Curcumin enhances elvitegravir concentration and alleviates oxidative stress and inflammatory response.

In this study, we investigated the potential of using curcumin (CUR) as an adjuvant to enhance the delivery of antiretroviral drug elvitegravir (EVG) across the BBB, and alleviate oxidative stress and inflammatory response, which are the major hallmark of HIV neuropathogenesis. In a mouse model, we compared the biodistribution of EVG alone and in combination with CUR using intraperitoneal (IP) and intranasal (IN) routes. IN administration showed a significantly higher accumulation of EVG in the brain, while both IP and IN routes led to increased EVG levels in the lungs and liver. The addition of CUR further enhanced EVG brain delivery, especially when administered via the IN route. The expression of neural marker proteins, synaptophysin, L1CAM, NeuN, and GFAP was not significantly altered by EVG or CUR alone or their combination, indicating preserved neural homeostasis. After establishing improved brain concentration and safety of CUR-adjuvanted EVG in mice in acute treatment, we studied the effect of this treatment in HIV-infected U1 macrophages. In U1 macrophages, we also observed that the addition of CUR enhanced the intracellular concentration of EVG. The total area under the curve (AUCtot) for EVG was significantly higher in the presence of CUR. We also evaluated the effects of CUR on oxidative stress and antioxidant capacity in EVG-treated U1 macrophages. CUR reduced oxidative stress, as evidenced by decreased reactive oxygen species (ROS) levels and elevated antioxidant enzyme expression. Furthermore, the combination of CUR and EVG exhibited a significant reduction in proinflammatory cytokines (TNFα, IL-1ß, IL-18) and chemokines (RANTES, MCP-1) in U1 macrophages. Additionally, western blot analysis confirmed the decreased expression of IL-1ß and TNF-α in EVG + CUR-treated cells. These findings suggest the potential of CUR to enhance EVG permeability to the brain and subsequent efficacy of EVG, including HIV neuropathogenesis.

Curcumin enhances elvitegravir concentration and alleviates oxidative stress and inflammatory response.

In this study, we investigated the potential of using curcumin (CUR) as an adjuvant to enhance the delivery of antiretroviral drug elvitegravir (EVG) across the BBB, and alleviate oxidative stress and inflammatory response, which are the major hallmark of HIV neuropathogenesis. In a mouse model, we compared the biodistribution of EVG alone and in combination with CUR using intraperitoneal (IP) and intranasal (IN) routes. IN administration showed a significantly higher accumulation of EVG in the brain, while both IP and IN routes led to increased EVG levels in the lungs and liver. The addition of CUR further enhanced EVG brain delivery, especially when administered via the IN route. The expression of neural marker proteins, synaptophysin, L1CAM, NeuN, and GFAP was not significantly altered by EVG or CUR alone or their combination, indicating preserved neural homeostasis. After establishing improved brain concentration and safety of CUR-adjuvanted EVG in mice in acute treatment, we studied the effect of this treatment in HIV-infected U1 macrophages. In U1 macrophages, we also observed that the addition of CUR enhanced the intracellular concentration of EVG. The total area under the curve (AUCtot) for EVG was significantly higher in the presence of CUR. We also evaluated the effects of CUR on oxidative stress and antioxidant capacity in EVG-treated U1 macrophages. CUR reduced oxidative stress, as evidenced by decreased reactive oxygen species (ROS) levels and elevated antioxidant enzyme expression. Furthermore, the combination of CUR and EVG exhibited a significant reduction in proinflammatory cytokines (TNFα, IL-1ß, IL-18) and chemokines (RANTES, MCP-1) in U1 macrophages. Additionally, western blot analysis confirmed the decreased expression of IL-1ß and TNF-α in EVG + CUR-treated cells. These findings suggest the potential of CUR to enhance EVG permeability to the brain and subsequent efficacy of EVG, including HIV neuropathogenesis.

Extracellular vesicles released from macrophages modulates interleukin-1ß in astrocytic and neuronal cells.

