背景:人参(PanaxginsengC.A.Meyer)是一种具有多种药理作用的珍贵中药。人参皂苷Rg1是从人参中提取的主要活性成分,以延缓衰老和抗氧化作用而闻名。越来越多的证据表明,Rg1在许多疾病中表现出抗炎特性,并且可以改善许多慢性肝病中的氧化损伤和炎症。
目的:肝细胞慢性炎症损伤是许多肝脏疾病的重要病理基础。然而,其机制尚不清楚,需要进一步探讨预防其发展的治疗策略.因此,本研究旨在探讨Rg1对脂多糖(LPS)诱导的慢性肝脏炎症损伤的保护作用及其机制。
方法:通过腹腔注射LPS(200μg/kg)21天建立小鼠慢性肝损伤模型。血清肝功能指标与IL-1β水平,使用相应的试剂盒检测IL-6和TNF-α。苏木精和伊红(H&E),周期酸希夫(PAS),和Masson染色用于可视化肝组织病理学损伤,糖原沉积,和肝纤维化。通过IF检测肝脏中p-Nrf2的核输入和Col4的产生,同时采用IHC检测NLRP3和AIM2在肝脏中的表达。Westernblot和q-PCR检测纤维化和凋亡相关蛋白和mRNA的表达,Keap1,p-Nrf2和NLRP3,NLRP1,AIM2炎性体相关蛋白在小鼠肝脏中的表达。细胞计数试剂盒-8检测人肝癌细胞(HepG2)的细胞活力,选择LPS的作用浓度,使用试剂盒检测细胞内ROS的产生。Westernblot检测HepG2细胞核Nrf2、HO-1、NQO1和NLRP3、NLRP1、AIM2炎性体相关蛋白的表达。最后,通过分子对接证明了Rg1和Nrf2分子互连的可行性。
结果:Rg1治疗21天降低了ALT水平,AST,血清IL-1β和炎症因子,LPS诱导小鼠IL-6和TNF-α的表达。病理结果提示Rg1治疗能明显减轻肝细胞损伤和凋亡,LPS刺激小鼠的炎症细胞浸润和肝纤维化。Rg1促进了LPS引起的肝脏中Keap1的降解,并增强了p-Nrf2,HO-1的表达,并降低了NLRP1,NLRP3,AIM2,caspase-1,IL-1β和IL-6的水平。此外,Rg1有效抑制LPS诱导的HepG2细胞中ROS的升高,而Nrf2的抑制逆转了Rg1在减少LPS刺激的HepG2细胞中ROS和NLRP3,NLRP1和AIM2表达的产生中的作用。最后,分子对接表明Rg1对Nrf2表现出强亲和力。
结论:结果表明,Rg1对LPS诱导的慢性肝损伤和纤维化有明显的改善作用。其机制可能是通过促进Nrf2与Keap1的解离,然后激活Nrf2信号,进一步抑制肝细胞中的NLRP3,NLRP1和AIM2炎性体介导的。
BACKGROUND: Ginseng (Panax ginseng C. A. Meyer) is a precious traditional Chinese medicine with multiple pharmacological effects. Ginsenoside Rg1 is a main active ingredient extracted from ginseng, which is known for its age-delaying and antioxidant effects. Increasing evidence indicates that Rg1 exhibits anti-inflammatory properties in numerous diseases and may ameliorate oxidative damage and inflammation in many chronic liver diseases.
OBJECTIVE: Chronic inflammatory injury in liver cells is an important pathological basis of many liver diseases. However, its mechanism remains unclear and therapeutic strategies to prevent its development need to be further explored. Thus, our study is to delve the protective effect and mechanism of Rg1 against chronic hepatic inflammatory injuries induced by lipopolysaccharide (LPS).
METHODS: The chronic liver damage model in mice was build up by injecting intraperitoneally with LPS (200 μg/kg) for 21 days. Serum liver function indicators and levels of IL-1β, IL-6 and TNF-α were examined by using corresponding Kits. Hematoxylin and Eosin (H&E), Periodic acid-Schiff (PAS), and Masson stains were utilized to visualize hepatic histopathological damage, glycogen deposition, and liver fibrosis. The nuclear import of p-Nrf2 and the generation of Col4 in the liver were detected by IF, while IHC was employed to detect the expressions of NLRP3 and AIM2 in the hepatic. The Western blot and q-PCR were used to survey the expressions of proteins and mRNAs of fibrosis and apoptosis, and the expressions of Keap1, p-Nrf2 and NLRP3, NLRP1, AIM2 inflammasome-related proteins in mouse liver. The cell viability of human hepatocellular carcinoma cells (HepG2) was detected by Cell Counting Kit-8 to select the action concentration of LPS, and intracellular ROS generation was detected using a kit. The expressions of Nuclear Nrf2, HO-1, NQO1 and NLRP3, NLRP1, and AIM2 inflammasome-related proteins in HepG2 cells were detected by Western blot. Finally, the feasibility of the molecular interlinking between Rg1 and Nrf2 was demonstrated by molecular docking.
RESULTS: Rg1 treatment for 21 days decreased the levels of ALT, AST, and inflammatory factors of serum IL-1β, IL-6 and TNF-α in mice induced by LPS. Pathological results indicated that Rg1 treatment obviously alleviated hepatocellular injury and apoptosis, inflammatory cell infiltration and liver fibrosis in LPS stimulated mice. Rg1 promoted Keap1 degradation and enhanced the expressions of p-Nrf2, HO-1 and decreased the levels of NLRP1, NLRP3, AIM2, cleaved caspase-1, IL-1β and IL-6 in livers caused by LPS. Furthermore, Rg1 effectively suppressed the rise of ROS in HepG2 cells induced by LPS, whereas inhibition of Nrf2 reversed the role of Rg1 in reducing the production of ROS and NLRP3, NLRP1, and AIM2 expressions in LPS-stimulated HepG2 cells. Finally, the molecular docking illustrated that Rg1 exhibits a strong affinity towards Nrf2.
CONCLUSIONS: The findings indicate that Rg1 significantly ameliorates chronic liver damage and fibrosis induced by LPS. The mechanism may be mediated through promoting the dissociation of Nrf2 from Keap1 and then activating Nrf2 signaling and further inhibiting NLRP3, NLRP1, and AIM2 inflammasomes in liver cells.