PDF下载 ( 43390 KB)
黄芪皂苷Ⅰ和毛蕊异黄酮在胆汁淤积性肝纤维化小鼠模型中的调控作用分析
DOI: 10.12449/JCH260513
Regulatory effect of astragaloside Ⅰ and calycosin on a mouse model of cholestatic liver fibrosis
-
摘要:
目的 筛选黄芪皂苷Ⅰ（ASⅠ）与毛蕊异黄酮（CY）抗胆汁淤积性肝纤维化最佳配伍剂量并进行验证。 方法 采用3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶（DDC）饮食诱导建立小鼠肝纤维化模型，以黄芪中皂苷成分ASⅠ和黄酮类成分CY为研究对象，运用均匀设计法筛选最佳配比；均匀设计实验选用80只C57/BL6J雄性小鼠，按随机数字表法分为Ctrl组（正常组，n=8）、DDC组（模型组，n=8）、TAS组（n=8）、均匀设计（JY）A～F组（每组n=8）、OCA组（n=8）；经多元回归分析，建立最优回归方程，获得潜在最佳剂量配比。进一步比较均匀设计实验中药效最优组及多元回归方程拟合的最佳剂量组药效，完成体内药效验证实验。计量资料多组间比较采用单因素方差分析，方差齐性检验采用Levene检验，若方差齐，采用LSD-t检验进行两两比较；若方差不齐，采用Dunnett T3检验。 结果 在均匀设计方案中，JYB组（3.125 mg/kg ASⅠ与50 mg/kg CY）可显著降低DDC诱导的胆汁淤积性肝纤维化小鼠血清丙氨酸氨基转移酶（ALT）、天冬氨酸氨基转移酶（AST）、碱性磷酸酶（ALP）、总胆汁酸（TBA）、总胆红素（TBil）和间接胆红素（IBil）水平（P值均<0.05），并可降低肝组织羟脯氨酸（Hyp）含量（P<0.01）、胶原面积半定量值（P<0.001）以及平滑肌肌动蛋白α-2基因（Acta2）、Ⅰ型胶原蛋白α1链（Col1a1）、细胞角蛋白7（Ck7）、细胞角蛋白19（Ck19）、表皮生长因子样激素受体1（Adgre1）、Toll样受体4（TLR4）、肿瘤坏死因子α（TNF-α）、趋化因子配体5（CCL5）的mRNA表达水平（P值均<0.05）。回归方程提示，50 mg/kg ASⅠ与50 mg/kgCY是潜在最佳配伍（即配伍P1组）。验证实验表明，配伍P1组仅对DDC小鼠血清AST、IBil水平及肝组织Hyp含量有显著改善（P值均<0.05），对肝组织胶原沉积、Acta2、Ck7、Ck19、Adgre1、CCL5的mRNA表达水平无显著影响（P值均>0.05）；而JYB组对小鼠血清ALP、ALT、AST、TBA、TBil、IBil水平，肝组织胶原沉积及Hyp含量，Acta2、Col1a1、Ck7、Ck19、Adgre1、TLR4、TNF-α、CCL5的mRNA表达水平均有显著改善（P值均<0.05）。 结论 3.125 mg/kgASⅠ与50 mg/kg CY组合可显著改善DDC小鼠肝纤维化，疗效与黄芪总皂苷相当，且在改善血清ALP、TBA水平方面较ASⅠ和CY单独给药有更显著的调控作用。 -
关键词:
- 胆汁淤积 /
- 肝纤维化 /
- 黄芪皂苷 /
- 毛蕊异黄酮 /
- 小鼠，近交C57BL
Abstract:Objective To identify and validate the optimal compatibility dosage of astragaloside Ⅰ （ASⅠ） and calycosin （CY） in the treatment of cholestatic liver fibrosis. Methods A 3，5-diethoxycarbonyl-1，4-dihydrocollidine （DDC） diet was used to establish a mouse model of liver fibrosis， and the uniform design method was used to identify the optimal combination ratio of the saponin component ASⅠ and the flavonoid component CY in Astragalus membranaceus. In the uniform design experiment， 80 male C57/BL6J mice were divided into normal group， model group， total astragalosides （TAS） group， groups A — F with a uniform design， and obeticholic acid （OCA） group using a random number table， with 8 mice in each group. The multiple regression analysis was used to establish the optimal regression equation and obtain the potential optimal combination ratio. The in vivo efficacy of the empirically optimal combination identified in the uniform design and the optimal dose combination predicted by the regression equation were compared for validation. A one-way analysis of variance was used for comparison of continuous data between multiple groups； the Levene test was used to determine the homogeneity of variance， and the least significant difference t-test was used for comparison of data with homogeneity of variance between two groups， while the Dunnett T3 test was used for comparison of data with heterogeneity of