四氯化碳影響兔肝傳入與傳出神經元之nNOS與NADPH-d的活性表現

Carbon tetrachloride influences expression of nNOS and NADPH-d in the hepatic afferent and efferent neurons of the rabbit

黃威靈(Wei-Ling Huang)

指導教授 ： 張宏名 博士 ； 藍琴臺 博士

中山醫學大學/醫學院/醫學研究所/碩士(2007年)

https://doi.org/10.6834/CSMU.2007.00109

摘要

肝硬化是國人十大死因的第六名，因此醫學界無不積極研發各種探討肝硬化的動物實驗模式。本研究採行沿用經年的餵食四氯化碳模式以引發動物肝硬化，進一步探討一氧化氮性神經元在肝臟病變過程中所扮演的角色。與日俱增的研究顯示，一氧化氮在肝臟除了具備擴張血管功能以及參與免疫和炎症反應外，也擔任著神經傳導或調節物質，甚至影響肝細胞的增生。因此，吾等使用菸鹼醯胺腺嘌呤雙核苷酸磷酸鹽-雙磷酶(NADPH-d)組織化學以及神經性一氧化氮合成酶(nNOS)免疫組織化學染色法，檢測兔肝在四氯化碳誘發肝硬化的狀態下，其傳入與傳出神經元的(NADPH-d)與nNOS之視密度質（Optical density）變化情形。結果顯示：在餵食四氯化碳四週起，其交感神經迴路上的T7-T10背根神經節、T7-T10脊髓側角中間外側細胞柱之交感節前神經元以及位於腹腔神經節的交感節後神經元所含之一氧化氮合成酶下降；而副交感神經迴路上的小結神經節、孤立徑核以及迷走神經背側運動核所含之一氧化氮合成酶亦下降。而直到長期（十七週）的投與四氯化碳後，交感神經的T7-T10背根神經元以及副交感神經的孤立徑核有含量微微回升的現象，其他的核區則一直保持一氧化氮合成酶含量下降的狀態。先前研究指出已經，自主（傳出）神經在調控肝內血液動態、膽汁分泌、碳水化合物及脂質的代謝，還有肝實質組織的再生上，扮演著重要角色。肝臟感覺（傳入）神經能偵測肝醣合成速率、脂肪酸與胺基酸的代謝率、血液中血糖濃度或是能量改變、肝門靜脈及肝內微循環的血壓、滲透壓、溫度，而回饋給自主神經系統，參與反射性活動，進而影響及調控肝臟功能，進行局部性或一般性的恆定控制。根據本研究結果推測，四氯化碳在短期內即可造成肝臟傷害，導致上述功能下降，並以一氧化氮合成酶製造量的減低反應出來，而此影響隨著毒物性肝纖維化至肝硬化的不可逆病理連續過程中，一直持續至長期（十七週）都沒有回復的現象。

關鍵字

四氯化碳 ； 肝臟 ； 一氧化氮 ； 自主神經 ； 自由基

並列摘要

Cirrhosis is the sixth in the top ten causes of death in Taiwan. Carbon tetrachloride (CCl4) animal model is proved as a good method to induce cirrhosis for studying the liver injury. Nitric oxide (NO) is an important regulator of some liver functions, such as enhancement of vasodilation, control of immune and inflammatory reactions, and affection of cell proliferation. It is also an important neurotransmitter or neuromodulator in the liver. To investigate whether CCl4–induced cirrhosis would have an influence on the nerve innervations in the rabbit liver, we studied the effect of CCl4 on the expression of nicotinamine adenine dinucleotide phosphate-diaphorase (NADPH-d) and neuronal NO-synthase (nNOS) in the hepatic afferent and efferent neurons. After treatment of CCl4 through oral route for four weeks, we have observed an interesting phenomenon in the animal model. That is the significantly decreased condition of nitric oxide synthase. The locations are sympathetic T7-T10 dorsal root ganglion, T7-T10 intermediolateral cell column, celiac ganglion, parasympathetic nodose ganglion, solitary tract nucleus, and dorsal motor vagal nucleus. However, after administration of CCl4 for seventeen weeks, the amount of nitric oxide synthase in the T7-T10 dorsal root ganglion and solitary tract nucleus is mild upregulated simultaneously but the others we previously described are still in the downregulated situation. Earlier studies have indicated that the autonomic efferents play important roles in intrahepatic haemodynamic and bile flow regulation, control of carbohydrate and lipid metabolism, and parenchymal cell regeneration. The hepatic sensory innervations (afferents) can detect the rate of glycogen synthesis, metabolism of lipid acid and amino acid, changes of blood glucose concentration and energy, blood pressure and temperature of portal vein, and intrahepatic microcirculation that may be able to elicit reflex activities and contribute to liver functional regulation and local or general homeostatic control. Our data showed that exposure to CCl4 for a short period could damage the liver function badly as indicated by the down-regulation of NO production in the hepatic afferent and efferent neurons. The result we observed is not a recovery process. The process is a gradually damaged event for liver injury, causing liver fibrosis even to cirrhosis. The duration we proceeded the experiment is at least seventeen weeks.

並列關鍵字

carbon tetrachloride ； liver ； nitric oxide ； autonomic nerve system ； free radical

參考文獻

immunohistochemical study of aminergic, cholinergic and peptidergic innervation in rat, hamster, guinea pig, dog and human livers. Liver 18.5: 352-59, 1998.

Alm P, Uvelius B, Ekstrom J, Holmqvist B, Larsson B, Andersson KE. Nitric oxide synthase-containing neurons in rat parasympathetic, sympathetic and sensory ganglia: a comparative study. Histochem.J. 27.10: 819-31, 1995.

Anderson CR. The distribution of nitric oxide synthase-containing autonomic preganglionic terminals in the rat. Brain Res. 614.1-2: 78-85, 1993.

Aqrawal HC, Aqrawal D. Tumor promoters accentuate phosphorylation of PO: evidence for the presence of protein kinase C in purified PNS myelin. Neurochem Res 14: 409-13, 1989.

Atkinson L, Batten TFC, Corbett EKA, Sinfield JK, Deuchars J. Subcellular localization of neuronal nitric oxide synthase in the rat nucleus of the solitary tract in relation to vagal afferent inputs. Neuroscience 118: 115-122, 2003.