Mechanism and experimental validation of a network pharmacology-based approach of Huoluo Yinao formula in treating chronic cerebral hypoperfusion.

Abstract

Chronic cerebral hypoperfusion (CCH) is a cerebral dysfunction caused by insufficient cerebral perfusion, and Huoluo Yinao (HLYN) formula is a traditional Chinese medicinal formula used for CCH treatment. We investigated the specific mechanism of HLYN formula on CCH through network pharmacological analysis and in vivo experiments. The active ingredients and drug targets of HLYN formula obtained through network pharmacological analysis were subjected to molecular docking with disease targets of CCH. Wistar male rats (six weeks) were used to establish a CCH rat model by the two-vessel occlusion (2VO) method. The rats were treated with HLYN alone or in combination with the HSP90AA1 overexpression vector before behavior tests. The groups were as follows: Sham group, CCH group, CCH+HLYN group, CCH+HLYN+oe-NC group, and CCH+HLYN+oe-HSP90AA1 group, with six rats in each group. The hippocampal tissues were assessed after Hematoxylin and eosin (H&E) and Nissl stainings. Autophagosomes were observed using immunofluorescence and transmission electron microscopy. There were 83 active ingredients and 1339 potential pharmacological targets in HLYN formula, while 3175 genes were found associated with CCH. A total of 235 overlapped genes were obtained. Rats in the CCH+HLYN group showed shortened escaping latency, increased times in the target quadrant, and increased number of platform crossings, along with attenuated tissue injury, elevated Nissl-positive neurons and less apoptosis. In addition, inflammation and oxidative stress (OS) were decreased in the CCH+HLYN group, evidenced by reduced malondialdehyde (MDA), Tumor Necrosis Factor-alpha (TNF-α), Interleukin (IL)-6 and IL-1β, and increased superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-PX), and IL-10 expression. HLYN formula led to suppressed HSP90AA1 expression and autophagy, along with increased p-PI3K/p-AKT/p-mTOR protein expression. The phenotypes in the CCH+HLYN group can be reversed by further treatment with HSP90AA1 overexpression. HLYN formula can inhibit HSP90AA1 expression to inhibit autophagy, inflammation, and OS, which in turn contributes to attenuating cognitive impairment in CCH rats.