熊去氧胆酸对α-萘异硫氰酸酯诱导大鼠急性肝损伤的保护作用

Protective effects of ursodeoxycholic acid on α-naphthylisothi-induced acute liver injury in rats

目的探讨熊去氧胆酸（uDcA）对α-萘异硫氰酸酯（ANIT）诱导大鼠胆汁淤积性肝损伤的保护作用及作用机制。方法取SD大鼠48只，其中42只以ANIT100mg／kg灌胃造成急性肝损伤，在肝损伤后24h处死6只，其余均分为对照组和UDCA组各18只。对照组予0．9％NaCl溶液灌胃，UDCA组予20mg／kgUDCA灌胃，分别在造模后48、72、96h各处死6只，另6只不予处理为空白对照组。所有大鼠处死留取血清和肝组织，检测血清ALT、AST、TBil、总胆汁酸（TBA），ELISA法检测血清IL-10、IL-6、TNF-α，实时荧光定量PCR检测肝组织多药耐药相关蛋白2（Mrp2）mRNA，HE染色观察肝组织炎性反应活动度。结果肝损伤造模后48h，UDCA组和对照组血清TBilE（143．80±12．08）μmol／L比（178．50±15．19）μmol／L]、TBAE（13．15±3．81）μmol／L比（21．68±7．93）μmol／L]、IL-10[（44．13±3．68）ng／L比（37．15±6．25）ng／L、IL-6[（50．80±2．09）ng／L比（57．32±4．63）ng／L]、TNF-α [（17．53±0．84）ng／L比（19．10±1．64）ng／L]比较，差异均有统计学意义（P〈0．01或P〈0．05）；肝损伤造模后72h，UDCA组和对照组血清ALT[（721．67±97．54）U／L比（929．50±148．29）U／L]、IL-10[（54．68±6．79）ng／L比（43．85±4．08）ng／L]比较，差异均有统计学意义（P〈0．01或P〈0．05）；肝损伤造模后96h，UDCA组和对照组血清ALTE（156．83±14．99）U／L比（250．67±42．29）U／L]、AST[（143．67±27．45）U／L比（206．00±63．94）U／L]、TBilr[（23．53±5．08）μmol／L比（34．02±9．98）μmol／L]比较，差异有统计学意义（P〈0．01或P〈0．05）。肝损伤造模后UDCA组和对照组肝组织Mrp2mRNA表达在48h（0．77±0．21、0．46±0．25）、72h（2．27±0．84、1．10±0．38）、96h（3．64±0．54、2．75±0．69）各时间点差异均有统计学意义（P〈0．01或P〈0．05）。结论UDCA对ANIT诱导的胆汁淤积性肝损伤保护作用机制可能和调控血清细胞因子及肝脏Mrp2表达有关。

Objective To investigate the protective effects and mechanism of ursodeoxycholic acid （UDCA） on α-naphthylisothi （ANIT）-indueed cholestatic liver injury in rats. Methods A total of 48 Sprague-Dawley （SD） rats were selected. Fouty-two rats were gavaged with ANIT （100 mg/kg） to induce acute liver injury, six rats were sacrificed 24 hours after the liver injury and the rats left were evenly divided into control group which were gavaged with saline and UDCA group which were gavaged with UDCA （20 mg/kg）. Six rats were sacrificed at 48 hours, 72 hours and 96 hours after modeling. The six untreated rats were set as blank control group. Serum and liver tissues of all rats were kept after sacrificed. Serum levels of alanine transaminase （ALT）, aspartate transaminase （AST）, total bilirubin （TBil） and total bile acid （TBA） were tested, interleukin-10 （IL-10）, interleukin-6 （IL-6）, and tumor necrosis factor-α （TNF-α） were detected by enzyme linked immunosorbent assay （ELISA）. The expression of muhidrug resistance associated protein2 （Mrp2） at mRNA level in liver tissue was measured by quantitative real-time polymerase chain reaction （qRT-PCR） and the inflammatory reaction activity of liver tissues was inspected with Haematoidin-Eosin （HE） staining under microscope. Results At 48 hours after liver injury modeling, serum TBil （143. 80±12.08） μmol/L vs. （178.50±15. 19） μmol/L, TBA （13. 15±3.81） μmol/L vs. （21. 68±7. 93） μmol/L, IL-10 （44.13±3.68,37.15±6.25 ng/L）, IL-6（50.80±2.09, 57.32±4.63 ng/L） and TNF-a （17.53± 0.84） ng/L vs. （19. 10 ± 1. 64） ng/L of UDCA group and control group were compared, and the differences were statistically significant （P〈 0. 01 or P〈 0. 05）. At 72 hours after liver injury modeling, serumALT （721.67±97.54） U/Lvs. （929.50±148.29） U/LandIL-10 （54.68±6.79） ng/L vs. （43.85±4.08） ng/L of UDCA group and control group were compared, and the differences were statistically significant （P〈0.01 or P〈0.05）. At 96 hours after liver injury modeling, serum ALT （156.83±14.99） U/Lvs. （250.67±42.29） U/L, AST （143. 67±27. 45） U/Lvs. （206.00± 63.94） U/L and TBil （23. 53±5.08） μ/L vs. （34. 02±9. 98） μmol/L of UDCA group and control group were compared, and the differences were statistically significant （P〈0.01 or P〈0.05）. The differences of Mrp2 expression at mRNA level in liver tissues between UDCA group and control group at 48 hours （0.77±0.21,0.46±0.25）, 72 hours （2.27±0.84,1. 10±0.38） and 96 hours （3.64±0.54,2.75±0.69） after liver injury modeling were statistically significant （P〈0.01 or P〈 0.05）. Conclusion The mechanism of the protective effects of UDCA on ANIT-induced liver injury may be related with the regulation of serum cytokines and liver Mrp2 expression.