重症急性胰腺炎大鼠肝损伤机制及褪黑素保护

目的 观察重症急性胰腺炎(SAP)大鼠肝组织核因子κB表达活性与肝脏损伤的关系,探讨褪黑素(Mel)在大鼠急性胰腺炎肝脏损伤中的保护作用.方法 SD大鼠随机分为重症急性胰腺炎(SAP)组,Mel干预(Mel)组及假手术(Sham)组,各32只.分别于造模成功后4、12、24、48 h处死大鼠,各时点各组8只.血样检测血清淀粉酶及丙氨酸氨基转移酶(ALT),ELISA法检测血清肿瘤坏死因子(TNF)α的含量,免疫组化evision二步法检测肝脏组织核因子κB的表达,缺口末端标记技术(TUNEL法)检测肝脏细胞凋亡情况,留取肝组织苏木精咿红染色下观察肝组织病理变化.结果 4 h点Sham组、SAP组、Mel组TNF-α表达量差异均无统计学意义(pg/ml:161±9、166±14、161±4、均P>0.05),SAP组淀粉酶、ALT、TNF-α表达量和核因子κB活性及肝细胞凋亡指数各时点均高于Sham组(均P<0.05),并随着时间推移逐渐升高,于24 h达高峰(24 h点SAP组和Sham组核因子κB活性和肝细胞凋亡指数分别为62.8±4.4 vs 7.8±0.6,22.33±2.43 vs 0.81±0.16,均P<0.05),此时肝组织病理损伤以肝细胞凋亡为主;至48 h,SAP组核因子κB活性和肝细胞凋亡指数(30.5±2.1、11.67±0.55)较24 h有所下降,而血清TNF-α、淀粉酶及ALT的含量(342±10)pg/ml、(3357±117)U/L、(574±32)U/L]仍继续升高,同时肝组织病理出现大片出血坏死及炎细胞浸润.Mel组上述指标各时间点均低于SAP组(均P<0.05),但高于Sham组(均P<0.05);Mel组相应各时点病理损伤亦较SAP组轻.结论 肝组织核因子κB的活化介导了重症急性胰腺炎肝脏损伤.Mel可抑制核因子κB活性和TNF-α表达,减轻了肝细胞凋亡和坏死,减轻了急性胰腺炎的肝脏损伤.

Mechanism of liver injury in severe acute pancreatitis rats and role of melatonin

Objective To investigate the activation level of nuclear factor-κB(NF-κB) in liver injury caused by severe acute pancreatitis (SAP) and the protective role of melatonin against liver injury. Methods Ninety-six Sprague-Dawley (SD) rats were randomly divided into 3 equal groups: SAP group undergoing injection of sodium taurocholate to establish SAP models, melatonin (Mel) treatment group undergoing intraperitoneal injection 50 mg/kg 30 minutes before the establishment of SAP models, and sham operation group (Sham group). 4, 12, 24, and 48 hours after the onset of operation, blood samples were collected from the inferior vena easa of 8 rats from each group to measure the serum level of amylase (AMY) and alanine transaminase (ALT) by iodine colorimetry, and to detect the serum level of tumor necrosis faetor-α (TNF-α) by ELISA. The livers were taken out to undergo pathological examination. Immunohistochemistry was used to examine the percentage of nuclear factor (NF)-κB in the hepatocytes. Terminal deoxynucleatidyl transferase mediated dUTP nick end labeling (TUNEL) was used to determine the extent of hepatic apoptosis. Results The AMY and ALT levels at different time points of the SAP and Mel subgroups were all significantly higher than those of the sham operation subgroups (all P<0.05), and the AMY and ALT levels at different time points of the Mel subgroup were all significantly lower than those of the SAP subgroups (all P < 0.05). The liver NF-κB activation level and hepatocellular apoptosis index of the SAP group increased since the fourth hour after the operation, and peaked at the time point of 24 hour, all significantly higher than those of the sham operation group (all P < 0. 05), and then declined. The TNF-α level at the time points of 12, 24, and 48 h in the SAP group were all significantly higher than those of the other 2 groups (all P<0.05). The levels of TNF-α, AMY, and ALT, the activity of NF-κB, and the extent of hepatocellular apoptosis at any time points of the Mel group were all significantly lower than those of the SAP group, but significantly higher than those of the sham operation group. Microscopy showed that the liver pathological damages of the Mel group were milder than those of the SAP group. Conclusion SAP with liver injury is associated with the hepatic NF-κB activation leading to the production of NF-κB dependent cytokines and chemokiues such as TNF-α. Melatonin reduces the apoptosis and necrosis in liver by inhibiting the activity of NF-κB and decreasing the expression of TNF-α.