2-BFI Provides Neuroprotection Against Fluorosis by Stabilizing Endoplasmic Reticulum-Mitochondria Contact Sites and Inhibiting Activation of the NLRP3 Inflammasome.

2-(2-benzofu-ranyl)-2-imidazoline (2-BFI) is a drug that has attracted much attention in recent years. It has a therapeutic effect on brain diseases in animal models such as Alzheimer's disease and cerebral infarction. However, whether 2-BFI affords neuroprotection against the toxicity of fluoride, which can cross the blood-brain barrier and cause neurological dysfunction is not known. We investigated the cell viability and apoptosis of SH-SY5Y cells and primary cultures of cortical neurons exposed to fluoride, and 2-BFI was used to protect both two kinds of cells against the effects of fluoride. We found that 2-BFI can provide neuroprotection on SH-SY5Y cells and primary cultures of cortical neurons upon fluorosis by maintaining the stability of endoplasmic reticulum-mitochondria contact sites and inhibiting activation of the NLR family pyrin domain containing 3 (NLRP3) inflammasome. This study may provide a new method for protecting against the neurotoxicity induced by fluoride exposure.

2-(2-Benzofuranyl)-2-Imidazoline Attenuates the Disruption of the Blood-Brain Barrier in EAE via NMDAR.

Blood-brain barrier (BBB) disruption has been recognized as an early hallmark of multiple sclerosis (MS) pathology. Our previous studies have shown that 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) protected against experimental autoimmune encephalomyelitis (EAE), a classic animal model of MS. However, the potential effects of 2-BFI on BBB permeability have not yet been evaluated in the context of EAE. Herein, we aimed to investigate the effect of 2-BFI on BBB permeability in both an animal model and an in vitro BBB model using TNF-α to imitate the inflammatory damage to the BBB in MS. In the animal model, 2-BFI reduced neurological deficits and BBB permeability in EAE mice compared with saline treatment. The Western blot results indicated that 2-BFI not only alleviated the loss of the tight junction protein occludin caused by EAE but also inhibited the activation of the NR1-ERK signaling pathway. In an in vitro BBB model, 2-BFI (100 µM) alleviated the TNF-α-induced increase in permeability and reduction in expression of occludin in monolayer bEnd.3 cells. Similar protective effects were also observed after treatment with the NMDAR antagonist MK801. The Western blot results showed that the TNF-α-induced BBB breakdown and increase in NMDAR subunit 1 (NR1) levels and ERK phosphorylation could be blocked by pretreatment with 2-BFI or MK801. However, no additional effect was observed on BBB permeability or the expression of occludin and p-ERK after pretreatment with both 2-BFI and MK801. Our study indicates that 2-BFI alleviates the disruption of BBB in the context of inflammatory injury similar to that of MS by targeting NMDAR1, as well as by likely activating the subsequent ERK signaling pathway. These results provide further evidence for 2-BFI as a potential drug for the treatment of MS.

2-BFI attenuates ischemic injury by modulating mTOR signaling and neuroinflammation in rats.

Ischemic stroke is one of the major diseases that cause mortality and morbidity of human beings, but there is still lack of effective treatment and prevention. We found that 2-(2-Benzofuranyl)-2-Imidazoline (2-BFI) is potently protective against stroke and acute inflammatory immune disease. Moreover, the mammalian target of rapamycin (mTOR) signaling contributes effectively to the modulation of post-stroke neuroinflammatory response. However, whether the protection of 2-BFI against ischemic injury is through mTOR-mediated neuroinflammatory response remains unestablished. Here, we used 2-BFI to treat ischemic rats induced by distal middle cerebral artery occlusion (dMCAO). We found that 2-BFI administration after dMCAO improved the neurological deficits and decreased the infarct volume. 2-BFI reduced phosphorylation of mTOR and p70S6, increased IL-10 and TGF-ß, and decreased IFN-γ levels in ischemic rats. Our results demonstrated that 2-BFI attenuates ischemic injury by inhibiting the activation of mTOR signaling and modulating neuroinflammation after stroke in rats.

