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化痰祛湿活血方对非酒精性脂肪性肝炎大鼠模型肝细胞线粒体超微结构的影响
DOI: 10.3969/j.issn.1001-5256.2022.08.012
伦理学声明：本研究方案于2016年7月经由河南中医药大学审批，批号: DWLL15010018，符合实验室动物管理与使用准则，通过河南中医药大学动物伦理审查。
利益冲突声明：本研究不存在研究者、伦理委员会成员以及与公开研究成果有关的利益冲突。
作者贡献声明：赵文霞负责课题设计；刘素彤负责资料分析，撰写论文；张丽慧参与收集数据，修改论文；刘鸣昊负责拟定写作思路；顾亚娇、郭敏、赵晨露负责指导撰写文章并最后定稿。
Effect of Huatan Qushi Huoxue prescription on the ultrastructure of hepatocyte mitochondria in rats with nonalcoholic steatohepatitis
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摘要:
目的 探讨化痰祛湿活血方对非酒精性脂肪性肝炎大鼠模型肝细胞形态的影响。 方法 48只雄性SD大鼠随机分为空白组、模型组、易善复组、化痰祛湿活血方组，每组12只。模型组和药物组大鼠第2周开始给药及造模，空白组给予普通饲料，另外3组给予高脂饲料。依据人与动物剂量折算法：化痰祛湿活血方等效剂量为1.26 g/100 g体质量，多烯磷脂酰胆碱胶囊(易善复)等效剂量为0.014 18 g/100 g体质量。模型组灌胃0.9% 氯化钠溶液，易善复组灌胃多烯磷脂酰胆碱混悬液，中药组给予灌胃化痰祛湿活血方颗粒混悬液，1次/d，连续10周。透射电子显微镜观察各组大鼠肝脏超微结构级定量分析。计量资料多组间比较采用单因素方差分析，进一步两两比较，方差齐者采用LSD-t检验，方差不齐者采用Dunnett’s T3检验。 结果 模型组可见肝细胞内有大量脂滴积累，线粒体形态和结构改变，数量减少，内质网数量减少。化痰祛湿活血方组肝细胞内脂滴明显减少，线粒体和内质网数量较模型组明显增加，线粒体膜及结构完整。易善复组肝细胞脂滴相对减少，线粒体数量增加，内质网数量减少，线粒体膜及结构相对完整。定量分析结果显示，与空白组相比，模型组脂滴面积明显增多，线粒体减少，体密度比较差异均有统计学意义(P值均＜0.01)；药物干预后，易善复组脂滴面积减少、线粒体数目增多，与模型组体密度比较差异均有统计学意义(P值均＜0.01)；中药组较易善复组脂滴面积减少更多、线粒体数目明显增加，体密度比较差异均有统计学意义(P值均＜0.05)。 结论 化痰祛湿活血方可改善脂质积累，增加线粒体体密度，保护线粒体结构和功能且疗效高于易善复。 -
关键词:
- 非酒精性脂肪性肝病 /
- 化痰祛湿活血方 /
- 线粒体, 肝 /
- 大鼠, Sprague-Dawley
Abstract:Objective To investigate the effect of Huatan Qushi Huoxue prescription on the ultrastructure of hepatocyte mitochondria in a rat model of nonalcoholic steatohepatitis (NASH). Methods A total of 48 male Sprague-Dawley rats were randomly divided into blank group, model group, Yishanfu group, and Huatan Qushi Huoxue prescription group, with 12 rats in each group. The rats in the model group and the drug groups were administered and modeled since week 2; the rats in the blank group were given normal diet, and those in the other three groups were given high-fat diet. Based on dose conversion between human and animal, the equivalent dose of Huatan Qushi Huoxue prescription was 1.26 g/100 g body weight, and the equivalent dose of polyene phosphatidylcholine capsules (Yishanfu) was 0.014 18 g/100 g body weight. The rats in the model group were given 0.9% sodium chloride by gavage, those in the Yishanfu group were given polyene phosphatidylcholine suspension by gavage, and those in the traditional Chinese medicine group were given the granules of Huatan Qushi Huoxue prescription by gavage, once a day for 10 consecutive weeks. A transmission electron microscope was used to observe liver ultrastructure and perform a quantitative analysis. A one-way analysis of variance was used for comparison of continuous data between multiple groups; for further pairwise comparison, the least significant difference t-test was used for data with homogeneity of variance, and the Dunnett's T3 was used for data with heterogeneity of variance. Results The