variance. Results In the uniform design regimen， the JYB combination （3.125 mg/kg ASI+50 mg/kg CY） significantly reduced the serum levels of alanine aminotransferase （ALT）， aspartate transaminase （AST）， alkaline phosphatase （ALP）， total bile acid （TBA）， total bilirubin （TBil）， and indirect bilirubin （IBil） in mice with DDC-induced cholestatic liver fibrosis （all P<0.05）， and it also reduced hepatic Hyp content （P<0.01）， semi-quantified collagen deposition area （P<0.001）， and the mRNA expression levels of Acta2， Col1a1， Ck7， Ck19， Adgre1， TLR4， TNF-α， and CCL5 in liver tissue （all P<0.05）. The regression equation showed that 50 mg/kg ASⅠ+50 mg/kg CY was the potential optimal combination， which was named as P1 combination. However， subsequent validation experiments showed that P1 combination only significantly improved the serum levels of AST and IBil and hepatic Hyp content in DDC mice （all P<0.05）， with no significant impact on hepatic collagen deposition and the mRNA expression levels of Acta2， Ck7， Ck19， Adgre1， and CCL5 （all P>0.05）. In contrast， the JYB combination significantly improved the serum levels of ALP， ALT， AST， TBA， TBil， and IBil， hepatic collagen deposition， hepatic Hyp content， and the mRNA expression levels of Acta2， Col1a1， Ck7， Ck19， Adgre1， TLR4， TNF-α， and CCL5 （all P<0.05）. Conclusion This study shows that the JYB combination （3.125 mg/kg ASⅠ+50 mg/kg CY） can significantly alleviate DDC-induced liver fibrosis， with comparable efficacy to total saponins from Astragalus membranaceus， and compared with ASⅠ or CY administered alone， the JYB combination has a significantly better regulatory effect on the serum levels of ALP and TBA. -
Key words:
- Cholestasis /
- Hepatic Fibrosis /
- Astragaloside /
- Calycosin /
- Mice， Inbred C57BL
-
注： a，各组小鼠血清肝功能指标；b，各组小鼠肝组织病理HE染色（×200）。与Ctrl组相比，***P<0.001；与DDC组相比，#P<0.05，##P<0.01。DDC，3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶；TAS，黄芪总皂苷；OCA，奥贝胆酸；HE，苏木素-伊红。
图 1 均匀设计各组小鼠血清肝功能及肝组织病理HE染色
Figure 1. Serum liver function and HE staining in mice from uniform design groups
注： a，肝组织病理SR染色（×100）；b，SR染色面积半定量；c，肝组织Hyp含量。与Ctrl组相比，***P<0.001；与DDC组相比，#P<0.05，##P<0.01，###P<0.001。DDC，3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶；TAS，黄芪总皂苷；OCA，奥贝胆酸；SR，天狼星红；Hyp，羟脯氨酸。
图 2 均匀设计各组小鼠肝组织SR染色及Hyp含量测定
Figure 2. Hepatic sirius red staining and hydroxyproline content assay in uniform design groups of mice
注： 与Ctrl组相比，**P<0.01，***P<0.001；与DDC组相比，#P<0.05，##P<0.01，###P<0.001。DDC，3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶；TAS，黄芪总皂苷；OCA，奥贝胆酸。
图 3 均匀设计各组小鼠肝组织纤维化及炎症相关基因mRNA相对表达水平
Figure 3. Expression levels of hepatic fibrosis and inflammation-related genes in uniform design groups of mice
注： a，各组小鼠血清肝功能指标；b，各组小鼠肝组织病理HE染色（×200）。与Ctrl组相比，***P<0.001；与DDC组相比，#P<0.05，##P<0.01，###P<0.001。DDC，3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶；TAS，黄芪总皂苷；OCA，奥贝胆酸；HE，苏木素-伊红。
