Evaluation of the Anti-Hepatitis B Virus Activity of Compound Huzhang Prescription in vivo and in vitro
-
摘要:
目的 研究复方虎杖方体内外抗乙肝病毒作用。 方法 体外实验采用CCK-8染色法检测复方虎杖方水提物、醇提物对HepG2.2.15细胞株的抗增殖活性，采用化学发光微粒子免疫检测法测定HBsAg、HBeAg含量，采用实时荧光定量PCR法检测HBV-DNA含量；体内实验采用血清斑点杂交法(Dot blot)测定给药5、10 d后及停药3 d后鸭血清中的DHBV-DNA含量。 结果 体外结果显示，复方虎杖方水提物、醇提物能有效抑制HepG2.2.15细胞增殖活性，IC50分别为51.28 mg/mL和46.78 mg/mL；复方虎杖方能有效抑制HepG2.2.15细胞分泌HBsAg、HBeAg，具有明显的剂量依赖关系，醇提物对HBeAg分泌的抑制作用明显强于水提物；复方虎杖方醇提物能有效抑制HepG2.2.15细胞HBV-DNA复制，但水提物效果不明显。体内结果显示，复方虎杖方能有效抑制鸭血清中DHBV-DNA复制，且能显著抑制停药后的病毒反跳。 结论 复方虎杖方体内和体外均有一定的抗乙肝病毒作用，研究为该方的开发应用奠定了基础。 Abstract: OBJECTIVE To investigate the anti-hepatitis B virus (HBV) effect of Compound Huzhang Prescription in vivo and in vitro.METHODS In vitro, CCK-8 staining method was used to determine the anti-proliferative activity of Compound Huzhang Prescription against HepG2.2.15 cell lines expressing HBV, chemiluminescent microparticle immunoassay method was used to determine hepatitis B surface antigen (HBsAg) and hepatitis B e antigen (HBeAg) levels, and fluorogenic quantitative PCR method was used to determine HBV-DNA level. In vivo, Dot blot assay was used to investigate the content of DHBV-DNA in serum at the 5th, 10th day after administration and the 3th day after drug withdrawal.RESULTS The results showed that water extract and alcohol extract of Compound Huzhang Prescription had a good inhibitory effect on the growth of HepG2.2.15 cells, with a half inhibitory concentration (IC50) of 51.28 mg/mL and 46.78 mg/mL, respectively. Compound Huzhang Prescription significantly inhibited HBsAg and HBeAg expressions and it showed a significant dose effect relationship. The inhibition of alcohol extract was stronger than that of water extract. The alcohol extract had an inhibitory effect on HBV-DNA replication, but the effect of water extract was not obvious. Compound Huzhang Prescription effectively inhibited DHBV-DNA replication, and significantly inhibited the virus rebound after drug withdrawal in vivo.CONCLUSION Compound Huzhang Prescription shows a certain anti-hepatitis B virus effect in vivo and in vitro, which lays a foundation for the development and application.-
Key words:
- Compound Huzhang Prescription /
- HepG2.2.15 cells /
- DHBV-DNA /
- HBsAg /
- HBeAg /
- anti-HBV activity
-
表 1 复方虎杖方提取物对HepG2.2.15的增殖抑制作用(x±s，n=5)
