Background:Hua-Zhuo-Kai-Yu decoction (HZKY) is an empirical formula in traditional Chinese medicine that is derived from the classic ancient prescription Da-Chai-Hu decoction. It has been demonstrated to have good clinical effects on nonalcoholic fatty liver disease (NAFLD). However, the mechanism by which HZKY acts on NAFLD remains unclear. In this study, network pharmacology was used to predict the potential targets of HZKY in NAFLD. Additionally, in vivo studies were conducted to validate the crucial pathways determined using network pharmacology. Methods: Active compounds in HZKY were screened using the Traditional Chinese Medicine Systems Pharmacology and Analysis Platform and Traditional Chinese Medicine Integrated Database, and the potential targets of compounds in HZKY were predicted using Traditional Chinese Medicine Systems Pharmacology and Analysis Platform, Traditional Chinese Medicine Integrated Database, Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine, and PUBCHEM. In addition, targets involved in NAFLD were obtained from the GeneCards and Online Mendelian Inheritance in Man databases, and the potential targets of HZKY in NAFLD were identified based on the common potential targets between HZKY and NAFLD. Cytoscape 3.7.2 was used to visualize crosstalk and identify the key genes from the potential targets of HZKY in NAFLD. Kyoto Encyclopedia of Genes and Genomes analysis was conducted to predict the pathways by which HZKY acts on NAFLD. Rats were fed with a high-fat diet for 12 weeks to induce NAFLD and were then orally administered HZKY. Serum lipid levels and hematoxylin and eosin and oil red O staining results were assessed to determine the effects of HZKY in NALFD. Furthermore, the mechanisms of action of HZKY in NAFLD, as determined using network pharmacology, were validated based on the inhibition of apoptosis in the liver using Western blotting. Results:A total of 269 potential targets of 130 active compounds in HZKY were identified (oral bioavailability≥30％and drug-like≥0.18), and 62 targets were selected after being compared with the targets of NAFLD. Bcl-2-associated X protein (BAX), caspase3 (CASP3), and caspase9 (CASP9) were the key genes with the highest values of network connectivity. In addition, 45 Kyoto Encyclopedia of Genes and Genomes pathways, including apoptosis, fatty acid synthesis, and estrogen signaling, were enriched according to the selected genes of HZKY. In vivo studies showed that the serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol were significantly elevated and the serum level of high-density lipoprotein cholesterol was decreased in the model group compared with those in the control group (P<0.01 for all). The expressions of BAX, CASP9, and CASP3 were upregulated in the model group compared with those in the control group (P<0.05, P<0.01, and P<0.01, respectively), while HZKY treatment decreased the body weights and serum levels of total cholesterol, triglyceride, and low-density lipoprotein cholesterol and increased the serum levels of high-density lipoprotein cholesterol in NAFLD model rats (P<0.05, P<0.01, P<0.05, and P<0.05, respectively). Hematoxylin and eosin and oil red O staining indicated that HZKY treatment reduced steatosis in the hepatocytes. Moreover, HZKY treatment inhibited apoptosis in the liver by downregulating the expressions of BAX, CASP3, and CASP9 (P <0.05, P<0.01, and P<0.05, respectively). Conclusion:Our study demonstrates that HZKY improves NAFLD by inhibiting apoptosis in the liver by reducing the levels of BAX, CASP3, and CASP9.