■作为中药配方,养阴活血汤(YYHXD)在临床上用于治疗肝纤维化。YYHXD的药理学特征包括作用于许多靶标和途径的多种组分,但是其功效背后的药理机制尚未得到彻底阐明。本研究旨在探讨YYHXD治疗肝纤维化的药理机制。
■制备YYYHXD水性提取物并使用HPLC-MS指纹图谱分析进行质量控制。建立CCl4诱导大鼠肝纤维化模型,动物被随机分为六组:对照组,低剂量YYHXD(L-YYHXD),中等剂量YYHXD(M-YYHXD),高剂量YYHXD(H-YYHXD),CCl4型号,和秋水仙碱组。治疗组的大鼠每天口服YYHXD(5、10或20g/kg)或秋水仙碱(0.2mg/kg),持续6周,对照组和模型组接受蒸馏水。组织学分析,包括苏木精和伊红(HE)和Masson三色染色,进行评估肝纤维化。血清生化标志物,如AST,ALT,HA,LN,被测量。炎症因子(IL-6和TNF-α)和氧化应激指标(SOD,GSH-Px,还评估了肝组织中的MDA)。此外,使用RNA测序进行转录组学分析,以鉴定对照之间的差异表达基因(DEG),CCl4型号,和H-YYHXD组。生物信息学分析,包括差异表达分析,蛋白质-蛋白质相互作用分析,和功能富集分析,进行了研究YYHXD的药理机制。油红O染色进一步证实了YYHXD对脂肪酸代谢和生物合成的调节作用,酶活性测定,qPCR,和西方印迹。Western印迹和免疫荧光染色也证实了AMPK信号通路参与肝纤维化的发生和进展。
■HE和Masson三色染色显示,与CCl4模型组相比,YYHXD组的胶原蛋白沉积减少,肝脏结构改善。血清生化标志物,包括AST,ALT,HA,LN,与CCl4模型组相比,YYHXD治疗组显著改善。炎症因子(IL-6和TNF-α)和氧化应激指标(降低SOD和GSH-Px,与CCl4模型组相比,YYHXD治疗可显着改善肝组织中MDA的增加。此外,从转录组数据中筛选出YYHXD治疗肝纤维化的96个基因,主要富含脂肪酸代谢和生物合成等生物途径,和AMPK信号通路。油红O染色显示YYHXD以剂量依赖性方式减少肝脂质积累,随着血清TG的降低,TC,和LDL-C水平。此外,qPCR和Westernblot分析表明,参与脂肪酸代谢和生物合成的关键酶的mRNA和蛋白质表达上调,Fasn和Fads2,由YYHXD调节。YYHXD还剂量依赖性地增强AMPK的磷酸化,如通过Western印迹和免疫荧光测定所证明的。
■YYHXD通过涉及多种靶点和途径的药理机制改善CCl4诱导的大鼠肝纤维化,包括脂肪酸合成和代谢途径以及AMPK信号通路。本研究为YYHXD的进一步研究和临床应用提供了参考和依据。
UNASSIGNED: As a traditional Chinese medicine formula, Yangyinghuoxue Decoction (YYHXD) is used clinically for therapy of hepatic fibrosis. The pharmacological profile of YYHXD comprises multiple components acting on many targets and pathways, but the pharmacological mechanisms underlying its efficacy have not been thoroughly elucidated. This study aimed at probing the pharmacological mechanisms of YYHXD in the treatment of hepatic fibrosis.
UNASSIGNED: YYHXD aqueous extract was prepared and quality control using HPLC-MS fingerprint analysis was performed. A CCl4-induced rat model of hepatic fibrosis was established, and animals were randomly assigned to six groups: control, low-dose YYHXD (L-YYHXD), medium-dose YYHXD (M-YYHXD), high-dose YYHXD (H-YYHXD), CCl4 model, and colchicine group. Rats in the treatment groups received daily oral administration of YYHXD (5, 10, or 20 g/kg) or colchicine (0.2 mg/kg) for 6 weeks, while the control and model groups received distilled water. Histological analysis, including hematoxylin and eosin (HE) and Masson\'s trichrome staining, was performed to evaluate hepatic fibrosis. Serum biochemical markers, such as AST, ALT, HA, and LN, were measured. Inflammatory cytokines (IL-6 and TNF-α) and oxidative stress indicators (SOD, GSH-Px, and MDA) in hepatic tissue were also assessed. Additionally, transcriptomic analysis using RNA-sequencing was conducted to identify differentially expressed genes (DEGs) between the control, CCl4 model, and H-YYHXD groups. Bioinformatics analysis, including differential expression analysis, protein-protein interaction analysis, and functional enrichment analysis, were performed to probe the pharmacological mechanisms of YYHXD. The regulatory effects of YYHXD on fatty acid metabolism and biosynthesis were further confirmed by Oil Red O staining, enzyme activity assays, qPCR, and Western blotting. Western blotting and immunofluorescence staining also validated the involvement of the AMPK signaling pathway in the occurrence and progression of hepatic fibrosis.
UNASSIGNED: HE and Masson\'s trichrome staining revealed reduced collagen deposition and improved liver architecture in YYHXD groups compared to the CCl4 model group. Serum biochemical markers, including AST, ALT, HA, and LN, were significantly improved in the YYHXD-treated groups compared to the CCl4 model group. The levels of inflammatory cytokines (IL-6 and TNF-α) and oxidative stress indicators (decreased SOD and GSH-Px, increased MDA) in hepatic tissue were significantly ameliorated by YYHXD treatment compared to the CCl4 model group. Moreover, 96 genes implicated in YYHXD therapy of hepatic fibrosis were screened from the transcriptomic data, which were principally enriched in biological pathways such as fatty acid metabolism and biosynthesis, and the AMPK signaling pathway. Oil Red O staining showed reduced hepatic lipid accumulation by YYHXD in a dose-dependent manner, along with decreased serum TG, TC, and LDL-C levels. Additionally, qPCR and Western blot analyses demonstrated upregulated mRNA and protein expression of key enzymes involved in fatty acid metabolism and biosynthesis, Fasn and Fads2, modulated by YYHXD. YYHXD also dose-dependently enhanced phosphorylation of AMPK as evidenced by Western blotting and immunofluorescence assays.
UNASSIGNED: YYHXD ameliorated CCl4-induced hepatic fibrosis in rats through pharmacological mechanisms that involved manifold targets and pathways, including aliphatic acid synthesis and metabolism pathways and the AMPK signaling pathway. This study provided a reference and basis for further research and clinical utilization of YYHXD.