高劑量N-乙醯半胱氨酸治療舒疼消熱劑誘導急性肝損傷小鼠的副作用與毒性機制

The side-effects and toxic mechanisms of high-dose N-acetylcysteine therapy in mice with propacetamol-induced acute liver damage

謝承錡(Cheng-Chi Hsieh)

指導教授 ： 王淑紅

中山醫學大學/醫學科技學院/生物醫學科學學系/碩士(2021年)

https://doi.org/10.6834/csmu202100080

摘要

乙醯胺酚（acetaminophen, APAP）是一種常見的鎮痛解熱藥，但在過量使用下會導致急性肝損傷或衰竭，且有一定的死亡率。N-乙醯半胱氨酸（N-acetyl cysteine, NAC）為臨床上用於治療APAP中毒唯一的解毒劑，但是NAC標準治療劑量並無法適用於每個APAP中毒病人，尤其是高劑量APAP中毒者；也沒有證據顯示提高NAC的治療劑量可以提高治療效果。舒疼消熱注射劑（Acetamol Injection，舒疼）是APAP的前驅物，常用於手術後減輕疼痛。舒疼引起的急性肝損傷或衰竭的作用機制與APAP一樣，而且APAP誘導小鼠的急性肝損傷或衰竭的病理機制與人類相似，因此，我們以小鼠研究高劑量NAC治療舒疼誘導急性肝損傷或衰竭小鼠的可能副作用與NAC毒性可能機制。我們首先測試不同品系小鼠在不同劑量舒疼處理後168小時的存活率，以舒疼處理BALB/cByJNarl（BALB/c）與C57BL/6JNarl（C57BL/6）小鼠48小時的致死劑量分別為1400 mg/kg與1600 mg/kg。以不同劑量NAC治療1200 mg/kg舒疼誘導急性肝損傷並分析168小時存活率的實驗中，發現以275 mg/kg的NAC（N275）治療兩種品系小鼠急性肝損傷的存活率皆為100%，但以400和800 mg/kg的NAC（N400和N800）治療皆會造成兩種品系小鼠的存活率反而下降，因此給予N275治療對兩種品系小鼠是一個最好的治療劑量。我們選用對於舒疼較為敏感的BALB/c小鼠作為我們後續研究過量NAC治療分子機制的實驗動物。給予小鼠1200 mg/kg舒疼1.5小時後分別以125 mg/kg的NAC（N125）、N275、N400和N800治療後的12、24和48小時的小鼠肝臟外觀與病理切片分析及血清中的肝臟功能指標‒麩丙酮酸轉氨酶（Alanine aminotransferase, ALT）與麩草醋酸轉氨酶（aspartate aminotransferase, AST）活性後，我們發現使用N125和N275治療有劑量效應。雖然N400與N800治療組的ALT與AST數值與細胞壞死比例皆相當，但N400與N800處理會造成舒疼中毒小鼠與正常小鼠的死亡，且N800處理顯著降低舒疼中毒小鼠與正常小鼠的肝臟穀胱甘肽（Glutathione, GSH）含量，並誘導發炎因子‒白細胞介素-6（Interleukin-6）的上升和肝臟微泡脂肪變性（microvesicle steatosis）。此外，N275和N400處理降低了正常小鼠血清中的三酸甘油酯（Triglyceride, TG），但增加了肝臟中的TG，而 N800處理明顯地增加了肝臟中的游離脂肪酸與TG及血清中總膽固醇含量。使用N800治療急性肝損傷小鼠168小時後，會造成兩種品系小鼠血清的腎臟功能指標‒肌酸酐（creatinine, CREA）的降低，使用N800治療急性肝損傷小鼠的48小時後，也會使CREA降低；而使用N800處理24小時會使小鼠血清的腎功能指標‒血尿素氮（blood urea nitrogen）及CREA升高，但N800處理組與N800治療組小鼠的腎臟病理切片均沒有發現異常，這說明高劑量的NAC可能會造成小鼠的代謝異常。總結以上結果，過量NAC會抑制肝臟GSH含量，干擾肝臟代謝，促進肝臟脂肪肝形成，誘發系統性發炎，進而造成N800處理組與N800治療組小鼠的死亡。然而高劑量NAC的詳細毒性分子機制有待未來進一步研究。