2-BFI Provides Neuroprotection Against Inflammation and Necroptosis in a Rat Model of Traumatic Brain Injury.

Inflammation and programmed necrosis (necroptosis) are the two hallmark pathological changes after traumatic brain injury (TBI) that contribute to aggravated brain damage. 2-(2-Benzofuranyl)-2-imidazoline (2-BFI) has been shown to exert both anti-inflammatory and programmed cell death effects. Therefore, the aim of the present study was to evaluate the potential beneficial effects of 2-BFI in a rat model of TBI induced by a weight-drop device. 2-BFI or vehicle was given via intraperitoneal injection starting at 30 min post trauma and then twice daily for three consecutive days. Following a neurofunctional test at 72 h after injury, histological, molecular, and immunohistochemistry analyses were performed on the pericontusional areas of the brain. 2-BFI treatment significantly attenuated neurological deficits, brain edema and blood-brain barrier permeability after TBI. Also, treatment with 2-BFI significantly reduced microglial activation, neutrophil infiltration, and proinflammatory cytokine interleukin (IL)-1ß secretion, which is related to nucleotide binding and oligomerization domain-like receptor family pyrin domain-containing 3 (NLRP3) inflammasome activation after TBI. In addition, 2-BFI treatment markedly reduced cortical tissue loss as well as repressed TBI-induced increases in necroptosis and necroptosis-associated proteins, including receptor-interacting protein (RIP1), RIP3, and mixed linkage kinase domain-like (MLKL) in the pericontusional brain tissue. Taken together, these findings indicate that 2-BFI may be an effective neuroprotectant after brain trauma and warrants further study.

Treatment With 2-BFI Attenuated Spinal Cord Injury by Inhibiting Oxidative Stress and Neuronal Apoptosis via the Nrf2 Signaling Pathway.

Previous reports showed that 2-(-2-benzofuranyl)-2-imidazoline (2-BFI) has antioxidant, anti-inflammatory and anti-apoptotic effects on neuroprotection in numerous disorders. However, the precise mechanisms remain elusive. The nuclear factor c factor 2 (Nrf2)/antioxidant response element (ARE) signaling pathway plays an important and essential role in the antioxidant and anti-inflammatory responses of the cell. Therefore, the purpose of this study was to investigate the potential neuroprotective effects of 2-BFI in a rat model of spinal cord injury (SCI) and to determine whether its neuroprotective effects are associated with the activation of Nrf2. To test this hypothesis, we examined the potential roles of 2-BFI in SCI models which were established in rats using a clip-compression injury method. Our results showed that treatment with 2-BFI twice daily improved locomotion recovery from SCI, which increased Nrf2 expression in both neurons and astrocytes, meanwhile, the level of heme oxygenase-1 (HO-1) also significantly enhanced. In addition, after the treatment with 2-BFI increased levels of superoxidase dismutase (SOD) and glutathione peroxidase (GPx) indicated the antioxidant effect of the drug. Furthermore, the upregulation of Bcl-2 and downregulation of Bax and caspase-3 implied antiapoptotic effects on neuroprotection of 2-BFI, which were verified by the Fluoro-Jade B (FJB) staining and TUNEL staining. Collectively, these results add to a growing body of evidence supporting that 2-BFI may attenuate SCI mediated by activation of the Nrf2/HO-1 signaling pathway.

Protective effects of 2-(2-benzonfuranyl)-2-imidazoline combined with tissue plasminogen activator after embolic stroke in rats.

