Knockdown of Smox protects the integrity of the blood-brain barrier through antioxidant effect and Nrf2 pathway activation in stroke.

Once an ischemic stroke occurs, reactive oxygen species (ROS) and oxidative stress degrade the tight connections between cerebral endothelial cells resulting in their damage. The expression of antioxidant genes may be enhanced, and ROS formation may be reduced following Nrf2 activation, which is associated with protection against ischemic stroke. Overexpression of spermine oxidase (Smox) in the neocortex led to increased H2O2 production. However, how Smox impacts the regulation of the blood-brain barrier (BBB) through antioxidants has not been examined yet. We conducted experiments both in the cell level and in the transient middle cerebral artery occlusion (tMCAO) model to evaluate the effect of Smox siRNA lentivirus (si-Smox) knockdown on BBB protection against ischemic stroke. Mice treated with si-Smox showed remarkably decreased BBB breakdown and reduced endothelial inflammation following stroke. The treatment with si-Smox significantly elevated the Bcl-2 to Bax ratio and decreased the production of cleaved caspase-3 in the tMCAO model. Further investigation revealed that the neuroprotective effect was the result of the antioxidant properties of si-Smox, which reduced oxidative stress and enhanced CD31+ cells in the peri-infarct cortical areas. Of significance, si-Smox activated Nrf2 in both bEnd.3 cells and tMCAO animals, and blocking Nrf2 with brusatol diminished the protective effects of si-Smox. The study findings suggest that si-Smox exerts neuroprotective effects and promotes angiogenesis by activating the Nrf2 pathway, thus decreasing oxidative stress and apoptosis caused by tMCAO. As a result, si-Smox may hold potential as a therapeutic candidate for preserving BBB integrity while treating ischemic stroke.

The Aldose Reductase Inhibitor Epalrestat Maintains Blood-Brain Barrier Integrity by Enhancing Endothelial Cell Function during Cerebral Ischemia.

Excessive activation of aldose reductase (AR) in the brain is a risk factor for aggravating cerebral ischemia injury. Epalrestat is the only AR inhibitor with proven safety and efficacy, which is used in the clinical treatment of diabetic neuropathy. However, the molecular mechanisms underlying the neuroprotection of epalrestat remain unknown in the ischemic brain. Recent studies have found that blood-brain barrier (BBB) damage was mainly caused by increased apoptosis and autophagy of brain microvascular endothelial cells (BMVECs) and decreased expression of tight junction proteins. Thus, we hypothesized that the protective effect of epalrestat is mainly related to regulating the survival of BMVECs and tight junction protein levels after cerebral ischemia. To test this hypothesis, a mouse model of cerebral ischemia was established by permanent middle cerebral artery ligation (pMCAL), and the mice were treated with epalrestat or saline as a control. Epalrestat reduced the ischemic volume, enhanced BBB function, and improved the neurobehavior after cerebral ischemia. In vitro studies revealed that epalrestat increased the expression of tight junction proteins, and reduced the levels of cleaved-caspase3 and LC3 proteins in mouse BMVECs (bEnd.3 cells) exposed to oxygen-glucose deprivation (OGD). In addition, bicalutamide (an AKT inhibitor) and rapamycin (an mTOR inhibitor) increased the epalrestat-induced reduction in apoptosis and autophagy related protein levels in bEnd.3 cells with OGD treatment. Our findings suggest that epalrestat improves BBB function, which may be accomplished by reducing AR activation, promoting tight junction proteins expression, and upregulating AKT/mTOR signaling pathway to inhibit apoptosis and autophagy in BMVECs.

IFN-γ regulates the transformation of microglia into dendritic-like cells via the ERK/c-myc signaling pathway during cerebral ischemia/reperfusion in mice.