We have recently demonstrated that long-term exposure of cigarette smoke condensate (CSC) to HIV-uninfected (U937) and -infected (U1) macrophages induce packaging of pro-inflammatory molecules, particularly IL-1ß, in extracellular vesicles (EVs). Therefore, we hypothesize that exposure of EVs derived from CSC-treated macrophages to CNS cells can increase their IL-1ß levels contributing to neuroinflammation. To test this hypothesis, we treated the U937 and U1 differentiated macrophages once daily with CSC (10 µg/ml) for 7 days. Then, we isolated EVs from these macrophages and treated these EVs with human astrocytic (SVGA) and neuronal (SH-SY5Y) cells in the absence and presence of CSC. We then examined the protein expression of IL-1ß and oxidative stress related proteins, cytochrome P450 2A6 (CYP2A6), superoxide dismutase-1 (SOD1), catalase (CAT). We observed that the U937 cells have lower expression of IL-1ß compared to their respective EVs, confirming that most of the produced IL-1ß are packaged into EVs. Further, EVs isolated from HIV-infected and uninfected cells, both in the absence and presence of CSC, were treated to SVGA and SH-SY5Y cells. These treatments showed a significant increase in the levels of IL-1ß in both SVGA and SH-SY5Y cells. However, under the same conditions, the levels of CYP2A6, SOD1, and catalase were only markedly altered. These findings suggest that the macrophages communicate with astrocytes and neuronal cells via EVs-containing IL-1ß in both HIV and non-HIV setting and could contribute to neuroinflammation.

Intranasal delivery of darunavir improves brain drug concentrations in mice for effective HIV treatment.

Despite the availability of combined antiretroviral therapy (cART), which reduces the HIV replication in chronically HIV-infected patients, HIV associated neurocognitive disorders (HAND) persists in the brain. The blood-brain barrier (BBB) is the major barrier for the penetration of drugs including antiretrovirals, limiting the drug penetration to the brain. In the present study, we have shown improved brain drug concentration in mice for darunavir (DRV), an FDA-approved drug, using an intranasal (IN) delivery method that bypasses the BBB. Here, we compared the time-dependent biodistribution of DRV at two different concentrations, high (25 mg/kg) and low (2.5 mg/kg), using two administration routes intravenous (IV) and intranasal (IN) in brain, liver, lungs, and plasma. Compared with IV administration, IN administration demonstrated a significantly improved DRV penetration in the brain at both low and high DRV concentrations (IV vs IN: at 2.5 mg/kg: 6.91 ± 1.69 ng/g vs 12.08 ± 2.91 ng/g, at 25 mg/kg: 12.84 ± 2.88 ng/g vs 19.74 ± 1.80 ng/g). As expected, IN administration showed significantly lower DRV concentrations in plasma (IV vs IN: at 2.5 mg/kg: 81.37 ± 22.04 ng/g vs 19.91 ± 12.65 ng/g, at 25 mg/kg: 899.12 ± 136.93 ng/g vs 320.56 ± 40.04 ng/g) and liver (IV vs IN: at 2.5 mg/kg: 118.39 ± 28.13 ng/g vs 29.27 ± 4.17 ng/g at 25 mg/kg: 1085.18 ± 255.0 ng/g vs 833.83 ± 242.4 ng/g). The IN administration did not show significant change in lungs compared to the IV administration. As a result, these findings suggest that the IN route can increase the DRV level in the brain, suppressing HIV in the brain reservoirs. Additionally, it could also reduce off-target effects, especially in peripheral organs.

Effect of benzo(a)pyrene on oxidative stress and inflammatory mediators in astrocytes and HIV-infected macrophages.

BACKGROUND: Benzo(a)pyrene (BaP), an important polycyclic aromatic hydrocarbons (PAH) component of cigarette/tobacco smoking, is known to cause adverse health effects and is responsible for various life-threatening conditions including cancer. However, it is not yet clear whether BaP contributes to the macrophage- and astrocyte-mediated inflammatory response. METHODS: We examined the acute (up to 72 h) effects of BaP on the expression of antioxidant enzymes (AOEs), cytokines/chemokines, and cytochromes P450 (CYP) enzymes in astrocytic cell lines, SVGA, and chronically HIV-infected U1 macrophage. The treated cells were examined for mRNA, protein levels of CYPs, AOEs superoxide dismutase-1 (SOD1) and catalase (CAT), cytokines/chemokines, using Western blot, multiplex ELISA, and reactive oxygen species (ROS) by flow cytometry analysis. RESULTS: Upon acute exposure, BaP (1 µM) showed a significant increase in the mRNA levels of CYPs (CYP1A1 and CYP1B1), and pro-inflammatory cytokine IL-1ß in SVGA cells following BaP for 24, 48, and 72h. In addition, we observed a significant increase in the mRNA levels of SOD1 and CAT at 24h of BaP treatment. In contrast, BaP did not exert any change in the protein expression of AOEs and CYP enzymes. In U1 cells, however, we noticed an interesting increase in the levels of MCP-1 as well as a modest increase in TNFα, IL-8 and IL-1ß levels observed at 72 h of BaP treatment but could not reach to statistically significant level. CONCLUSIONS: Overall, these results suggest that BaP contributes in part to macrophage and astrocyte-mediated neuroinflammation by mainly inducing IL-1ß and MCP-1 production, which is likely to occur with the involvement of CYP and/or oxidative stress pathways.

