探討台灣本土大蟬花Wu-BFP-14的急毒性與對於乙醯胺酚誘導急性肝損傷的保護機制

乙醯胺酚(N-acetyl-p-aminophenol, Acetaminophen, APAP)是一種被廣泛使用的鎮痛及解熱用藥，當APAP使用過量則會造成急性肝損傷(Acute liver injury, ALI)。舒疼消熱注射劑(Acetamol Injection)的成分‒對乙酰氨基酚(Propacetamol)是APAP的前驅物，在肝臟中的代謝及誘導損傷方式與APAP一致。大蟬花(Cordyceps cicadae)為一種寄生於蟬科幼蟲的真菌，含有多醣、蟲草酸、麥角甾醇及核苷，具有腎臟保護、免疫調節、神經保護、抗癌及抗糖尿病等功效。因此，本研究探討台灣新北市分離且人工培養的大蟬花Wu-BFP-14子實體(Fruiting body, FB) 或菌絲體(Mycelium, M) 之生物安全性，以及對Propacetamol誘導小鼠ALI的保護效果及可能機制。首先分析大蟬花的急毒性，利用管餵實驗小鼠1 g/kg或5 g/kg的FB或M，觀察實驗小鼠的存活率與行為，並在14天後犧牲進行分析。結果發現所有實驗小鼠皆存活，也沒有明顯的行為異常。1 g/kg的FB或M皆不會影響小鼠的體重增長，不過攝食量、飲水量卻有明顯增加；而管餵5 g/kg FB或M的小鼠體重增長明顯減少，但其中只有5 g/kg M組的小鼠攝食量、飲水量有明顯增加。管餵FB或M的兩個劑量小鼠的心、肝、脾、肺、腎及睪丸外觀皆正常，血清肝、腎功能指數包括：天門冬胺酸轉胺酶(Aspartate amino transferase, AST)、丙胺酸轉胺酶(Alanine amino transferase, ALT)、血尿素氮(Blood urea nitrogen, BUN)都在正常範圍，而5 g/kg F組別血清中肌酐酸(Creatinine, CREA)則有明顯增加，不過肝臟和腎臟病理切片都沒有顯示出明顯毒性。接著我們先管餵200 mg/kg FB或M的95%乙醇萃取物(A)或純水萃取物(W)，再使用600 mg/kg Propacetamol誘導小鼠ALI，進行不同大蟬花萃取物的護肝效果測試。結果顯示四種大蟬花萃取物都能顯著降低APAP誘導ALI造成的血清ALT活性上升，不過只有M的乙醇萃物(MA)及水萃物(MW)能夠顯著降低血清中的AST活性上升，其中又以MW的效果最好，肝臟病理切片結果也顯示MW確實減少APAP誘導的ALI。因此，我們選用MW進行後續使用劑量與作用機制分析。在連續管餵三天不同劑量的MW(62.5、125、250 mg/kg)後，使用600 mg/kg Propacetamol誘導小鼠產生ALI，探討最佳保護劑量以及護肝可能作用機制。結果發現62.5 mg/kg及125 mg/kg MW具有護肝效果，而餵食250 mg/kg MW的小鼠肝臟損傷的嚴重程度與Propacetamol組相當，沒有護肝的效果。在護肝可能作用機制的部分，MW能透過抑制促發炎因子包括：腫瘤壞死因子-α(Tumor necrosis factor-α)、白細胞介素-1β(Interleukin-1β)，降低發炎相關指標一氧化氮(Nitric oxide)、髓過氧化物酶(Myeloperoxidase)，減少APAP誘導的發炎反應；透過提升超氧化物歧化酶(Superoxide dismutase)、穀胱甘肽過氧化物酶(Glutathione peroxidase)、過氧化氫酶(Catalase)活性，維持穀胱甘肽(Glutathione)在肝臟中的濃度，減少脂質過氧化產物–丙二醛 (Malondialdehyde)及活性氮產物–3-硝化酪胺酸(3-Nitrotyrosine)產生。另外，藉由降低磷酸化的細胞外調節蛋白激酶(Extracellular regulated protein kinases), c-Jun N端激酶(c-Jun N-terminal kinase)及抑制細胞凋亡Bcl-2相關X蛋白(Bcl-2 associated X protein)表現量，達到減少肝臟細胞的凋亡。從以上結果可以得知，大蟬花Wu-BFP-14菌絲體水萃取物能夠透過抗發炎、抗氧化以抗凋亡機制，來達到對Propacetamol誘導ALI的保護效果，具有顯著護肝功效。後續大蟬花的生物醫學應用值得進一步探討。

