摘要
目的合成8-溴乙氧基大黄酸,探讨8-溴乙氧基大黄酸对肝癌Hep G2.2.15细胞分泌的乙型肝炎病毒表面抗原(HBs Ag)和e抗原(HBe Ag)的抑制作用和机制。方法以大黄酸为结构基础,化学全合成8-溴乙氧基大黄酸,采用红外光谱(IR)、核磁共振氢谱(~1H-NMR)及碳谱(^(13)C-NMR)对其结构进行表征;MTT法测定8-溴乙氧基大黄酸对细胞生长的抑制作用;ELISA检测Hep G2.2.15细胞分泌的HBs Ag和HBe Ag;Western blot法检测细胞内乙肝病毒X基因(HBx)蛋白的表达;流式细胞仪分析细胞周期;激光共聚焦显微镜测定细胞内游离钙离子浓度。结果 IR、~1H-NMR和13C-NMR的结果确证合成产物为8-溴乙氧基大黄酸,合成产物和大黄酸对Hep G2.2.15细胞具有较好的抑制细胞生长的作用,作用72 h后IC_(50)分别为14.29 mg·L^(-1)和11.59mg·L^(-1)。采用无细胞毒剂量的8-溴乙氧基大黄酸处理细胞后,对细胞分泌的HBs Ag和HBe Ag有抑制作用,其抑制作用随着浓度的增加,呈现剂量依赖性,且合成产物的抑制效果优于大黄酸;8-溴乙氧基大黄酸可降低HBx蛋白表达水平和细胞内钙离子浓度,阻碍乙型肝炎病毒(HBV)复制,但不影响细胞周期。结论本研究合成的8-溴乙氧基大黄酸显示了比大黄酸更为优越的抑制HepG2.2.15细胞HBsAg和HBeAg的分泌作用的活性,其作用机制可能是通过降低HBx蛋白表达、减少钙离子浓度而实现。
Aim To synthesize 8-bromo-ethoxy Rhein and investigate its mechanisms and inhibition effect on hepatitis B surface antigen （ HBsAg ） and e antigen （ HBeAg） in HepG2.2.15 cells.Methods 8-bromo-ethoxy Rhein was synthesized based on the chemical structure of Rhein , and its structure was identified by IR,1 H-NMR and 13 C-NMR spectra.MTT assay was used to test the inhibitory effect of 8-bromo-ethoxy Rhein on HepG2.2.15 cells.After the cells treatment by 8-bromo-ethoxy Rhein , the HBsAg and HBeAg in cell supernatant were detected by ELISA .The expres-sion of hepatitis B virus X gene （ HBx） was detected by Western blot .The cell cycles were examined with flow cytometry.The intracellular free calcium concentration was detected by laser scanning confocal microscopy . Results The structure of 8-bromo-ethoxy Rhein was confirmed by IR,1 H-NMR and 13 C-NMR.MTT results showed that synthetic product and Rhein could inhibit the cell proliferation in HepG2.2.15 cells.After trea-ted with 8-bromo-ethoxy Rhein and Rhein for 72 h,the half inhibitory concentration 50%（ IC50 ） was 14.29 mg？ L-1 and 11.59 mg？ L-1 , respectively .Using non-cytotoxic dose of 8-bromo-ethoxy Rhein , the inhibitory effect on HBsAg and HBeAg was gradually enhanced with increasing 8-bromo-ethoxy Rhein concentration . The inhibitory effect of synthetic product on hepatitis B virus was better than that of Rhein .8-bromo-ethoxy Rhein could down-regulate the expression of HBx , in-tracellular calcium ion concentration and block the hepatitis B virus （ HBV ） replication.Flow cytometry results showed 8-bromo-ethoxy Rhein didn′t affect the cell cycle .Conclusions Compare with Rhein , the synthesis of 8-bromo-ethoxy Rhein shows stronger inhi-bition on hepatitis B virus in HepG2.2.15, and its mechanisms may involve down-regulating the expres-sion of HBx and reducing calcium ion concentration .
出处
《中国药理学通报》
CAS
CSCD
北大核心
2016年第8期1175-1180,共6页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81460561)
广西高校重点实验室-壮医方药基础与应用研究重点实验室(No桂教科研〔2014〕6号)
