8-溴乙氧基大黄酸的合成及其对肝癌HepG2.2.15细胞乙肝病毒抑制作用的研究
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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( HBs Ag) and e antigen( HBe Ag) in Hep G2. 2. 15 cells. Methods 8-bromoethoxy Rhein was synthesized based on the chemical structure of Rhein,and its structure was identified by IR,~1H-NMR and~(13)C-NMR spectra. MTT assay was used to test the inhibitory effect of 8-bromo-ethoxy Rhein on Hep G2. 2. 15 cells. After the cells treatment by 8-bromo-ethoxy Rhein,the HBs Ag and HBe Ag in cell supernatant were detected by ELISA. The expression 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,~1H-NMR and~(13)C-NMR. MTT results showed that synthetic product and Rhein could inhibit the cell proliferation in Hep G2. 2. 15 cells. After treated with 8-bromo-ethoxy Rhein and Rhein for 72 h,the half inhibitory concentration 50%( IC_(50)) was 14. 29 mg·L~(-1)and 11. 59 mg·L~(-1),respectively. Using noncytotoxic dose of 8-bromo-ethoxy Rhein,the inhibitory effect on HBs Ag and HBe Ag 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,intracellular 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 inhibition on hepatitis B virus in Hep G2. 2. 15,and its mechanisms may involve down-regulating the expression of HBx and reducing calcium ion concentration.
Aim To synthesize 8-bromo-ethoxy Rhein and investigate its mechanisms and inhibition effect on hepatitis B surface antigen( HBs Ag) and e antigen( HBe Ag) in Hep G2. 2. 15 cells. Methods 8-bromoethoxy Rhein was synthesized based on the chemical structure of Rhein,and its structure was identified by IR,~1H-NMR and~(13)C-NMR spectra. MTT assay was used to test the inhibitory effect of 8-bromo-ethoxy Rhein on Hep G2. 2. 15 cells. After the cells treatment by 8-bromo-ethoxy Rhein,the HBs Ag and HBe Ag in cell supernatant were detected by ELISA. The expression 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,~1H-NMR and~(13)C-NMR. MTT results showed that synthetic product and Rhein could inhibit the cell proliferation in Hep G2. 2. 15 cells. After treated with 8-bromo-ethoxy Rhein and Rhein for 72 h,the half inhibitory concentration 50%( IC_(50)) was 14. 29 mg·L~(-1)and 11. 59 mg·L~(-1),respectively. Using noncytotoxic dose of 8-bromo-ethoxy Rhein,the inhibitory effect on HBs Ag and HBe Ag 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,intracellular 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 inhibition on hepatitis B virus in Hep G2. 2. 15,and its mechanisms may involve down-regulating the expression of HBx and reducing calcium ion concentration.
引文
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[17]Bouchard M J,Wang L H,Schneider R J.Calcium signaling by HBx protein in hepatitis B virus DNA replication[J].Science,2001,294(5550):2376-8.
[18]Sells M A,Chen M L,Acs G.Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J].Proc Nat Acad Sci,1987,84(4):1005-9.
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[2]赫晓林,黄建炜,许瑞安,崔秀灵.HBV病毒复制机制及慢性乙型肝炎药物靶点[J].中国药理学通报,2015,31(3):152-6.[2]He X L,Huang J W,Xu R A,Cui X L.Hepatitis B virus replication mechanisms and drug targets of chronic hepatitis B[J].Chin Pharmacol Bull,2015,31(3):152-6..
[3]Lai W W,Yang J S,Lai K C,et al.Rhein induced apoptosis through the endoplasmic reticulum stress,caspase-and mitochondria-dependent pathways in SCC-4 human tongue squamous cancer cells[J].In Vivo,2009,23(2):309-16.
[4]Shi P,Huang Z,Chen G.Rhein induces apoptosis and cell cycle arrest in human hepatocellular carcinoma BEL-7402 cells[J].Am J Chin Med,2008,36(4):805-13.
