A combined molecular biology and network pharmacology approach to investigate the multi-target mechanisms of Chaihu Shugan San on Alzheimer's disease.

Chaihu Shugan San (CSS) is a well-known herbal formula used to nourish liver and blood, promote blood circulation and Qi flow in Traditional Chinese Medicine. Modern pharmacological studies and clinical uses showed that CSS could ameliorate cognitive dysfunction of Alzheimer's disease (AD). The present study aimed to elucidate the multi-target mechanisms of CSS on AD using network pharmacology analysis and verify its effect by biological experiments. Firstly, a total of 152 active compounds in CSS, 520 predicted biological targets and 160 AD-related targets were identified. Subsequently, the networks including herb-compound-target network, AD-target network, and CSS potential target-AD target network were constructed. 60 key targets highly responsible for the beneficial effect of CSS on AD were identified by central network topological analysis. They were significantly characterized as nuclear or cytoplasmic proteins with molecular function of protein binding. They were also enriched in various biological processes through PI3K-Akt signaling pathway, MAPK signaling pathway and HIF signaling pathway by GO function and KEGG pathway enrichment analysis. Pretreatment with CSS ameliorated Aβ-induced neural cell death and reduced the number of apoptotic cells in differentiated PC12 cells. Moreover, increased phosphorylation of Akt accompanied with decreased Bax expression was found after CSS pretreatment, suggesting that Akt signaling pathway was involved in the protective effect of CSS against neural cells death. The present study systematically revealed the multi-target mechanisms of CSS on AD using network pharmacology approach, as well as validated the protective effect of CSS against Aβ-induced neural cells death through Akt signaling pathway. It provided indications for further mechanistic studies and also for the development of CSS as a potential treatment for AD patients.