model group had a large number of lipid droplets accumulated in hepatocytes, changes in mitochondrial morphology and structure, and reductions in the number of mitochondria and endoplasmic reticulum. The Huatan Qushi Huoxue prescription group had a significant reduction in lipid droplets in hepatocytes and significant increases in the number of mitochondria and endoplasmic reticulum compared with the model group, with intact mitochondrial membrane and structure. The Yishanfu group had a reduction in lipid droplets in hepatocytes, an increase in the number of mitochondria, and a reduction in the number of endoplasmic reticulum, with relatively intact mitochondrial membrane and structure. The quantitative analysis showed that compared with the blank group, the model group had a significant increase in the area of lipid droplets and a significant reduction in mitochondria, with a significant difference in mitochondrial density between the two groups (all P < 0.01); after drug intervention, the Yishanfu group had a significant reduction in the area of lipid droplets and a significant increase in the number of mitochondria, with a significant difference in mitochondrial density between the Yishanfu group and the model group (all P < 0.01); compared with the Yishanfu group, the traditional Chinese medicine group had a significantly greater reduction in the area of lipid droplets and a significant increase in the number of mitochondria, with a significant difference in mitochondrial density between the two groups (all P < 0.05). Conclusion Huatan Qushi Huoxue prescription can improve lipid accumulation, increase mitochondrial density, and protect mitochondrial structure and function, with a better clinical effect than Yishanfu. -
图 1 电镜下空白组大鼠肝细胞超微结构
注：a，空白组大鼠肝细胞形态正常(×5000)，箭头为脂滴；b，放大倍数后可见清晰完整线粒体及大量内质网(×50 000)，箭头为线粒体。
Figure 1. Ultrastructure of liver cells in blank group under electron microscope
图 2 电镜下模型组大鼠肝细胞超微结构
注：a, 模型组大鼠肝细胞内大量脂滴积累(×2500)，箭头为脂滴; b, 放大倍数后可见线粒体结构破坏、内质网数量减少(×50 000)，箭头为线粒体。
Figure 2. Ultrastructure of rat liver cells in model group under electron microscope
图 3 电镜下中药组大鼠肝细胞超微结构
注：a，中药组大鼠肝细胞内脂滴明显减少(×2500)；b，放大倍数后可见线粒体膜较完整(×50 000)，箭头为线粒体。
Figure 3. Ultrastructure of rat liver cells in Chinese medicine group under electron microscope
图 4 电镜下易善复组肝细胞超微结构
注：a, 对照组大鼠肝细胞内脂滴减少(×2500)，箭头为脂滴; b，放大倍数后可见线粒体结构较完整(×50 000)，箭头为线粒体。
Figure 4. Ultrastructure of Essentiale control liver cells under electron microscope
表 1 各组大鼠脂滴和线粒体体密度比较
Table 1. Lipid droplet and mitochondrial body density were compared in each group
组别 例数 脂滴体密度 线粒体体密度 空白组 30 0.12 ±0.10 49.76±2.19 模型组 30 42.11 ±2.341) 20.84±5.011) 易善复组 30 13.74 ±9.722) 32.18±2.922) 中药组 30 1.55 ±0.902)3) 34.02±2.962)3) F值 451.4 360.0 P值 ＜0.000 1 ＜0.000 1 注：与空白组相比，1)P＜0.01；与模型组相比，2)P＜0.01；与易善复组相比，3)P＜0.05。 
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