PDF下载 ( 1395 KB)
植物雌激素鹰嘴豆芽素A（BCA）对肝纤维化去势小鼠模型的改善作用及机制
DOI: 10.12449/JCH240114
伦理学声明：本课题实验方案于2021年7月9日获得贵州医科大学实验动物伦理委员会审批，批号：2100313，符合实验室动物管理与使用准则。
利益冲突声明：本文不存在任何利益冲突。
作者贡献声明：谭超容参与课题的设计，负责课题实施，数据分析，撰写论文；李小飘、冉俊艳、熊英、廖尚高参与课题实施，数据收集，修改论文；张金娟、何迅负责课题设计，指导实验实施及文章撰写并定稿。
Role and mechanism of action of phytoestrogen biochanin A in improving liver fibrosis in ovariectomized mice
-
摘要:
目的 探究植物雌激素鹰嘴豆芽素A（BCA）对CCl4诱导的雌性双侧卵巢切除（去势）小鼠肝纤维化的影响及其机制。 方法 取50只去势雌性昆明小鼠，腹腔注射CCl4建立肝纤维化模型，将建模小鼠按体质量随机分成模型组、阳性对照组、BCA低、中、高剂量组各10只，同时选取10只同批雌性小鼠切除双侧卵巢旁少量脂肪组织作为假手术组。假手术组和模型组灌胃等体积的0.5%羧甲基纤维素钠溶液，阳性对照组用雌二醇2 mg/kg灌胃，BCA低、中、高剂量组分别按25、50、100 mg/kg BCA灌胃，1次/d，连续7周。给药结束后麻醉处死小鼠取材，测定肝指数和子宫指数，HE及Masson染色观察肝组织病理改变，生化法检测AST、ALT活性，ELISA法检测肝组织中IL-6、TNF-α水平，Western Blot法测定肝组织中Ⅰ型胶原蛋白（Collagen Ⅰ）、转化生长因子-β1（TGF-β1）、α-平滑肌肌动蛋白（α-SMA）、雌激素受体β（ERβ）、p-NF-κBp65/NF-κBp65蛋白的相对表达量。计量资料两组间比较采用成组t检验，多组间比较采用单因素方差分析；进一步两两比较采用LSD-t检验。非正态分布的计量资料多组间比较及进一步两两比较均采用Kruskal-Wallis H检验。 结果 与假手术组相比，模型组肝指数升高，子宫指数降低，血清AST和ALT活性、肝组织中IL-6、TNF-α水平及Collagen Ⅰ、TGF-β1、α-SMA、p-NF-κBp65/NF-κBp65蛋白表达均升高（P值均<0.05），肝组织ERβ表达变化不明显（P>0.05），模型组肝组织明显纤维化病变，出现肝细胞水肿、脂肪样变、坏死并伴有炎细胞浸润，胶原纤维增生沉积、交错分布。与模型组比较，BCA各剂量组肝指数降低，血清ALT和AST活性、肝组织IL-6、TNF-α水平及Collagen Ⅰ、TGF-β1、α-SMA、p-NF-κBp65/NF-κBp65的蛋白表达均降低（P值均<0.05），子宫指数无明显变化（P>0.05），肝组织ERβ蛋白表达升高（P<0.05），肝组织的纤维化病变有不同程度改善。 结论 BCA可有效改善CCl4诱导的雌性去势小鼠肝纤维化，其作用机制可能是通过上调ERβ抑制NF-κB信号通路，减轻炎症反应而实现的。 Abstract:Objective To investigate the effect of phytoestrogen biochanin A （BCA） on liver fibrosis induced by CCl4 in female mice with bilateral oophorectomy （ovariectomized） and its mechanism. Methods A total of 50 ovariectomized Kunming mice were selected and given intraperitoneal injection of CCl4 to establish a model of liver fibrosis， and then according to body weight， they were randomly divided into model group， positive control group， and low-， middle-， and high-dose BCA groups， with 10 mice in each group. In addition， 10 female mice in the same litter were given resection of a small amount of adipose tissue near both ovaries to establish the sham-operation group. The mice in the positive control group were given estradiol 2 mg/kg by gavage， and those in the low-， middle-， and high-dose BCA groups were given BCA by gavage at a dose of 25， 50， and 100 mg/kg， respectively， once a day for 7 consecutive weeks； the mice in the sham-operation group and the model group were given an equal volume of 0.5% sodium carboxymethyl cellulose solution by gavage. The mice were anesthetized and sacrificed after administration to collect samples. Liver index and uterus index were measured； HE staining and Masson staining were used to observe liver histopathological changes； the biochemical analysis was used to measure the activity of aspartate aminotransferase （AST） and alanine aminotransferase （ALT）； ELISA was