组别 浓度/ (mg·mL-1) 醇提物 水提物 A 抑制率/% A 抑制率/% 空白对照组 - 1.625±0.088 - 1.607±0.067 - 拉米夫定组 0.10 1.052±0.067** 29.1 1.052±0.067** 29.1 复方虎杖方组 0.39 1.564±0.046 3.8 1.572±0.088 2.2 0.78 1.554±0.071 4.4 1.542±0.084 4.0 1.56 1.496±0.058* 7.9 1.532±0.079 4.7 3.12 1.486±0.076* 8.5 1.513±0.042* 5.9 6.25 1.457±0.086** 10.3 1.489±0.087* 7.4 12.5 1.314±0.077** 19.1 1.402±0.058**# 12.8 25 1.103±0.099** 32.1 1.110±0.080** 30.9 50 0.766±0.040** 52.8 0.822±0.076** 48.9 100 0.543±0.024** 66.6 0.624±0.055**## 61.1 注：与空白对照组比较，*P＜0.05，**P＜0.01；与醇提物组比较，#P＜0.05，##P＜0.01。 
下载: 导出CSV
表 2 复方虎杖方提取物对HepG2.2.15细胞分泌HBsAg、HBeAg的抑制作用(x±s, n=3)
组别 浓度/ (mg·mL-1) HBsAg HBeAg 含量/ (×10-6 U·mL-1) 抑制率/% 含量/ (×10-6 U·mL-1) 抑制率/% 空白对照组 - 40.65±3.53 - 143.27±2.97 - 拉米夫定组 0.05 27.67±0.15**## 31.93 84.05±5.70**## 41.33 醇提物组 6.25 16.71±2.22** 58.89 108.77±3.99** 24.08 12.5 10.88±1.02** 73.23 105.20±1.89** 26.57 25 7.58±0.24** 81.35 90.98±7.43** 36.50 50 2.68±0.28** 93.41 27.64±2.90** 80.71 100 2.64±0.11** 93.51 24.47±2.09** 82.92 水提物组 6.25 13.01±1.17** 68.00 94.97±7.43** 33.71 12.5 10.15±0.11** 75.03 92.93±8.24** 35.14 25 8.92±0.49**# 78.06 96.79±2.50** 32.44 50 5.24±0.36**## 87.11 91.67±8.75**## 36.02 100 4.22±0.23**## 89.62 84.64±6.57**## 40.92 注：与空白对照组比较，**P＜0.01；与醇提物组比较，#P＜0.05，##P＜0.01。
下载: 导出CSV
表 3 复方虎杖方在HepG2 2.2.15细胞培养中对HBV-DNA含量的影响(x±s，n=3)
组别 浓度/ (mg·mL-1) HBV-DNA/ lg(copies·mL-1) 抑制率/% 空白对照组 - 5.493±0.096 - 拉米夫定组 0.05 3.200±0.164**# 41.74 醇提物组 6.25 4.993±0.141* 9.10 12.5 4.453±0.138** 18.93 25 4.099±0.154** 25.38 50 3.736±0.203** 31.99 100 3.601±0.255** 34.44 水提物组 6.25 5.658±0.106## - 12.5 5.342±0.100## 2.75 25 5.315±0.198## 3.24 50 5.059±0.094**## 7.90 100 5.342±0.100## 2.75 注：与空白对照组比较，*P＜0.05, **P＜0.01；与醇提物组比较，#P＜0.05，##P＜0.01。
下载: 导出CSV
表 4 复方虎杖方对体内DHBV-DNA的影响(x±s)
组别 剂量/ (g·kg-1) n 灰度值 抑制率/% D0 D5 D10 P3 D5 D10 P3 病毒对照组 - 7 878 484±141 937 1 059 275±154 578 993 071±141 462 1 012 491±178 209 -24.5 -17.6 -18.3 醇提物组 45 6 801 851±18 090 658 557±116 629* 679 085±261 114 411 827±292 410* 18.0 15.3 48.6 15 6 801 643±50 351 753 145±186 445 655 449±208 628 5 017 168±215 876* 5.5 17.9 37.7 5 6 824 137±18 037 448 840±283 521* 461 559±245 227* 260 467±193 381** 45.8 43.8 68.3 拉米夫定组 0.2 4 841 472±30 679 114 331±47 396** 211 554±84 580** 441 942±357 087 86.6 75.0 48.2 注：与D0比较，*P＜0.05，**P＜0.01。
下载: 导出CSV
-
[1] LI JQ, BAO MR, GE J, et al. Research progress of therapeutic vaccines for treating chronic hepatitis B[J]. Hum Vaccin Immunother, 2017, 13(5): 986-997. doi: 10.1080/21645515.2016.1276125 [2] WANG FS, FAN JG, ZHANG Z, et al. The global burden of liver disease: The major impact of China[J]. Hepatology, 2014, 60(6): 2099-2108. doi: 10.1002/hep.27406 [3] KANG L, PAN JQ, WU JF, et al. Anti-HBV drugs: Progress, unmet needs, and new hope[J]. Viruses, 2015, 7(9): 4960-4977. doi: 10.3390/v7092854 [4] 汪露露, 何丹丹, 王满, 等. 