Pre-test Cortisol Levels in Predicting Short Synacthen Test Outcome: A Retrospective Analysis.

Objective: Short Synacthen tests (SSTs) are expensive, dependent on Synacthen availability, and need supervision. To reduce SST testing, we examined the utility of pre-test cortisol (Cort0) and related parameters in predicting outcome. Design and Measurements: We retrospectively examined the following in all SSTs; (i) Cort0 (ii) indications (iii) and time and place of testing. Receiver operated characteristic (ROC) curves were devised for Cort0 to obtain the best cut-off for outcome prediction in those who had SSTs between 8 and 10 am (Group 1) and at other times (Group 2). Results: Of 506 SSTs, 13 were unsuitable for analysis. 111/493 SSTs (22.5%) were abnormal. (1) ROC curves predicted - (a) SST failure with 100% specificity when Cort0 was ⩽124 nmol/L (Group 1), or ⩽47 (Group 2); (b) a normal SST with 100% sensitivity when Cort0 ⩾314 nmol/L (Group 1) and ⩾323 nmol/L (Group 2). (2) There was significant correlation between Cort0 and 30-minute cortisol (rs = 0.65-0.78, P < .001). (3) Median Cort0 was lower in those who failed SSTs compared to those who passed (147 vs 298 nmol/L respectively, P < .001). (4) SST failure was commoner in Group 1 vs 2 (P = .001). (5) There was no difference in outcome between out-patient and inpatient SSTs. (6) SST failure was most common for 'steroid related' indications (39.6%, P < .001). Conclusions: This study indicates that (1) Cort0 ⩾ 323 (Group1) and ⩾314 nmol/L (Group 2) predicted a normal SST with 100% sensitivity; (2) Using these cut offs 141/493 (28.6%) tests may have been avoided; (3) supporting evidence should be considered in those with a lower pre-test predictability of failure.

Targeted Drug Delivery to the Central Nervous System Using Extracellular Vesicles.

The blood brain barrier (BBB) maintains the homeostasis of the central nervous system (CNS) and protects the brain from toxic substances present in the circulating blood. However, the impermeability of the BBB to drugs is a hurdle for CNS drug development, which hinders the distribution of the most therapeutic molecules into the brain. Therefore, scientists have been striving to develop safe and effective technologies to advance drug penetration into the CNS with higher targeting properties and lower off-targeting side effects. This review will discuss the limitation of artificial nanomedicine in CNS drug delivery and the use of natural extracellular vesicles (EVs), as therapeutic vehicles to achieve targeted delivery to the CNS. Information on clinical trials regarding CNS targeted drug delivery using EVs is very limited. Thus, this review will also briefly highlight the recent clinical studies on targeted drug delivery in the peripheral nervous system to shed light on potential strategies for CNS drug delivery. Different technologies engaged in pre- and post-isolation have been implemented to further utilize and optimize the natural property of EVs. EVs from various sources have also been applied in the engineering of EVs for CNS targeted drug delivery in vitro and in vivo. Here, the future feasibility of those studies in clinic will be discussed.

Nutraceuticals in HIV and COVID-19-Related Neurological Complications: Opportunity to Use Extracellular Vesicles as Drug Delivery Modality.