used to measure the levels of interleukin-6 （IL-6） and tumor necrosis factor-α （TNF-α） in liver tissue， and Western blot was used to measure the relative protein expression levels of collagen Ⅰ， transforming growth factor-beta 1 （TGF-β1）， alpha-smooth muscle actin （α-SMA）， estrogen receptor beta （ERβ）， and p-NF-κBp65/NF-κBp65 in liver tissue. The t-test was used for comparison of continuous data between two groups； a one-way analysis of various was used for comparison between multiple groups， and the least significant difference t-test was used for further comparison between two groups. The Kruskal-Wallis H test was used for comparison of non-normally distributed continuous data between multiple groups and further comparison between two groups. Results Compared with the sham-operated group， the model group had a significant increase in liver index and a significant reduction in uterus index， as well as significant increases in the activities of serum AST and ALT， the levels of IL-6 and TNF-α in liver tissue， and the protein expression levels of collagen Ⅰ， TGF-β1， α-SMA， and p-NF-κBp65/NF-κBp65 in liver tissue （all P<0.05）， with no significant change in the expression of ERβ in liver tissue （P>0.05）， and the model group showed significant fibrosis lesions in the liver， such as hepatocyte edema， steatosis， and necrosis with inflammatory cell infiltration and hyperplasia， deposition， and staggered distribution of collagen fibers. Compared with the model group， the low-， middle-， and high-dose BCA groups had significant reductions in liver index， the activities of serum AST and ALT， the levels of IL-6 and TNF-α， and the protein expression levels of collagen Ⅰ， TGF-β1， α-SMA， and p-NF-κBp65/NF-κBp65 in liver tissue （all P<0.05）， with no significant change in uterine index （P>0.05）， as well as a significant increase in the protein expression level of ERβ in liver tissue （P<0.05） and varying degrees of improvement in liver fibrosis lesions. Conclusion BCA can effectively improve CCL4-induced liver fibrosis in ovariectomized female mice， possibly by upregulating ERβ to inhibit the NF-κB signaling pathway and then alleviating inflammatory response. -
Key words:
- Hepatic Fibrosis /
- Phytoestrogens /
- Ovariectomy /
- Mice
-
图 1 BCA对CCl4诱导肝纤维化小鼠子宫、肝脏形态学及肝脏病理的影响
Figure 1. Effects of BCA on the morphological and pathological changes of uterus and liver in CCl4-induced liver fibrosis mice
图 2 BCA对CCl4诱导肝纤维化小鼠蛋白表达的影响
Figure 2. Effects of BCA on protein expressions in CCl4-induced liver fibrosis mice
表 1 BCA对CCl4诱导肝纤维化小鼠肝、子宫指数的影响
Table 1. Effects of BCA on liver and uterus weight ratios in CCl4-induced liver fibrosis mice