Stroke is the third leading cause of death and disability in developing countries. The effective therapy for acute ischemic stroke is thrombolysis with recombinant tissue plasminogen activator (rt-PA) within 4.5 h of stroke onset. An effective post-ischemic neuroprotectant would extend the advantages of rt-PA, and protect against complications of thrombolysis. We previously reported that 2-(2-benzofuranyl)-2-imidazoline (2-BFI), a newly discovered ligand for high-affinity type 2 imidazoline receptor (I2R), provides neuroprotection against ischemic stroke in rats. Here we investigated the protective effects of 2-BFI in combination with delayed intravenous rt-PA after stroke induced by embolic middle cerebral artery occlusion (eMCAO) in rats. Infarct size was determined using 2,3,5-triphenyltrazolium chloride staining, while neurological deficit was assessed based on neurological score. Numbers of apoptotic cells in vivo were estimated using TUNEL stain, and expression of the pro-apoptotic protein BAX and anti-apoptotic protein BCL-2 were quantified by Western blotting. The results showed that 2-BFI (3 mg/kg) administered at 0.5 h after embolic MCAO combined with rt-PA (10 mg/kg) administered at 6 h reduced brain infarct size, mitigated neurological deficit, decreased the number of TUNEL-positive cells, down-regulated BAX expression, and up-regulated BCL-2 expression. These findings suggest that 2-BFI may extend the therapeutic window of rt-PA to 6 h after embolic stroke onset in rats.

Protective effect of the imidazoline I2 receptor agonist 2-BFI on oxidative cytotoxicity in astrocytes.

Astrocytes perform a variety of functions that are important for normal neuronal activity and recovery after brain injury. Because astrocytes are very vulnerable to H2O2, protection of astrocytes from oxidative damage in various neurological diseases is important in maintaining brain function and preventing brain damage. In this study, we investigated the characteristics and mechanisms of a specific imidazoline I2 receptor agonist 2-BFI-mediated cytoprotection using a rat astrocyte cultures of H2O2-exposed oxidative stress. Here we show that 2-BFI in H2O2-exposed astrocytes protects cell death through increased lysosomal membrane stability, LC3-II conversion, and subsequently suppresses accumulation of p62. These effects of 2-BFI were significantly reversed after treatment with the lysozyme activity inhibitor Bafilomycin A1. These results suggest that the cytoprotective effects of 2-BFI, which increases lysosomal stability in oxidative stress, may involve regulation of lysosomal-associated membrane protein-dependent autophagy and autolysosome degradation in astrocytes.

Neuroanatomical characterization of imidazoline I2 receptor agonist-induced antinociception.

Chronic pain is a significant public health problem with a lack of safe and effective analgesics. The imidazoline I2 receptor (I2 R) is a promising analgesic target, but the neuroanatomical structures involved in mediating I2 R-associated behaviors are unknown. I2 Rs are enriched in the arcuate nucleus, dorsal raphe (DR), interpeduncular nucleus, lateral mammillary body, medial habenula, nucleus accumbens (NAc) and paraventricular nucleus; thus, this study investigated the antinociceptive and hypothermic effects of microinjections of the I2 R agonist 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI). In rats, intra-DR microinjections produced antinociception in complete Freund's adjuvant- and chronic constriction injury-induced pain models. Intra-NAc microinjections produced antinociception and increased noxious stimulus-associated side time in a place escape/avoidance paradigm. Intra-NAc pretreatment with the I2 R antagonist idazoxan but not the D1 receptor antagonist SCH23390 or the D2 receptor antagonist raclopride attenuated intra-NAc 2-BFI-induced antinociception. Intra-NAc idazoxan did not attenuate systemically administered 2-BFI-induced antinociception. Microinjections into the other regions did not produce antinociception, and in none of the regions produced hypothermia. These data suggest that I2 R activation in some but not all I2 R-enriched brain regions is sufficient to produce antinociception and supports the theory that different I2 R-associated effects are mediated via distinct receptor populations, which may in turn be distributed differentially throughout the CNS.

The imidazoline I2 receptor agonist 2-BFI attenuates hypersensitivity and spinal neuroinflammation in a rat model of neuropathic pain.

