Zhong Jian, Deaciuc Ion V, Burikhanov Ravshan, de Villiers Willem J S
Division of Digestive Diseases and Nutrition, Department of Internal Medicine, University of Kentucky Medical Center, MN649-0298, Lexington, KY 40536, USA.
Biochim Biophys Acta. 2006 Apr;1762(4):468-77. doi: 10.1016/j.bbadis.2005.12.012. Epub 2006 Feb 13.
Although IL-10 down-regulates pro-inflammatory cytokine secretion by hepatic Kupffer cells, the mechanisms underlying its hepatoprotective effects are not fully clear. This study tested the hypothesis that IL-10 protects the liver against pro-inflammatory cytokines by counteracting their pro-apoptotic effects. Wild type and IL-10 knockout mice were treated with bacterial lipopolysaccharide and sacrificed 1, 4, 8, and 12 h later. Plasma ALT activity was measured as a marker of liver injury. Liver pathology and TUNEL response were assessed by histology. Plasma levels and whole liver mRNA levels were measured for TNF-alpha, IL-1 beta, TGF-beta1, IL-10, and their respective receptors. Hepatic mRNA levels were measured for several pro-apoptotic adaptors/regulators, including FasL, Fas receptor, FADD, TRADD, Bad, Bak, Bax, and Bcl-X(S), and anti-apoptotic regulators, including Bcl-w, Bcl-X(L), Bcl-2, and Bfl-1. Caspase-3 activity in the liver was determined as well as immunohistochemistry for IL-1RII, TGF-betaRII and Fas receptor. At all time points the livers from IL-10 knockout mice displayed a significantly increased number of apoptotic nuclei compared to wild type mice. Changes in plasma cytokine levels and their liver mRNA levels were consistent with suppression by IL-10 of pro-inflammatory cytokine secretion. In addition, pro-inflammatory cytokine receptor mRNA levels (TNF-alpha, TGF-beta, and IL-1 beta) were markedly up-regulated by LPS at all time points in IL-10 knockout mice as compared to wild type mice. Expression of the pro-inflammatory cytokine receptor IL-1RII was similarly increased as shown by immunostaining. The mRNA levels of a typical pro-apoptotic cytokine, TRAIL, were increased and LPS also up-regulated the mRNA expression of other apoptotic factors to a larger extent in IL-10 knockout mice than in their wild type counterparts, suggestive of an IL-10 anti-apoptotic effect. In the livers of knockout mice, markedly increased caspase-3 activity was already evident at the 1-h time point following LPS administration, while in the wild type animals this increase was delayed. Immunostaining also indicated that LPS increased hepatic expression of the pro-apoptotic receptors Fas and TGF-betaRII in IL-10 knockout mice. The data presented in this study show that: (i) IL-10 modulates not only the secretion of pro-inflammatory cytokines, but also the receptors of these cytokines, and ii) IL-10 protects the liver against LPS-induced injury at least in part by counteracting pro-inflammatory cytokine-induced liver apoptosis.
尽管白细胞介素-10（IL-10）可下调肝库普弗细胞促炎细胞因子的分泌，但其肝保护作用的潜在机制尚不完全清楚。本研究验证了以下假说：IL-10通过抵消促炎细胞因子的促凋亡作用来保护肝脏免受其侵害。野生型和IL-10基因敲除小鼠用细菌脂多糖处理，1、4、8和12小时后处死。测定血浆谷丙转氨酶（ALT）活性作为肝损伤的标志物。通过组织学评估肝脏病理和TUNEL反应。测定血浆中肿瘤坏死因子-α（TNF-α）、白细胞介素-1β（IL-1β）、转化生长因子-β1（TGF-β1）、IL-10及其各自受体的水平以及全肝mRNA水平。测定几种促凋亡衔接蛋白/调节因子的肝脏mRNA水平，包括Fas配体（FasL）、Fas受体、Fas相关死亡结构域蛋白（FADD）、肿瘤坏死因子受体相关死亡结构域蛋白（TRADD）、促凋亡蛋白Bad、Bak、Bax和Bcl-XS，以及抗凋亡调节因子，包括Bcl-w、Bcl-XL、Bcl-2和Bfl-1。测定肝脏中半胱天冬酶-3的活性以及IL-1受体II型（IL-1RII）、TGF-β受体II型（TGF-βRII）和Fas受体的免疫组化情况。在所有时间点，与野生型小鼠相比，IL-10基因敲除小鼠肝脏中的凋亡细胞核数量显著增加。血浆细胞因子水平及其肝脏mRNA水平的变化与IL-10对促炎细胞因子分泌的抑制作用一致。此外，与野生型小鼠相比，在所有时间点，脂多糖均使IL-10基因敲除小鼠促炎细胞因子受体mRNA水平（TNF-α、TGF-β和IL-1β）显著上调。免疫染色显示促炎细胞因子受体IL-1RII的表达同样增加。典型促凋亡细胞因子肿瘤坏死因子相关凋亡诱导配体（TRAIL）的mRNA水平升高，并且与野生型小鼠相比，脂多糖在IL-10基因敲除小鼠中还更大程度地上调了其他凋亡因子的mRNA表达，提示IL-10具有抗凋亡作用。在基因敲除小鼠肝脏中，脂多糖给药后1小时半胱天冬酶-3活性就已显著增加，而野生型动物中这种增加则延迟出现。免疫染色还表明，脂多糖增加了IL-10基因敲除小鼠肝脏中促凋亡受体Fas和TGF-βRII的表达。本研究呈现的数据表明：（i）IL-10不仅调节促炎细胞因子的分泌，还调节这些细胞因子的受体；（ii）IL-10至少部分地通过抵消促炎细胞因子诱导的肝脏凋亡来保护肝脏免受脂多糖诱导的损伤。
