Hydroxysafflor yellow A attenuates the inflammatory response in cerebral ischemia-reperfusion injured mice by regulating microglia polarization per SIRT1-mediated HMGB1/NF-κB signaling pathway.

Hydroxysafflor yellow A (HSYA), an active component isolated from Carthamus tinctorius L., has demonstrated potent protective effects against cerebral ischaemia/reperfusion (I/R) injury. Microglial polarisation plays a crucial role in I/R. However, the mechanism by which HSYA regulates microglial polarisation remains unclear. To explore the mechanism of action of HSYA on the phenotypic polarisation of microglia stimulated by lipopolysaccharide (LPS) in a mouse model of I/R injury. BV2 cells injured by LPS and a modified middle cerebral artery occlusion/reperfusion (MCAO/R) model were used to mimic I/R in vitro and in vivo, respectively. BV2 cell morphology was assessed by optical microscopy, and cell viability was evaluated using the CCK-8 assay. The effect of HSYA on MCAO/R mice was assessed using the Longa assay, brain index, triphenyl tetrazolium chloride, and haematoxylin and eosin staining. LDH, NO, IL-6, TNF-α, and IL-10 levels were measured using corresponding ELISA kits following the manufacturers' protocols. M1 and M2 type microglia markers, including CD86, CD16/32, iNOS, YM1/2, TGF-β, and Arg, were detected by western blotting. M1 and M2 cell surface markers (CD86 and CD206) were detected using immunofluorescence. Molecular docking, DARTS, and CETSA were applied to investigate the interactions between HSYA and SIRT1. The role of HSYA in regulating the binding of HMGB1 to SIRT1 was tested using co-immunoprecipitation. Proteins related to the HMGB1/NF-κB pathway were also analysed by western blotting. HSYA promoted microglial polarisation from M1 to M2 type in LPS-induced BV2 cells and MCAO/R mice. HSYA significantly reduced M1 polarisation markers, including IL-6, TNF-α, CD86, CD16/32, while increasing the expression of IL-10, Arg, YM1/2, TGF-β. Furthermore, compared to the MCAO/R group, HSYA significantly improved neurological scores, brain index, and infarct volume and normalised nucleolar arrangement. Molecular docking assessment showed that HSYA exhibited strong binding SIRT1 and significantly improved the interactions between SIRT1 and HMGB1. HSYA also decreased the expression of cytoplasm-HMGB1 and reduced the P-P65/P65 ratio. HSYA attenuates LPS-induced and MCAO/R-induced inflammatory responses by modulating microglia polarisation. This effect is associated with the SIRT1-mediated HMGB1/NF-κB signalling pathway.

Yao M ，Liu Y ，Meng D ，Zhou X ，Chang D ，Li L ，Wang N ，Huang Q ... -  《-》

Lactate modulates microglial inflammatory responses through HIF-1α-mediated CCL7 signaling after cerebral ischemia in mice.

Lactate is a potent regulator of neuroinflammation. We recently demonstrated that lactate alleviated neuronal injury via HIF-1α-regulated microglial inflammation after oxygen-glucose deprivation (OGD). However, the underlying mechanisms and the effect of lactate on microglial responses after ischemic stroke remained unknown. Mouse acute cerebral ischemia-reperfusion injury was induced by middle cerebral artery occlusion (MCAO). L-lactate (100 mM, 2 μl) was intracerebroventricularly administrated 30 min after the reperfusion. Microglia responses were evidenced by the expression of multiple markers such as CD86, iNOS, arginase-1, CD206 and Ym1 in the peri-infarction 24 h after MCAO using western blot analysis and quantitative real-time PCR. Inflammatory factors IL-6, TNF-α, TGF-β and IL-10, as well as NF-κB signaling were also detected. Infarct size and neuronal apoptosis in the peri-infarction at 24 h, mice survival within 7 days and long-term neurobehavioral function were evaluated. The involvement of HIF-1α in lactate-mediated microglial inflammation after MCAO was assessed using a HIF-1α inhibitor. Additionally, transcriptome analysis was used to identify the potential lactate targets in BV2 cells after OGD. The recombinant product of the identified CCL7 gene was used to verify its effect on cerebral ischemia-reperfusion injury in vivo. Lactate supplementation reduced infarction volume, neuronal apoptosis and neurological deficits. Lactate reduced the expression of CD86, iNOS, IL-6, TNF-α and elevated the expression of arginase-1, CD206, Ym1, TGF-β and IL-10 in the peri-infarction at 24 h after reperfusion. Consistently, lactate inhibited the NF-κB signaling. Additionally, lactate upregulated HIF-1α in microglia 24 h after reperfusion, while inhibition of HIF-1α reversed the effects of lactate on brain damage and neuroinflammation after cerebral ischemia. Furthermore, CCL7 was identified as the top down-regulated inflammatory gene induced by lactate in OGD-treated BV2 cells. It was also found high expression of CCL7 in the peri-infarction at 24 h after reperfusion and lactate treatment inhibited CCL7 expression. However, HIF-1α inhibitor reversed the effect of lactate treatment on CCL7 expression. Finally, supplementation of recombinant CCL7 reversed the mitigated neuroinflammation and neuroprotective effect rendered by lactate treatment after MCAO. We concluded that treatment with lactate modulated the microglia inflammatory responses and alleviated cerebral ischemia injury. The inhibition of CCL7/NF-κB signaling by HIF-1α might be involved in the beneficial effect of lactate treatment.

