摘要
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.
出处
《中华医学杂志》
CAS
CSCD
北大核心
2011年第47期3358-3362,共5页
National Medical Journal of China
基金
国家自然科学基金(81070357、30660066)