Curcumin enhances elvitegravir concentration and alleviates oxidative stress and inflammatory response

Abstract 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 (AUC tot ) 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-1β, IL-18) and chemokines (RANTES, MCP-1) in U1 macrophages. Additionally, western blot analysis confirmed the decreased expression of IL-1β 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.

The eradication of latent HIV reservoirs from the CNS and the management of neuronal complications present signi cant challenges in the treatment of HAND. Poor penetration of ART drugs into the CNS, viral replication-induced neuronal damage, and potential neurotoxicity associated with ART drugs contribute to suboptimal viral suppression and inadequate neuronal protection 5,6 . Additionally, the selective nature of the BBB, e ux transporters, e.g., P-glycoprotein (P-gp) in BBB and myeloid cells, and the presence of metabolic enzymes, e.g., cytochrome P4503A4 in myeloid cells further limit the concentrations of ART drugs in the CNS 5,7−9 . To address these challenges, recent advancements in the eld have explored novel strategies such as nanoparticulate ART, ART nanosuspensions, and monocyte/macrophage targeted ART approaches [10][11][12] . These approaches have shown promising results in humanized HIV-infected mice, suggesting that enhancing CNS ART delivery can have neuroprotective effects. Re-formulated ART preparations with longer-lasting effective drug release are also being considered for new human clinical trials, speci cally targeting HIV-associated neurocognitive impairment 13 . Interestingly, despite the initiation of combination ART (cART), the overall prevalence of HAND remains unchanged 3 . This suggests that viral load alone is not the crucial driver for the development of HAND. Emerging evidence suggests that self-fueling in ammatory processes within the CNS contribute to neurodegeneration 14 .
Thus, targeting these in ammatory processes, independent of viral load, with immunomodulatory drugs in addition to cART, presents an intriguing approach to reduce chronic in ammation and maintain cognitive function in HAND patients.
In recent years, there has been a growing interest in exploring natural compounds or nutraceuticals with anti-HIV activity to enhance therapeutic outcomes in neuroHIV and HAND [15][16][17] . While studies investigating natural compounds as potential anti-in ammatory agents for HAND treatment are limited, CUR has emerged as a promising candidate. CUR, the primary curcuminoid derived from the rhizome of Curcuma longa, possesses diverse biological properties, including anti-in ammatory, antioxidant, and antimicrobial effects 18,19 . It has demonstrated anti-viral activity against a wide range of viruses, including HIV, and has shown neuroprotective effects in the context of neurodegenerative diseases 20,21 . CUR's ability to reduce neuroin ammation, protect against oxidative damage, and inhibit the formation of amyloid brils associated with neurocognitive impairments makes it a promising adjuvant. Our hypothesis is that CUR, especially using IN delivery, enhances the transmigration of the ART drug EVG in macrophages and in rodent brain. We also hypothesize that CUR alone and with EVG combination result in reduced oxidative stress and in ammation, the major hallmarks of HIV neuropathogenesis and HAND.

Results:
1. Effect of CUR on biodistribution of EVG at high dose via IP and IN route in mice The primary aim of this study is to compare the in vivo biodistribution of EVG, both as a standalone drug and in combination with CUR, through the utilization of two different administration routes: IP and IN. In the rst phase of the experiment, we investigated the distribution of EVG (25 mg/kg) in combination with CUR (20 mg/kg) following IP administration in the brain, liver, lungs, and plasma for 12h. The results demonstrated that the addition of CUR signi cantly increased the accumulation of EVG in the plasma for IP administration ( Fig. 1a and 1b, #p ≤ 0.05). There was a pattern of increase in EVG concentrations by CUR in the plasma for IN administration, however, they were not statistically signi cant ( Fig. 1c and 1d).
Although not statistically signi cant, a similar trend of relatively high EVG concentrations in combination therapy was observed in the brain (Fig. 1e), lungs (Fig. 1f), and liver ( Fig. 1g) when compared to EVG (IP) treatment alone. In the subsequent phase of the experiment, we evaluated the impact of IN administration on the biodistribution of EVG in the presence of CUR. Our ndings indicated a signi cant increase in EVG accumulation in the brain (Fig. 1e) when compared to EVG (IN) only group (###p ≤ 0.001) and EVG (IP) only group ($$$p ≤ 0.001). EVG concentration in lungs (Fig. 1f, ##p ≤ 0.01, $$p ≤ 0.01) also appeared to be increased in EVG + CUR (IN) group. A similar trend, with increased EVG concentrations in combination therapy, was also observed in plasma and liver, although the effect was not statistically signi cant. These results highlight the potential of CUR to enhance the brain delivery of EVG, especially for IN administration.

