大麻素受体在肝星状细胞活化中的作用及姜黄素干预效应  

Research on the role of cannabinoid receptors in hepatic stellate cell activation and interfering effects of curcumin

作　　者：张自力[1,2] 张涉[1,2] 郭瑶[1,2] 王妤清[1,2] 倪雯霞[1,2] 张衍[1,2] 孔德松[1,2] 郑仕中[1,2,3] 

机构地区：[1]南京中医药大学药学院,江苏南京210023 [2]南京中医药大学中药学一级学科,江苏南京210023 [3]江苏省中药药效与安全性评价重点实验室,江苏南京210023

出　　处：《中国药理学通报》2013年第5期626-631,共6页Chinese Pharmacological Bulletin

基　　金：国家自然科学基金资助项目(No 81270514;30873424);十一五科技支撑计划(No 2008BAI51B02);教育部博士点博导基金(No 20103237110010);江苏省自然科学基金(NoBK2008456);江苏省针灸学重点实验室开放课题(NoKJA200801);江苏省六大人才基金(No 2009-B-010);江苏高校优势学科建设工程资助项目PAPD(No ysxk-2010);南京中医药大学中药学一级学科开放课题资助(No2011ZYX4-008);江苏省普通高校研究生科研创新计划项目(No CXLX110768)

摘　　要：目的探究大麻素受体在肝星状细胞(HSCs)活化中的作用及姜黄素的干预效应,为阐明肝纤维化发生发展机理及姜黄素抗肝纤维化机制提供依据。方法 MTT法检测CBR1激动剂NADA与拮抗剂AM251,CBR2激动剂JWH015与拮抗剂AM630对HSCs增殖的影响;Western blot实验检测AM251对HSCs中ERK、JNK、p38蛋白表达及磷酸化水平的影响;Western bolt与细胞免疫荧光检测了姜黄素对HSCs中两种类型大麻素受体CBR1与CBR2蛋白表达的影响;Western bolt检测在姜黄素对CBR1激动剂与拮抗剂的刺激下HSCs产生的细胞外基质(ECM)成分α1(I)collagen和Fibronectin的影响。结果激动CBR1可促进HSCs的增殖,相反拮抗CBR1后则可以抑制HSCs的增殖(P<0.05);而激动CBR2也可抑制HSCs的增殖(P<0.05),但抑制CBR2对HSCs的增殖没有明显的影响(P>0.05)。CBR1拮抗剂AM251能够明显抑制ERK与JNK的磷酸化,并呈剂量依赖性的关系(P<0.05,P<0.01),但对p38蛋白表达的影响较小(P>0.05)。姜黄素可抑制HSCs中CBR1的表达(P<0.05),对CBR2蛋白表达的影响则差异没有统计学意义(P>0.05)。姜黄素可呈剂量依赖性的抑制CBR1激动剂导致的ECM成分表达的上升,并可协同CBR1拮抗剂抑制HSCs表达ECM成分的作用(P<0.05,P<0.01)。结论大麻素受体在HSCs的增殖活化过程中具有重要作用。Aim To explore the role of cannabinoid receptors in the activation of hepatic stellate cells （HSCs） and the interfering effects of curcumin in the hope of providing basis for elucidating the mechanism of liver fibrosis and curcumin inhibition of liver fibrosis. Methods Influence of CBR1 agonist NADA and antagonist AM630, CBR2 agonist JWH015 and antagonist AM251 on the proliferation of HSCs was evaluated by MIT assay. Western blot assays were used to detect the expression of ERK, JNK and p38 and their phosphorylation levels in HSCs treated with AM251. The effect of cureumin on the expression of two types of cannabinoid receptors CBR1 and CBR2 in HSCs was determined by Western blot and immunofluorescence. The effect of eureumin on extracellular matrix （ECM） components α1 （I） collagen and fibronectin in HSCs stimulating by CBR1 agonist and antagonist was also examined by Western blot. Results Activating CBR1 promoted the proliferation of HSCs; on the contrary, CBR1 antagonism inhibited HSCs proliferation （P 〈 0. 05 ）. At the same time, activating CBR2 also inhibited the proliferation of HSCs （ P 〈 0.05 ）, but CBR2 inhibition had no apparent effect on the proliferation of HSCs （ P 〉 0. 05 ）. CBR1 antagonist AM251 signifi- cantly inhibited the phosphorylation of ERK and JNK in a dose-dependent manner （P 〈 0.05, P 〈 0.01 ）, but p38 was not affected （P 〉 0. 05）. Curcumin inhibited the expression of CBR1 （ P 〈 0. 05 ）, but at the same time, it had no significant effect on CBR2 （P 〉 0. 05 ）. .Curcumin inhibited the expression of ECM components upregulated by CBR1 agonist dose-dependently in HSCs, and eollaboratively inhibited the expression of ECM components in HSCs exposed to CBR1 antagonist （ P 〈 0. 05, P 〈 0. 01 ）. Conclusions Cannabinoid receptors play an important role in the process of HSCs activation. Curcumin may achieve the purpose of treating liver fibrosis through the intervention of cannabinoid receptor pathways.

关 键 词：肝纤维化 肝星状细胞 姜黄素 大麻素受体 增殖 MAPK信号通路 细胞外基质 

分 类 号：R282.71[医药卫生—中药学] R329.24[医药卫生—中医学]