Abstract. Huoxue Huayu (HXHY) has been widely used in traditional Chinese medicine (TCM) as a key therapeutic principle for osteoarthritis (OA), and related herbs have been widely prescribed to treat OA in the clinic. The aims of the present study were to explore a multi-target therapy for OA using 10 common HXHY herbs and to investigate their potential applications for treatment of other diseases. A novel computational simulation approach that integrates chemical structure, ligand clusters, chemical space and drug-likeness evaluations, as well as docking and network analysis, was used to investigate the properties and effects of the herbs. The compounds contained in the studied HXHY herbs were divided into 10 clusters. Comparison of the chemical properties of these compounds to those of other compounds described in the DrugBank database indicated that the properties of the former are more diverse than those of the latter and that most of the HXHY-derived compounds do not violate the 'Lipinski's rule of five'. Docking analysis allowed for the identification of 39 potential bioactive compounds from HXHY herbs and 11 potential targets for these compounds. The identified targets were closely associated with 49 diseases, including neoplasms, musculoskeletal, nervous system and cardiovascular diseases. Ligand-target (L-T) and ligand-target-disease (L-T-D) networks were constructed in order to further elucidate the pharmacological effects of the herbs. Our findings suggest that a number of compounds from HXHY herbs are promising candidates for mult-target therapeutic application in OA and may exert diverse pharmacological effects, affecting additional diseases besides OA. IntroductionOsteoarthritis (OA) is a degenerative joint disease, which causes chronic pain and functional restrictions in the affected joints (1). At present, there is no effective treatment for reversing or preventing its onset. Non-steroidal anti-inflammatory drugs (NSAIDs) are mainly used for treating OA, particularly in the early stages of the disease, but these drugs are often associated with clinically adverse effects (2). Furthermore, the social and economic cost related to OA remains particularly high (3). Therefore, ongoing research attempts to develop improved therapeutic strategies for OA.Several lines of evidence suggest that treatment that aims at a number of targets at once may be more effective against complex diseases such as OA (4,5). On the other hand, traditional Chinese medicine (TCM), a complex system employing multiple components and targets, has been recognized in Western countries as a popular complementary and alternative medicinal approach. TCM has been used in the treatment of OA, and often has fewer side-effects than those reported for NSAIDs (6-8). Those reports indicate that TCM may provide a novel promising strategy for treatment of OA.Huoxue Huayu (HXHY) has been widely used in TCM as a key therapeutical principle for OA in China (9,10). Ten common HXHY herbs, Ligusticum chuanxiong, Salvia miltiorrhiza, Strychnos nux-v...