Adjudin protects against cerebral ischemia reperfusion injury by inhibition of neuroinflammation and blood-brain barrier disruption.
ABSTRACT: Neuroinflammation mediated by activation of microglia and interruption of the blood-brain barrier (BBB) is an important factor that contributes to neuron death and infarct area diffusion in ischemia reperfusion injury. Finding novel molecules to regulate neuroinflammation is of significant clinical value. We have previously shown that adjudin, a small molecule compound known to possess antispermatogenic function, attenuates microglia activation by suppression of the NF-?B pathway. In this study we continued to explore whether adjudin could be neuroprotective by using the transient middle cerebral artery occlusion (tMCAO) model. Adjudin treatment after reperfusion significantly decreased the infarction volume and neuroscore compared to the vehicle group. Staining of CD11b showed that adjudin markedly inhibited microglial activation in both the cortex and the striatum, accompanied by a reduction in the expression and release of cytokines TNF-?, IL-1? and IL-6. Concomitantly, adjudin noticeably prevented BBB disruption after ischemia and reperfusion, as indicated by the reduction of IgG detection in the brain cortex and striatum versus the vehicle group. This finding was also corroborated by immunofluorescence staining and immunoblotting of tight junction-related proteins ZO-1, JAM-A and Occludin, where the reduction of these proteins could be attenuated by adjudin treatment. Moreover, adjudin obviously inhibited the elevated MMP-9 activity after stroke. Together these data demonstrate that adjudin protects against cerebral ischemia reperfusion injury, and we present an effective neuroinflammation modulator with clinical potential.
Project description:Neuroinflammation caused by microglial activation plays a key role in ischemia, neurodegeneration and many other CNS diseases. In this study, we found that Adjudin, a potential non-hormonal male contraceptive, exhibits additional function to reduce the production of proinflammatory mediators. Adjudin significantly inhibited LPS-induced IL-6 release and IL-6, IL-1?, TNF-? expression in BV2 microglial cells. Furthermore, Adjudin exhibited anti-inflammatory properties by suppression of NF-?B p65 nuclear translocation and DNA binding activity as well as ERK MAPK phosphorylation. To determine the in vivo effect of Adjudin, we used a permanent middle cerebral artery occlusion (pMCAO) mouse model and found that Adjudin could reduce ischemia-induced CD11b expression, a marker of microglial activation. Furthermore, Adjudin treatment attenuated brain edema and neurological deficits after ischemia but did not reduce infarct volume. Thus, our data suggest that Adjudin may be useful for mitigating neuroinflammation.
Project description:Transplantation of neural stem cells (NSCs) has been proposed as a promising therapeutic strategy for the treatment of ischemia/reperfusion (I/R)-induced brain injury. However, existing evidence has also challenged this therapy on its limitations, such as the difficulty for stem cells to survive after transplantation due to the unfavorable microenvironment in the ischemic brain. Herein, we have investigated whether preconditioning of NSCs with adjudin, a small molecule compound, could enhance their survivability and further improve the therapeutic effect for NSC-based stroke therapy.We aimed to examine the effect of adjudin pretreatment on NSCs by measuring a panel of parameters after their transplantation into the infarct area of ipsilateral striatum 24 hours after I/R in mice.We found that pretreatment of NSCs with adjudin could enhance the viability of NSCs after their transplantation into the stroke-induced infarct area. Compared with the untreated NSC group, the adjudin-preconditioned group showed decreased infarct volume and neurobehavioral deficiency through ameliorating blood-brain barrier disruption and promoting the expression and secretion of brain-derived neurotrophic factor. We also employed H2O2-induced cell death model in vitro and found that adjudin preconditioning could promote NSC survival through inhibition of oxidative stress and activation of Akt signaling pathway.This study showed that adjudin could be used to precondition NSCs to enhance their survivability and improve recovery in the stroke model, unveiling the value of adjudin for stem cell-based stroke therapy.
