偶氮鎓二醇盐衍生物JS-K通过MEK/ERK通路抑制肝纤维化抗大鼠肝癌  

Containment of hepatocellular carcinoma by diazeniumdiolate derivative JS-K via inhibition of liver fibrosis along MEK/ERK pathway

作　　者：刘玲[1] 曹梦瑶 陈晶晶 徐静蕾 黄紫乐 LIU Ling;CAO Meng-yao;CHEN Jing-jing;XU Jing-lei;HUANG Zi-le(Department of Pharmacy,School of Basic Medical Sciences,Henan University of Science and Technology,Luoyang 471003,China)

机构地区：[1]河南科技大学基础医学院药学系,河南洛阳471003

出　　处：《中国药理学与毒理学杂志》2020年第6期428-435,共8页Chinese Journal of Pharmacology and Toxicology

基　　金：河南省科技攻关项目(202102310439)。

摘　　要：目的探讨偶氮鎓二醇盐衍生物O2(2,4-二硝基苯基)1-((4-乙氧羰基)哌嗪-1-yl)偶氮-1-鎓-1,2-二醇(JS-K)对大鼠原发性肝癌的影响。方法Wistar雄性大鼠40只,随机分为正常对照组(ip,生理盐水10 mL·kg^-1,连续16周)、模型组(ip,二乙基亚硝胺(DEN)50 mg·kg^-1,前4周每周2次,4周后每周1次,连续16周)、JS-K 0.25和0.50 mg·kg^-1组((ip,DEN 50 mg·kg^-1)+(尾静脉注射,JS-K 0.25或0.50 mg·kg^-1),每周2次,在DEN注射后第2天给药,连续16周)。计数大鼠肝肿瘤结节数并称量瘤质量;比色法测定血清γ-谷氨酰转肽酶(γ-GT)和碱性磷酸酶(AKP)活性及羟脯氨酸含量;HE染色观察肝组织病理变化;ELISA检测血清甲胎蛋白(AFP)、甲胎蛋白异质体3(AFP-L3)和异常凝血酶原(APT)含量;Gomori银染法检测肝组织网状纤维水平;免疫组化法检测肝组织中磷酸化细胞外信号调节激酶(p-ERK)蛋白的表达;Western印迹法检测肝组织中促细胞丝裂原细胞外激酶(MEK)/细胞外信号调节激酶(ERK)信号通路相关蛋白表达水平。结果给药16周后,与正常对照组相比,模型组肝肿瘤结节数和瘤质量显著增加,血清γ-GT和AKP活性及羟脯氨酸含量明显升高(P<0.01);肝小叶结构破坏区域增加,细胞出现多核、核固缩、核内空泡等现象;AFP,AFP-L3和APT水平明显升高(P<0.05,P<0.01);网状纤维塌陷、融合、包裹形成假小叶;肝组织ERK,MEK和P90RSK磷酸化水平均明显升高(P<0.01)。与模型组相比,JS-K各给药组大鼠肝肿瘤结节数和瘤质量显著减少(P<0.05,P<0.01);血清中γ-GT和AKP活性及羟脯氨酸含量明显降低(P<0.01);肝小叶结构相对较好,癌细胞分化程度高;AFP,AFP-L3和APT含量明显降低(P<0.05,P<0.01);网状纤维阳性表达区显著减少(P<0.01),大鼠肝组织ERK,MEK和P90RSK蛋白磷酸化水平显著降低(P<0.01)。结论偶氮鎓二醇盐衍生物通过抑制MEK/ERK通路干预肝纤维化进程,从而对大鼠原发性肝癌具有一定的抑制作用。OBJECTIVE To investigate the regulatory effects of diazeniumdiolate derivative(JS-K),O2-(2,4-dinitrophenyl)1-[(4-ethoxycarbonyl)piperazin-1-yl]diazen 1-ium-1,2-diolate on hepatocellular carcinoma of rats.METHODS Forty Wistar male rats were randomly divided into normal control group(ip,saline 10 mL·kg^-1),model group[ip diethylnitrosamine(DEN)50 mg·kg^-1 twice a week for 4 weeks followed by once a week for 12 weeks],and JS-K groups[(ip DEN 50 mg·kg^-1)+(caudal vein injection,JS-K 0.25 and 0.5 mg·kg^-1,twice a week)].JS-K was injected for 16 weeks starting on the second day after DEN injection.The number of hepatocellular carcinoma nodules and the tumor mass were calculated.Colorimetric methods were used to determine activities of γ-glutamyl transpeptidase(γ-GT)and alkaline phosphatase(AKP)and contents of hydroxyproline.Hematoxylin-eosin(HE)staining was used to detect liver damage in rats while ELISA was used to detect the contents of alpha-fetoprotein(AFP),alpha-fetoprotein heterosome 3(AFP-L3),and abnormal prothrombin(APT)in the serum.Gomori-staining was employed to determine the level of reticular fiber in the liver tissue.The immunohistochemical method was employed to detect the expression of extracellular signal-regluated kinase(p-ERK)protein in liver tissue while Western blotting was used to detect MEK/ERK pathway-related proteins in liver tissue.RESULTS Compared with the normal control group,tumor nodule numbers and tumor mass in the model group were significantly increased and the activities ofγ-GT and AKP and contents of hydroxyproline were increased(P<0.01)16 weeks after administration.Meanwhile,the damaged areas of the liver lobular structure were increased.Multiple nucliei,nuclear shrinkage,intranuclear vacuoles,and the increase in reticular fibers occurred in the model group compared with the normal control group.Furthermore,the contents of AFP,AFP-L3 and APT in the serum were significantly increased in the model group(P<0.05,P<0.01).Reticular fiber manifested collapse,fusion,and was wrapped to

关 键 词：偶氮鎓二醇盐 JS-K 细胞外信号调节激酶 细胞凋亡 

分 类 号：R975[医药卫生—药品]