内皮素对肝硬化大鼠Kupffer细胞血小板活化因子生成水平的影响

Effects of endothelin on platelet activating factor (PAF) synthesis and release by cultured Kupffer cells from CCl_4-induced cirrhotic rats

目的探讨肝硬化时Kupffer细胞与血小板活化因子(PAF)生成的关系及内皮素-1(ET-1)在其中的作用。方法30只SD大鼠随机分为正常对照组与CCl4诱导的肝硬化模型组,每组15只。分离、培养Kupffer细胞,培养24h后,分别用0、1、10、100和1000nmol/L ET-1刺激,测定Kupffer细胞产生和释放的PAF水平。通过RT-PCR、受体饱和结合试验分析Kupffer细胞PAF、ET-1受体(ETA、ETB)和内皮素前体原(ppET-1)mRNA表达情况。结果肝硬化大鼠Kupffer细胞合成与释放PAF明显增加,分别为对照组的1.48倍(1.02±0.06pg/g DNAvs0.69±0.07pg/g DNA,P<0.01)和2.15倍(1.42±0.14pg/g DNAvs0.66±0.04pg/g DNA,P<0.01)。ET-1刺激增强了Kupffer细胞生成与释放PAF的效应,并呈剂量依赖性,在肝硬化组尤为明显。肝硬化组Kupffer细胞PAF及ETB受体mRNA水平及受体密度明显增加,而受体亲和力无变化,无ETA受体和ppET-1 mRNA表达。结论Kupffer细胞是肝硬化时PAF生成的一个重要来源,ET-1通过表达增加的ETB受体进一步促进Kupffer细胞生成并释放PAF,提示Kupffer细胞参与了肝硬化门脉高压的形成。

Objective To investigate the correlation between Kupffer cells and the synthesis of platelet activating factor （PAF） in experimental hepatic cirrhosis, and to elucidate the effects of endothelin （ET） on portal hypertension. Methods Thirty SD rats were randomly assigned to two groups： control group and CCl4-induced hepatic cirrhotic group. Kupffer cells, isolated from the livers of animals in both groups, were cultured for 24h. ET 1-induced PAF synthesis, and mRNA expression of PAF, ET-1 receptor and preproendothelin-1 in Kupffer cells were determined by rapid 3H-PAF scintillation proximity assay, saturation binding technique and semi-quantitative reverse transcriptase polymerase chain reaction （RT-PCR）, respectively. Results Cell associated PAF synthesis and release increased about 1.48 folds and two-folds, respectively, by cirrhotic Kupffer cells as compared to the control （1.02 ± 0. 06 vs 0. 69 ± 0. 07 pg / mg DNA in Kupffer cells and 1.42± 0. 14 vs 0. 66 ± 0. 04 pg/mg DNA in medium）. Endotbelin-1 enhanced Kupffer cells to stimulate PAF synthesis in a concentration-dependent manner, and for cirrhotic Kupffer cells, the effect was more significant than control. Cirrhotic Kupffer cells also had increased densities of functional receptors for both PAF and ET-1 （exclusively ETB）, but did not chan_ge the affinity of these receptors. No mRNA transcripts for the ETA receptor or preproET-1 were detected. Conclusion Kupffer cell is the main source of PAF in the cirrhotic rats. ET-1 stimulates PAF synthesis in activated Kupffer cells via ETB receptor. Since both ET-1 and PAF individually cause portal hypertension, Kupffer cells may play a role in portal hypertension associated with liver cirrhosis.