臺灣博碩士論文加值系統

發炎時巨噬細胞及肝細胞均會產生一氧化氮 NO，NO 為自由基，具有
毒殺細菌之作用。體外實驗中巨噬細胞 J774A.1 接受 interferon-r 與
lipopolysaccharide 刺激時，細胞內的鐵濃度調節 NO 的生合成；鐵濃
度低時，增加誘發型 nitric oxide synthase（iNOS）活性；鐵的濃度高
時，減少該酵素活性。肝臟是鐵的主要儲存器官，當體內鐵堆積且感染發
炎時，體內的 NO 生成會改變嗎？會使肝臟受傷程度加劇嗎？為探究上述
問題，本研究採用 Propionbacterium acnes 誘發大鼠發炎之動物模式，
共進行兩個實驗。結果發現高鐵會使發炎時之 NO 量減少。
以離乳之 Sprague-Dawley 大鼠為對象，先分兩組餵以兩種飼料：正常組
是 AIN-76 飼料含鐵 35 (g/g，高鐵組是 AIN-76 飼料加 2.5% carbonyl
iron，餵養 8 至 10 週後，高鐵組的肝鐵濃度可達正常組的 10 倍以上
；此時以尾巴靜脈注射熱處理過之細菌 Propionbacterium acnes (28
mg/kg) 以誘發大鼠肝臟慢性發炎。實驗中共包含六組動物：正常及高鐵
發炎組（CP 及 IOP），正常及高鐵發炎抑制組（CPN 及 IOPN）採腹腔注
射 NOS 抑制劑 L-NAME（10 mg/kg），正常及高鐵對照組（CC 及 IOC）
以 PBS 或生理食鹽水代替菌液。在菌液注射後一週內追蹤大鼠尿中 NO
代謝爭排泄量，並定期犧牲大鼠，收取組織以供生化分析。
體內 NO 生成量之評量方法包括：用 electron paramagnetic resonance
直接分析紅血球 Hb-NO complex 生成量，以酵素法間接偵測血漿硝酸根
和亞硝酸根總濃度及尿液硝酸根和亞硝酸根總排泄量。另外依實驗需要而
分析血漿 AST 及 ALT 做為肝臟損傷指標，血紅素、血球比容、血清鐵、
攜鐵容量、攜鐵蛋白飽和度、肝臟與脾臟的鐵濃度、血漿鐵蛋白、肝臟與
脾臟鐵蛋白含量等為鐵營養代謝指標，TBARS 值為氧化傷害指標。
實驗一探討發炎過程高鐵對 NO 產量之影響。大鼠於菌液注射後第二、三
、四及七天犧牲。尿液方面，正常發炎組六天硝酸根和亞硝酸根排泄總量
為 218+/-38 mmole/mg creatinine，高鐵發炎組111+/-16 mmole/mg
creatinine，高鐵組顯著較低。每日尿液硝酸根和亞硝酸根總排泄量，正
常發炎組以發炎第四天最高，高鐵發炎組則無一致表現，可見鐵堆積之動
物發炎時 NO 之生成減少，並且其排泄模式改變。血液方面，發炎第七天
正常發炎組血漿濃度達 1137+/-483 mM，高鐵發炎組僅 181+/-48 mM，紅
血球 Hb-NO complex 生成具相同現象，血液的表現也反應高鐵組的 NO
量較少。發炎第二和三天每日腹腔注射 10 mg/kg L-NAME，對兩組血漿及
紅血球 NO 量無抑制作用。
肝臟損傷方面，沒有發炎時的正常與高鐵兩組之間的 AST 及 ALT 沒有差
異，高鐵發炎組與正常發炎組也無顯著差異，在發炎第二和三天接受抑制
劑時，高鐵發炎抑制組的 AST 及 ALT 比發炎組高，反映肝臟損傷更嚴重
，但正常組則不因抑制劑而有差別。
實驗二的設計大致與實驗一相似，不過抑制劑於發炎第二天至第六天給予
。大鼠於注射菌液後第三及七天犧牲。尿液六天硝酸根和亞硝酸根的排泄
總量在正常發炎組為 183+/- 26 mmole/mg creatinine，在高鐵發炎組
為 104+/-11 mmole/mg creatinine，高鐵發炎組顯著較低；正常發炎抑
制組為 101+/-13 mmole/mg creatinine，高鐵發炎抑制組為 81+/-14
mmole/mg creatinine，抑制劑使 NO 代謝物排泄總量在正常組減少為
55%，高鐵組只減少為 78%，高鐵時抑制劑的作用較不明顯。血液方面，
發炎第七天正常組血漿硝酸根和亞硝酸根總濃度為 1867+/-950 mM，高鐵
發炎組為 321+/-6 mM，正常發炎抑制組為 1101+/- 598 mM，高鐵發炎
抑制組為 268+/-95 mM，紅血球 Hb-NO complex 生成具相同現象，血液
的表現也反映高鐵組 NO 量較少，受抑制劑的作用較弱。肝臟組織病理學
觀察到高鐵組有鐵沈積現象，及發炎時正常組及高鐵組之肝臟受損。
本研究確定體內鐵高量時，減少發炎時NO之生成，給予NOS抑制劑 L-NAME
使一氧化氮產生較少時，肝臟受損程度較嚴重。

Macrophage and hepatic cells are the principle sources of nitric
oxide in inflammation. NO is a free radical, which is directly
toxic to the bacterium. NO synthesis has been shown to be
regulated by intracellular iron levels in a macrophage cell line
J774A.1 stimulated by interferon-r and lipopolysaccharide. The
activity of inducible nitric oxide synthase was increased at low
iron level and decreased at high iron level. Liver is the major
iron storage organ, will high iron status affect in vivo
production onitric oxide and enhance liver damage during
inflammation?In order to answer this question, a murine model of
chronic hepatic inflammation induced by Propionbacterium acnes
was used. Two experiments were included in this study and the
results indicate that nitric oxide production in iron-loaded
rats was decreased. Wealing Spague-
Dawley rats were rendered iron-loaded by feeding an AIN76 diet
supplemented with 2.5% carbonyl iron, while control rats were
fed a AIN76 diet. After hepatic iron content was increased to
more than ten times of the control, inflammation was induced by
injection of heated inactivated Propionbacterium acnes (28 mg/kg
body weight) via the tail vein. Rats received PBS or saline
served as non-inflammatory controls. Rats of inhibition groups