Project description:<h4>Background and purpose</h4>Inflammatory reaction plays a crucial role in cerebral ischemia reperfusion (IR) injury. It has been shown that activated microglia long-term existed in cerebral ischemia and induced second injury. Therefore, we hypothesize that prepared phosphatidylserine (PS)-modified microbubbles (PS-MBs) combined with ultrasound-targeted microbubble destruction (UTMD) can safely open the blood-brain barrier (BBB) and target activated microglia for inflammatory area in the later stage of ischemia reperfusion.<h4>Methods</h4>To verify our hypothesis, rat model of IR was established, then the change of activated microglia/macrophage (M/M) and permeability of BBB at 1, 7, 14, and 21?days could be clearly observed post IR. And the activated M/M still can be observed during the whole experiment.<h4>Results</h4>The Evans blue extravasation of BBB gradually declined from day 1 to day 21. Compared to the control group, microbubbles containing PS were taken up more by activated M/M (approximately twofold) both in vitro and in vivo.<h4>Conclusions</h4>PS-MBs combined with ultrasound (US) exposure could safely open BBB, and the resulting PS nanoparticles (PS-NPs) could further target activated M/M in the neuroinflammation.
Project description:Injury due to brain ischemia followed by reperfusion (I/R) may be an important therapeutic target in the era of thrombectomy. FTY720, a widely known sphingosine-1-phosphate receptor agonist, exerts various neuroprotective effects. The aim of this study was to examine the protective effect of FTY720 with respect to I/R injury, especially focusing on blood-brain barrier (BBB) protection and anti-inflammatory effects. Male rats were subjected to transient ischemia and administered vehicle or 0.5 or 1.5 mg/kg of FTY720 immediately before reperfusion. Positron emission tomography (PET) with [18F]DPA-714 was performed 2 and 9 days after the insult to serially monitor neuroinflammation. Bovine and rat brain microvascular endothelial cells (MVECs) were also subjected to oxygen-glucose deprivation (OGD) and reperfusion, and administered FTY720, phosphorylated-FTY720 (FTY720-P), or their inhibitor. FTY720 dose-dependently reduced cell death, the infarct size, cell death including apoptosis, and inflammation. It also ameliorated BBB disruption and neurological deficits compared to in the vehicle group. PET indicated that FTY720 significantly inhibited the worsening of inflammation in later stages. FTY720-P significantly prevented the intracellular redistribution of tight junction proteins but did not increase their mRNA expression. These results suggest that FTY720 can ameliorate I/R injury by protecting the BBB and regulating neuroinflammation.
Project description:Ischemic stroke can induce rapid disruption of blood-brain barrier (BBB). It has been suggested that increased BBB permeability can affect the pathological progression of ischemic tissue. However, the impact of increased BBB permeability on microglial activation and synaptic structures following reperfusion after ischemia remains unclear. In this study, we investigated microglial activation, dendritic damage and plasticity of dendritic spines after increasing BBB permeability following transient global cerebral ischemia in the somatosensory cortices in mice. Bilateral common carotid artery ligation (BCAL) was used to induce transient global cerebral ischemia. Mannitol was used to increase the BBB permeability. Intravital two-photon imaging was performed to image the dendritic structures and BBB extravasation. Microglial morphology was quantitated using a skeletonization analysis method. To evaluate inflammation of cerebral cortex, the mRNA expression levels of integrin alpha M (CD11b), CD68, chemokine (C-X-C motif) ligand 10 (IP10) and tumor necrosis factor alpha (TNF-α) were measured by fluorescent quantitative PCR. Intravital two-photon imaging revealed that mannitol caused a drastic increase in BBB extravasation during reperfusion after transient global ischemia. Increased BBB permeability induced by mannitol had no significant effect on inflammation and dendritic spines in healthy mice but triggered a marked de-ramification of microglia; importantly, in ischemic animals, mannitol accelerated de-ramification of microglia and aggravated inflammation at 3 h but not at 3 days following reperfusion after ischemia. Although mannitol did not cause significant change in the percentage of blebbed dendrites and did not affect the reversible recovery of the dendritic structures, excessive extravasation was accompanied with significant decrease in spine formation and increase in spine elimination during reperfusion in ischemic mice. These findings suggest that increased BBB permeability induced by mannitol can lead to acute activation of microglia and cause excessive loss of dendritic spines after transient global cerebral ischemia.
