菌絲體水萃物(MW)進行成分分析，結果顯示 FW 中的腺苷、胞苷、蟲草素、鳥苷、肌苷、胸苷、尿嘧啶及尿苷的含量皆高於 MW，而腺嘌呤、胞嘧啶、蟲草酸、次黃嘌呤、蛋白質、多糖及胸腺嘧啶則低於 MW。接著以 1 g/kg 的 FW 與 MW 對小鼠進行急毒性分析，結果顯示，FW 及 MW 組與生理食鹽水(PBS)對照組在體重、飲食與飲水量方面均沒有明顯差異，血清的肝、腎功能指標—丙胺酸轉胺酶(Alamine aminotransferase, ALT)、天門冬胺酸轉胺酶(Aspartate aminotransferase, AST)、血尿素氮(Blood urea nitrogen, BUN)及肌酸酐(Creatinine, CREA)與對照組相比無明顯提高，肝腎組織切片也未觀察到明顯的病理性變化，證實 FW 與 MW 具良好生物安全性。進一步管餵不同劑量(12.5、25、50、100 mg/kg) FW，分析 FW 保護 APAP 誘導肝損傷的效果，根據組織外觀結果顯示使用25 mg/kg (FW25)的小鼠肝臟損傷程度最低，同時 FW 25、FW50 組降低肝功能指數 ALT、AST 的效果顯著。而肝臟組織切片染色結果顯示 FW25、FW50 組明顯降低出血，其中 FW25 顯著減少凝血性壞死。綜合以上結果顯示 FW25 與 FW50 護肝效果最好。在護肝分子機制方面，首先對氧化壓力進行分析，實驗結果顯示，FW25、FW50 組顯著降低 APAP 誘導的脂質過氧化產物—丙二醛(Malondialdehyde, MDA)及蛋白質氧化產物— 3-硝基酪胺酸(3-Nitrotyrosine, 3-NT)。抗氧化酵素活性方面，FW25顯著提升過氧化氫酶(Catalase, CAT)與超氧歧化酶(Superoxide Dismutase, SOD)活性，維持肝臟中穀胱甘肽(Glutathione, GSH)濃度；與此同時，抗氧化相關基因血鐵質氧化酶(Heme Oxygenase-1, HO-1)之 mRNA 及蛋白表現量在 FW25、FW50中顯著降低。由以上結果得知 FW 可透過活化抗氧化機制來降低 APAP 過量所帶來的氧化壓力。在發炎反應方面，FW25、FW50 可顯著抑制 APAP 誘導的血清中腫瘤壞死因子-α (Tumor Necrosis Factor-α, TNF-α)、介白素-6 (Interleukin-6, IL-6)與介白素-1β (Interleukin-1β, IL-1β)含量，顯示 FW 具有良好的抗發炎能力。此外，FW25 顯著抑制胞外訊號調節激酶(Extracellular signal-Regulated Kinase, ERK)與 c-Jun 氨基末端激酶(c-Jun N-terminal Kinase, JNK)的磷酸化，推測 FW 之護肝作用與抑制絲裂原活化蛋白激酶(Mitogen-Activated Protein Kinase, MAPK) pathway 有關。綜合以上結果，管餵1 g/kg 劑量的 FW 與 MW 對小鼠肝腎功能無不良影響；人工養殖大蟬花子實體水萃物。FW25與 FW50具有較好的護肝效果，FW 主要透過減緩氧化壓力、促進抗氧化酵素活性，並透過調控 MAPK pathway 來減緩下游的發炎發生，最後還能夠透過減緩細胞凋亡，進而保護 APAP 誘導的急性肝損傷。

Cordyceps cicadae is a parasitic fungus that contains multiple bioactive components, including polysaccharides, cordycepin, adenosine, and various nucleosides, which are similar to those found in Ophiocordyceps sinensis. As Cordyceps cicadae can be artificially cultivated, it presents a potential substitute for O. sinensis. Acetaminophen (APAP), also known as N-acetyl-p-aminophenol, is a widely used analgesic and antipyretic drug. However, APAP overdose is one of the most common causes of drug-induced acute liver injury (ALI). In this study, we evaluated the hepatoprotective effects and underlying mechanisms of the water extract from artificially cultivated fruiting bodies of Cordyceps cicadae (FW) against APAP-induced ALI in mice. First, we analyzed the chemical composition of water extracts from both the fruiting bodies (FW) and mycelium (MW) of Cordyceps cicadae. The results showed that FW contained higher levels of adenosine, thymine, uracil, cytidine, inosine, and guanosine compared to MW, while MW had higher levels of cordycepin, uridine, cytosine, hypoxanthine, adenine, and cordycepic acid. Acute toxicity testing with 1 g/kg FW or MW showed no significant differences in body weight, food and water intake, or serum biochemical indicators Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Blood urea nitrogen (BUN) and Creatinine (CREA) compared to the control group. Histological analysis of liver and kidney tissues showed no pathological abnormalities, indicating the good biosafety profile of FW and MW. To investigate the hepatoprotective effects of FW, mice were pretreated with different doses (12.5, 25, 50, and 100 mg/kg) for three days before ALI induction through APAP overdose. Among all groups, both 25 mg/kg (FW25) and 50 mg/kg (FW50) showed the best protective effect, as evidenced by decreased serum ALT and AST levels and less severe liver damage in histological staining, including reduced hemorrhage and coagulative necrosis. Mechanistic studies showed that FW25 and FW50 significantly reduced the levels of oxidative stress markers such as malondialdehyde and 3-nitrotyrosine while enhancing the activity of antioxidant enzymes, including catalase and superoxide dismutase, and maintaining glutathione levels in the liver. Furthermore, FW25 and FW50 significantly suppressed the expression of inflammatory cytokines, including tumor necrosis factor-α, interleukin-6, and interleukin-1β. FW25 also inhibited the phosphorylation of ERK and JNK, suggesting the involvement of the MAPK signaling pathway in its hepatoprotective effects. In conclusion, the water extract of artificially cultivated Cordyceps cicadae fruiting bodies (FW) is safe for use in mice and exhibits significant protective effects against APAP-induced acute liver injury, particularly at 25 mg/kg and 50 mg/kg doses. These effects are likely mediated through antioxidant, anti-inflammatory, and MAPK pathway regulatory mechanisms. FW demonstrates potential for further development as a hepatoprotective agent.