People living with HIV/AIDS (PLWHA) are at an increased risk of severe and critical COVID-19 infection. There is a steady increase in neurological complications associated with COVID-19 infection, exacerbating HIV-associated neurocognitive disorders (HAND) in PLWHA. Nutraceuticals, such as phytochemicals from medicinal plants and dietary supplements, have been used as adjunct therapies for many disease conditions, including viral infections. Appropriate use of these adjunct therapies with antiviral proprieties may be beneficial in treating and/or prophylaxis of neurological complications associated with these co-infections. However, most of these nutraceuticals have poor bioavailability and cannot cross the blood-brain barrier (BBB). To overcome this challenge, extracellular vesicles (EVs), biological nanovesicles, can be used. Due to their intrinsic features of biocompatibility, stability, and their ability to cross BBB, as well as inherent homing capabilities, EVs hold immense promise for therapeutic drug delivery to the brain. Therefore, in this review, we summarize the potential role of different nutraceuticals in reducing HIV- and COVID-19-associated neurological complications and the use of EVs as nutraceutical/drug delivery vehicles to treat HIV, COVID-19, and other brain disorders.

Racial Health Disparity and COVID-19.

The infection by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) and resultant coronavirus diseases-19 (COVID-19) disproportionally affects minorities, especially African Americans (AA) compared to the Caucasian population. The AA population is disproportionally affected by COVID-19, in part, because they have high prevalence of underlying conditions such as obesity, diabetes, and hypertension, which are known to exacerbate not only kidney diseases, but also COVID-19. Further, a decreased adherence to COVID-19 guidelines among tobacco smokers could result in increased infection, inflammation, reduced immune response, and lungs damage, leading to more severe form of COVID-19. As a result of high prevalence of underlying conditions that cause kidney diseases in the AA population coupled with tobacco smoking make the AA population vulnerable to severe form of both COVID-19 and kidney diseases. In this review, we describe how tobacco smoking interact with SARS-CoV-2 and exacerbates SARS-CoV-2-induced kidney diseases including renal failure, especially in the AA population. We also explore the role of extracellular vesicles (EVs) in COVID-19 patients who smoke tobacco. EVs, which play important role in tobacco-mediated pathogenesis in infectious diseases, have also shown to be important in COVID-19 pathogenesis and organ injuries including kidney. Further, we explore the potential role of EVs in biomarker discovery and therapeutics, which may help to develop early diagnosis and treatment of tobacco-induced renal injury in COVID-19 patients, respectively.

Nicotine self-administration with menthol and audiovisual cue facilitates differential packaging of CYP2A6 and cytokines/chemokines in rat plasma extracellular vesicles.

In this study, we investigated whether intravenously self-administered nicotine with menthol and audiovisual cue modulates nicotine-metabolizing CYP2A6, oxidative stress modulators, and cytokines/chemokines in plasma extracellular vesicles (EVs) in rats. We assigned rats to self-administered nicotine with: (a) audiovisual cue (AV), (b) menthol, and (c) menthol and AV cue. We found increased levels of CD9 in plasma EVs after self-administered nicotine with menthol and AV cue. Moreover, expression of CYP2A6 in plasma EVs was significantly increased after self-administered nicotine in response to menthol and AV cue. However, despite an upward trend on SOD1 and catalase, increase was not found to be statistically significant, while total antioxidant capacity was found to be significantly increased in plasma and plasma EVs obtained after self-administered nicotine with menthol and AV cue. Among cytokine and chemokine profiling, we found a significant increase in the levels of MCP-1 after self-administered nicotine with menthol and AV cue and complete packaging of IL-1ß in EVs. Taken together, the study provides evidence that nicotine in response to menthol and AV cues can package altered levels of CYP2A6, and cytokines/chemokines in plasma EVs that may contribute to cell-cell communication, nicotine metabolism, and inflammation upon cigarette smoking.

Anti-HIV Activity of Cucurbitacin-D against Cigarette Smoke Condensate-Induced HIV Replication in the U1 Macrophages.

Chemodietary agents are emerging as promising adjuvant therapies in treating various disease conditions. However, there are no adjuvant therapies available to minimize the neurotoxicity of currently existing antiretroviral drugs (ARVs). In this study, we investigated the anti-HIV effect of a chemodietary agent, Cucurbitacin-D (Cur-D), in HIV-infected macrophages using an in-vitro blood-brain barrier (BBB) model. Since tobacco smoking is prevalent in the HIV population, and it exacerbates HIV replication, we also tested the effect of Cur-D against cigarette smoke condensate (CSC)-induced HIV replication. Our results showed that Cur-D treatment reduces the viral load in a dose-dependent (0-1 µM) manner without causing significant toxicity at <1 µM concentration. Further, a daily dose of Cur-D (0.1 µM) not only reduced p24 in control conditions, but also reduced CSC (10 µg/mL)-induced p24 in U1 cells. Similarly, Cur-D (single dose of 0.4 µM) significantly reduced the CSC (single dose of 40 µg/mL)-induced HIV replication across the BBB model. In addition, treatment with Cur-D reduced the level of pro-inflammatory cytokine IL-1ß. Therefore, Cur-D, as an adjuvant therapy, may be used not only to suppress HIV in the brain, but also to reduce the CNS toxicity of currently existing ARVs.

