CAJ | 학술논문

目的探讨脂多糖诱导D-氨基半乳糖胺致敏大鼠急性肝损伤肝细胞凋亡、炎性因子表达情况及其发生机制.方法 56只大鼠分为0 h对照组与1、2、4、6、24和48 h脂多糖+D-氨基半乳糖胺处理组.在相应时间点处死大鼠后收集肝组织及血清,肝组织苏木精-伊红染色后光学显微镜下观察ELISA法检测血清细胞因子表达;反转录(RT)-PCR法检测TNF-α、IL-β、诱导型一氧化氮合酶(iNOS)和p53基因表达;收集24 h肝组织用底物显色法检测Caspase-3、8、9,12活性.组间比较用方差分析.结果经药物处理后,肝组织出现碎片状坏死、大量炎性细胞浸润等表现,从6 h开始,24 h和48 h显著加重.血清TNF-α浓度在1 h处理组为(727.8±261.3)ng/L,显著高于对照组及其他处理组(F=49.82,P<0.01),2 h处理组为(156.4±52.2)ng/L,显著低于1 h组,但高于对照组(F:30.23,P<0.01);血清IL-β浓度逐渐上升,24 h处理组最高,为(360.5±121.6)ng/L(F=18.61,P<0.01).24 h处理组肝组织Caspase-3、8、9、12活性明显高于对照组(F=84.96,P<0.01).iNOS基因在对照组无表达,药物作用后6 h达最高,24 h和48 h则显著下降(F=34.07,P<0.01);p53基因在24 h和48 h处理组表达明显增高(F=37.43,P<0.01);TNF-α和IL-1β基因表达均较对照组升高(F=2.94,P<0.05),其峰值均出现在1 h处理组.结论小剂量脂多糖可诱导D-氨基半乳糖胺致敏大鼠发生急性肝损伤;Caspase-3、8、9、12活性明显增强是其特征性改变之一;肝损伤的发生与TNF-α、iNOS和p53基因早期高水平表达有密切关系.
목적 탐토지다당유도D-안기반유당알치민대서급성간손상간세포조망、염성인자표체정황급기발생궤제.방법 56지대서분위0 h대조조여1、2、4、6、24화48 h지다당+D-안기반유당알처리조.재상응시간점처사대서후수집간조직급혈청,간조직소목정-이홍염색후광학현미경하관찰ELISA법검측혈청세포인자표체;반전록(RT)-PCR법검측TNF-α、IL-β、유도형일양화담합매(iNOS)화p53기인표체;수집24 h간조직용저물현색법검측Caspase-3、8、9,12활성.조간비교용방차분석.결과 경약물처리후,간조직출현쇄편상배사、대량염성세포침윤등표현,종6 h개시,24 h화48 h현저가중.혈청TNF-α농도재1 h처리조위(727.8±261.3)ng/L,현저고우대조조급기타처리조(F=49.82,P<0.01),2 h처리조위(156.4±52.2)ng/L,현저저우1 h조,단고우대조조(F:30.23,P<0.01);혈청IL-β농도축점상승,24 h처리조최고,위(360.5±121.6)ng/L(F=18.61,P<0.01).24 h처리조간조직Caspase-3、8、9、12활성명현고우대조조(F=84.96,P<0.01).iNOS기인재대조조무표체,약물작용후6 h체최고,24 h화48 h칙현저하강(F=34.07,P<0.01);p53기인재24 h화48 h처리조표체명현증고(F=37.43,P<0.01);TNF-α화IL-1β기인표체균교대조조승고(F=2.94,P<0.05),기봉치균출현재1 h처리조.결론 소제량지다당가유도D-안기반유당알치민대서발생급성간손상;Caspase-3、8、9、12활성명현증강시기특정성개변지일;간손상적발생여TNF-α、iNOS화p53기인조기고수평표체유밀절관계.
Objective To observe hepatocellular apoptosis and inflammatory cytokines expression and their mechanisms for lipopolysaeeharide (LPS)-induced acute liver failure in D-ga|actosamine (D-GalN)-sensitized rats. Methods Fifty-six rats were randomly divided into following groups: 0, 1, 2, 4, 6, 24 and 48 hours. 0 hour group served as control group and the rest did as treatment groups. The rats in the treatment groups received intraperitoneal injections of LPS (50 ng/g) and D-GaIN (300 μg/g) dissolved in 1 mL sterile 0.9% sodium chloride solution, while the rats in control group were treated with 1 mL sterile 0.9% sodium chloride solution only. The rats were sacrificed in the corresponding time points and their sera and liver tissues were collected. Liver tissues were fixed and stained with hematoxylin and eosin for optical microscopy examination. The serum cytokine expressions were detected by enzyme-linked immunosorbent assay (ELISA). The expressions of tumor necrosing factor (TNF)-α, interleukin (IL)-1β, inducible nitric oxide synthase (iNOS) and p53 gene were detected by reverse transcriptase-polymerase chain reaction, and the 24 hours treated rats liver Caspase-3,8,9,12 activity were detected by chromogenie substrate method. Data for the experiments were expressed as x±s, and differences among means were compared using the analysis of variance. Results After drug treatment, liver tissues showed piecemeal necrosis and inflammatory cell infiltration, which significantly increased from 6 hours, 24 hours to 48 hours. The 1 hour treatment group with the highest concentration of TNF-α (727. 8 ± 261. 3) ng/L were significantly higher than the control group and other treatment groups(F= 49.82, P<0.01), 2 hours treatment group (156.4 ± 52.2) ng/L was significantly lower than the 1 hour group, but significantly higher than the control group (F = 30. 23, P< 0.01 ). But serum concentrations of IL-1β gradually increased, reaching the highest level in 24 hours group (360.5±121.6)ng/L (F= 18. 61, P<0. 01). Liver Caspase-3,8,9, 12 activity in 24 hours treatment group was significantly higher than in the control group (F= 84.96, P<0.01). The mRNA expression of iNOS gene, which were not detected in normal controls, reached the peak at 6 hours group after drug treatment and notably dropped in 24 hours and 48 hours groups(F=34.07,P<0.01), p53 gene expression significantly upregulated at 24 hours and 48 hours groups(F=37.43,P<0.01). TNF-α and IL-1β gene expression in the treatment group were higher than in the control group(F=2.94,P<0.05), and both reached the peak at 1 hour treatment group. Conclusions Acute liver failure can be induced by low dose LPS in D-GaiN-sensitized rats. One of the features changes is that Caspase-3,8,9,12 activities are markedly enhanced, and the occurrence of liver injury may be associated with the early high expression of TNF-α, iNOS and p53 gene.