内源性胆红素对外周血白细胞寿命及功能的调节

Leukocyte，which can prevent the occurrence of inflammatory diseases, plays an important role of the human immune system. Although the life-span of peripheral blood leukocytes has been proved, how these cells maintain themselves remains unclear. Ubiquitin-proteasome system (UPS) and autophagy are two of the most important mechanisms for intracellular protein degradation, while UPS is responsible for more than 80% protein degradation. Inducing the aggregation of large amount of ubiquitinated protein, the ubiquitin-dependent protein degradation inhibition can regulate cell survival state by influencing pro-apoptotic related protein degradation. Bilirubin is a small molecule with human endogenous, which has certain toxicity and antioxidant effects. Our previous work found that bilirubin is associated with ubiquitin-dependent protein degradation inhibition and excluded the direct effect of bilirubin on autophagy. Furthermore, similar inhibition of ubiquitin-dependent protein degradation has also been found in the cells from patients with hyperbilirubinemia compared to the normal ones, which is an important new discovery of endogenous bilirubin. We speculate that the physiological and pathological concentration of bilirubin also inhibits the degradation of ubiquitinated proteins function in the peripheral blood leukocytes. In this project, we will prove the effect of endogenous bilirubin on the life-time and function of leukycotes, and further clarify its molecular mechanism, for instance, whether it relates to 19S proteasome-mediated de-ubiquitination enzymes (UCHL5 and USP14) function inhibition or not.

白细胞是机体防御系统的重要组成部分，可阻止炎症疾病发生。尽管外周血白细胞寿命已被学界明确，但其相关机制仍未见报道。泛素蛋白酶体系统（UPS）和自噬是细胞内两个最重要的蛋白降解途径，其中UPS负责细胞内80%以上蛋白的降解。抑制蛋白酶体降解功能会导致大量泛素化蛋白聚集，通过影响促凋亡相关蛋白降解进而调节细胞生存状态。胆红素是人体内源性小分子物质，具有一定的毒性和抗氧化作用。我们前期发现，胆红素能够抑制蛋白酶体相关去泛素化酶活性，并排除胆红素对细胞自噬的直接作用。更进一步发现，相比于正常人，高胆红素血症患者的细胞泛素化蛋白高度聚集，这是内源性胆红素作用的一个重要新发现。我们推测生理和病理浓度胆红素对外周血白细胞同样具有抑制泛素化蛋白降解功能。本项目全面论证内源性胆红素对白细胞寿命与功能的影响，并进一步明确其分子机制，是否与其介导的19S蛋白酶体去泛素化酶(UCHL5及USP14)功能抑制有关。

我们前期发现，胆红素能够抑制蛋白酶体相关去泛素化酶活性，并排除胆红素对细胞自噬的直接作用。白细胞是机体防御系统的重要组成部分，尽管外周血白细胞寿命已被学界明确，但其相关机制仍未见报道。本项目旨在探讨内源性胆红素对白细胞寿命与功能的影响，并进一步明确其分子机制，是否与其介导的19S蛋白酶体去泛素化酶(UCHL5及USP14)功能抑制有关。本项目在研究过程中，被获赠高胆红素血症小鼠模型（Ugt1敲除鼠）。研究结果发现：①高胆红素血症小鼠与野生型小鼠相比，白细胞总数无差异，但中性粒细胞比例下降，淋巴细胞比例上升；腹腔注射白蛋白2周后，白细胞总数无差异，中性粒细胞比例上升，淋巴细胞比例下降；并进一步统计临床黄疸新生儿数据，发现高浓度胆红素组的新生儿与正常组的新生儿相比，中性粒细胞比例下降，淋巴细胞比例升高。以上结果表明内源性高胆红素浓度确实影响了体内白细胞比例。②进一步探讨高胆红素浓度对小鼠白细胞功能的影响，发现高胆红素小鼠组其胸腺、脾脏CD4+T、CD8+T细胞上调，说明高胆红素浓度影响了小鼠的免疫功能；进一步发现其对小鼠体液免疫不影响。③为更进一步研究其相关机制，体外实验研究发现高浓度胆红素在1%血清条件下，能引起白细胞、Raw264.7、THP-1和Ana-1细胞发生凋亡，并引起Ub泛素化堆积；④ 另外，泛素化堆积，引起细胞内蛋白分子通路Mek-Erk磷酸化增加，蛋白不能被降解，进而促进细胞凋亡。