received i. p. injection of L-NAME (10 mg/kg body weight),
competitive inhibitor for nitric oxide synthase, in addition to
i.v. injection. Six groups of rats were included: non-
inflammatory control (CC) and iron-loaded (IOC), inflammatory
control (CP) and iron-loaded (IOP), and inhibition control (CPN)
and iron -loaded (IOPN) groups. After injection, urinary
excretion of NO metabolites was monitored for a week. During the
week, rats were also killed at specified days and tissues were
collected for biochemical analysis.
NO production was monitored by measuring urinary and plasma
contents of nitrite and nitrate with a colorimetric method using
Griess reagent and nitrate reductase and by measuring complex of
hemoglobin and NO (Hb-NO) using electron paramagnetic resonance
method. Biochemical analysis included indicators of liver
damage, such as plasma ASL and ALT, indicators of iron status,
such as hemoglobin, hematocrit, serum iron, total iron binding
capacity, transferrin saturation, liver and spleen iron contents
and tissuferritin contents, and TBARS served as a indicator of
lipid peroxidation. Experiment one was a time-
course study and rats were killed at the 2nd, 3rd, 4th and 7th
day after injection. Six-day excretion of urinary nitrite and
nitrate was significantly less in IOP (111+/-16 umole/mg
creatinine) than in CP (218+/-38 umole/mg creatinine). Urinary
nitrate/nitrite excretion peaked at the 4th day in CP, while
there was no consistent trend in IOP, implying that pattern of
nitrate/nitrite excretion was altered in iron-loaded rats.
Plasma concentration of nitrate/nitrite was significant lor in
IOP (181+/-48 uM) than in CP.(1137+/-483 uM) at the 7th day, and
similar results were observed for Hb-NO. There were no
significant reduction in plasma concentration and Hb-NO in both
CPN and IOPN, in which inhibitor was dosed for only two days
beginning at the 2nd day.
Plasma AST and ALT between CC and IOC, CP and IOP, and between
CPN and CP were not significantly different, but were
significantly higher in IOPN then in IOP, implying that liver
damage was more severe in iron-loaded rats when NO production
was reduced during inflammation.
Experiment two had similar designs as experiment one with slight
modifications that the inhibitor was dosed for five days
beginning at the 2nd day. Rats were killed at the 3rd and the
7th day after injection. Six-day excretion of urinary nitrite
and nitrate was significantly less in IOP (104+/-11 umole/mg
creatinine) than in CP (183+/-26 umole/mg creatinine), and this
excretion was reduced by the inhibitor to 55% in CPN (101+/-13
umole/mg creatinine) and to 78% in IOPN (81+/-14 umole/mg
creatinine). Plasma ncentration of nitrate/nitrite was
significant lower in IOP (321+/-336 uM) than in CP (1867+/-950
uM) at the 7th day, and was reduced by the inhibitor in both CPN
(1101+/-598 uM) and IOPN (268+/-95 uM). Similar results were
observed for Hb-NO complex. The effect of iron loading on NO was
reconfirmed in this experiment. Histological examination
observed high hepatic iron content in iron-loaded rats and liver
damage in both inflammatory control and iron-loaded rats.
In conclusion, NO production was reduced in iron-loading rats,
and liver damage was enhanced when NO production was reduced.