[5]李丽,李勇文,唐爱存.甲基阿魏酸对Hep G2.2.15细胞HBs Ag和HBe Ag的抑制作用[J].中药药理与临床,2011,27(3):14-6.[5]Li L,Li Y W,Tang A C.The inhibitory effect of methferulic acid on HBs Ag and HBe Ag in the Hep G2.2.15 cells[J].Pharmacol Clin Med,2011,27(3):14-6.
[6]卫生部.原发性肝癌诊疗规范(2011年版)[J].临床肿瘤学杂志,2011,16(10):929-46.[6]Ministry of Public Health.Treatment protocols of primary liver cancer(2011 edition)[J].Chin Clin Oncol,2011,16(10):929
[7]Zhang Y H,Wu Y Q,Ye S Q,et al.The response to interferon is influenced by hepatitis B virus genotype in vitro and in vivo[J].Virus Res,2013,171(1):65-70.
[8]杨凯,管世鹤,王爱华,等.拉米夫定联合α干扰素序贯处理增强干扰素诱导跨膜蛋白表达的研究[J].中国药理学通报,2013,29(11):1568-72.[8]Yang K,Guan S H,Wang A H,et al.Enhancement of interferoninduced transmembrane protein expression in Hep G2.2.5 cells sequentially treated with lamivudine and interferon-α[J].Chin Pharmacol Bull,2013,29(11):1568-72.
[9]陆豫,黄志纾,谭嘉恒,等.大黄素衍生物的合成及细胞毒性研究[J].有机化学,2005,25(8):944-8.[9]Lu Y,Huang Z S,Tan J H,et al.Synthesis and cytotoxicity of emodin derivatives[J].Chin J Org Chem,2005,25(8):944-8.
[10]Capranico G,Palumbo M,Tinelli S,et al.Conformational drug determinants of the sequence specificity of drug-stimulated topoisomerase II DNA cleavage[J].J Mol Biol,1994,235(4):1218-30.
[11]Glisson B S,Killary A M,Merta P,et al.Dissociation of cytotoxicity and DNA cleavage activity induced by topoisomeraseⅡ-reactive intercalating agents in hamster-human somatic cell hybrids[J].Cancer Chemother Pharmacol,1992,31(2):131-8.
[12]Zunino F,Capranico G.DNA topoisomerase II as the primary target of anti-tumor anthracyclines[J].Anticancer Drug Des,1990,5(4):307-17.
[13]Ben D D,Palumbo M,Zagotto G,et al.DNA topoisomerase II structures and anthracycline activity:insights into ternary complex formation[J].Curr Pharm Des,2007,13(27):2766-80.
[14]Kim C M,Koike K,Saito I,et al.HBx gene of hepatitis B virus induces liver cancer in transgenic mice[J].Nature,1991,351(6324):317-20.
[15]Yu D Y,Moon H B,Son J K,et al.Incidence of hepatocellular carcinoma in transgenic mice expressing the hepatitis B virus Xprotein[J].J Hepatol,1999,31(1):123-32.
[16]Yang N,Tang Y,Wang F,et al.Blockade of store-operated Ca2+entry inhibits hepatocarcinoma cell migration and invasion by regulating focal adhesion turnover[J].Cancer Lett,2013,330(2):163-9.
[17]Bouchard M J,Wang L H,Schneider R J.Calcium signaling by HBx protein in hepatitis B virus DNA replication[J].Science,2001,294(5550):2376-8.
[18]Sells M A,Chen M L,Acs G.Production of hepatitis B virus particles in Hep G2 cells transfected with cloned hepatitis B virus DNA[J].Proc Nat Acad Sci,1987,84(4):1005-9.
[19]Sells M A,Zelent A Z,Shvartsman M,et al.Replicative intermediates of hepatitis B virus in Hep G2 cells that produce infectious virions[J].J Virol,1988,62(8):2836-44.