Docosahexaenoic acid promotes M2 microglia phenotype via activating PPARγ-mediated ERK/AKT pathway against cerebral ischemia-reperfusion injury.

In ischemia-reperfusion stroke, microglia play a dual role in brain injury as well as brain repair, and promoting their switch from a pro-inflammatory M1 phenotype to an anti-inflammatory M2 phenotype is considered to be a potential therapeutic strategy. Docosahexaenoic acid (DHA) is an essential long-chain omega-3 polyunsaturated fatty acid that exhibits potent anti-inflammatory properties in the acute phase of ischemic stroke, but its effect on microglia polarization is unknown. Thus, the objective of this study was to investigate the neuroprotective effects of DHA on rat brain following ischemia-reperfusion injury, and to investigate the mechanism by which DHA regulates microglia polarization. We administered DHA 5 mg/kg intraperitoneally daily for 3 d following a transient middle cerebral artery occlusion reperfusion model in rats. The protective effects of DHA on cerebral ischemia-reperfusion injury were detected by TTC staining, HE staining, Nissler staining, and TUNEL staining. Quantitative real-time PCR, immunofluorescence, western blot, and enzyme-linked immunosorbent assay were used to detect the expression of M1 and M2 microglia-associated markers and PPARγ-mediated ERK/AKT signaling pathway proteins. We found that DHA significantly improved brain injury by decreasing the expression of the M1 phenotypic marker (iNOS, CD16) and increasing the expression of the M2 phenotypic marker (Arg-1, CD206). DHA also increased the expression of peroxisome proliferator-activated receptor gamma (PPARγ) mRNA and protein, increased the expression of the pathway protein AKT, and decreased the expression of ERK1/2. In addition, DHA promoted the expression of anti-inflammatory factor IL-10 and decreased the expression of pro-inflammatory factors TNF-α and IL-1β. However, the PPARγ antagonist GW9662 greatly blocked these beneficial effects. These results suggest that DHA may activate PPARγ to inhibit ERK and activate AKT signaling pathways to regulate microglia polarization, thereby reducing neuroinflammation and promoting neurological recovery to alleviate cerebral ischemia-reperfusion injury.

Lv H ，Jia S ，Sun Y ，Pang M ，Lv E ，Li X ，Meng Q ，Wang Y ... -  《-》

Astragaloside IV promotes microglia/macrophages M2 polarization and enhances neurogenesis and angiogenesis through PPARγ pathway after cerebral ischemia/reperfusion injury in rats.

Microglia/macrophages play a dual role in brain injury and repair following cerebral ischemia/reperfusion. Promoting microglia/macrophage polarization from pro-inflammatory M1 to anti-inflammatory M2 phenotype has been considered as a potential treatment for ischemic stroke. Astragaloside IV (AS-IV) is a primary active ingredient of Chinese herb Radix Astragali, which protects against acute cerebral ischemic/reperfusion injury through its antioxidant, anti-inflammatory, and anti-apoptotic properties. However, it remains unknown whether AS-IV improves ischemic brain tissue repair and its underlying mechanism. A transient middle cerebral artery occlusion (tMCAO) rat model was used in this study. The results showed that AS-IV significantly improved long-term brain injury, reduced the expression of M1 microglia/macrophage markers and increased the expression of M2 microglia/macrophage markers 14 days after cerebral ischemia/reperfusion. AS-IV also increased peroxisome proliferator-activated receptor γ (PPARγ) mRNA and protein expression. Moreover, AS-IV promoted neurogenesis and angiogenesis, and increased the protein expression of brain-derived growth factor (BDNF), insulin-like growth factor-1 (IGF-1) and vascular endothelial growth factor (VEGF). However, these beneficial effects were greatly blocked by PPARγ antagonist T0070907. These results together suggest that AS-IV could enhance neurogenesis, angiogenesis and neurological functional recovery, which may be partially through transforming microglia/macrophage from M1 to M2 phenotype in a PPARγ-dependent manner after cerebral ischemia/reperfusion injury. Therefore, AS-IV can be considered as a promising therapeutic agent for ischemic stroke.

Li L ，Gan H ，Jin H ，Fang Y ，Yang Y ，Zhang J ，Hu X ，Chu L ... -  《-》

Quercetin improves cerebral ischemia/reperfusion injury by promoting microglia/macrophages M2 polarization via regulating PI3K/Akt/NF-κB signaling pathway.