图 4 配伍验证各组小鼠血清肝功能及肝组织病理HE染色
Figure 4. Serum liver function and HE staining in mice from combination verification groups
注： a，肝组织病理SR染色（×100）；b，SR染色面积半定量；c，肝组织Hyp含量。与Ctrl组相比，***P<0.01；与DDC组相比，#P<0.05，##P<0.01。DDC，3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶；TAS，黄芪总皂苷；OCA，奥贝胆酸；SR，天狼星红；Hyp，羟脯氨酸。
图 5 配伍验证各组小鼠肝组织SR染色及Hyp含量测定
Figure 5. Hepatic sirius red staining and hydroxyproline content assay in combination verification groups of mice
注： 与Ctrl组相比，**P<0.01，***P<0.001；与DDC组相比，#P<0.05，##P<0.01，###P<0.001。DDC，3，5-二乙氧基羰基-1，4-二氢-2，4，6-三甲基吡啶；TAS，黄芪总皂苷；OCA，奥贝胆酸。
图 6 配伍验证各组小鼠肝组织纤维化及炎症相关基因mRNA相对表达水平
Figure 6. Expression levels of hepatic fibrosis and inflammation-related genes in combination verification groups of mice
表 1 均匀设计实验中每日给药剂量
Table 1. Dosing regimens in uniform design experiment
组别 ASⅠ（mg/kg） CY（mg/kg） JYA组 1.560 6.250 JYB组 3.125 50.000 JYC组 6.250 3.125 JYD组 12.500 25.000 JYE组 25.000 1.560 JYF组 50.000 12.500 注：ASⅠ，黄芪皂苷Ⅰ；CY，毛蕊异黄酮。
下载: 导出CSV
表 2 配伍验证实验中每日给药剂量
Table 2. Dosing regimens in compatibility verification experiment
组别 每日给药剂量 Ctrl组 0.3% 羧甲基纤维素钠（10 mL/kg） DDC组 0.3% 羧甲基纤维素钠（10 mL/kg） TAS组 TAS （56 mg/kg） P1组 ASⅠ（50 mg/kg）+CY（50 mg/kg） JYB组 ASⅠ（3.125 mg/kg）+CY（50 mg/kg） ASⅠ组 ASⅠ （50 mg/kg） CY组 CY （50 mg/kg） OCA组 OCA （10 mg/kg） 注：TAS，黄芪总皂苷；ASⅠ，黄芪皂苷Ⅰ；CY，毛蕊异黄酮；OCA，奥贝胆酸。
下载: 导出CSV
表 3 引物序列
Table 3. Primer sequences
基因 引物序列（5'-3'） 引物长度
（bp）Acta2 F：GTCCCAGACATCAGGGAGTAA 21 R：TCGGATACTTCAGCGTCAGGA 21 Col1a1 F：GCTCCTCTTAGGGGCCACT 19 R：CCACGTCTCACCATTGGGG 19 Ck7 F：TCAGGATGGTAAGCGGATGTT 21 R：AAGGGCTCCACGTAGGTAATC 21 Ck19 F：GGGGGTTCAGTACGCATTGG 20 R：GAGGACGAGGTCACGAAGC 19 Adgre1 F：TGACTCACCTTGTGGTCCTAA 21 R：CTTCCCAGAATCCAGTCTTTCC 22 TLR4 F：AGCTCCTGACCTTGGTCTTG 20 R：CGCAGGGGAACTCAATGAGG 20 TNF-α F：GGAACACGTCGTGGGATAATG 21 R：GGCAGACTTTGGATGCTTCTT 21 CCL5 F：GCTGCTTTGCCTACCTCTCC 20 R：TCGAGTGACAAACACGACTGC 22 注：Acta2，平滑肌肌动蛋白α-2基因；Col1a1，Ⅰ型胶原蛋白α1链；Ck7，细胞角蛋白7；Ck19，细胞角蛋白19；Adgre1，表皮生长因子样激素受体1；TLR4，Toll样受体4；TNF-α，肿瘤坏死因子α；CCL5，趋化因子配体5。
下载: 导出CSV
-
[1] CHEN RL, MA X. Pathogenesis of cholestasis-induced liver fibrosis and thoughts for blockade[J]. J Clin Hepatol, 2019, 35( 2): 247- 251. DOI: 10.3969/j.issn.1001-5256.2019.02.002.陈瑞玲, 马雄. 胆汁淤积导致肝纤维化的机制及其阻断策略[J]. 临床肝胆病杂志, 2019, 35( 2): 247- 251. DOI: 10.3969/j.issn.1001-5256.2019.02.002. [2] TRAUNER M, FUCHS CD. Novel therapeutic targets for cholestatic and fatty liver disease[J]. Gut, 2022, 71( 1): 194- 209. DOI: 10.1136/gutjnl-2021-324305. [3] JANSEN PLM, GHALLAB A, VARTAK N, et al. The ascending pathophysiology of cholestatic liver disease[J]. Hepatology, 2017, 65( 2): 722- 738. DOI: 10.1002/hep.28965. [4] ZHANG LZ, SHI JW, SHEN Q, et al. Astragalus saponins protect against extrahepatic and intrahepatic cholestatic liver fibrosis models by activation