Pinocembrin protects hemorrhagic brain primarily by inhibiting toll-like receptor 4 and reducing M1 phenotype microglia

Abstract

Neuroinflammation is a major contributor to intracerebral hemorrhage (ICH) progression, but no drug is currently available to reduce this response and protect against ICH-induced injury. Recently, the natural product pinocembrin has been shown to ameliorate neuroinflammation and is undergoing a phase II clinical trial for ischemic stroke treatment. In this study, we examined the efficacy of pinocembrin in an ICH model, and further examined its effect on microglial activation and polarization. In vivo, pinocembrin dose-dependently reduced lesion volume by 47.5% and reduced neurologic deficits of mice at 72 h after collagenase-induced ICH. The optimal dose of pinocembrin (5 mg/kg) suppressed microglial activation as evidenced by decreases in CD68-positive microglia and reduced proinflammatory cytokines tumor necrosis factor (TNF)-α, interleukin (IL)-1β, and IL-6. Pinocembrin also reduced the number of classically activated M1-like microglia without affecting M2-like microglia in the perilesional region. Additionally, pinocembrin decreased the expression of toll-like receptor (TLR)4 and its downstream target proteins TRIF and MyD88. The protection by pinocembrin was lost in microglia-depleted mice and in TLR4lps-del mice, and pinocembrin failed to decrease the number of M1-like microglia in TLR4lps-del mice. In lipopolysaccharide-stimulated BV-2 cells or primary microglia, pinocembrin decreased M1-related cytokines and markers (IL-1β, IL-6, TNF-α, and iNOS), NF-κB activation, and TLR4 expression, but it did not interfere with TLR4/MyD88 and TLR4/TRIF interactions or affect microglial phagocytosis of red blood cells. Inhibition of the TLR4 signaling pathway and reduction in M1-like microglial polarization might be the major mechanism by which pinocembrin protects hemorrhagic brain. With anti-inflammatory properties, pinocembrin could be a promising new drug candidate for treating ICH and other acute brain injuries.

Modulators of microglial activation and polarization after intracerebral haemorrhage

“…These results suggest that the ratio of IFNγ to M2-like cytokine levels might be a more reliable marker than IFNγ levels alone for the assessment of inflammatory status in the chronic phase after ICH. Our group has shown that M1 markers such as CD16, CD32 and iNOS are highly expressed on microglia on days 1 and 3 after ICH 62 , suggesting that M1-like microglial polarization occurs early in the acute phase.…”

Cerebroprotection by salvianolic acid B after experimental subarachnoid hemorrhage occurs via Nrf2- and SIRT1-dependent pathways

“…In addition, we employed Nrf2 KO mice to investigate whether the neuroprotective effects of SalB occur via Nrf2 signaling. Mice were anesthetized with isoflurane (4.0% for induction, 2.0% for maintenance) and ventilated with oxygen-enriched air (20%:80%) via a nose cone, as in our previous studies [38,39]. After anesthetization, a burr hole was drilled into the skull 4.5 mm anterior to the bregma.…”

Alpha-7 Nicotinic Receptor Signaling Pathway Participates in the Neurogenesis Induced by ChAT-Positive Neurons in the Subventricular Zone

“…Cerebral edema was reflected by the brain water content, which was calculated as described previously [47, 48]. We sacrificed six mice per group to assess brain water content at day 7.…”