Sanpian decoction ameliorates cerebral ischemia-reperfusion injury by regulating SIRT1/ERK/HIF-1α pathway through in silico analysis and experimental validation

Abstract

Ethnopharmacological relevance: Cerebral ischemia-reperfusion injury (CIRI) is a complex pathophysiological process involving multiple factors, and becomes the footstone of rehabilitation after ischemic stroke. Sanpian decoction (SPD) has exhibited protective effects against CIRI, migraine, and other cerebral vascular diseases. However, the underlying mechanisms have not been completely elucidated.

Aim of the study: This study sought to explore the potential mechanisms underlying the effect of SPD against CIRI.

Materials and methods: High-performance liquid chromatography (HPLC) and ultra-high-performance liquid chromatography (UPLC) were carried out to determine the chemical constituents of SPD. A network pharmacology approach combined with experimental verification was conducted to elucidate SPD's multi-component, multi-target, and multi-pathway mechanisms in CIRI occurrence. The pharmacodynamics of the decoction was evaluated by establishing the rat model of middle cerebral artery occlusion/reperfusion (MCAO/R). In vivo and in vitro experiments were carried out, and the therapeutic effects of SPD were performed using 2,3,5-triphenyltetrazolium chloride (TTC) staining, hematoxylin-eosin (HE) staining, and Nissl staining. We used terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) staining and flow cytometry to evaluate cortex apoptosis. The quantification of mRNA and corresponding proteins were performed using real-time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot respectively.

Results: Our research showed that pretreatment with SPD improved neurological function and inhibited CIRI. Network pharmacology revealed that the hypoxia-inducible factor-1 (HIF-1) signaling pathway and mitogen-activated protein kinase (MAPK) signaling pathway-mediated apoptosis may be associated with CIRI. In vivo and in vitro experiments, we confirmed that SPD increased cerebral blood flow, improved neural function, and reduced neural apoptosis via up-regulating the expression of sirtuin 1 (SIRT1) and down-regulating phospho-extracellular regulated protein kinases (p-ERK)/ERK and HIF-1α levels in CIRI rats.

Conclusion: Taken together, the present study systematically revealed the potential targets and signaling pathways of SPD in the treatment of CIRI using in silico prediction and verified the therapeutic effects of SPD against CIRI via ameliorating apoptosis by regulating SIRT1/ERK/HIF-1α.

Keywords: Apoptosis; Cerebral ischemia-reperfusion injury; HIF-1 signaling pathway; MAPK signaling pathway; Network pharmacology; Sanpian decoction.