Effects of neuroendocrine factors on the exaggeration of liver injury: Study using ex vivo and in vitro system

本研究旨在探讨肝交感神经在肝损伤加重中的作用机制。采用大鼠肝脏体外灌流系统和体外肝细胞培养系统，我们得到了以下新的发现：1)在氨基半乳糖处理后的大鼠肝脏中，电刺激肝交感神经增加了LDH和AST的漏出。不仅灌注儿茶酚胺、三磷酸腺苷，而且内皮素等肝窦细胞分泌的因子也观察到类似的变化，提示肝交感神经的作用至少部分是通过肝窦细胞起作用的。2)在半乳糖胺诱导的肝损伤中，我们不仅观察到肝细胞的坏死，而且还观察到肝细胞的凋亡，因此我们研究了诱导细胞凋亡的细胞因子的参与。在氨基半乳糖处理后的灌流大鼠肝脏中，肝神经刺激使肝脏分泌的肿瘤坏死因子和白介素6增加。肝大部切除后再生肝组织中肿瘤坏死因子和白介素6的分泌增加，提示细胞因子可刺激肝再生。由于肿瘤坏死因子和白介素6的释放被格拉预处理的大鼠显著抑制，这些细胞因子是由正弦细胞产生的。3)培养的肝细胞和分离的大鼠肝非实质细胞在培养上清液中可见半乳糖胺诱导的细胞死亡，且呈剂量依赖性。加入去甲肾上腺素或三磷酸腺苷可增加氨基半乳糖诱导的细胞死亡。肝实质细胞与非实质细胞共培养也有类似的变化。这些结果提示，肝神经刺激所致肝损伤的加重受包括肝窦细胞在内的复杂机制的调节。

The present study was designed to explore the mechanism of the action of hepatic sympathetic nerves on aggravation of liver injury. Using ex vivo system with perfused rat liver, and in vitro system with cultured hepatocytes, we have had the new findings as follows:1) In perfused rat liver after treatment with galactosamine, the electrical stimulation of hepatic sympathetic nerve increased the leakage of LDH and AST from the liver. Similar changes were observed not only by the infusion of catecholamine, ATP but also by the factors secreted from sinusoidal cells like endothelin, suggesting that the effects of hepatic sympathetic nerves were at least partly by hepatic sinusoidal cells. 2) Because not only necrosis but also apoptosis was observed in galacttfsamine-induced liver injury, we examined the involvement of cytokines which induce apoptosis. In perfused rat liver after treatment with galactosamine, hepatic nerve stimulation increased the secretion of TNF and IL-6 from the liver. The increased secretion of TNF and IL-6 was observed in regenerating liver after partial hepatectomy, indicating that the cytokines may stimulate liver regeneration. Since the release of TNF and IL-6 was significantly inhibited by pretreatment of rat with gadolinium, these cytokines were produced by sinusoidal cells. 3) Cultured hepatocytes and non-parenchymal cells isolated from rat liver showed cell death by galactosamine in the cultured medium in a dose-dependent manner. The cell death induced by galactosamine increased by the addition of noradrenaline or ATP in the medium. The co-culture of hepatic parenchymal cells and non-parenchymal cells showed similar changes. These results suggested the aggravation of liver injury induced by hepatic nerve stimulation was regulated by the complicated mechanism including hepatic sinusoidal cells.