褪黑素对卡介苗加脂多糖诱导的小鼠肝损伤的保护作用。
Protective effect of melatonin against liver injury in mice induced by Bacillus Calmette-Guerin plus lipopolysaccharide.
作者信息
Wang Hua, Wei Wei, Shen Yu-Xian, Dong Chen, Zhang Ling-Ling, Wang Ni-Ping, Yue Li, Xu Shu-Yun
机构信息
Institute of Clinical Pharmacology, Anhui Medical University, Hefei 230032, Anhui Province, China.
出版信息
World J Gastroenterol. 2004 Sep 15;10(18):2690-6. doi: 10.3748/wjg.v10.i18.2690.
AIM
To investigate the effects and mechanisms of melatonin on immunological liver injury in mice.
METHODS
A model of liver injury was induced by tail vein injection of Bacillus Calmette Guerin (BCG) and lipopolysaccharide (LPS) in mice. Kupffer cells and hepatocytes were isolated and cultured according to a modified two-step collagenase perfusion technique. Levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) and nitric oxide (NO), content of malondiadehyde (MDA), activity of superoxide dismutase (SOD), were measured by biochemical methods. Tumor necrosis factor-alpha (TNF-alpha) activity was determined by RIA. Interleukin (IL)-1 activity was measured by thymocyte proliferation bioassay. Hepatic tissue sections were stained with hematoxylin and eosin and examined under a light microscope.
RESULTS
Immunological liver injury induced by BCG+LPS was successfully duplicated. Serum transaminase (ALT, AST) activities were significantly decreased by melatonin (0.25, 1.0, 4.0 mg/kg bm). Meanwhile, MDA content was decreased and SOD in liver homogenates was upregulated. Furthermore, pro-inflammatory mediators (TNF-alpha, IL-1, NO) in serum and liver homogenates were significantly reduced by melatonin. Histological examination demonstrated that melatonin could attenuate the area and extent of necrosis, reduce the immigration of inflammatory cells. In in vitro experiment, TNF-alpha was inhibited at the concentrations of 10(-8)-10(-6) mol/L of melatonin, while IL-1 production of Kupffer cells induced by LPS (5 microg/mL) was decreased only at the concentration of 10(-6) mol/L of melatonin, but no effect on NO production was observed. Immunological liver injury model in vitro was established by incubating hepatocytes with BCG- and LPS-induced Kupffer cells. Activities of ALT, TNF-alpha, IL-1, and MDA in supernatant were significantly increased. Melatonin had little effect on the level of ALT, but reduced the content of TNF-alpha and MDA at concentrations of 10(-7)-10(-5) mol/L and decreased the content of IL-1 at concentrations of 10(-6)-10(-5) mol/L.
CONCLUSION
Melatonin could significantly protect liver injury in mice, which was related to free radical scavenging, increased SOD activity and pro-inflammatory mediators.
目的
探讨褪黑素对小鼠免疫性肝损伤的影响及其机制。
方法
通过尾静脉注射卡介苗（BCG）和脂多糖（LPS）诱导小鼠肝损伤模型。采用改良的两步胶原酶灌注技术分离并培养库普弗细胞和肝细胞。采用生化方法检测丙氨酸转氨酶（ALT）、天冬氨酸转氨酶（AST）和一氧化氮（NO）水平、丙二醛（MDA）含量、超氧化物歧化酶（SOD）活性。采用放射免疫分析法测定肿瘤坏死因子-α（TNF-α）活性。采用胸腺细胞增殖生物测定法检测白细胞介素（IL）-1活性。肝组织切片进行苏木精-伊红染色，在光学显微镜下观察。
结果
成功复制了BCG+LPS诱导的免疫性肝损伤。褪黑素（0.25、1.0、4.0mg/kg体重）可显著降低血清转氨酶（ALT、AST）活性。同时，肝匀浆中MDA含量降低，SOD上调。此外，褪黑素可显著降低血清和肝匀浆中促炎介质（TNF-α、IL-1、NO）水平。组织学检查表明，褪黑素可减轻坏死面积和程度，减少炎症细胞浸润。在体外实验中，褪黑素浓度为10^(-8)-10^(-6)mol/L时可抑制TNF-α，而LPS（5μg/mL）诱导的库普弗细胞IL-1产生仅在褪黑素浓度为10^(-6)mol/L时降低，对NO产生无影响。通过将肝细胞与BCG和LPS诱导的库普弗细胞共同孵育建立体外免疫性肝损伤模型。上清液中ALT、TNF-α、IL-1和MDA活性显著升高。褪黑素对ALT水平影响较小，但在浓度为10^(-7)-10^(-5)mol/L时可降低TNF-α和MDA含量，在浓度为10^(-6)-10^(-5)mol/L时可降低IL-1含量。
结论
褪黑素可显著保护小鼠肝损伤，这与清除自由基、提高SOD活性及降低促炎介质有关。
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