Integrating network pharmacology and in vivo experimental validation to reveal the alleviation of mailuoning oral liquid on non-alcoholic fatty liver disease

Non-alcoholic fatty liver disease (NAFLD) especially the later stage non-alcoholic steatohepatitis (NASH) seriously endangers human's health and has become a global public health issue in recent years. Mailuoning Oral Liquid (MLN) is a modern traditional Chinese medicine prescription composed by Lonicerae japonicae flos, Achyranthis bidentatae radix, Scrophulariae radix and Dendrobium Caulis. MLN is generally used to treat the syndrome of blood stasis in clinical practice.To observe the alleviation of MLN on NASH in vivo, and explore the possible underlying mechanism. Furthermore, this study also aims to find which Chinese medicinal drug contained in MLN exerts the main pharmacological activity.NASH model was induced in mice by feeding with methionine and choline deficient (MCD) diet. The effects of MLN on hepatic lipids accumulation, liver inflammation, hepatic fibrosis, and the expression of some molecules were investigated by histological observation, biochemical index analysis, quantitative real-time PCR and western blot. Network pharmacology was applied to predict those involved molecular targets and potential mechanisms, which was further validated in vivo. BODIPY fluorescence staining assay was used to detect cellular lipids accumulation.MLN (7.8, 23.4 ml/kg) improved NASH in MCD-fed mice. Network pharmacology results demonstrated that peroxisome proliferator-activated receptor α (PPARα) signaling pathway was crucially involved in the MLN-provided alleviation on NASH. Further experimental validation results showed that MLN increased the expression of peroxisome proliferator-activated receptor gamma coactivator-1α (PGC-1α) and restored the decreased expression of nuclear PPARα in MCD-fed mice. Further results displayed that Achyranthis bidentatae radix and Lonicerae japonicae flos contributed greatly to the MLN-provided alleviation on NASH in vivo. BODIPY fluorescence staining assay showed that 25R-inokosterone and cynaroside, two compounds from Achyranthis bidentatae radix and Lonicerae japonicae flos, obviously reduced intracellular lipids accumulation in hepatocytes stimulated by non-esterified fatty acid (NEFA).MLN improved NASH in MCD-fed mice, and the PGC-1α-PPARα signaling pathway was involved in this process. Moreover, Lonicerae japonicae flos and Achyranthis bidentatae radix contained in MLN contributed greatly to the MLN-provided improvement on NASH.