兩種阿醭酚生物鹼類藥物在內毒素血症保護作用之研究

Abstract

阻斷細胞介素或氧自由基的釋出被認為是可以有效的減少敗血症/敗血性休克造成的多重器官傷害，並可提高存活率。因此在本篇論文中，我們分兩部分探討來自於樟科植物中兩種具有抗氧化活性的阿醭酚生物鹼類藥物thaliporphine及合成之次阿醭酚化合物N-allylsecoboldine在敗血症的保護作用及其可能機轉。 第一部份 Thaliporphine增加脂多醣 (lipopolysaccharide, LPS) 誘導內毒素血症的存活率及減少多重器官損傷之作用 Thaliporphine是一種來自於中藥草的芳香環阿醭酚類生物鹼，具有抗氧化及a1受體拮抗劑的活性。給予大白鼠內毒素 (E. coli lipopolysaccharide, LPS) 可誘導嚴重低血壓及心搏過速，同時亦可看到以正腎上腺素測試的血管低反應性發生。預先給予thaliporphine，發現其可以有意義減少LPS誘導大白鼠所造成的後期的低血壓反應，而在較高劑量的thaliporphine (1 mg/kg) 則能減少LPS誘導大白鼠所產生的心搏過速。LPS有意義的增加一氧化氮 (nitric oxide, NO•) 及超氧游離基 (superoxide anion, O2•-) 亦可被預先給予的1 mg/kg thaliporphine減少。內毒素血症動物組於整個實驗240分鐘期間，造成血漿腫瘤壞死因子-a(tumor necrosis factor-alpha,TNF-alpha 呈現一鐘型曲線，且於實驗後的60分鐘達到最高；預先給予1 mg/kg thaliporphine則能有意義減少此60分鐘的高TNF-alpha血漿濃度。除此之外，LPS造成血糖呈現兩期變化，而thaliporphine可以改善後期血糖下降情形。內毒素血症誘導肝、腎及心臟等多重器官壞死的現象可由aspartate aminotransferase (GOT) , alanine aminotransferase (GPT) , creatinine (CRE) , lactate dehydrogenase (LDH) and creatine phosphate kinase muscle-brain (CKMB) 等血漿值增加而得知，thaliporphine不僅可以有意義減少這些生化值的減少，亦可以減少受傷組織導致發炎細胞浸潤現象。另外，thaliporphine以劑量依賴 (dose-dependence) 關係增加了LPS小鼠的存活率。因此，由本篇的結果認為thaliporphine可以作為一個新藥用於減少內毒素誘導造成的循環衰竭、多重器官損傷及增加存活率﹔而thaliporphine的貢獻可能在於抑制TNF-alpha、NO•及O2•-的產生。 第二部分 N-Allylsecoboldine可作為預防內毒素血症所導致之急性腎臟衰竭的新型作用劑 N-Allylsecoboldine是一種具有抗氧化及alpha1受體拮抗劑活性的secoapophine衍生物。預先給予N-allylsecoboldine可以有意義的減少因LPS造成大白鼠後期血壓下降、低血糖及血漿TNF-alpha增加的情形。因內毒素血症而過量產生的血漿NO•並不能被N-allylsecoboldine所改變，但是因內毒素血症而持續減少的尿中NO•含量有部分能被N-allylsecoboldine所恢復。然而，N-allylsecoldine可抑制內毒素血症鼠腎皮質的inducible nitric oxide synthase (iNOS) 蛋白質表現。從N-allylsecoboldine明顯恢復LPS鼠的許多生化酵素標記來看，可知N-allylsecoboldine亦具有改善內毒素血症所導致的器官衰竭能力。內毒素血症與腎功能不良的關聯可由腎血流、尿中鉀的排除及腎臟nitrate的清除率減少得知。然而，預先給予N-allylsecoboldine可以有意義的減少腎功能不良的發生。另外，給予低劑量的N-allylsecoboldine可以減少LPS小鼠的死亡率。此部分實驗證明了N-allylsecoboldine具有幫助對抗內毒素血症造成的急性腎衰竭及增加存活的能力。而其作用可能是由於抑制iNOS的蛋白質表現，TNF-alpha的產生，以及自由基清除的活性。然而，在敗血症發生時，N-allylsecoboldine具有保護的作用，但是對於其中alpha1受體拮抗作用所扮演的角色仍然不十分清楚。