基于指纹图谱与主成分分析相结合的复方虎杖方提取工艺研究[J]. 中草药, 2017, 48(2): 278-282. https://www.cnki.com.cn/Article/CJFDTOTAL-ZCYO201702015.htm [5] 刘克冕, 刘超, 鞠建明, 等. 一种具有治疗肝功能异常功效的中药组合物: CN201310343132. X[P]. 2013-11-19. [6] 杨雪, 范尧夫, 曹琳, 等. 复方虎杖方治疗非酒精性脂肪性肝病的理论机制探讨[J]. 中医药学报, 2018, 46(6): 17-20. https://www.cnki.com.cn/Article/CJFDTOTAL-ZYXB201806005.htm [7] 狄红杰. 复方虎杖方治疗非酒精性脂肪性肝病的疗效和机制研究[D]. 南京: 南京中医药大学, 2017. [8] 罗宏伟, 张四喜. 白藜芦醇抗乙型肝炎病毒及肝炎所致纤维化的初步研究[J]. 中国生化药物杂志, 2016, 36(12): 29-32. https://www.cnki.com.cn/Article/CJFDTOTAL-SHYW201612007.htm [9] 韩彦琪, 曹勇, 董亚楠, 等. 基于神经网络分析的疏风解毒胶囊抗炎作用谱效关系研究[J]. 中草药, 2019, 50(15): 3526-3533. doi: 10.7501/j.issn.0253-2670.2019.15.002 [10] 青艳萍, 张晓明, 周博. 喜炎平注射液与盐酸小檗碱联用对小儿轮状病毒肠炎疗效及对外周血中性粒细胞的影响[J]. 药物评价研究, 2019, 42(12): 2414-2417. https://www.cnki.com.cn/Article/CJFDTOTAL-YWPJ201912024.htm [11] 汪露露. 复方虎杖方提取工艺及抗乙肝物质基础研究[D]. 合肥: 安徽中医药大学, 2018. [12] YANG HY, ZHENG NQ, LI DM, et al. Entecavir combined with furin inhibitor simultaneously reduces hepatitis B virus replication and e antigen secretion[J]. Virol J, 2014, 11: 165. doi: 10.1186/1743-422X-11-165 [13] LIU S, WEI WX, SHI KC, et al. In vitro and in vivo anti-hepatitis B virus activities of the lignan niranthin isolated from Phyllanthus niruri L. [J]. J Ethnopharmacol, 2014, 155(2): 1061-1067. doi: 10.1016/j.jep.2014.05.064 [14] CUI XY, INAGAKI Y, WANG DL, et al. The supercritical CO2 extract from the skin of Bufo bufo gargarizans Cantor blocks hepatitis B virus antigen secretion in HepG2.2.15 cells[J]. Biosci Trend, 2014, 8(1): 38-44. doi: 10.5582/bst.8.38 [15] 杨俊, 许军, 刘燕华, 等. 白花蛇舌草抗乙肝病毒化合物体外筛选[J]. 时珍国医国药, 2013, 24(6): 1402-1403. doi: 10.3969/j.issn.1008-0805.2013.06.049 [16] ZHANG ZW, ZHANG J, PEI XY, et al. An aptamer targets HBV core protein and suppresses HBV replication in HepG2.2.15 cells[J]. Int J Mol Med, 2014, 34(5): 1423-1429. doi: 10.3892/ijmm.2014.1908 [17] ZHANG HL, DAI LH, WU YH, et al. Evaluation of hepatocyteprotective and anti-hepatitis B virus properties of Cichoric acid from Cichorium intybus leaves in cell culture[J]. Biol Pharm Bull, 2014, 37(7): 1214-1220. doi: 10.1248/bpb.b14-00137 [18] 杨坤, 贾雷, 魏思思, 等. 苷泰胶囊抗乙肝病毒的实验研究[J]. 中成药, 2011, 33(11): 1969-1972. doi: 10.3969/j.issn.1001-1528.2011.11.033 [19] 施恬树, 曹佳莉, 杨艺楠, 等. HBV cccDNA检测方法的研究进展[J]. 科学通报, 2020, 65(16): 1529-1545. https://www.cnki.com.cn/Article/CJFDTOTAL-KXTB202016003.htm [20] 刘志红, 零小樟, 江建宁. 慢性乙型肝炎抗病毒治疗终点标志物研究进展[J]. 中国实用内科杂志, 2020, 40(6): 451-456. https://www.cnki.com.cn/Article/CJFDTOTAL-SYNK202006004.htm -
点击查看大图
图(1) / 表(4)
计量
- 文章访问数: 390
- HTML全文浏览量: 73
- PDF下载量: 310
- 被引次数: 0
