Carvedilol attenuates inflammatory reactions of lipopolysaccharide-stimulated BV2 cells and modulates M1/M2 polarization of microglia via regulating NLRP3, Notch, and PPAR-γ signaling pathways.

Khoshnavay Foumani M ，Amirshahrokhi K ，Namjoo Z ，Niapour A ... -  《-》

Kaempferol improves depression-like behaviors through shifting microglia polarization and suppressing NLRP3 via tilting the balance of PPARγ and STAT1 signaling.

The pathogenesis of depression is largely influenced by dyshomeostasis of neuroinflammation regulated by microglia M1/M2 polarization, and NLRP3 inflammasome acts critical roles in shifting microglia polarization. Kaempferol (Kae), a major flavonoid in edible plants, possesses anti-inflammation and anti-depression capacity, but its underlying cellular and molecular mechanisms of antidepressive effect have not yet fully explored. In vivo studies with lipopolysaccharide (LPS)-induced depressive mice were carried out to evaluate antidepressant effect of Kae. In vitro, BV2 microglia cell line stimulated by LPS along with IFN-γ to detect pharmacological effects of Kae on microglia polarization and NLRP3. Based on two depression-related GEO datasets (GSE54570 and GSE54568) and the potential targets of Kae obtained from GeneCards database, enrichment analysis and protein-protein interaction (PPI) network construction reveal potential therapeutic targets of Kae for depression. Then the precise antidepressant mechanisms of Kae were verified by western blot and immunofluorescent staining in vivo and vitro. Our results showed that Kae significantly improves LPS-induced depressive behaviors and alleviates neuroinflammation in prefrontal cortex. Moreover, Kae obviously shifted microglia polarization to M2 phenotype, and also suppressed NLRP3 in prefrontal cortex and BV2. Enrichment analysis and PPI network construction suggested PPARγ and STAT1 signaling are related to regulation of NLRP3 in depression. Furtherly, Kae remarkably enhanced PPARγ activation and inhibited nuclear translocation of p-STAT1 in microglia of prefrontal cortex and BV2. Importantly, pre-incubation with PPARγ antagonist T0070907 or overexpression with CASTAT1 (constitutively active STAT1) both prevented pharmacologic effects of Kae on shifting microglia polarization and suppressing NLRP3 in BV2. Noteworthily, T0070907 significantly blocked the inhibitory effect of Kae on STAT1 while overexpression with CASTAT1 abolished the effect of Kae on PPARγ activation in BV2. Above results suggested that pharmacologic effects of Kae on microglia polarization and NLRP3 are dependent on the balance of counter-regulatory PPARγ and STAT1 signaling. Our results indicated that the shifting microglia polarization and suppression of NLRP3 via tilting the balance of PPARγ and STAT1 signaling may be the antidepressant mechanism of Kae, which provides a novel perspective for elucidating antidepressive effect of Kae.

Su P ，Liu L ，Gong Y ，Peng S ，Yan X ，Bai M ，Xu E ，Li Y ... -  《-》

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 ... -  《-》

USP22 inhibits microglial M1 polarization by regulating the PU.1/NLRP3 inflammasome pathway.

This study aimed to investigate the effect of Ubiquitin-Specific Peptidase 22 (USP22) on the inflammatory response mediated by BV-2 mouse microglia and explore the role of the PU box binding protein 1 (PU.1)/NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome in the USP22-induced polarization of BV-2 cells. The BV-2 mouse microglia line was cultured in vitro, and plasmid and siRNA transfection was performed to overexpress or knockdown USP22. Subsequently, BV-2 cells were treated with lipopolysaccharide (LPS) and interferon-gamma (IFN-γ) and interleukin (IL)-4 to induce M1 and M2 polarization, respectively. Western blot was used to detect the expression levels of USP22, PU.1, M1 polarization markers [inducible nitric oxide synthase (iNOS), and cluster of differentiation (CD) 86], M2 polarization markers [arginase 1 (Arg1), and CD206], in BV-2 cells from different treatment groups. Additionally, measurement was performed on the inflammasome NLRP3, and its activation-related proteins [NIMA-related kinase7 (NEK7), cleaved-caspase 1, apoptosis-associated speck-like protein containing a CARD (ASC)]. Enzyme-linked immunosorbent (ELISA) assay was employed to determine the levels of inflammatory cytokines tumor necrosis factor-alpha (TNF-α), IL-1 β, and IL-10 in the cells. Furthermore, immunofluorescence was utilized to analyze the levels of iNOS and Arg1-positive BV-2 cells in different treatment groups. Moreover, the ubiquitination level of PU.1 was detected using immunoprecipitation. The protein expression level of USP22 was significantly down-regulated in BV-2 cells treated with M1 polarization. Overexpression of USP22 remarkably reduced the protein levels of iNOS and CD86, but markedly increased the protein levels of Arg1 and CD206 in cells. Besides, there was a notable reduction in the levels of TNF-α and IL-1 β in the cell culture medium, while a remarkable increase was observed in the level of IL-10. Additionally, the level of iNOS-positive cells was significantly decreased, while the level of Arg1-positive cells was considerably increased. However, up-regulation of PU.1 expression could reverse the above results and promoted the expression of NLRP3 and its activation-related proteins. Notably, overexpression of USP22 significantly down-regulated the protein expression and ubiquitination level of PU.1. USP22 inhibits the M1 polarization of BV-2 mouse microglia. The PU.1/NLRP3 inflammasome pathway may be a critical regulatory pathway of USP22 in BV-2 cell polarization.

