Salvianolic acid C improves cerebral ischemia reperfusion injury through suppressing microglial cell M1 polarization and promoting cerebral angiogenesis

Abstract

This study aimed to investigate the mechanism of salvianolic acid C (SAC), the active ingredient in Salvia miltiorrhiza, in improving cerebral ischemia injury. The mouse microglial cells BV2 and mouse endothelial cells bEnd.3 were used as the objects of study. LPS/IFN-γ was applied to simulate the BV2 polarization, and bEnd.3 cells were treated under hypoxic condition. The BV2 cell polarization level was measured through flow cytometry (FCM), the TLR4 and MyD88 expression levels were detected by fluorescence staining, whereas the expression of inflammatory factors TNF-α, IL-6 and IL-1β was analyzed through ELISA. Tubule formation assay was also conducted to observe the tubule formation ability of bEnd.3 cells in vitro, and the level of VEGFR2 was detected by fluorescence staining. Cells were treated with the PKM2 inhibitor IN3, aiming to observe the influence of SAC on glycolysis of BV2 cells. In addition, the mouse model of cerebral ischemia was constructed through the middle cerebral artery occlusion (MCAO) method, and the pathological changes in brain tissues were detected after SAC intervention. Meanwhile, the levels of IBA-1, CD31 and ZO-1 were determined through histochemical staining. Nissl staining to detect nerve cell damage. In BV2 cell experiment, SAC suppressed the M1 polarization of BV2 cells, reduced the inflammatory factor levels, and inhibited the activation of TLR4 signal through suppressing glycolysis. When PKM2 was suppressed, the effects of SAC were antagonized. In the bEnd.3 model, SAC promoted tubule formation in bEnd.3 cells under hypoxic condition, and increased the expression of VEGFR2 and Notch1. In the mouse model, SAC improved the neurological function in MCAO mice, and inhibited the activation of microglial cells and the expression of inflammatory factors. At the same time, SAC up-regulated the expression of ZO-1 and CD31, and maintained the blood-brain barrier (BBB) function. As a major component of Salvia miltiorrhiza, SAC can suppress microglial cell polarization and promote tubule formation in endothelial cells to exert the neurological repair function in cerebral ischemia. SAC is a multi-functional neuroprotective small molecule.

Keywords: Cerebral ischemia; Endothelial cells; Inflammation; Microglial cells; Polarization; Salvianolic acid C.