Cerebral ischemia-reperfusion injury induces a secondary immune inflammatory reaction that exacerbates brain injury and clinical prognosis. Dendritic cells (DCs) and microglia are both important regulators of neuroinflammation. Studies have confirmed that a large number of cells express the DC surface marker CD11c in the ischemic area, and some of these cells also express microglial markers. However, the specific mechanism of transformation between microglia and DCs and their roles in the process of cerebral ischemia-reperfusion injury are still not clear. In this study, we established a mouse model and flow cytometry was used to detect the expression of mature DC surface molecules in activated microglia. IFN-γ knockout mice were used to determine the regulatory effect of IFN-γ on microglial transformation. We found that CD11c+ cells were derived from microglia after ischemia-reperfusion injury, and this group of cells highly expressed MHC-II molecules and other costimulatory molecules, such as CD80 and CD86, which were regulated by IFN-γ and its downstream signaling molecules ERK/c-myc. In summary, our results showed in cerebral ischemia-reperfusion injury, IFN-γ regulates the transformation of microglia to DC-like cells. Microglial-derived DC-like cells possess the ability to present antigens and activate naïve T cells which is regulated by the ERK/c-myc signaling pathway.

MicroRNA-182 exacerbates blood-brain barrier (BBB) disruption by downregulating the mTOR/FOXO1 pathway in cerebral ischemia.

Cerebral ischemia causes damage to the structure and function of the blood-brain barrier (BBB) and alleviating BBB destruction will be of great significance for the treatment and prognosis of ischemic stroke. Recently, microRNAs have been shown to play a critical role in BBB integrity. However, the potential mechanism by which microRNA-182 (miR-182) affects the BBB in ischemic stroke remains unclear. We demonstrated for the first time that cerebral ischemia leads to a significant progressive increase in miR-182 after pMCAO, and bEnd.3 cells are the primary target cells of miR-182. In miR-182 KD transgenic mice, infarct volume, and BBB permeability were attenuated, and tight junction (TJ) proteins increased. Inhibition of miR-182 with an antagomir reduced OGD-induced apoptosis of bEnd.3 cells and the loss of ZO-1 and Occludin. To further explore the mechanism by which miR-182 regulates BBB integrity, we detected the apoptotic proteins Bcl-2/Bax and demonstrated that mTOR and FOXO1 were the targets of miR-182. Inhibition of mTOR/FOXO1 by rapamycin/AS1842856 decreased the ratio of Bcl-2/Bax and exacerbated TJ protein loss. Taken together, inhibition of miR-182 protects BBB integrity by reducing endothelial cell apoptosis through the mTOR/FOXO1 pathway. Thus, miR-182 may be a potential target for the treatment of BBB disruption during cerebral ischemia.

Excess salt intake promotes M1 microglia polarization via a p38/MAPK/AR-dependent pathway after cerebral ischemia in mice.

A high salt diet (HSD) is among the most important risk factors for many diseases. One mechanism by which HSD aggravates cerebral ischemic injury is independent of blood pressure changes. The direct role of HSD in inflammation after cerebral ischemia is unclear. In this research, after twenty-one days of being fed a high salt diet, permanent focal ischemia was induced in mice via operation. At 12 h and 1, 3 and 5 days postischemia, the effects of HSD on the lesion volume, microglia polarization, aldose reductase (AR) expression, and inflammatory processes were analyzed. We report that in mice, surplus dietary salt promotes inflammation and increases the activation of classical lipopolysaccharide (LPS)-induced microglia/macrophages (M1). This effect depends on the expression of the AR protein in activated microglia after permanent middle cerebral artery ligation (pMCAL) in HSD mice. The administration of either the AR inhibitor Epalrestat or a p38-neutralizing antibody blocked the polarization of microglia and alleviated stroke injury. In conclusion, HSD promotes polarization in pro-inflammatory M1 microglia by upregulating the expression of the AR protein via p38/MAPK, thereby exacerbating the development of ischemia stroke.

Enhancement of T Follicular Helper Cell-Mediated Humoral Immunity Reponses During Development of Experimental Autoimmune Myasthenia Gravis.