Inhibition of extracellular vesicle pathway using neutral sphingomyelinase inhibitors as a neuroprotective treatment for brain injury.

Traumatic brain injury is a sudden trauma or blow on the head, and severe traumatic brain injury is a major cause of death and disability worldwide. The acute and chronic consequences following traumatic brain injury can lead to progressive secondary neurodegenerative changes and cognitive dysfunction. To date, there is no effective pharmaceutical products for the treatment to reduce secondary damage after brain injury. The discovery of extracellular vesicles has attracted considerable scientific attention due to their role in cell-to-cell communication. Extracellular vesicles have shown their potential to carry not only biological molecules but also as a drug delivery vehicle. As a carrier of molecular information, extracellular vesicles have been involved in physiological functions as well as in the modulation of immune responses. Here, we aim to provide new insights into the contrasting role of extracellular vesicles in the propagation of inflammatory responses after brain injury. As a carrier of pro-inflammatory molecules, their role as functional mediators in the pathophysiology of brain injury is discussed, addressing the inhibition of the extracellular vesicle pathway as an anti-inflammatory or neuroprotective approach to improve the outcome of both acute and chronic inflammation following brain injury. Here, we summarize therapeutic strategies to diminish the risk the neurodegeneration post brain injury and propose that neutral sphingomyelinase inhibitors could be used as potentially useful therapeutic agents for the treatment of brain injury associated neuroinflammation.

Challenges in Biomaterial-Based Drug Delivery Approach for the Treatment of Neurodegenerative Diseases: Opportunities for Extracellular Vesicles.

Biomaterials have been the subject of numerous studies to pursue potential therapeutic interventions for a wide variety of disorders and diseases. The physical and chemical properties of various materials have been explored to develop natural, synthetic, or semi-synthetic materials with distinct advantages for use as drug delivery systems for the central nervous system (CNS) and non-CNS diseases. In this review, an overview of popular biomaterials as drug delivery systems for neurogenerative diseases is provided, balancing the potential and challenges associated with the CNS drug delivery. As an effective drug delivery system, desired properties of biomaterials are discussed, addressing the persistent challenges such as targeted drug delivery, stimuli responsiveness, and controlled drug release in vivo. Finally, we discuss the prospects and limitations of incorporating extracellular vesicles (EVs) as a drug delivery system and their use for biocompatible, stable, and targeted delivery with limited immunogenicity, as well as their ability to be delivered via a non-invasive approach for the treatment of neurodegenerative diseases.

Intervention and Improved Well-Being of Basic Science Researchers During the COVID 19 Era: A Case Study.

The coronavirus disease-19 (COVID-19) pandemic has affected individuals of all categories, irrespective of their geographical locations, professions, gender, or race. As a result of full or partial lock-down and stay-at-home orders, the well-being and productivity of individuals were severely affected. Since basic science research requires laboratory experiments, the work-from-home strategy hurt their productivity. In addition, the combination of decreased productivity and staying at home is likely to compromise their well-being by causing stress and anxiety. In this case study, a strategy was developed to engage researchers through listening and learning, motivation, and empowerment, using regular virtual sessions. Through these virtual sessions, research work was prioritized and coordinated, from idea conception to writing research papers and grant proposals. Perceived stress scores (PSS) and COVID-19-related stress (COVID-SS) scores were measured to evaluate general and COVID-19-induced stress, respectively, every month from March to July 2020 during the COVID-19 era. The result showed a significant improvement in both the PSS and the COVID-SS scores of the intervention group compared to the control group. In addition, while there was no/minimal change in PSS and COVID-SS scores from March to subsequent months until July for the control group, the intervention groups showed significant and consistent improvement in both scores in the intervention group. Overall, the intervention strategy showed improved well-being for basic science researchers, which was also consistent with their improved productivity during the COVID-19 era.

Extracellular Vesicles in Viral Replication and Pathogenesis and Their Potential Role in Therapeutic Intervention.

