绿原酸抗石胆酸胆汁淤积模型肝损伤的作用与机制研究

PROTECTION OF CHLOROGENIC ACID AGANIST CHOLESTATIC LIVER INJURY INDUCED BY LITHOCHOLIC ACID AND THE UNDERLYING MECHANISM

目的探究绿原酸(chlorogenic acid,CGA)对抗石胆酸(lithocholic acid,LCA)致胆汁淤积肝损伤的作用和机制。方法选用雄性健康ICR小鼠40只,分为8组,阴性对照组(CN)连续灌胃给予玉米油5 d,1/d;阳性对照组(M)造模前灌胃给予玉米油3 d,3个保护组每天灌胃低,中,高剂量的CGA[5、15、50 mg/(kg·bw),LCA+L-CGA、LCA+M-CGA、LCA+H-CGA],1/d,共计5 d;D4,M组和保护组分别给予LCA灌胃[300 mg/(kg·bw),2/d,间隔12 h];3个CGA组(L-CGA,M-CGA,H-CGA)每日给予上述保护组相同的三个剂量的CGA,1/d,连续5 d。M组和保护组在末次给予LCA 12 h后处死,其余各组也同时处死。收集血清和肝脏、检测肝功能、肝组织病理学变化以及胆汁酸代谢转运基因的表达。结果 LCA+M-CGA组小鼠血清血生化水平为ALT(208±53)U/L,AST(274±87)U/L,ALP(76±42)U/L,TBA(199±103)μmol/L,ALT,AST,TBA水平均显著低于M组[(390±132)U/L,(646±168)U/L,(347±19)μmol/L]。组织病理学分析显示,LCA+M-CGA组小鼠肝细胞的坏死程度与M组比较显著减轻。Q-PCR检测显示,给予中剂量CGA治疗后,胆汁酸代谢相关基因m RNA的变化较M组发生代偿性逆转,而单独给予CGA的组别中Oatp1表达升高,其余代谢转运基因的转录未见剂量相关性改变。结论 CGA对LCA引起的胆汁淤积性肝损伤具有明显的保护作用;CGA可调节转运体基因Oatp1的转录,但对其它胆汁酸合成和转运基因无直接调节作用,其调节代谢转运对抗胆汁淤积性肝损伤的作用微弱;CGA对抗LCA模型胆於性肝损伤作用可能基于抗炎作用,确切机制有待进一步研究。

Objective To explore the protective effects of chlorogenic acid（CGA） against cholestatic liver injury induced by lithocholic acid（LCA） and the mechanism involved. Methods Forty male ICR mice were randomly assigned into 8 groups. The normal control group was gavaged corn oil once a day for 5 days and so did the positive control group for the first 3 days. Three protection groups were gavaged low, medium, high doses of CGA（5, 15, 50 kg/mg·bw, LCA＋L-CGA,LCA＋M-CGA,LCA＋H-CGA） once daily for 5 consecutive days. To induce acute intrahepatic cholestasis, positive groups and protection groups were gavaged LCA（300 kg/（mg·bw）, twice daily, 12 h intervals） from day 4. Another three CGA groups were also gavaged the same doses of CGA（5, 15, 50 kg/（mg·bw）, L-CGA,M-CGA,H-CGA） for 5 days. Mice in the positive and protection groups were killed 12 h after the third LCA gavage. Mice in other groups were killed at the same time. The liver function, pathological changes and bile acid metabolism were assayed. Results Serum ALT, AST, ALP, TBA in LCA＋M-CGA group were（208±53） U/L,（274±87） U/L,（76±42） U/L and（199±103） μmol/L respectively, in which the ALT, AST, TBA were lower than those in the positive group（（390±132） U/L,（646±168） U/L,（347±19） μmol/L）. Hematoxylin-eosin staining showed that the necrosis and infiltration of inflammation in LCA＋M-CGA group were improved compared with those in the positive group. Q-PCR analysis revealed that the expression of bile acid metabolism genes was normalized by the treatment of medium-dose of CGA. The expression of Oatp1 increased in three CGA groups while the expression of other genes was not significantly changed compared to the normal control group. Conclusion CGA can protect against cholestatic liver injury caused by LCA. The protection is related to anti-inflammatory action of CGA, and not direct regulation of bile acid synthesis and transport genes is observed.