Zhang Y ，Zhang S ，Yang L ，Zhang Y ，Cheng Y ，Jia P ，Lv Y ，Wang K ，Fan P ，Zhang P ，Wei H ... -  《-》

AEBP1 Silencing Protects Against Cerebral Ischemia/Reperfusion Injury by Regulating Neuron Ferroptosis and Microglia M2 Polarization Through PRKCA-PI3K-Akt Axis.

Cerebral ischemia/reperfusion injury is one of the main causes of neuronal damage. Neuron ferroptosis and microglia polarization are considered as critical processes during cerebral ischemia/reperfusion. Adipocyte enhancer-binding protein 1 (AEBP1) usually acts as a transcriptional repressor which is involved in various diseases. However, it is still remains unknown whether AEBP1 could have important roles in regulating the neuron ferroptosis and microglia polarization in cerebral ischemia/reperfusion injury. The oxygen-glucose deprivation and reperfusion (OGD/R)-treated cells and middle cerebral artery occlusion (MCAO)-treated mice were used as in vitro and in vivo models. The differentially expressed factors were analyzed according to GEO datasets. Relative mRNA and protein expression levels were detected by qRT-PCR and western blot analysis. Cell viability was measured by CCK-8 assay. ROS, GSH and iron contents were detected using specifical assay kits. CD26 and CD206 levels were measured by immunofluorescence assay. Inflammatory cytokines were detected by ELISA. The association between AEBP1 and PRKCA was assessed by luciferase reporter and ChIP analyses. The neuron damage in mice was analyzed by TTC staining and neurological deficit score. Transcription factor AEBP1 was increased in OGD/R-treated HT22 and BV2 cells. AEBP1 silencing attenuated OGD/R-induced HT22 cell ferroptosis through increasing cell viability, GSH and GPX4 levels, and decreasing ROS, iron and ACSL4 levels. AEBP1 knockdown promoted microglia M2 polarization by increasing CD206-positive cells and Arg-1 level, and reducing iNOS, TNF-α, IL-1β and IL-6 levels in BV2 cells. AEBP1 transcriptionally repressed PRKCA expression, and further regulated PI3K/Akt signaling activation. Inhibition of PRKCA or PI3K/Akt reversed the effects of AEBP1 silencing on neuron ferroptosis and microglia M2 polarization. AEBP1 downregulation attenuated neuronal damage by decreasing infarct size and deficit scores in MCAO-treated mice. AEBP1 silencing mitigated neuron ferroptosis and promoted microglia M2 polarization through increasing PRKCA and activating PI3K/Akt signaling, indicating the potentially protective action of AEBP1 knockdown in cerebral ischemia/reperfusion injury.

Zhang Y ，Li Y ，Liu F 《-》

Hydroxysafflor Yellow A promotes angiogenesis of brain microvascular endothelial cells from ischemia/reperfusion injury via glycolysis pathway in vitro.