Blockades of cytokine and oxygen free radicals release are considered to be beneficial in reducing multiple organ injury and increase the survival rate in sepsis/septic shock, so we discussed in the two parts about aporphine alkaloid agents – thaliporphine and N-allylsecoboldine, which were obtained from Chinese herb. Also, we attempted to elucidate the protective effects and the probably contributive mechanisms. Part One: Thaliporphine increases survival rate and attenuates multiple organ injury in lipopolysaccharide-induced endotoxemia. Thaliporphine, a phenolic aporphine alkaloid obtained from Chinese herbs and possessing antioxidant and alpha1 adrenoceptor antagonistic activity, has protective effects in endotoxemic rats. Rats injected with endotoxin (E. coli lipopolysaccharide, LPS) developed severe hypotension and tachycardia as well as vascular hyporeactivity to norepinephrine. Pretreatment of LPS-treated rats with thaliporphine attenuated the delayed hypotension significantly whilst only a higher dose (1 mg/kg) of thaliporphine decreased LPS-induced tachycardia. LPS significantly increased nitric oxide (NO•) and superoxide anion (O2•-) levels, a response that was reduced by pretreatment with 1 mg/kg thaliporphine. Endotoxemia for 240 min resulted in a bell-shaped time course for the change of serum tumor necrosis factor-alpha (TNF-alpha) level with a peak at 60 min. Pretreatment of LPS-treated rats with 1 mg/kg thaliporphine significantly reduced the serum TNF-alpha level at 60 min. In addition, LPS caused a biphasic change in blood glucose and thaliporphine attenuated the late-phase decrease in blood glucose. Endotoxemia induced multiple organ injury in the liver, kidney and heart, as indicated by increases of aspartate aminotransferase (GOT), alanine aminotransferase (GPT), creatinine (CRE), lactate dehydrogenase (LDH) and creatine phosphate kinase muscle-brain (CKMB) levels in serum. These increases of biochemical markers and inflammatory cell infiltration into injured tissues were reduced significantly by treatment with thaliporphine. In addition, thaliporphine increased the survival rate of LPS treated mice dose-dependently. In conclusion, our results suggest that thaliporphine could be a novel agent for attenuating endotoxin-induced circulatory failure and multiple organ injury and may increase the survival rate. These beneficial effects of thaliporphine may be attributed to the suppression of TNF-alpha, NO• and O2•- production. Part Two: N-Allylsecoboldine as a novel agent prevents acute renal failure resulted from endotoxemia. N-Allylsecoboldine is a secoaporphine derivative with antioxidant and alpha1-adrenoceptor blocking activities. Pretreatment of LPS-treated rats with N-allylsecoboldine significantly attenuated the late-phase hypotension, hypoglycemia and incremental plasma tumor necrosis factor (TNF)-alpha. Overproduction of plasma nitrate in endotoxemia was not changed but the continuous decrease of urinary nitrate appeared to be partially ameliorated by N-allylsecoboldine. However, N-allylsecoboldine inhibited the inducible nitric oxide synthase (iNOS) protein expression in the renal cortex of endotoxemic rats. N-Allylsecoboldine also improved the endotoxemia-induced organ injury as demonstrated from the conspicuous recovery of marker enzymes in the LPS-treated rats. Endotoxemia was associated with renal dysfunctions as indicated by decreases in renal blood flow, urinary potassium excretion, and renal nitrate clearance. However, pretreatment with N-allylsecoboldine showed significant alleviation of these renal dysfunctions. In addition, a lower dose of N-allylsecoboldine ameliorated the mortality of LPS-treated mice. This study demonstrates N-allylsecoboldine's ability to avail against acute renal failure and increase survival rate during endotoxemia. These beneficial effects may be attributed to the inhibition of iNOS expression, TNF-alpha production, and free radical scavenging activities. However, the role of alpha1-adrenoceptor antagonism for N-allylsecoboldine in sepsis remains unclear.