Extracellular vesicles (EVs) have shown their potential as a carrier of molecular information, and they have been involved in physiological functions and diseases caused by viral infections. Virus-infected cells secrete various lipid-bound vesicles, including endosome pathway-derived exosomes and microvesicles/microparticles that are released from the plasma membrane. They are released via a direct outward budding and fission of plasma membrane blebs into the extracellular space to either facilitate virus propagation or regulate the immune responses. Moreover, EVs generated by virus-infected cells can incorporate virulence factors including viral protein and viral genetic material, and thus can resemble noninfectious viruses. Interactions of EVs with recipient cells have been shown to activate signaling pathways that may contribute to a sustained cellular response towards viral infections. EVs, by utilizing a complex set of cargos, can play a regulatory role in viral infection, both by facilitating and suppressing the infection. EV-based antiviral and antiretroviral drug delivery approaches provide an opportunity for targeted drug delivery. In this review, we summarize the literature on EVs, their associated involvement in transmission in viral infections, and potential therapeutic implications.

An update on drug-drug interactions between antiretroviral therapies and drugs of abuse in HIV systems.

INTRODUCTION: While considerable progress has been made in the fight against HIV/AIDS, to date there has not been a cure, and millions of people around the world are currently living with HIV/AIDS. People living with HIV/AIDS have substance abuse disorders at higher rates than non-infected individuals, which puts them at an increased risk of drug-drug interactions. AREAS COVERED: Potential drug-drug interactions are reviewed for a variety of potential drugs of abuse, both licit and illicit. These drugs include alcohol, cigarettes or other nicotine delivery systems, methamphetamine, cocaine, opioids, and marijuana. Potential interactions include decreased adherence, modulation of drug transporters, or modulation of metabolic enzymes. We also review the relative incidence of the use of these drugs of abuse in People living with HIV/AIDS. EXPERT OPINION: Despite considerable improvements in outcomes, disparities in outcomes between PLWHA who use drugs of abuse, vs those who do not still exist. It is of critical necessity to improve outcomes in these patients and to work with them to stop abusing drugs of abuse.

Circulatory Astrocyte and Neuronal EVs as Potential Biomarkers of Neurological Dysfunction in HIV-Infected Subjects and Alcohol/Tobacco Users.

The diagnosis of neurocognitive disorders associated with HIV infection, alcohol, and tobacco using CSF or neuroimaging are invasive or expensive methods, respectively. Therefore, extracellular vesicles (EVs) can serve as reliable noninvasive markers due to their bidirectional transport of cargo from the brain to the systemic circulation. Hence, our objective was to investigate the expression of astrocytic (GFAP) and neuronal (L1CAM) specific proteins in EVs circulated in the plasma of HIV subjects, with and without a history of alcohol consumption and tobacco smoking. The protein expression of GFAP (p < 0.01) was significantly enhanced in plasma EVs obtained from HIV-positive subjects and alcohol users compared to healthy subjects, suggesting enhanced activation of astrocytes in those subjects. The L1CAM expression was found to be significantly elevated in cigarette smokers (p < 0.05). However, its expression was not found to be significant in HIV subjects and alcohol users. Both GFAP and L1CAM levels were not further elevated in HIV-positive alcohol or tobacco users compared to HIV-positive nonsubstance users. Taken together, our data demonstrate that the astrocytic and neuronal-specific markers (GFAP and L1CAM) can be packaged in EVs and circulate in plasma, which is further elevated in the presence of HIV infection, alcohol, and/or tobacco. Thus, the astroglial marker GFAP and neuronal marker L1CAM may represent potential biomarkers targeting neurological dysfunction upon HIV infection and/or alcohol/tobacco consumption.

Bifenthrin insecticide promotes oxidative stress and increases inflammatory mediators in human neuroblastoma cells through NF-kappaB pathway.