of farnesoid X receptor[J]. J Ethnopharmacol, 2024, 318( Pt A): 116833. DOI: 10.1016/j.jep.2023.116833. [5] ZHANG LZ, HU YH, QI SL, et al. Astragalus saponins and its main constituents ameliorate ductular reaction and liver fibrosis in a mouse model of DDC-induced cholestatic liver disease[J]. Front Pharmacol, 2022, 13: 965914. DOI: 10.3389/fphar.2022.965914. [6] GUO T, LIU ZL, ZHAO Q, et al. A combination of astragaloside I, levistilide A and calycosin exerts anti-liver fibrosis effects in vitro and in vivo[J]. Acta Pharmacol Sin, 2018, 39( 9): 1483- 1492. DOI: 10.1038/aps.2017.175. [7] DUAN XP, MENG Q, WANG CY, et al. Effects of calycosin against high-fat diet-induced nonalcoholic fatty liver disease in mice[J]. J Gastroenterol Hepatol, 2018, 33( 2): 533- 542. DOI: 10.1111/jgh.13884. [8] YI W. Clinical and experimental study of Lidan mixture against oxidative stress in the treatment of cholestatic liver fibrosis[D]. Wuhan: Hubei University of Chinese Medicine, 2022. DOI: 10.27134/d.cnki.ghbzc.2022.000006.易巍. 利胆合剂拮抗氧化应激治疗胆汁淤积性肝纤维化的临床及实验研究[D]. 武汉: 湖北中医药大学, 2022. DOI: 10.27134/d.cnki.ghbzc.2022.000006. [9] BANALES JM, HUEBERT RC, KARLSEN T, et al. Cholangiocyte pathobiology[J]. Nat Rev Gastroenterol Hepatol, 2019, 16( 5): 269- 281. DOI: 10.1038/s41575-019-0125-y. [10] HOHENESTER S, de BUY WENNIGER LM, PAULUSMA CC, et al. A biliary HCO3 - umbrella constitutes a protective mechanism against bile acid-induced injury in human cholangiocytes[J]. Hepatology, 2012, 55( 1): 173- 183. DOI: 10.1002/hep.24691. [11] QIU JN, YAN JY, LIU W, et al. Metabolomics analysis delineates the therapeutic effects of Huangqi decoction and astragalosides on α-naphthylisothiocyanate(ANIT)-induced cholestasis in rats[J]. J Ethnopharmacol, 2021, 268: 113658. DOI: 10.1016/j.jep.2020.113658. [12] KENNEDY L, CARPINO G, OWEN T, et al. Secretin alleviates biliary and liver injury during late-stage primary biliary cholangitis via restoration of secretory processes[J]. J Hepatol, 2023, 78( 1): 99- 113. DOI: 10.1016/j.jhep.2022.07.034. [13] WANG JJ, XIANG SX, XIE ZS, et al. Research progress on antifibrotic effect and molecular mechanisms of chemical components from astragali Radix[J]. Tradit Chin Drug Res Clin Pharmacol, 2023, 34( 12): 1799- 1805. DOI: 10.19378/j.issn.1003-9783.2023.12.018.王佳俊, 向世勰, 谢治深, 等. 黄芪化学成分抗纤维化作用及分子机制研究进展[J]. 中药新药与临床药理, 2023, 34( 12): 1799- 1805. DOI: 10.19378/j.issn.1003-9783.2023.12.018. [14] LIU W, LI CH, ZHANG LZ, et al. Analysis of rationality of quality control for astragaloside I based on pharmacokinetic characteristics of Astragalus saponins[J]. Shanghai J Tradit Chin Med, 2026, 60( 1): 73- 81. DOI: 10.16305/j.1007-1334.2026.z20250513006.刘伟, 李春晖, 张霖璋, 等. 基于黄芪皂苷类成分药代动力学特征解析黄芪皂苷I的质控合理性[J]. 