组别 动物数（只） 肝指数 子宫指数 假手术组 6 3.63%±0.35% 0.34%（0.27%～0.58%） 模型组 6 5.29%±0.47%1） 0.09%（0.05%～0.34%）1） 阳性对照组 6 4.97%±0.25% 0.49%（0.45%～0.65%）2） BCA低剂量组 6 4.51%±0.43%2） 0.11%（0.06%～0.33%） BCA中剂量组 6 4.54%±0.44%2） 0.08%（0.05%～0.32%） BCA高剂量组 6 4.64%±0.20%2） 0.10%（0.09%～0.11%） 统计值 F=19.942 H=24.059 P值 <0.000 1 <0.000 1 注：与假手术组比较，1）P<0.05；与模型组比较，2）P<0.05。 
下载: 导出CSV
表 2 BCA对CCl4诱导肝纤维化小鼠AST、ALT活性的影响
Table 2. Effects of BCA on serum activities of AST and ALT in CCl4-induced liver fibrosis mice
组别 动物数（只） AST（U/L） ALT（U/L） 假手术组 10 14.92±2.21 7.81±0.61 模型组 10 43.65±17.711） 32.44±9.141） 阳性对照组 10 20.56±5.322） 14.96±6.232） BCA低剂量组 10 21.17±3.872） 17.22±4.032） BCA中剂量组 10 20.72±6.482） 16.70±5.882） BCA高剂量组 10 19.66±4.682） 14.97±2.962） F值 13.703 19.488 P值 <0.000 1 <0.000 1 注：与假手术组比较，1）P<0.05；与模型组比较，2）P<0.05。
下载: 导出CSV
表 3 BCA对CCl4诱导肝纤维化小鼠IL-6、TNF-α水平的影响
Table 3. Effects of BCA on serum contents of IL-6 and TNF-α in CCl4-induced liver fibrosis mice
组别 动物数（只） IL-6（pg/mg） TNF-α（pg/mg） 假手术组 10 107.43±21.23 37.01±12.16 模型组 10 368.39±76.411） 94.49±26.511） 阳性对照组 10 123.40±43.222） 43.05±11.702） BCA低剂量组 10 208.26±37.812） 70.77±9.782） BCA中剂量组 10 172.18±55.652） 53.43±14.382） BCA高剂量组 10 113.15±17.512） 40.63±17.442） F值 31.623 12.645 P值 <0.000 1 <0.000 1 注：与假手术组比较，1）P<0.05；与模型组比较，2）P<0.05。
下载: 导出CSV
表 4 BCA对CCl4诱导肝纤维化小鼠肝组织中蛋白表达的影响
Table 4. Effects of BCA on protein expressions in CCl4-induced liver fibrosis mice
组别 动物数（只） α-SMA TGF-β1 Collagen Ⅰ ERβ p-NF-κBp65/NF-κBp65 假手术组 3 0.15±0.02 0.28±0.06 0.31±0.14 0.63±0.12 0.45±0.13 模型组 3 0.39±0.051） 0.72±0.071） 0.60±0.021） 0.59±0.13 0.90±0.211） 阳性对照组 3 0.26±0.102） 0.45±0.062） 0.42±0.032） 0.94±0.272） 0.41±0.182） BCA低剂量组 3 0.26±0.082） 0.56±0.092） 0.47±0.022） 0.83±0.15 0.64±0.12 BCA中剂量组 3 0.19±0.062） 0.49±0.082） 0.43±0.022） 0.95±0.152） 0.45±0.092） BCA高剂量组 3 0.18±0.032） 0.47±0.062） 0.42±0.032） 0.98±0.152） 0.53±0.082） F值 5.850 12.057 6.895 3.207 5.018 P值 <0.006 <0.0001 <0.003 <0.046 <0.01 注：与假手术组比较，1）P<0.05；与模型组比较，2）P<0.05。
下载: 导出CSV
-
[1] LI MB, LI JY, FENG DP. Research advances in the reversal of liver fibrosis[J]. J Clin Hepatol, 2023, 39( 1): 193- 198. DOI: 10.3969/j.issn.1001-5256.2023.01.030.李满彪, 李金玉, 冯对平. 肝纤维化逆转的研究进展[J]. 临床肝胆病杂志, 2023, 39( 1): 193- 198. DOI: 10.3969/j.issn.1001-5256.2023.01.030. [2] AYDIN MM, AKÇALI KC. Liver fibrosis[J]. Turk J Gastroenterol, 2018, 29( 1): 14- 21. DOI: 10.5152/tjg.2018.17330. [3] XU LM, LIU P, Guidelines for diagnosis and treatment of hepatic fibrosis with integrated traditional Chinese and Western medicine(2019 edition)[J]. J Integr Med, 2020, 18( 3): 203- 213. DOI: 10.1016/j.joim.2020.03.001. [4] YOON YJ, FRIEDMAN SL, LEE YA. Antifibrotic therapies: Where are we now?[J]. Semin Liver Dis, 2016, 36( 1): 87- 98. DOI: 10.1055/s-0036-1571295. [5] NASTA P. Immune activation, aging and gender and progression of liver disease[J]. Acta Biomed, 2011, 82( 2): 115- 123. [6] QIAO BK, MA XY, HAN LY, et al. Estrogen enhances liver repair by regulating hepatic cell regeneration[J]. J Southwest Univ Nat Sci Ed, 2020, 42( 8): 49- 58. DOI: 10.13718/j.cnki.xdzk.2020.08.007.