Project description:In response to stroke-induced injury, astrocytes can be activated and form a scar. Inflammation is an essential component for glial scar formation. Previous study has shown that adjudin, a potential Sirt3 activator, could attenuate lipopolysaccharide (LPS)- and stroke-induced neuroinflammation. To investigate the potential inhibitory effect and mechanism of adjudin on astrocyte activation, we used a transient middle cerebral artery occlusion (tMCAO) model with or without adjudin treatment in wild type (WT) and Sirt3 knockout (KO) mice and performed a wound healing experiment in vitro. Both our <i>in vivo</i> and <i>in vitro</i> results showed that adjudin reduced astrocyte activation by upregulating Sirt3 expression. In addition, adjudin treatment after stroke promoted functional and neurovascular recovery accompanied with the decreased area of glial scar in WT mice, which was blunted by Sirt3 deficiency. Furthermore, adjudin could increase Foxo3a and inhibit Notch1 signaling pathway via Sirt3. Both the suppression of Foxo3a and overexpression of N1ICD could alleviate the inhibitory effect of adjudin <i>in vitro</i> indicating that Sirt3-Foxo3a and Sirt3-Notch1 signaling pathways were involved in the inhibitory effect of adjudin in wound healing experiment.
Project description:Paradols are non-pungent and biotransformed metabolites of shogaols and reduce inflammatory responses as well as oxidative stress as shogaols. Recently, shogaol has been noted to possess therapeutic potential against several central nervous system (CNS) disorders, including cerebral ischemia, by reducing neuroinflammation in microglia. Therefore, paradol could be used to improve neuroinflammation-associated CNS disorders. Here, we synthesized paradol derivatives (2- to 10-paradols). Through the initial screening for anti-inflammatory activities using lipopolysaccharide (LPS)-stimulated BV2 microglia, 6-paradol was chosen to be the most effective compound without cytotoxicity. Pretreatment with 6-paradol reduced neuroinflammatory responses in LPS-stimulated BV2 microglia by a concentration-dependent manner, which includes reduced NO production by inhibiting iNOS upregulation and lowered secretion of proinflammatory cytokines (IL-6 and TNF-?). To pursue whether the beneficial in vitro effects of 6-paradol leads towards in vivo therapeutic effects on transient focal cerebral ischemia characterized by neuroinflammation, we employed middle cerebral artery occlusion (MCAO)/reperfusion (M/R). Administration of 6-paradol immediately after reperfusion significantly reduced brain damage in M/R-challenged mice as assessed by brain infarction, neurological deficit, and neural cell survival and death. Furthermore, as observed in cultured microglia, 6-paradol administration markedly reduced neuroinflammation in M/R-challenged brains by attenuating microglial activation and reducing the number of cells expressing iNOS and TNF-?, both of which are known to be produced in microglia following M/R challenge. Collectively, this study provides evidences that 6-paradol effectively protects brain after cerebral ischemia, likely by attenuating neuroinflammation in microglia, suggesting it as a potential therapeutic agent to treat cerebral ischemia.