關鍵字

乙醯胺酚；對乙醯氨基酚；大蟬花；菌絲體；肝損傷；抗氧化；抗發炎

並列摘要

Acetaminophen (N-acetyl-p-aminophenol, Acetaminophen, APAP) is a widely used analgesic and antipyretic drug, but overdose of APAP can cause acute liver injury (ALI). Propacetamol is a precursor drug of APAP, and the mechanisms of Propacetamol induced liver damage are as same as those of APAP. Cordyceps cicadae is a fungus parasitic on the larvae of Cicadae. It contains polysaccharides, cordycepin acid, ergosterol and nucleosides. It has kidney protection, immune regulation, neuroprotection, anticancer and antidiabetic effects. In this study, we investigated the biosafety of the C. cicadae Wu-BFP-14, which was isolated in the New Taipei city of Taiwan and artificially cultured, as well as its protective effect and possible mechanism(s) on APAP-induced ALI in mice. We firstly investigated the acute toxicity of the artificially cultured C. cicadae. The laboratory mice were fed with 1 g/kg or 5 g/kg of fruiting body (FB) or mycelium (Mycelium, M) of C. cicadae Wu-BFP-14, and the survival rate and behavior of the laboratory mice were observed and then mice were sacrificed for analyses after 14 days. It was found that all laboratory mice survived and had no obvious behavioral abnormalities. Treatments of 1 g/kg of FB or M didn’t affect the body weight gains of mice, the heart, liver, spleen, lung, kidney and testes were normal in appearance, but there was a significant increase in the food intakes, water intakes; while mice fed with 5 g/kg FB or M their body weight gains decreased, with 5 g/kg M showing a significant increase in food intake and water intake. Mice fed with FB or M serum liver and kidney biochemical indexes including aspartate amino transferase (AST), alanine amino transferase (ALT), and blood urea nitrogen (BUN) were all within the normal range, and the pathological sections of the liver and kidney did not show obvious damage. Next, we fed mice with 200 mg/kg 95% ethanol extract (A) or pure water extract (W) of FB or M for 1 hour, and then induced ALI in mice by injection of 600 mg/kg Propacetamol to evaluate the liver protection effect of different C. cicadae extracts. The results showed that all of four C. cicadae extracts could significantly reduce the increases in serum ALT activity caused by APAP, but only the ethanol extract (MA) and water extract (MW) of M could significantly reduce the increase in serum AST activity caused by APAP. Among these four extracts, the MW showed the best hepatoprotective effect. The results of liver pathological sections also confirmed that the MW did reduce APAP-induced ALI. Therefore, we selected the MW for subsequent dose and mechanism analyses. After three days of continuous feeding mice with different doses of MW (62.5, 125, 250 iv mg/kg), 600 mg/kg Propacetamol was injected to induce ALI, and then the optimal protective dose and possible hepatoprotective mechanism(s) of MW were deciphered. The results showed that 62.5 mg/kg and 125 mg/kg MW had hepatoprotective effects, while the severity of liver damage in mice fed with 250 mg/kg MW was similar to that in the group injected with Propacetamol only. MW could inhibit pro-inflammatory cytokines including tumor necrosis factor-α, interleukin-1β, nitric oxide and myeloperoxidase, to reduce APAP-induced inflammatory responses. By increasing the activity of superoxide dismutase, catalase, glutathione peroxidase, and maintaining the concentration of glutathione in the liver, MA could reduce APAP-induced Malondialdehyde and 3-Nitrotyrosine. MA could reduce phosphorylation of extracellular regulated protein kinases and c-Jun N-terminal kinase, and inhibit expression levels of apoptosis Bcl-2 associated X protein to reduce APAP induce apoptosis of liver cells. In conclusion, the water extract of C. cicadae Wu-BFP14 mycelium can protect APAP-induced ALI in mice through anti-inflammatory, antioxidative and anti-apoptotic mechanisms. The biomedical applications of C. cicadae Wu-BFP-14 will be further studied.

並列關鍵字

Acetaminophen ； Propacetamol ； Cordyceps cicadae ； Mycelium ； Liver injury ； Anti-oxidation ； Anti-inflammation

參考文獻

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