乔冰珂, 马兴宇, 韩凌云, 等. 雌激素通过调控肝细胞更新促进CCl4诱导的肝损伤修复[J]. 西南大学学报(自然科学版), 2020, 42( 8): 49- 58. DOI: 10.13718/j.cnki.xdzk.2020.08.007. [7] CODES L, ASSELAH T, CAZALS-HATEM D, et al. Liver fibrosis in women with chronic hepatitis C: Evidence for the negative role of the menopause and steatosis and the potential benefit of hormone replacement therapy[J]. Gut, 2007, 56( 3): 390- 395. DOI: 10.1136/gut.2006.101931. [8] YANG WW, LU Y, XU YC, et al. Estrogen represses hepatocellular carcinoma(HCC) growth via inhibiting alternative activation of tumor-associated macrophages(TAMs)[J]. J Biol Chem, 2012, 287( 48): 40140- 40149. DOI: 10.1074/jbc.M112.348763. [9] XU JW, GONG J, FENG XL, et al. Effects of estrogen on the activation of hepatic stellate cells and collagen synthesis in liver fibrotic rats[J]. J Xi'an Jiaotong Univ Med Sci, 2003, 24( 6): 624- 626. DOI: 10.3969/j.issn.1671-8259.2003.06.032.许君望, 龚均, 冯新利, 等. 雌激素对肝纤维化大鼠肝星状细胞活化及胶原合成的影响[J]. 西安交通大学学报(医学版), 2003, 24( 6): 624- 626. DOI: 10.3969/j.issn.1671-8259.2003.06.032. [10] ROSSOUW JE, ANDERSON GL, Prentice RL, et al. Risks and benefits of estrogen plus progestin in healthy postmenopausal women: principal results From the Women's Health Initiative randomized controlled trial[J]. JAMA, 2002, 288( 3): 321- 33. DOI: 10.1001/jama.288.3.321 [11] WANG H, ZHUANG W, XUE XO. Research progress and clinical application of phytoestrogen activity in traditional Chinese Medicine[J]. Jilin J Chin Med, 2018, 38( 3): 364- 368. DOI: 10.13463/j.cnki.jlzyy.2018.03.033.王浩, 庄威, 薛晓鸥. 中药植物雌激素活性研究及其临床应用研究进展[J]. 吉林中医药, 2018, 38( 3): 364- 368. DOI: 10.13463/j.cnki.jlzyy.2018.03.033. [12] WANG YX, WU CY, ZHOU JH, et al. Overexpression of estrogen receptor β inhibits cellular functions of human hepatic stellate cells and promotes the anti-fibrosis effect of calycosin via inhibiting STAT3 phosphorylation[J]. BMC Pharmacol Toxicol, 2022, 23( 1): 77. DOI: 10.1186/s40360-022-00617-y. [13] DENG T, LIU J, ZHANG MM, et al. Inhibition effect of phytoestrogen calycosin on TGF-β1-induced hepatic stellate cell activation, proliferation, and migration via estrogen receptor B[J]. Can J Physiol Pharmacol, 2018, 96( 12): 1268- 1275. DOI: 10.1139/cjpp-2018-0474. [14] LI CW, WEI YL. Advances in studies on the characteristics and application of phytoestrogens[J]. Genom Appl Biol, 2020, 39( 3): 1264- 1269. DOI: 10.13417/j.gab.039.001264.李从文, 魏云林. 植物雌激素的特性及其应用研究进展[J]. 基因组学与应用生物学, 2020, 39( 3): 1264- 1269. DOI: 10.13417/j.gab.039.001264. [15] ZHAO Y, ZHENG HX, XU Y, et al. Research progress in phytoestrogens of traditional Chinese medicine[J]. China J Chin Mater Med, 2017, 42( 18): 3474- 3487. DOI: 10.19540/j.cnki.cjcmm.2017.0135.