Angiogenesis of brain microvascular endothelial cells (BMECs) after cerebral ischemia was conducive to improving the blood supply of ischemia tissues, which was upregulated by glycolysis. Hydroxysafflor Yellow A (HSYA) mends damaged tissues through increasing angiogenesis. HSYA treated proliferation, migration and angiogenesis of BMECs in vitro in vitro during OGD/R. HSYA regulated the key enzymes of glycolysis, such as hexokinase 2 (HK2) and 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), glucose uptake and products (pyruvate, ATP and lactate) were detected by western blot and kits, respectively. Scratch wound assay, transwell, tube formation and spheroid sprouting were used to explore the pathway that HSYA recovered migration and angiogenesis of BMECs. We evaluated the potential target of HSYA promoting glycolysis via molecular docking, drug affinity responsive target stability (DARTS) and cellular thermal shift assay (CETSA). HSYA promoted the proliferation, migration, tube formation and spheroid sprouting of BMECs during OGD/R, and stimulated the expression of tip phenotype marker protein (CD34), and the receptor (Notch-1) that regulated the differentiation of endothelial cells into tip/stalk phenotype. In glycolysis, PFKFB3 expression was upregulated by HSYA; HSYA also improved ATP and pyruvate levels, as well as lactate release after OGD/R. Finally, upregulating VEGFA and p-VEGFR2 of HSYA was weakened because of suppressing glycolysis; the HSYA's improvement of BMECs migration and angiogenesis was attenuated under the inhibition of glycolysis, which confirmed that HSYA were upregulating angiogenesis and expression of VEGFA/VEGFR2 by glycolysis pathway. The result about molecular docking, DARTS and CETSA suggested that PFKFB3 was the possible target of HSYA. HSYA promotes angiogenesis of BMECs in vitro through the glycolysis mediated VEGFA/VEGFR2 pathway, and PFKFB3 is the potential target of HSYA to heighten glycolysis.

Ruan J ，Wang L ，Wang N ，Huang P ，Chang D ，Zhou X ，Seto S ，Li D ，Hou J ... -  《-》

Preventive effects of matrine on LPS-induced inflammation in RAW 264.7 cells and intestinal damage in mice through the TLR4/NF-κB/MAPK pathway.

Matrine is a tetracyclic quinolizidine alkaloid with diverse bioactive properties, including anti-inflammatory and neuroprotective properties. However, the underlying anti-inflammatory mechanisms remain unclear. This study aimed to explore how matrine reduces Lipopolysaccharide (LPS)-induced inflammation in RAW 264.7 cells and to assess its protective effects against LPS-induced intestinal damage. The effect of matrine on cell viability was assessed using the cell counting kit-8 (CCK-8) assay. Additionally, its impact on inflammatory cytokines and macrophage polarization was assessed using enzyme-linked immunosorbent assay (ELISA), flow cytometry, and quantitative real-time polymerase chain reaction (qRT-PCR) analyses. The effects on intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), nitric oxide (NO) production, and oxidative stress were evaluated using 2',7'-dichlorodihydrofluorescein diacetate staining and JC-1 and Griess assays. Immunofluorescence staining was used to observe the translocation of the NF-κB p65 subunit. Western blotting (WB) and qRT-PCR were employed to analyze the expression levels of proteins related to the toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB)/mitogen-activated protein kinase (MAPK) pathway. An LPS-induced mouse model was established to study the intestinal inflammation and barrier injury. Mouse feces characteristics, colon length, and disease activity index (DAI) were recorded. Hematoxylin-eosin (H&E) and alcian blue/periodic acid schiff (AB/PAS) staining were used to observe morphological changes and barrier damage in the duodenum, jejunum, ileum, and colon and to measure villus length, crypt depth, goblet cell count, and positive areas in the duodenum, jejunum, and ileum. The content of short-chain fatty acids (SCFAs) in the colon was determined using gas chromatography (GC). Matrine inhibited LPS-induced inflammatory cytokine levels, suppressed macrophage M1 polarization, and promoted M2 macrophage polarization. Matrine reduced LPS-induced increases in ROS and NO levels and regulates oxidative stress. Additionally, matrine inhibited the nuclear translocation of the NF-κB p65 subunit and exerted anti-inflammatory effects by suppressing the activation of the TLR4/NF-κB/MAPK pathway. In vivo experiments indicated that matrine significantly alleviated LPS-induced diarrhea, increased DAI, and shortened the colon. Matrine reduced the production of the pro-inflammatory cytokine interleukin (IL)-6, IL-1β, and tumor necrosis factor (TNF)-α and the pro-inflammatory mediator NO in mouse intestinal tissues while promoting the content of the anti-inflammatory cytokine IL-10. Furthermore, it improved intestinal tissue structure and alleviated LPS-induced intestinal barrier damage. Finally, matrine increased the SCFA levels in the intestine. Matrine exerted its anti-inflammatory effects and protects against intestinal injury through the TLR4/NF-κB/MAPK signaling pathway.

Mao N ，Yu Y ，Lu X ，Yang Y ，Liu Z ，Wang D ... -  《-》