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。采用离体大鼠肝脏灌流系统和离体培养肝细胞系统，我们得到以下新的发现：1）在半乳糖胺处理的大鼠肝脏灌流系统中，电刺激肝交感神经可增加肝脏LDH和AST的漏出。不仅注入儿茶酚胺、ATP，而且肝窦细胞分泌的内皮素等因子也有类似的变化，提示肝交感神经的作用至少部分是通过肝窦细胞实现的。2)因为在半乳糖胺诱导的肝损伤中不仅观察到坏死而且还观察到凋亡，所以我们检测了诱导凋亡的细胞因子的参与。在灌流的大鼠肝脏后，半乳糖胺治疗，肝神经刺激增加分泌的TNF和IL-6从肝脏。肝部分切除后再生肝中TNF和IL-6的分泌增加，提示这些细胞因子可能促进肝再生。由于钆预处理可显著抑制大鼠肝窦细胞分泌TNF和IL-6，因此这些细胞因子由肝窦细胞产生。3)培养的肝细胞和从大鼠肝脏分离的非实质细胞显示出细胞死亡的半乳糖胺在培养基中以剂量依赖性的方式。半乳糖胺诱导的细胞死亡增加的培养基中加入去甲肾上腺素或ATP。肝实质细胞与非实质细胞共培养时也有类似的变化。提示肝神经刺激引起的肝损伤的加重是由包括肝窦细胞在内的复杂机制调节的。

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.