赵元, 郑红霞, 徐颖, 等. 中药植物雌激素的研究进展[J]. 中国中药杂志, 2017, 42( 18): 3474- 3487. DOI: 10.19540/j.cnki.cjcmm.2017.0135. [16] CHENG HL, WANG L, LUO Q, et al. Advances in studies on pharmacological effects of biochanin A[J]. J Liaoning Univ Tradit Chin Med, 2014, 16( 12): 92- 95. DOI: 10.13194/j.issn.1673-842x.2014.12.034.程海林, 王雷, 罗晴, 等. 鹰嘴豆芽素A基础药理作用研究进展[J]. 辽宁中医药大学学报, 2014, 16( 12): 92- 95. DOI: 10.13194/j.issn.1673-842x.2014.12.034. [17] ZHOU YM, NING HX, ZHANG XM, et al. Effects of biochanin A for osteoporosis in ovariecto-mized rats[J]. Chinese Pharmacological Bulletin, 2014, 30( 12): 1775- 1776. DOI: 10.3969/j.issn.1001-1978.2014.12.032.周延萌, 宁慧娴, 张小敏, 等. 鹰嘴豆芽素A对雌激素缺乏诱发大鼠骨质疏松症的作用[J]. 中国药理学通报, 2014, 30( 12): 1775- 1776. DOI: 10.3969/j.issn.1001-1978.2014.12.032. [18] KŘÍŽOVÁ L, DADÁKOVÁ K, KAŠPAROVSKÁ J, et al. Isoflavones[J]. Molecules, 2019, 24( 6): 1076. DOI: 10.3390/molecules24061076. [19] FAN Y, YAN LT, YAO Z, et al. Biochanin A regulates cholesterol metabolism further delays the progression of nonalcoholic fatty liver disease[J]. Diabetes Metab Syndr Obes, 2021, 14: 3161- 3172. DOI: 10.2147/DMSO.S315471. [20] TANG AC, WEI YF, LIU XH, et al. Protective effect and mechanism of tadehaginoside on hepatic fibrosis model mice induced by carbon tetrachloride[J]. China Pharm, 2020, 31( 2): 190- 195. DOI: 10.6039/j.issn.1001-0408.2020.02.12.唐爱存, 韦燕飞, 刘喜华, 等. 葫芦茶苷对四氯化碳致肝纤维化模型小鼠的保护作用及机制研究[J]. 中国药房, 2020, 31( 2): 190- 195. DOI: 10.6039/j.issn.1001-0408.2020.02.12. [21] SUN HT, CHEN GX, WEN B, et al. Oligo-peptide I-C-F-6 inhibits hepatic stellate cell activation and ameliorates CCl4-induced liver fibrosis by suppressing NF-κB signaling and Wnt/β-catenin signaling[J]. J Pharmacol Sci, 2018, 136( 3): 133- 141. DOI: 10.1016/j.jphs.2018.01.003. [22] WANG ZR, TIAN FH, RAO MY, et al. Research progress of mice models for hepatic fibrosis[J]. China J Mod Med, 2021, 31( 12): 46- 50. DOI: 10.3969/j.issn.1005-8982.2021.12.009.王梓睿, 田飞鸿, 饶木艳, 等. 小鼠肝纤维化模型复制方法的研究进展[J]. 中国现代医学杂志, 2021, 31( 12): 46- 50. DOI: 10.3969/j.issn.1005-8982.2021.12.009. [23] HUANG FJ, ZHANG JJ, DONG L, et al. Preliminary study on improvement effect of Tiarella polyphylla ethanol extract on CCl4-induced hepatic fibrosis in mice and its mechanism[J]. China Pharm, 2021, 32( 14): 1685- 1691. DOI: 10.6039/j.issn.1001-0408.2021.14.04.黄甫静, 张金娟, 董莉, 等. 黄水枝醇提物对CCl4致小鼠肝纤维化的改善作用及其机制初探[J]. 中国药房, 2021, 32( 14): 1685- 1691. DOI: 10.6039/j.issn.1001-0408.2021.14.04. [24] JIANG Y, XIANG C, ZHONG F, et al. Histone H3K27 methyltransferase EZH2 and demethylase JMJD3 regulate hepatic stellate cells activation and liver fibrosis[J]. Theranostics, 2021, 11( 1): 361- 378. DOI: 10.7150/thno.46360. [25] XIE JF. Effect and mechanism of CircRNA97 on reversing hepatic fibrosis through miRNA-146b-5p/HIPK1 axis[D]. Nanchang: Nanchang University, 2022. DOI: 10.27232/d.cnki.gnchu.2022.000617.