上海中医药杂志, 2026, 60( 1): 73- 81. DOI: 10.16305/j.1007-1334.2026.z20250513006. [15] YU Q, KODA S, XU N, et al. CsHscB derived from a liver fluke Clonorchis sinensis ameliorates cholestatic hepatic fibrosis in a mouse model of sclerosing cholangitis[J]. Curr Mol Med, 2024, 24( 4): 505- 515. DOI: 10.2174/1566524023666230418111949. [16] WANG ZL, JIN R, HAO M, et al. Treatment of ursodeoxycholic acid with glucocorticoids and immunosuppressants may improve the long-term survival rate in primary biliary cholangitis patients[J]. Medicine, 2022, 101( 46): e31395. DOI: 10.1097/MD.0000000000031395. [17] ZENG G, ESLICK GD, WELTMAN M. Systematic review and meta-analysis: Comparing hepatocellular and cholestatic patterns of drug-induced liver injury[J]. ILIVER, 2023, 2( 2): 122- 129. DOI: 10.1016/j.iliver.2023.05.002. [18] LI SD, WANG Z, LIU JK, et al. Research progress on Astragalus active ingredient in treatment of hepatic fibrosis based on cell signaling pathway[J]. Chin Pharmacol Bull, 2023, 39( 1): 18- 23. DOI: 10.12360/CPB202101088.李淑娣, 王振, 刘江凯, 等. 基于细胞信号通路探讨黄芪活性成分抗肝纤维化的研究进展[J]. 中国药理学通报, 2023, 39( 1): 18- 23. DOI: 10.12360/CPB202101088. [19] ZHU Y, CHAI YL, XIAO GJ, et al. Astragalus and its formulas as a therapeutic option for fibrotic diseases: Pharmacology and mechanisms[J]. Front Pharmacol, 2022, 13: 1040350. DOI: 10.3389/fphar.2022.1040350. [20] FU K, DAI S, YOU J, et al. Pharmacological action of astragaloside IV in the prevention and treatment of liver diseases and its mechanism[J]. J Clin Hepatol, 2025, 41( 10): 2174- 2179. DOI: 10.12449/JCH251032.付珂, 代书, 游娟, 等. 黄芪甲苷防治肝脏疾病的药理作用及其机制[J]. 临床肝胆病杂志, 2025, 41( 10): 2174- 2179. DOI: 10.12449/JCH251032. [21] YUAN X, GONG Z, WANG B, et al. Astragaloside inhibits hepatic fibrosis by modulation of TGF-β1/Smad signaling pathway[J]. Evid Based Complement Alternat Med, 2018: 3231647. DOI: 10.1155/2018/3231647. [22] LI Y, YI H, CAI YL, et al. Effect of total flavonoids of Astragalus membranaceus on hepatic fibrosis induced by carbon tetrachloride in rats[J]. Chin Tradit Pat Med, 2019, 41( 7): 1710- 1713. DOI: 10.3969/j.issn.1001-1528.2019.07.045.李艳, 伊航, 蔡轶伦, 等. 黄芪总黄酮对四氯化碳诱导大鼠肝纤维化的影响[J]. 中成药, 2019, 41( 7): 1710- 1713. DOI: 10.3969/j.issn.1001-1528.2019.07.045. [23] AN L, LIN YF, LI LY, et al. Integrating network pharmacology and experimental validation to investigate the effects and mechanism of Astragalus Flavonoids against hepatic fibrosis[J]. Front Pharmacol, 2021, 11: 618262. DOI: 10.3389/fphar.2020.618262. [24] MENG XT, YUE SJ, YANG ZR, et al. Investigation on biotransformation characteristics of astragalosides in human intestinal microbiota[J]. Food Drug, 2018, 20( 3): 161- 167. DOI: 10.3969/j.issn.1672-979X.2018.03.001.孟欣桐, 乐世俊, 杨智睿, 等. 黄芪皂苷类成分在人源肠道菌群中生物转化特征研究[J]. 食品与药品, 2018, 20( 3): 161- 167. DOI: 10.3969/j.issn.1672-979X.2018.03.001. -
本文二维码
计量
- 文章访问数: 9
- HTML全文浏览量: 5
- PDF下载量: 0
- 被引次数: 0