Project description:<h4>Background</h4>White matter injury is the major form of brain damage in very preterm infants. Selective white matter injury in the immature brain can be induced by lipopolysaccharide (LPS)-sensitized hypoxic-ischemia (HI) in the postpartum (P) day 2 rat pups whose brain maturation status is equivalent to that in preterm infants less than 30?weeks of gestation. Neuroinflammation, blood-brain barrier (BBB) damage and oligodendrocyte progenitor apoptosis may affect the susceptibility of LPS-sensitized HI in white matter injury. c-Jun N-terminal kinases (JNK) are important stress-responsive kinases in various forms of insults. We hypothesized that LPS-sensitized HI causes white matter injury through JNK activation-mediated neuroinflammation, BBB leakage and oligodendroglial apoptosis in the white matter of P2 rat pups.<h4>Methods</h4>P2 pups received LPS (0.05?mg/kg) or normal saline injection followed by 90-min HI. Immunohistochemistry and immunoblotting were used to determine microglia activation, TNF-?, BBB damage, cleaved caspase-3, JNK and phospho-JNK (p-JNK), myelin basic protein (MBP), and glial fibrillary acidic protein (GFAP) expression. Immunofluorescence was performed to determine the cellular distribution of p-JNK. Pharmacological and genetic approaches were used to inhibit JNK activity.<h4>Results</h4>P2 pups had selective white matter injury associated with upregulation of activated microglia, TNF-?, IgG extravasation and oligodendroglial progenitor apoptosis after LPS-sensitized HI. Immunohistochemical analyses showed early and sustained JNK activation in the white matter at 6 and 24?h post-insult. Immunofluorescence demonstrated upregulation of p-JNK in activated microglia, vascular endothelial cells and oligodendrocyte progenitors, and also showed perivascular aggregation of p-JNK-positive cells around the vessels 24?h post-insult. JNK inhibition by AS601245 or by antisense oligodeoxynucleotides (ODN) significantly reduced microglial activation, TNF-? immunoreactivity, IgG extravasation, and cleaved caspase-3 in the endothelial cells and oligodendrocyte progenitors, and also attenuated perivascular aggregation of p-JNK-positive cells 24?h post-insult. The AS601245 or JNK antisense ODN group had significantly increased MBP and decreased GFAP expression in the white matter on P11 than the vehicle or scrambled ODN group.<h4>Conclusions</h4>LPS-sensitized HI causes white matter injury through JNK activation-mediated upregulation of neuroinflammation, BBB leakage and oligodendrocyte progenitor apoptosis in the immature brain.
Project description:Adjudin, also known as AF-2364 and an analog of lonidamine (LND), is a male contraceptive acting through the induction of premature sperm depletion from the seminiferous epithelium when orally administered to adult rats, rabbits or dogs. It is also known that LND can target mitochondria and block energy metabolism in tumor cells. However, whether Adjudin exhibits any anti-cancer activity remains to be elucidated. Herein we described the anti-proliferative activity of Adjudin on cancer cells in vitro and on lung and prostate tumors inoculated in nude mice. We found that Adjudin induced apoptosis in cancer cells through a Caspase-3-dependent pathway. Further experiments revealed that Adjudin could trigger mitochondrial dysfunction in cancer cells, apparently affecting the mitochondrial mass, inducing the loss of mitochondrial membrane potential and reducing cellular ATP levels. Intraperitoneal administration of Adjudin to tumor-bearing athymic nude mice also significantly suppressed the lung and prostate tumor growth. When used in combination with cisplatin, Adjudin enhances the sensitivity to cisplatin-induced cancer cell cytotoxicity. Taken together, these findings have demonstrated that Adjudin may be a potential drug for cancer therapy.
Project description:Transient retinal ischemia is a major complication of retinal degenerative diseases and contributes to visual impairment and blindness. Evidences indicate that microglia-mediated neuroinflammation has a key role in the neurodegenerative process, prompting the hypothesis that the control of microglia reactivity may afford neuroprotection to the retina against the damage induced by ischemia-reperfusion (I-R). The available therapeutic strategies for retinal degenerative diseases have limited potential, but the blockade of adenosine A<sub>2A</sub> receptor (A<sub>2A</sub>R) emerges as candidate strategy. Therefore, we evaluated the therapeutic potential of a selective A<sub>2A</sub>R antagonist (KW6002) against the damage elicited by I-R. The administration of KW6002 after I-R injury reduced microglia reactivity and inflammatory response and afforded protection to the retina. Moreover, we tested the ability of caffeine, an adenosine receptor antagonist, in mediating protection to the retina in the I-R injury model. We demonstrated that caffeine administration dually regulated microglia reactivity and cell death in the transient retinal ischemic model, depending on the reperfusion time. At 24?h of reperfusion, caffeine increased microglial reactivity, inflammatory response and cell death elicited by I-R. However, at 7 days of reperfusion, caffeine administration decreased microglia reactivity and reduced the levels of proinflammatory cytokines and cell death. Together, these results provide a novel evidence for the use of adenosine A<sub>2A</sub>R antagonists as potential therapy for retinal ischemic diseases and demonstrate the effect of caffeine on the regulation of microglia-mediated neuroinflammation in the transient ischemic model.