關鍵字

乙醯胺酚 ； N-乙醯半胱氨酸 ； 舒疼消熱注射劑 ； 穀胱甘肽 ； 微泡脂肪變性

並列摘要

Acetaminophen (APAP) is a common analgesic and antipyretic, but APAP overdose induces acute liver damage, failure, and even death. N-acetyl cysteine (NAC) is the only antidote used clinically to treat APAP poisoning. The standard therapeutic dose of NAC can’t be applied to every patient, especially those with high-dose APAP poisoning. Furthermore, there is insufficient evidence to prove that increasing NAC dose can treat patients who failed in standard treatment. Acetamol Injection (propacetamol) is the precursor of APAP and is often used to relieve pain after surgery. The mechanism of acute liver damage and failure caused by the propacetamol is the same as that of APAP. The pathological mechanism of APAP-induced acute liver damage and failure in mice is similar to that of human patients. Therefore, we used laboratory inbred mice to study the potential side effect(s) and toxicity mechanism(s) of high-dose NAC in the treatments of normal mice and mice with propacetamol-induced acute liver damage. First, we tested the 168-hour survival rate of two different strains of mice at different doses of propacetamol. The lethal doses of BALB/cByJNarl (BALB/c) and C57BL/6JNarl (C57BL/6) mice treated with propacetamol for 48 hours were 1400 mg/kg and 1600 mg/kg, respectively. Two inbred mouse strains with different sensitivities to propacetamol-induced hepatotoxicity were therapied with different NAC doses. Results showed that 275 mg/kg NAC (N275) can completely rescue 1200 mg/kg propacetamol (P1200) poisoning in two mouse strains. However, 400 and 800 mg/kg NAC (N400 and N800) therapies decreased the survival rates in two mouse strains. Therefore, N275 is the best therapeutic dose for P1200 poisoning in two mouse strains. We used BALB/c mice that are more sensitive to propacetamol to explore the molecular mechanism(s) of different NAC doses in the therapy of propacetamol-induced acute liver damage. We analyzed the liver appearance and pathological hepatic sections and serum contents of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) until 12, 24 and 48 hours therapied with 125 mg/kg NAC (N125), N275, N400 and N800 for 1.5 hours after injection with propacetamol. Result showed that the therapeutic effects of NAC on propacetamol induced hepatotoxicity were dose-dependent from N125 to N275 according to pathological sections of liver and serum ALT/AST activities from 12 to 48 hours. In the N400 and N800 therapy groups, the serum ALT and AST activities and the percentages of necrosis of hepatocytes were equivalent. Elevated doses of NAC (N400 and N800) caused additional deaths in both propacetamol-injected and normal mice. N800 treatment significantly decreased hepatic GSH levels, induced inflammatory interleukin-6 (IL-6) levels and hepatic microvesicular steatosis in both propacetamol-injected and normal mice. Furthermore, both N275 and N400 treatments decreased serum triglyceride (TG) but increased hepatic TG, whereas N800 treatment significantly increased hepatic TG and free fatty acid, and serum total cholesterol levels. Furthermore, after using N800 to treat P1200 poisoning mice for 168 hours, it caused decreases in the serum creatinine (CREA) levels of the two strains of mice. After 48 hours of using N800 to treat P1200 poisoning mice, it also reduced the serum CREA levels. Treatment with N800 after 24 hours increased the serum blood urea nitrogen (BUN) and CREA levels. However, no abnormalities were found in the pathological renal sections of the mice in the N800 treatment group and the N800 therapy group. These results indicated that high doses of NAC may cause metabolic abnormalities in mice. In conclusion, NAC overdose decreases hepatic GSH level, induces hepatic and systemic inflammations, interferes with fatty acid metabolism, and then may cause the death of normal and propacetamol poisoning mice. The detailed molecular mechanism(s) of high-dose NAC toxicity need to be further elucidated.

並列關鍵字

Acetaminophen ； N-acetyl cysteine ； propacetamol ； Glutathione ； microvesicle steatosis

參考文獻

1. Ho, C.M.; Lee, C.H.; Wang, J.Y.; Lee, P.H.; Lai, H.S.; Hu, R.H. Nationwide longitudinal analysis of acute liver failure in Taiwan. Medicine (Baltimore) 2014, 93, e35.

2. Yang, R.; Miki, K.; He, X.; Killeen, M.E.; Fink, M.P. Prolonged treatment with n-acetylcystine delays liver recovery from acetaminophen hepatotoxicity. Crit Care 2009, 13, R55.

3. Stewart, J. Paracetamol. https://www.drugs.com/paracetamol.html

4. Larson, A.M.; Polson, J.; Fontana, R.J.; Davern, T.J.; Lalani, E.; Hynan, L.S.; Reisch, J.S.; Schiodt, F.V.; Ostapowicz, G.; Shakil, A.O., et al. Acetaminophen-induced acute liver failure: Results of a United States multicenter, prospective study. Hepatology 2005, 42, 1364-1372.

5. Ramachandran, A.; Jaeschke, H. Acetaminophen hepatotoxicity. Semin Liver Dis 2019, 39, 221-234.