Traditional Chinese medicine prescription Mailuoning alleviated liver fibrosis through inhibiting the TGFβ/Smad-mediated extracellular matrix production.

ETHNOPHARMACOLOGICAL RELEVANCE: In traditional Chinese medicine theory, the primary therapeutic principles for liver fibrosis involve clearing heat, resolving toxin, tonifying deficiency, invigorating blood and resolving stasis. Mailuoning (MLN) is a Chinese patent medicine composed of five herbal medicines: Lonicerae Japonicae Flos, Lonicerae Flos, Scrophulariae Radix, Dendrobii Caulis, and Achyranthis Bidentatae Radix. These herbs contribute synergistically: Lonicerae Japonicae Flos and Lonicerae Flos function to clear heat and remove toxin, Dendrobii Caulis nourish yin and clear heat, Achyranthis Bidentatae Radix eliminate stasis and unblock meridians, Scrophulariae Radix clear heat, remove toxin, nourish yin and resolve masses. Due to their activities, these herbs were widely used in various compound formulas for liver fibrosis management in China, and their synergistic effects make MLN a relevant therapeutic option.

AIM OF THE STUDY: To evaluate the therapeutic effects of MLN on liver fibrosis, explore the underlying mechanisms, and discover its potential active compounds.

METHODS: Mouse liver fibrosis model was established using CCl4 or a combination of high-fat diet (HFD) and CCl4. Serum biochemical parameters and liver histological observation were assessed to evaluate the anti-fibrotic efficacy of MLN. Transcriptomic analysis, real-time polymerase chain reaction (RT-PCR) and Western-blot (WB) were performed to identify key signaling pathways. "Recipe decomposition" experiments were conducted to identify the primary medicinal herb contributing to MLN's anti-fibrotic effect. Molecular docking and microscale thermophoresis (MST) analysis were conducted to discover active compounds.

RESULTS: In CCl4-induced liver fibrosis, both prophylactic or therapeutic administration of MLN (13, 40 g/kg) reduced hepatic collagen deposition and hydroxyproline content, as well as serum levels of hyaluronic acid (HA) and type IV collagen (Col IV). Meanwhile, MLN suppressed the expression of genes related to ECM synthesis. Transcriptomic analysis and validation experiments manifested that MLN's efficacy was associated with the regulation of ECM production, angiogenesis, and inflammation, mainly involving HIF-1α, TGFβ and NFκB signaling pathways. Results from "recipe decomposition" experiments indicated that Scrophulariae Radix was the primary active medicinal herb contributing to MLN's anti-fibrotic effect, as its removal markedly diminished MLN's efficacy against liver fibrosis. Moreover, in the HFD + CCl4 model, MLN (13, 40 g/kg) also reduced hepatic collagen deposition and serum levels of HA and Col IV, and suppressed ECM production by inhibiting TGFβ-Smad2/3 pathway was found to be mainly involved. Five compounds from Scrophulariae Radix including angoroside C (AC), harpagoside (HPG), aucubin (AU), sweroside (SWE), and geniposidic acid (GPA) were found to inhibit TGFβ-induced hepatic stellate cell (HSC) activation. AC and HPG exhibited a weak binding affinity to TGFβRII or TGFβRI. AC decreased both TGFβRI and TGFβRII mRNA expression, whereas HPG and GPA reduced TGFβRI mRNA expression in vitro. Consistent with this, MLN reduced hepatic TGFβRI and TGFβRII mRNA expression in vivo.

CONCLUSION: MLN alleviated liver fibrosis primarily by suppressing ECM production through inhibiting TGFβ signaling pathway. Scrophulariae Radix was identified as the main contributor to MLN's anti-fibrotic effect, with AC, HPG, and GPA proposed as the key active compounds responsible for this activity.