Myasthenia gravis (MG) is a prototypical antibody-mediated neurological autoimmune disease with the involvement of humoral immune responses in its pathogenesis. T follicular helper (Tfh) cells have been implicated in many autoimmune diseases. However, whether and how Tfh cells are involved in MG remain unclear. Here, we established and studied a widely-used and approved animal model of human MG, the rat model with acetylcholine receptor alpha (AChRα) subunit (R-AChR97-116)-induced experimental autoimmune myasthenia gravis (EAMG). This model presented mild body-weight loss 10 days after the first immunization (representing the early stage of disease) and more obvious clinical manifestations and body-weight loss 7 days after the second immunization (representing the late stage of disease). AChR-specific pre-Tfh cells and mature Tfh cells were detected in these two stages, respectively. In co-cultures of Tfh cells and B cells, the number of IgG2b-secreting B cells and the level of anti-AChR antibodies in the supernatant were higher in the cultures containing EAMG-derived Tfh cells. In immunohistochemistry and immunofluorescence assays, a substantial number of CD4+/Bcl-6+ T cells and a greater number of larger germinal centers were observed in lymph node tissues resected from EAMG rats. Based on these results, we hypothesize that an AChR-specific Tfh cell-mediated humoral immune response contributes to the development of EAMG.

Regulator of G protein signaling 5 (RGS5) inhibits sonic hedgehog function in mouse cortical neurons.

Regulator of G protein signaling 5 (RGS5) acts as a GTPase-activating protein (GAP) for the Gαi subunit and negatively regulates G protein-coupled receptor signaling. However, its presence and function in postmitotic differentiated primary neurons remains largely uncharacterized. During neural development, sonic hedgehog (Shh) signaling is involved in cell signaling pathways via Gαi activity. In particular, Shh signaling is essential for embryonic neural tube patterning, which has been implicated in neuronal polarization involving neurite outgrowth. Here, we examined whether RGS5 regulates Shh signaling in neurons. RGS5 transcripts were found to be expressed in cortical neurons and their expression gradually declined in a time-dependent manner in culture system. When an adenovirus expressing RGS5 was introduced into an in vitro cell culture model of cortical neurons, RGS5 overexpression significantly reduced neurite outgrowth and FM4-64 uptake, while cAMP-PKA signaling was also affected. These findings suggest that RGS5 inhibits Shh function during neurite outgrowth and the presynaptic terminals of primary cortical neurons mature via modulation of cAMP.

Irgm1 promotes M1 but not M2 macrophage polarization in atherosclerosis pathogenesis and development.

BACKGROUND AND AIMS: Atherosclerosis is a chronic inflammatory vascular disease related to macrophages uptake of low-density lipoprotein and their subsequent transformation into foam cells. M1 (inflammatory)/M2 (anti-inflammatory) balance was suggested to impact disease progression. In this study, we investigated whether the immunity related GTPase (Irgm1) regulates macrophage polarization during atherosclerosis development. METHODS: We used apolipoprotein E (ApoE) knockout and Irgm1 haplodeficient mice and induced atherosclerosis with high-cholesterol diet for the indicated months. Atherosclerotic arteries were collected from patients undergoing vascular surgery, to determine the lesional expression of Irgm1 and distribution of M1/M2 populations. RESULTS: Our results showed that IRGM/Irgm1 expression was increased in atherosclerotic artery samples (1.7-fold, p=0.0045) compared with non-atherosclerotic arteries, which was consistent with findings in the murine experimental atherosclerosis model (1.9-fold, p=0.0002). IRGM/Irgm1 expression was mostly found in lesional M1 macrophages. Haplodeficiency of Irgm1 in ApoE(-/-) mice resulted in reduced infiltrating M1 macrophages in atheroma (94%, p=0.0002) and delayed development of atherosclerotic plaques. In vitro experiments also confirmed that Irgm1 haplodeficiency reduced iNOS expression of polarized M1 macrophages (81%, p=0.0034), with negligible impact on the M2 phenotype. Moreover, we found that Irgm1 haplodeficiency in mice significantly reduced expression level of M1 function-related transcription factors, interferon regulatory factor (Irf) 5 and Irf8, but not Irf4, an M2-related transcription factor. CONCLUSIONS: This study shows that Irgm1/IRGM participates in the polarization of M1 macrophage and promotes development of atheroma in murine experimental atherosclerosis.