Yu MC ，Li XL ，Ning ML ，Yan ZZ ，Yu WT ... -  《-》

Buyang Huanwu Decoction prevents hemorrhagic transformation after delayed t-PA infusion via inhibiting NLRP3 inflammasome/pyroptosis associated with microglial PGC-1α.

Delayed tissue-type plasminogen activator (t-PA) thrombolysis, which has a restrictive therapeutic time window within 4.5 h following ischemic stroke (IS), increases the risk of hemorrhagic transformation (HT) and subsequent neurotoxicity. Studies have shown that the NLRP3 inflammasome activation reversely regulated by the PGC-1α leads to microglial polarization and pyroptosis to cause damage to nerve cells and the blood-brain barrier. The effect of Buyang Huanwu Decoction (BHD), a traditional Chinese medicine prescription widely used in the recovery of IS, on HT injury after delayed t-PA treatment had been found with clinical studies, while the underlying mechanisms are reminded to be further clarified. This study sought to investigate the therapeutic effect and the underlying mechanisms of BHD in ischemic rat brains with delayed t-PA treatment. The components of BHD extracts were identified by High Performance Liquid Chromatography (HPLC) and the effective components in the rat brains from BHD were analyzed by liquid chromatography-mass spectrometry (LC-MS). In vivo experiment was carried out by 5 h of middle cerebral artery occlusion (MCAO) following by t-PA infusion for 0.5 h plus reperfusion 19 h, while the in vitro BV2 cells were stimulated by lipopolysaccharide (LPS)-adenosine triphosphate (ATP) to activate microglia pyroptosis, of which the MCC950 (NLRP3 inhibitor) and NSA (GSDMD inhibitor) were adopted as reverse validation. PGC-1α siRNA was utilized to study the mechanisms of BHD against microglial polarization and pyroptosis in BV2 cells. HPLC analysis demonstrated the fingerprint of BHD with six reference standards (Hydroxysafflor yellow A, Calycosin-7-glucoside, Paeoniflorin, Formononetin, Ferulic acid and Amygdalin), the last two of which can be found in rat brains by LC-MS analysis. In the following experiments, we found the major discoveries as follow: (1) BHD improved the neurological outcomes, the structural integrity of the blood-brain barrier and the neuronal structure in HT rats with MCAO following by delayed t-PA infusion; (2) the presence of t-PA promoted the suppression of PGC-1α and the activation of microglial NLRP3 inflammasome and pyroptosis in the HT rats; (3) BHD promoted the transformation of microglia from M1 to M2 type for inhibiting inflammatory response; (4) BHD restrained NLRP3 inflammasome/pyroptosis activation in microglia, prevented the translocations of NF-κB into the nucleus, as well as enhanced microglia-specific PGC-1α in ischemic rats following t-PA delayed thrombolysis; (5) BHD suppressed NLRP3 inflammasome assembly and increased PGC-1α expression in the LPS-ATP-induced BV2 cells; (6) PGC-1α silencing withdrew the protective role of BHD against NLRP3 inflammasome/pyroptosis. Mechanistically, BHD existed the protective effect against HT injury after delayed t-PA treatment through up-regulating microglial PGC-1α to inhibit NLRP3 inflammasome and pyroptosis, and serves as a potential adjuvant therapy for HT injury.

Pan Y ，Nie L ，Chen W ，Guan D ，Li Y ，Yang C ，Duan L ，Wan T ，Zhuang L ，Lai J ，Li W ，Zhang Y ，Wang Q ... -  《-》