谢俊锋. CircRNA97通过miRNA-146b-5p/HIPK1轴逆转肝纤维化的作用及机制研究[D]. 南昌: 南昌大学, 2022. DOI: 10.27232/d.cnki.gnchu.2022.000617 [26] SUN ZC, ZHAN XL. Myrrhone inhibits the progression of hepatic fibrosis by regulating the abnormal activation of hepatic stellate cells[J]. J Biochem Mol Toxicol, 2022, 36( 11): e23177. DOI: 10.1002/jbt.23177. [27] LIN IY, CHIOU YS, WU LC, et al. CCM111 prevents hepatic fibrosis via cooperative inhibition of TGF-β, Wnt and STAT3 signaling pathways[J]. J Food Drug Anal, 2019, 27( 1): 184- 194. DOI: 10.1016/j.jfda.2018.09.008. [28] YANG JH, JU BW, XIANG XY, et al. Mechanism of inhibition of hepatic stellate cell activation by TGF-β1/Smad pathway regulated by Gentianella turkestanorum[J]. China J Tradit Chin Med Pharm, 2022, 37( 12): 7392- 7396.杨建华, 居博伟, 向雪滢, 等. 新疆假龙胆调控TGF-β1/Smad通路抑制肝星状细胞活化的作用机制[J]. 中华中医药杂志, 2022, 37( 12): 7392- 7396. [29] GE SF, YANG LX. Progress in the role of inflammation during the development of liver fibrosis[J]. Chin Hepatol, 2018, 23( 6): 546- 548. DOI: 10.14000/j.cnki.issn.1008-1704.2018.06.029.葛善飞, 杨丽霞. 炎症在肝纤维化发生中的作用研究进展[J]. 肝脏, 2018, 23( 6): 546- 548. DOI: 10.14000/j.cnki.issn.1008-1704.2018.06.029. [30] CUBERO FJ. Shutting off inflammation: A novel switch on hepatic stellate cells[J]. Hepatology, 2016, 63( 4): 1086- 1089. DOI: 10.1002/hep.28442. [31] ZHU H, PING J, XU LM. Role of the nuclear factor-kappa B signaling pathway on the progress of hepatic fibrosis and the anti-fibrotic mechanism of traditional Chinese medicine[J]. J Clin Hepatol, 2018, 34( 4): 858- 861. DOI: 10.3969/j.issn.1001-5256.2018.04.035.朱慧, 平键, 徐列明. NF-κB通路在肝纤维化进展和中药抗肝纤维化机制中的作用[J]. 临床肝胆病杂志, 2018, 34( 4): 858- 861. DOI: 10.3969/j.issn.1001-5256.2018.04.035. [32] LONG TT, LIU ZJ, SHANG JC, et al. Polygonatum sibiricum polysaccharides play anti-cancer effect through TLR4-MAPK/NF-κB signaling pathways[J]. Int J Biol Macromol, 2018, 111: 813- 821. DOI: 10.1016/j.ijbiomac.2018.01.070. [33] TU KF, XU JY, YI ZZ. Relationship of the expression of estrogen receptor β and nuclear factor-kappa B of triple negative breast cancer[J]. J Mod Oncol, 2016, 24( 7): 1042- 1045. DOI: 10.3969/j.issn.1672-4992.2016.07.006.涂开峰, 徐久元, 易子桢. 三阴性乳腺癌中ERβ和NF-κB的表达及其相关性[J]. 现代肿瘤医学, 2016, 24( 7): 1042- 1045. DOI: 10.3969/j.issn.1672-4992.2016.07.006. [34] YAO Y. The role of ERβ in the occurrence and development of breast cancer through NF-κB/IL-8 signaling pathway[D]. Qingdao: Qingdao University, 2022. DOI: 10.27262/d.cnki.gqdau.2022.001348.姚瑶. ERβ通过NF-κB/IL-8信号通路在乳腺癌发生发展中作用探讨[D]. 青岛: 青岛大学, 2022. DOI: 10.27262/d.cnki.gqdau.2022.001348 -
本文二维码
图(2) / 表(4)
计量
- 文章访问数: 110
- HTML全文浏览量: 32
- PDF下载量: 25
- 被引次数: 0
