urantide对小鼠急性肝细胞凋亡的影响及其机制

Effects of urantide, a urotensin receptor inhibitor, on acute hepatocyte apoptosis in mice

目的探讨urotensinⅡ（UⅢ）特异性受体（UT）特异性拮抗剂urantide对急性肝细胞凋亡的影响及其机制。方法雄性BALB／c小鼠按随机排列表法随机分为4组（每组6只）：健康对照组、预处理对照组、模型组和预处理模型组。预处理对照组和预处理模型组给予尾静脉注射0．6mg／kgurantide预处理，健康对照组和模型组则给予相同体积生理盐水。30min后模型组及预处理模型组立即以脂多糖联合D-半乳糖胺（D-GaIN）腹腔注射诱导急性肝细胞凋亡小鼠模型，健康对照组和预处理对照组则给予相应体积的生理盐水。用原位末端转移酶标记（TUNEL）技术分析和半胱氨酸蛋白酶（caspase）-3活性测定检测肝细胞凋亡程度；用反转录（RT）-PCR及酶联免疫吸附试验（ELISA）方法检测肝组织及血浆前炎细胞因子肿瘤坏死因子（TNF）-α、干扰素（IFN）．吖和白细胞介素（IL）-1β的表达与分泌水平。结果模型组可见大量肝细胞凋亡，而预处理模型组肝细胞凋亡指数和caspase-3活性均明显低于模型组[（26±11）％比（77±20）％，（2．50±0．83）pm01．min-1．mg。比（3．764-0．42）pmol·min-1·mg-1，均P〈0．01]；肝组织前炎细胞因子TNF-α、IL-1β和IFN．1的mRNA相对表达水平及蛋白分泌水平也均明显低于模型组[1．69±0．47比3．57±0．79、0．31±-0．02比0．46±0．06、2．81±0．72比3．35±0．84，（233±36）pg／ml比（441±157）pg／ml、（228±21）p∥ml比（364±20）pg／ml、（93．8±5．2）pg／ml比（180．34-4．3）pg／ml，均P〈0．01]。结论urantide可通过抑制前炎细胞因子的表达与分泌抑制脂多糖／D．GaIN诱导的急性肝细胞凋亡。这表明uII／UT受体系统在急性肝衰竭免疫炎性损伤中起关键性的作用，并有可能成为ALF药物治疗的新靶点。

Objective To explore the effects of urantide, a urotensinⅡ receptor （UⅡ） inhibitor, on lipopolysaccharide （LPS）/D-galactosamine （D-GalN）-induced acute hepatoeyte apoptosis in mice. Methods Male BALB/c mice were randomly divided into 4 groups （ n = 6 each） ： normal control, pre-treatment control, model and pre-treatment model. The pre-treatment control and pre-treatment model groups received urantide （0. 6 mg/kg body weight） by a caudal vein injection. At 30 minutes post-injection, the model and pre-treatment model groups were treated with LPS/D-GalN to induce acute hepatocyte apoptosis via an intraperitoneal injection. Hepatocyte apoptosis was examined by terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling （TUNEL） and caspase-3 colorimetric assay. The expressions of proinflammatory cytokines, such as tumor necrosis factor alpha （TNF-α）, interferon-α （IFN- y） and interleukin-1 beta （IL-1 β）, were detected by reverse transcription-polymerase chain reaction （RT-PCR） and enzyme-linked immunosorbent assay. Results Massive hepatocyte apoptosis were detected in the model group. The apoptotie index was clearly reduced in the pre-treatment model group [ （ 26 ± 11 ） % vs （ 77 ± 20 ） %, P 〈 0. 01 ]. And the aetivity of caspase-3 was also lower in the pre-treatment model group than that in the model group [ （2. 50 ±0. 83 ） pmol ~ rain - 1 . mg± vs （3.76 ±0. 42） pmol ~ rain -1· mg- 1, P 〈0. 01 ]. In addition, the serum and liver tissue levels of TNF-α, IL-I[3 and IFN--,/ in the pre-treatment model group were significantly lower than those in the model group [ 1.69 ± 0. 47 vs 3.57 ±0. 79, 0. 31 ± 0.02 vs 0.46 ±0.06, 2.8l ±0.72 vs 3.35 ±0.84, （233 ±36） pg/ml vs （441 ±157） pg/ml, （228 ＋ 21） pg/ml vs （364 ±20） pg/ml, （93.8 ±5.2） pg/ml vs （180.3±4.3） pg/ml, allP〈O. 01]. Conclusion LPS/D-GalN-induced acute hepatoeyte apoptosis can be inhibited by a pretreatment of urantide through an inhibition of expression and secretion of proinflammatory eytokines. The U I[/UT receptor system plays a pivotal role in the liver immuno-inflammatory injury of acute liver failure （ALF）. And it may become a new dru target of ALE therapy.