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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
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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
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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。 
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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。
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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。
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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。
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