Chronic pain is a large, unmet public health problem. Recent studies have demonstrated the importance of neuroinflammation in the establishment and maintenance of chronic pain. However, pharmacotherapies that reduce neuroinflammation have not been successfully developed to treat chronic pain thus far. Several preclinical studies have established imidazoline I2 receptor (I2R) agonists as novel candidates for chronic pain therapies, and while some I2R ligands appear to modulate neuroinflammation in certain scenarios, whether they exert anti-neuroinflammatory effects in models of chronic pain is unknown. This study examined the effects of the prototypical I2R agonist 2-(2-benzofuranyl)-2-imidazoline hydrochloride (2-BFI) on hypersensitivity and neuroinflammation induced by chronic constriction injury (CCI), a neuropathic pain model in rats. In CCI rats, twice-daily treatment with 10 mg/kg 2-BFI for seven days consistently increased mechanical and thermal nociception thresholds, reduced GFAP and Iba-1 levels in the dorsal horn of the spinal cord, and reduced levels of TNF-α relative to saline treatment. These results were recapitulated in primary mouse cortical astrocyte cultures. Incubation with 2-BFI attenuated GFAP expression and supernatant TNF-α levels in LPS-stimulated cultures. These results suggest that I2R agonists such as 2-BFI may reduce neuroinflammation which may partially account for their antinociceptive effects.

2-(2-benzofuranyl)-2-imidazoline (2-BFI) improved the impairments in AD rat models by inhibiting oxidative stress, inflammation and apoptosis.

Alzheimer's Disease (AD) is one of the commonest neural degeneration in aging population, and has become a global health challenge. 2-(2-benzofuranyl)-2-imidazoline (2-BFI) was reported to effectively improved the damage of patients with neuropathological disorders. In the present study, we investigated the effect of 2-BFI on the improvement of antioxidative, inflammation, and apoptosis in AD rats. Sprague-Dawley rats (2 months old, n=40) were used in this study and after injection of Aß1-42 into hippocampal CA1 (Cornu Ammonis) region, the rats were given high, moderate and low dose of 2-BFI though intraperitoneal (i.p.) injection. Then spatial memory and navigation ability were analyzed by Morrize Water Maze. For the molecular testing, chemical colorimetry, ELISA and immunoblotting were performed to measure the activities of antioxidative enzymes, the abundance of immune cytokines and expression of apoptotic proteins, respectively. Hematoxylin and Eosin staining was used to analyze the pathological changes. We observed that 2-BFI significantly ameliorated the learning and memory abilities in rat models with AD by dosage treatment, as demonstrated by the shorten learning latency and greater times of travel across the platform quadrant. Additionally, reactive oxygen species (ROS) and malondialdehyde (MDA), were decreased after treatment of 2-BFI with dosage dependency, while the activities of superoxidase dismutase (SOD) and (GPX) Glutathione peroxidase were in turn enhanced, suggesting that 2-BFI could protect the antioxidative enzymes and reduce the oxidative stress in the hippocampus. Moreover, the expression of inflammatory factors including TNF-a and IL-1ß were decreased after 2-BFI treatment. Additionally, the neuronal apoptosis was also attenuated, as shown by Western blot results. Taken together, the cognitive impairment in AD rats could be significantly improved by 2-BFI in a dose-dependent manner through suppressing oxidants accumulation, inhibiting of inflammatory response, as well as enhancing the neural viability.

Gender difference in epileptogenic effects of 2-BFI and BU224 in mice.

Imidazoline I2 receptors are involved in pain modulation and psychiatric disorders and its ligands may represent a new therapeutic strategy against pain and depression. In particular, 2-BFI and BU224 are the two most widely studied I2 receptor ligands and have antinociceptive and antidepressant-like activities in rodents. However, little is known of the toxicological effects and potential gender differences of these I2 receptor ligands. This study examined the epileptogenic activities of 2-BFI and BU224 in male and female mice and also examined their underlying receptor mechanisms. 2-BFI (10-40 mg/kg, i.p.) and BU224 (10-40 mg/kg) produced epileptic seizures in a dose-related manner, as did the epileptogenic agent, pentylenetetrazole (PTZ, 15-60 mg/kg). However, female mice were significantly more sensitive than male mice in all the measures. The commonly used I2 receptor antagonist, idazoxan (10mg/kg), did not block the onset and magnitude of the epileptic seizures or lethality induced by 2-BFI and BU224. When studied in combination, PTZ potentiated the epileptogenic effect of 2-BFI and BU224. The lack of antagonism by idazoxan of the epileptogenic activities of 2-BFI and BU224 suggests that the epileptogenic effects of 2-BFI and BU224 are mediated by non-imidazoline I2 receptors and that I2 receptors remain a viable therapeutic target for neurological disorders such as pain.