P2Y6 Receptor Activation Aggravates NLRP3-dependent Microglial Pyroptosis via Downregulation of the PI3K/AKT Pathway in a Mouse Model of Intracerebral Hemorrhage.

Pro-inflammatory signals generated after intracerebral hemorrhage (ICH) trigger a form of regulated cell death known as pyroptosis in microglia. White matter injury (WMI) refers to the condition where the white matter area of the brain suffers from mechanical, ischemic, metabolic, or inflammatory damage. Although the p2Y purinoceptor 6 (P2Y6R) plays a significant role in the control of inflammatory reactions in central nervous system diseases, its roles in the development of microglial pyroptosis and WMI following ICH remain unclear. In this study, we sought to clarify the role of P2Y6R in microglial pyroptosis and WMI by using an experimental mouse model of ICH. Type IV collagenase was injected into male C57BL/6 mice to induce ICH. Mice were then treated with MRS2578 and LY294002 to inhibit P2Y6R and phosphatidylinositol 3-kinase (PI3K), respectively. Bio-conductivity analysis was performed to examine PI3K/AKT pathway involvement in microglial pyroptosis. Quantitative Real-Time PCR, immunofluorescence staining, and western blot were conducted to examine microglial pyroptosis and WMI following ICH. A modified Garcia test, corner turning test, and forelimb placement test were used to assess neurobehavior. Hematoxylin-eosin staining (HE) was performed to detect cells damage around hematoma. Increases in the expression of P2Y6R, NLRP3, ASC, Caspase-1, and GSDMD were observed after ICH. P2Y6R was only expressed on microglia. MRS2578, a specific inhibitor of P2Y6R, attenuated short-term neurobehavioral deficits, brain edema and hematoma volume while improving both microglial pyroptosis and WMI. These changes were accompanied by decreases in pyroptosis-related proteins and pro-inflammatory cytokines both in vivo and vitro. Bioinformatic analysis revealed an association between the PI3K/AKT pathway and P2Y6R-mediated microglial pyroptosis. The effects of MRS2578 were partially reversed by treatment with LY294002, a specific PI3K inhibitor. P2Y6R inhibition alleviates microglial pyroptosis and WMI and ameliorates neurological deficits through the PI3K/AKT pathway after ICH. Consequently, targeting P2Y6R might be a promising approach for ICH treatment.

Bone marrow-mesenchymal stem cells alleviate microglial Pyroptosis after intracerebral hemorrhage in rat by secreting C1q/tumor necrosis factor-related protein 3.

BACKGROUND: There are no specific treatment methods for intracerebral hemorrhage (ICH). Neuroinflammation triggered by microglial pyroptosis plays an important role in ICH pathophysiology. Bone marrow mesenchymal stem cells (BMSCs) are widely used in the treatment of neurological diseases because of their paracrine function. In this study, we aimed to clarify whether BMSCs can alleviate microglial pyroptosis after ICH by secreting C1q/tumor necrosis factor-related protein 3 (CTRP3), a adiponectin paralog with established metabolic regulatory properties and neuroprotective effects. METHODS: In an in vitro study, microglia were stimulated with hemin for pyroptosis and then co-cultured with BMSCs, CTRP3, or CTRP3-small interfering RNA (siRNA)-BMSC; in an in vivo study, intracerebroventricular transplantation of BMSCs or siRNA-CTRP3-BMSCs was performed after ICH surgery. The expression of inflammation-related factors was detected by qRT-PCR and ELISA. Western blotting and immunofluorescence staining were performed to detect the expression of pyroptotic protein, and western blotting was used to detect the activation of phosphoinositide 3-kinase (PI3K), protein kinase B (AKT) and splenic tyrosine kinase (Syk). Behavioral changes were detected 7 days after transplantation. RESULTS: ELISA and qRT-PCR results showed that the production of inflammatory cytokines in hemin-stimulated microglia was significantly downregulated following pretreatment with BMSCs or CTRP3. The Caspase-1 activity assay kit and western blotting results showed that BMSCs attenuated microglial pyroptosis by secreting CTRP3. Furthermore, the modulation functions of BMSCs or CTRP3 involve the promotion of PI3K/AKT and inhibition of Syk signaling pathway activation. Neurological deficits, edema, and disruption of tight junction protein were completely alleviated, while inflammation-related factors and microglial pyroptosis after ICH were significantly downregulated after BMSCs administration. CONCLUSION: BMSCs can inhibit neuroinflammation by inhibiting microglial pyroptosis, thus alleviate ICH symptoms, likely by suppressing the Syk signaling pathway while promoting the PI3K/AKT signaling pathway activation through producing CTRP3.