The extensive application of bifenthrin (BF) insecticide in agriculture has raised serious concerns with regard to increased risks of developing neurodegenerative diseases. Recently, our group showed that BF exposure in rodent models induced oxidative stress and inflammation markers in various regions of the brain (frontal cortex, striatum and hippocampus) and this was associated with behavioral changes. This study aimed to confirm such inflammatory and oxidative stress in an in vitro cell culture model of SK-N-SH human neuroblastoma cells. Markers of oxidative stress (ROS, NO, MDA, H2O2), antioxidant enzyme activities (CAT, GPx, SOD) and inflammatory response (TNF-α, IL-6, PGE2) were analyzed in SK-N-SH cells after 24 h of exposure to different concentrations of BF (1-20 µM). Protein synthesis and mRNA expression of the enzymes implicated in the synthesis of PGE2 were also measured (COX-2, mPGES-1) as well as nuclear factor κappaB (NF-κBp65) and antioxidant nuclear erythroid-2 like factor-2 (Nrf-2). Cell viability was analyzed by MTT-tetrazolio (MTT) and lactate dehydrogenase (LDH) assays. Exposure of SK-N-SH cells to BF resulted in a concentration-dependent reduction in the number of viable cells (reduction of MTT and increase in LDH activity). There was also a BF concentration-dependent increase in oxidative stress markers (ROS release, NO, MDA and H2O2) and decrease in the activity of antioxidant enzymes (CAT and GPx activities). There was further a concentration-dependent increase in pro-inflammatory cytokines (TNF-α and IL-6) and inflammatory mediator PGE2, increase in protein synthesis and mRNA expression of inflammatory markers (COX-2, mPGES-1 and NF-κBp65) and decrease in protein synthesis and mRNA expression of antioxidant Nrf-2. Our data shows that BF induces various oxidative stress and inflammatory markers in SK-N-SH human neuroblastoma cells as well as the activation of NF-κBp65 signaling pathway. This is in line with prior results in brain regions of rodents exposed in vivo to BF showing increased oxidative stress in response to BF exposure, occurring in pro-inflammatory conditions and likely activating programmed cell death.

Neutral Sphingomyelinase Inhibition Alleviates LPS-Induced Microglia Activation and Neuroinflammation after Experimental Traumatic Brain Injury.

Neuroinflammation is one of the key secondary injury mechanisms triggered by traumatic brain injury (TBI). Microglial activation, a hallmark of brain neuroinflammation, plays a critical role in regulating immune responses after TBI and contributes to progressive neurodegeneration and neurologic deficits following brain trauma. Here we evaluated the role of neutral sphingomyelinase (nSMase) in microglial activation by examining the effects of the nSMase inhibitors altenusin and GW4869 in vitro (using BV2 microglia cells and primary microglia), as well as in a controlled cortical injury (CCI) model in adult male C57BL/6 mice. Pretreatment of altenusin or GW4869 prior to lipopolysaccharide (LPS) stimulation for 4 or 24 hours, significantly downregulated gene expression of the pro-inflammatory mediators TNF-α, IL-1ß, IL-6, iNOS, and CCL2 in microglia and reduced the release of nitric oxide and TNF-α These nSMase inhibitors also attenuated the release of microparticles and phosphorylation of p38 MAPK and ERK1/2. In addition, altenusin pretreatment also reduced the gene expression of multiple inflammatory markers associated with microglial activation after experimental TBI, including TNF-α, IL-1ß, IL-6, iNOS, CCL2, CD68, NOX2, and p22phox Overall, our data demonstrate that nSMase inhibitors attenuate multiple inflammatory pathways associated with microglial activation in vitro and after experimental TBI. Thus, nSMase inhibitors may represent promising therapeutics agents targeting neuroinflammation.

Inhibition of neuroinflammation by thymoquinone requires activation of Nrf2/ARE signalling.

Thymoquinone is an antioxidant phytochemical that has been shown to inhibit neuroinflammation. However, little is known about the potential roles of intracellular antioxidant signalling pathways in its anti-inflammatory activity. The objective of this study was to elucidate the roles played by activation of the Nrf2/ARE antioxidant mechanisms in the anti-inflammatory activity of this compound. Thymoquinone inhibited lipopolysaccharide (LPS)-induced neuroinflammation through interference with NF-κB signalling in BV2 microglia. Thymoquinone also activated Nrf2/ARE signalling by increasing nuclear localisation, DNA binding and transcriptional activity of Nrf2, as well as increasing protein levels of HO-1 and NQO1. Suppression of Nrf2 activity through siRNA or with the use of trigonelline resulted in the loss of anti-inflammatory activity by thymoquinone. Taken together, our studies show that thymoquinone inhibits NF-κB-dependent neuroinflammation in BV2 microglia, by targeting antioxidant pathway involving activation of both Nrf2/ARE. We propose that activation of Nrf2/ARE signalling pathway by thymoquinone probably results in inhibition of NF-κB-mediated neuroinflammation.