CAJ | 학술논문

目的探讨urotensinⅡ(UⅡ)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0.6 mg/kg urantide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-GalN)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)-γ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) pmol·min-1·mg-1比(3.76±0.42) pmol·min -·mg-1,均P＜0.01]；肝组织前炎细胞因子TNF-α、IL-1β和IFN-γ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) pg/ml比(364±20) pg/ml、(93.8±5.2) pg/ml比(180.3±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-GalN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.
목적 탐토urotensinⅡ(UⅡ)특이성수체(UT)특이성길항제urantide대급성간세포조망적영향급기궤제.방법 웅성BALB/c소서안수궤배렬표법수궤분위4조(매조6지):건강대조조、예처리대조조、모형조화예처리모형조.예처리대조조화예처리모형조급여미정맥주사0.6 mg/kg urantide예처리,건강대조조화모형조칙급여상동체적생리염수.30min후모형조급예처리모형조립즉이지다당연합D-반유당알( D-GalN)복강주사유도급성간세포조망소서모형,건강대조조화예처리대조조칙급여상응체적적생리염수.용원위말단전이매표기(TUNEL)기술분석화반광안산단백매(caspase)-3활성측정검측간세포조망정도；용반전록(RT)-PCR급매련면역흡부시험( ELISA)방법검측간조직급혈장전염세포인자종류배사인자(TNF)-α、간우소(IFN)-γ화백세포개소(IL)-1β적표체여분비수평.결과 모형조가견대량간세포조망,이예처리모형조간세포조망지수화caspase-3활성균명현저우모형조[(26±11)％비(77±20)％,(2.50±0.83) pmol·min-1·mg-1비(3.76±0.42) pmol·min -·mg-1,균P＜0.01]；간조직전염세포인자TNF-α、IL-1β화IFN-γ적mRNA상대표체수평급단백분비수평야균명현저우모형조[1.69±0.47비3.57±0.79、0.31±0.02비0.46±0.06、2.81±0.72비3.35±0.84,(233±36) pg/ml비(441±157) pg/ml、(228 ±21) pg/ml비(364±20) pg/ml、(93.8±5.2) pg/ml비(180.3±4.3) pg/ml,균P＜0.01].결론 urantide가통과억제전염세포인자적표체여분비억제지다당/D-GalN유도적급성간세포조망.저표명UⅡ/UT수체계통재급성간쇠갈면역염성손상중기관건성적작용,병유가능성위ALF약물치료적신파점.
Objective To explore the effects of urantide,a urotensin Ⅱ receptor (UT) inhibitor,on lipopolysaccharide (LPS)/D-gaiactosamine (D-GalN)-induced acute hepatocyte 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-t),interferon-γ ( 1FN-γ) and interleukin-1 beta (IL-1β),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 apoptotic index was clearly reduced in the pre-treatment model group [ ( 26 ± 11 ) ％ vs (77 ±20) ％,P ＜0.01 ].And the activity of caspase-3 was also lower in the pre-treatment model group than that in the model group [ ( 2.50 ± 0.83 ) pmol · min- 1 · mg- 1 vs ( 3.76 ± 0.42 ) pmol · min -1 · mg- 1,P ＜0.01 ].In addition,the serum and liver tissue levels of TNF-α,IL-1β and IFN-γin 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 ± 0.06,2.81 ± 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＜0.01].Conclusion LPS/D-GalN-induced acute hepatocyte apoptosis can be inhibited by a pretreatment of urantide through an inhibition of expression and secretion of proinflammatory cytokines.The U Ⅱ/UT receptor system plays a pivotal role in the liver immuno-inflammatory injury of acute liver failure (ALF).And it may become a new drug target of ALF therapy.