Inhibition of Dectin-1 Alleviates Neuroinflammatory Injury by Attenuating NLRP3 Inflammasome-Mediated Pyroptosis After Intracerebral Hemorrhage in Mice: Preliminary Study Results.

Background: Neuroinflammation plays an important role following intracerebral hemorrhage (ICH). NLRP3 inflammasome-mediated pyroptosis contributes to the mechanism of neuroinflammation. It has been reported that dendritic cell-associated C-type lectin-1 (Dectin-1) activation triggers inflammation in neurological diseases. However, the role of Dectin-1 on NLRP3 inflammasome-mediated pyroptosis after ICH remains unclear. Here, we aimed to explore the effect of Dectin-1 on NLRP3 inflammasome-mediated pyroptosis and neuroinflammation after ICH. Methods: Adult male C57BL/6 mice were used to establish the ICH model. Laminarin, an inhibitor of Dectin-1, was administered for intervention. Expression of Dectin-1 was evaluated by Western blot and immunofluorescence. Brain water content and neurobehavioral function were tested to assess brain edema and neurological performance. Western blot was conducted to evaluate the level of GSDMD-N. ELISA kits were used to measure the levels of IL-1ß and IL-18. qRT-PCR and Western blot were performed to evaluate the expressions of NLRP3 inflammasome, IL-1ß, and IL-18. Results: The expression of Dectin-1 increased following ICH, and Dectin-1 was expressed on microglia. In addition, inhibition of Dectin-1 by laminarin decreased brain edema and neurological impairment after ICH. Moreover, inhibition of Dectin-1 decreased the expression of pyroptosis-related protein, GSDMD-N, and inflammatory cytokines (IL-1ß and IL-18). Mechanistically, Dectin-1 blockade inhibits NLRP3 inflammasome activation, thereby alleviating neuroinflammatory injury by attenuating NLRP3 inflammasome-mediated pyroptosis both in vivo and in vitro. Conclusion: Our study indicates that the inhibition of Dectin-1 alleviates neuroinflammation by attenuating NLRP3 inflammasome-mediated pyroptosis after ICH.

The enhancement of dermal papilla cell aggregation by extracellular matrix proteins through effects on cell-substratum adhesivity and cell motility.

Generally, cells tend to aggregate on a substratum with lower cell adhesivity. However, it also leads to compromised cell growth and higher cell loss after seeding. This study is aimed at tackling this dilemma by extracellular matrix (ECM) protein coating of a lower adhesive substratum poly(ethylene-co-vinyl alcohol) (EVAL) that has been shown to facilitate hair follicle dermal papilla (DP) spheroid formation. We found that coating with either fibronectin (Fn), collagen I, or collagen IV yields higher adhesivity and cell growth than that with laminin. However, cells can only aggregate on uncoated or Fn-coated EVAL. Quantitatively, Fn coating increases the number of spheroids by 67%. Analysis of cell migration reveals that collagen I, collagen IV and laminin coatings reduce cell motility, while Fn coating keeps cells highly motile. Inhibition of cell migration hinders spheroid formation. In addition, disruption of Fn function does not significantly compromise intercellular adhesion. Hence, Fn enhances cell aggregation by enhancing cell attachment, cell growth and cell motility. Our study demonstrates that intercellular organization as spheroids or flat monolayers is switchable by specific ECM protein coating and preserving cell motility is vital to cell aggregation. In addition to generation of spheroidal DP microtissues for hair follicle regeneration and large-scale production of aggregates of other cells, this strategy can help to regulate the tissue-substrate adhesivity and tissue spreadability on the surface of implantable materials.