The modulation of microglial polarization from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype shows promise as a therapeutic strategy for ischemic stroke. Quercetin, a natural flavonoid abundant in various plants, possesses anti-inflammatory, anti-apoptotic, and antioxidant properties. Nevertheless, its effect and underlying mechanism on microglia/macrophages M1/M2 polarization in the treatment of cerebral ischemia/reperfusion injury (CI/RI) remain poorly explored. In the current study, we observed that quercetin ameliorated neurological deficits, reduced infarct volume, decreased the number of M1 microglia/macrophages (CD16/32+/Iba1+), and enhanced the number of M2 microglia/macrophages (CD206+/Iba1+) after establishing the CI/RI model in rats. Subsequent in vivo and in vitro experiments indicated that quercetin downregulated M1 markers (CD86, iNOS, TNF-α, IL-1β, and IL-6) and upregulated M2 markers (CD206, Arg-1, IL-10, and TGF-β). Network pharmacology analysis and molecular docking revealed that the PI3K/Akt/NF-κB signaling pathway emerged as the core pathway. Western blot confirmed that quercetin upregulated the phosphorylation of PI3K and Akt, while alleviating the phosphorylation of IκBα and NF-κB both in vivo and in vitro. However, the PI3K inhibitor LY294002 reversed the effects of quercetin on M2 polarization and the expression of key proteins in the PI3K/Akt/NF-κB pathway in primary microglia after oxygen-glucose deprivation/reoxygenation (OGD/R) in vitro. Collectively, our findings demonstrate that quercetin facilitates microglia/macrophages M2 polarization by modulating the PI3K/Akt/NF-κB signaling pathway in the treatment of CI/RI. These findings provide novel insights into the therapeutic mechanisms of quercetin in ischemic stroke.

Li L ，Jiang W ，Yu B ，Liang H ，Mao S ，Hu X ，Feng Y ，Xu J ，Chu L ... -  《-》

Lipoxin A4 regulates microglial M1/M2 polarization after cerebral ischemia-reperfusion injury via the Notch signaling pathway.

Microglia are rapidly activated after acute ischemic stroke, and the polarization of microglial is associated with the prognosis of acute ischemic stroke. Lipoxin A4 (LXA4), an anti-inflammatory agent, has a protective effect against ischemic stroke. However, the role of LXA4 on the polarization of microglial after acute ischemic stroke remains undetermined. We hypothesized that LXA4 may exert the neuroprotective effect though regulating the polarization of microglial. In this study, clinical features of acute ischemic stroke were simulated using a rat model of model of middle cerebral artery occlusion (MCAO) in vivo and the BV2 microglia oxygen-glucose deprivation/reoxygenation model (OGD/R) in vitro. The protective effects of LXA4 on cerebral ischemia-reperfusion injury were determined using TTC staining, HE staining, and TUNEL staining. The expression of targeted genes was assayed using quantitative real-time PCR (qRT-PCR), immunofluorescence, and western blot to investigated the regulation of LXA4 on microglia polarization after acute ischemic stroke. We found that LXA4 exerted protective effects on focal cerebral ischemia-reperfusion injury and reduced the expression of the pro-inflammatory cytokines IL-1β and TNF-α. Furthermore, LXA4 inhibited the expression of Notch-1, Hes1, iNOS and CD32 all of which are associated with the differentiation into M1 microglia. By contrast, LXA4 upregulated the expression of Hes5, Arg-1 and CD206 all of which are associated with M2 phenotype in microglia. In addition, blocking the Notch signaling pathway with the inhibitor DAPT significantly mitigated the effect of LXA4 on microglia differentiation. These data suggest that LXA4 may regulate the polarization of microglia after cerebral ischemia-reperfusion injury through the Notch signaling pathway.

Li QQ ，Ding DH ，Wang XY ，Sun YY ，Wu J ... -  《-》

Electroacupuncture Alleviates Neuroinflammation by Regulating Microglia Polarization via STAT6/PPARγ in Ischemic Stroke Rats.

Previous study showed that electroacupuncture (EA) produced a protective effect on cerebral ischemia-reperfusion injury (CIRI) in rats and may correlate with the anti-inflammatory effects of microglia. This study aimed to investigate further whether EA could modulate neuroinflammation by targeting the Signal Transducer and Activator of Transcription 6 (STAT6) and Peroxisome Proliferator-Activated Receptor γ (PPARγ) pathway, the key regulator of microglia. Middle cerebral artery occlusion (MCAO) rats were used, and 6 h after reperfusion, EA interventions were performed in Chize (LU 5), Hegu (LI 4), Sanyinjiao (SP 6), and Zusanli (ST 36) on the affected side of the rats, the group that received EA + STAT6 phosphorylation inhibitor AS1517499 was used as a parallel control. The degree of neurological impairment, infarct volume, microglia polarization, inflammation levels and activity of STAT6/PPARγ pathway were then assessed by neurological deficit score, triphenyl tetrazolium chloride (TTC) staining, immunofluorescence, western blotting (WB), quantitative real-time PCR (qPCR) and Enzyme linked immunosorbent assay (ELISA). The data showed that EA significantly alleviated nerve injury, reduced infarct volume, enhanced the expression and activity of STAT6/PPARγ pathway, inhibited NF-κB activity, increased M2 microglia numbers and anti-inflammatory factor release, and inhibited microglia M1-type polarization and pro-inflammatory factor expression. In contrast, inhibition of STAT6 phosphorylation exacerbated neural damage, inhibited STAT6/PPARγ pathway activity, promoted microglia M1-type polarization and exacerbated neuroinflammation, resulting in an attenuated positive effect of EA intervention. Therefore, we concluded that EA intervention could attenuate microglia-associated neuroinflammation by enhancing the expression and activity of STAT6/PPARγ pathway, thereby reducing CIRI in MCAO rats.

Yao Z ，Cai L ，Zhao A ，Yang L ，Chen Z ，Zhang Y ，Liang G ，Luo M ，Xu X ，Zhou G ... -  《-》