Excess salt exacerbates blood-brain barrier disruption via a p38/MAPK/SGK1-dependent pathway in permanent cerebral ischemia.

High salt diet (HSD) is one of the most important risk factors that contribute to many vascular diseases including ischemic stroke. One proposed mechanism underlying the disruption of blood-brain barrier (BBB) mediated by HSD is indirectly through enhancing blood pressure. The direct role of HSD on BBB integrity is unclear. Our purpose is to determine whether and how HSD might be involved in BBB breakdown during ischemia. To test that, we induced model of cerebral ischemia by permanent middle cerebral artery ligation (pMCAL) in either normal diet or HSD fed mice. We observed that HSD significantly enhanced ischemic brain damage which was associated with enhanced BBB disruption, increased leukocytes infiltration and loss of tight junction (TJ) proteins expression without apparently altering blood pressure. Our in vitro experiment also revealed that sodium chloride (NaCl) treatment down-regulated TJ protein expression by endothelial cells and substantially increased BBB permeability during starvation. Inhibition of p38/MAPK/SGK1 pathway eliminated the effect of NaCl on BBB permeability in vitro. In addition, we noticed a positive correlation between urinary sodium levels and ischemic lesion size in stroke patients. Together, our study demonstrates a hypertension-independent role of HSD during ischemia and provides rationale for post cerebral ischemic attack management.

Anti-Citrullinated Protein Antibodies Induce Macrophage Subset Disequilibrium in RA Patients.

We used samples from rheumatoid arthritis (RA) patients to examine whether Anti-citrullinated protein antibodies (ACPAs) alter macrophage subset distribution and promote RA development. Macrophage subset distributions and interferon regulatory factor 4 (IRF4) and IRF5 expressions were analyzed. ACPAs were purified by affinity column. After RA and osteoarthritis (OA) patients' macrophages were cocultured with ACPAs, macrophage subsets and IRF4 and IRF5 expressions were measured. Small interfering RNAs (siRNAs) were transfected into ACPA-activated cells to suppress IRF4 or IRF5. Fluorescence-activated cell sorting (FACS), Western blot, and immunohistochemistry were performed. Macrophage subset disequilibrium occurred in RA patient synovial fluids. IRF4 and IRF5 were all expressed in the synovial fluid and synovium. ACPAs (40 IU/ml) could induce macrophages to polarize to M1 subsets, and the percentage of increased M1/M2 ratio of RA patients was higher than that of the OA patients. ACPAs also induce IRF4 and IRF5 protein expressions. IRF5 siRNA transfection impaired ACPA activity significantly. We demonstrated that macrophage subset disequilibrium occurred in RA patients. ACPAs induced IRF5 activity and led to M1 macrophage polarization.

Toll-like receptor-mediated IRE1α activation as a therapeutic target for inflammatory arthritis.

In rheumatoid arthritis (RA), macrophage is one of the major sources of inflammatory mediators. Macrophages produce inflammatory cytokines through toll-like receptor (TLR)-mediated signalling during RA. Herein, we studied macrophages from the synovial fluid of RA patients and observed a significant increase in activation of inositol-requiring enzyme 1α (IRE1α), a primary unfolded protein response (UPR) transducer. Myeloid-specific deletion of the IRE1α gene protected mice from inflammatory arthritis, and treatment with the IRE1α-specific inhibitor 4U8C attenuated joint inflammation in mice. IRE1α was required for optimal production of pro-inflammatory cytokines as evidenced by impaired TLR-induced cytokine production in IRE1α-null macrophages and neutrophils. Further analyses demonstrated that tumour necrosis factor (TNF) receptor-associated factor 6 (TRAF6) plays a key role in TLR-mediated IRE1α activation by catalysing IRE1α ubiquitination and blocking the recruitment of protein phosphatase 2A (PP2A), a phosphatase that inhibits IRE1α phosphorylation. In summary, we discovered a novel regulatory axis through TRAF6-mediated IRE1α ubiquitination in regulating TLR-induced IRE1α activation in pro-inflammatory cytokine production, and demonstrated that IRE1α is a potential therapeutic target for inflammatory arthritis.