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Nonalcoholic fatty liver disease (NAFLD) is the most common type of liver disease in developed countries. Oxidative stress plays a critical role in the progression of NAFLD. Modern pharmacological study and clinical trials have demonstrated the remarkable antioxidant activity of Gynostemma pentaphyllum (GP) in chronic liver disease. One aim of this study was to explore the potential protective effects and mechanisms of action of GP extract on NAFLD. The in vivo results showed that GP extract could alleviate fatty degeneration and haptic fibrosis in NAFLD mice. For exploring the hepatoprotective mechanisms of GP, we used network pharmacology to predict the potential active components of GP and their intracellular targets in NAFLD. Based on the network pharmacology results, we further utilized biomedical assays to validate this in silico prediction. The results showed that Gypenoside XL could upregulate the protein level of PPARα in NAFLD; the transcription level of several PPARα downstream target genes such as acyl-CoA oxidase (ACO) and carnitine palmitoyltransferase-1 (CPT-1) also increased after Gypenoside XL treatment. The overexpression of ACO and CPT-1 may involve the hepatoprotective effects of GP and Gypenoside XL on NAFLD by regulating mitochondrial fatty acid β-oxidation. [ABSTRACT FROM AUTHOR]