Wu Jianjun, Zhang Dingqi, Zhu Bo, Wang Siqi, Xu Yongbin, Zhang Congcong, Yang Hailing, Wang Shunchun, Liu Ping, Qin Luping, Liu Wei
College of Pharmaceutical Sciences, Zhejiang Chinese Medical University, Hangzhou, 310053, Zhejiang, China.
Key Laboratory of Liver and Kidney Diseases (Ministry of Education), Institute of Liver Diseases, Shuguang Hospital, Shanghai University of Traditional Chinese Medicine, 528 Zhangheng Road, Shanghai, 201203, China.
Chin Med. 2022 May 12;17(1):56. doi: 10.1186/s13020-022-00607-6.
The unripe fruits of Rubus chingii Hu. ("Fu-peng-zi" in Chinese) is a well-known herbal tonic in traditional Chinese medicine (TCM) for tonifying liver and kidney. However, little is known regarding its therapeutic efficacy against liver fibrosis and the underlying mechanism.
The current research aims to explore the potential of Rubus chingii Hu. unripe fruits extract (RF) in the treatment of liver fibrosis and explore the underlying mechanism. RF was administered (450 and 900 mg·kg of body weight per day) orally to male C57BL/6 mice with CCl-induced liver fibrosis for 3 weeks. The histopathological changes and fibrosis stage in liver tissue were assessed using hematoxylin and eosin (H&E) and Sirius red staining. The distribution of α-SMA and Col1A1 in the liver was analyzed to determine the hepatic stellate cells (HSCs) activation using immunohistochemistry and immunofluorescent analysis. Various biochemical markers in serum (ALT, AST) and liver (Hyp, IL1-β, IL6, TNF-α and MCP-1) were observed to assess the liver's injury, fibrosis, and inflammation. In liver tissue, fibrosis-associated proteins including α-SMA, TGF-β1, Smad2/3, p-Smad2/3, and Smad4 were detected through a Western blot assay. Pyrosequencing-based analysis of bacterial 16 S ribosomal RNA from variable regions V3-V4 of fecal samples characterized the gut microbiota. Spearman's rank correlation analysis was performed for the association between altered bacterial genera by RF and pharmacodynamics parameters.
Three weeks of RF treatment can significantly lower liver inflammatory levels, pathological abnormalities, and collagen fibrous deposition in mice with CCl-induced liver fibrosis. The expressions of α-SMA and Col1A1 were lowered by RF, while the expression levels of TGF-β/Smads signaling pathway-related proteins, including TGF-β1, p-Smad2/3, and Smad4, were dramatically decreased by RF. The RF treatment significantly increased or reduced 18 different bacterial species, restoring the CCl-induced gut microbiota imbalance to the normal group's levels. According to correlation analysis, the bacterial genera Bifidobacterium and Turicibacter were the most significant in restoring CCl-induced liver fibrosis.
RF can reduce liver damage and delay the onset of liver fibrosis through modulating TGF-β/Smads signaling pathway. Furthermore, RF's anti-liver fibrosis effect was related to balancing the gut microbial community, partly attained by increasing Bifidobacterium and Turicibacter in liver fibrosis.
掌叶覆盆子（中药名为“覆盆子”）的未成熟果实是传统中药中一种著名的滋补肝肾的草药。然而，关于其抗肝纤维化的治疗效果及潜在机制知之甚少。
本研究旨在探讨掌叶覆盆子未成熟果实提取物（RF）治疗肝纤维化的潜力并探究其潜在机制。将RF（每天450和900mg·kg体重）口服给予CCl4诱导肝纤维化的雄性C57BL/6小鼠，持续3周。使用苏木精和伊红（H&E）染色及天狼星红染色评估肝组织的组织病理学变化和纤维化阶段。采用免疫组织化学和免疫荧光分析，分析肝脏中α-SMA和Col1A1的分布，以确定肝星状细胞（HSCs）的激活情况。观察血清（ALT、AST）和肝脏（Hyp、IL1-β、IL6、TNF-α和MCP-1）中的各种生化标志物，以评估肝脏损伤、纤维化和炎症。通过蛋白质免疫印迹法检测肝组织中包括α-SMA、TGF-β1、Smad2/3、p-Smad2/3和Smad4在内的纤维化相关蛋白。基于焦磷酸测序分析粪便样本可变区V3-V4的细菌16S核糖体RNA，对肠道微生物群进行特征分析。对RF改变的细菌属与药效学参数之间的关联进行Spearman等级相关分析。
RF治疗3周可显著降低CCl4诱导的肝纤维化小鼠的肝脏炎症水平、病理异常和胶原纤维沉积。RF降低了α-SMA和Col1A1的表达，同时显著降低了TGF-β/Smads信号通路相关蛋白（包括TGF-β1、p-Smad2/3和Smad4）的表达水平。RF治疗显著增加或减少了18种不同的细菌种类，将CCl4诱导的肠道微生物群失衡恢复到正常组水平。根据相关性分析，双歧杆菌属和Turicibacter属在恢复CCl4诱导的肝纤维化方面最为显著。
RF可通过调节TGF-β/Smads信号通路减轻肝脏损伤并延缓肝纤维化的发生。此外，RF的抗肝纤维化作用与平衡肠道微生物群落有关，部分是通过增加肝纤维化中的双歧杆菌属和Turicibacter属来实现的。