Effect of CUR on biodistribution of EVG at low dose via IP and IN route in mice
This study was performed to evaluate the in vivo biodistribution of EVG (5 mg/kg) in the presence of CUR (4 mg/kg) at low dose using both the IP and IN routes (Fig. 2, a-g). The results indicate that the IP or IN administration of EVG in the presence of CUR does not lead to an increased concentration of EVG in plasma (Fig. 2, a-d). However, like high dose study, the results indicated that CUR addition to combination treatment signi cantly increases EVG accumulation in the brain upon IN administration when compared to EVG + CUR (IP) group (Fig. 2e, $$$p ≤ 0.001). The biodistribution of EVG in the lungs and liver did not exhibit any signi cant alterations upon the addition of CUR in the treatment, regardless of the administration route (IP or IN) ( Fig. 2f and 2g). Overall, although low dose treatment was less effective in increasing EVG concentrations in combination therapy in plasma or organs, it was still effective via IN compared with IP route in brain.
3. Effect of CUR on neural marker proteins in EVG treated mice HAND involves key events such as neuronal apoptosis, dysregulation of neuronal support cells, and loss of dendritic arbor. Therefore, we aimed to examine whether speci c neuronal markers (synaptophysin, L1CAM, NeuN, and GFAP) in mice brains are altered when treated with high dose IN EVG + CUR (25 and 20 mg/kg, respectively) for 12 hours. Our ndings showed that neither EVG or CUR alone nor addition of CUR to the EVG treatment altered the expression of these neuronal markers compared to the control group ( Fig. 3, a-d). This suggests that the neural homeostasis is not affected by the addition of CUR to the EVG treatment, at least upon acute exposure.

Effect of CUR on intracellular EVG concentration in U1 macrophages
In our 48h experiment involving HIV-infected U1 macrophages, we administered EVG (1 µM) either alone or in combination with CUR (5 µM), and the intracellular EVG levels were measured (represented as percentage of the initial concentration). Our results consistently demonstrated higher intracellular EVG concentrations in the EVG + CUR group compared to the EVG only group (Fig. 4a, #p ≤ 0.05 at 10 min). Moreover, to assess the overall exposure to EVG, we calculated the total area under the curve (AUC tot ) for EVG in both treatment groups. Notably, the AUC tot of EVG + CUR was signi cantly higher than that of the EVG only group (Fig. 4b, #p ≤ 0.05). These ndings highlight the ability of CUR to enhance the intracellular delivery and retention of EVG in U1 macrophages, which could subsequently enhance EVG e cacy.

Effects of CUR on oxidative stress and antioxidant capacity in EVG-treated U1 macrophages
To determine whether the addition of CUR to the EVG treatment results in alteration of oxidative stress, we measured the reactive oxygen species (ROS) levels in the EVG + CUR treated U1 macrophages. Our results showed that 24h treatment with EVG + CUR does not alter the ROS levels when compared to control treatment ( Fig. 5a, b). While the biological effects resulting from ROS-induced oxidative stress are widely utilized for monitoring purposes, it is equally crucial to assess the antioxidant capacity of biological uids and cells. In this study, we assessed the overall antioxidant capacity of U1 macrophages. The ndings revealed that CUR alone signi cantly elevated the antioxidant capacity in comparison to the control group (Fig. 5c, **p ≤ 0.01). Moreover, the combination of CUR with EVG also enhanced the antioxidant capacity compared to the control (Fig. 5c, *p ≤ 0.05). Furthermore, we examined the protein expression of two important antioxidant enzymes, catalase and SOD1, in U1 macrophages after 24h exposure to EVG + CUR. Our ndings revealed that EVG treatment alone led to a signi cant decrease in SOD1 expression, and a similar trend was observed with catalase expression ( Fig. 5d and 5e, **p ≤ 0.01). However, the addition of CUR to the EVG treatment resulted in increased SOD1 expression (Fig. 5e, #p ≤ 0.01). Although the trend for catalase expression followed a similar pattern with EVG and CUR treatment, it did not reach statistical signi cance. Taken together, the results suggest that although EVG alone increases/shows no difference in oxidative stress or antioxidant capacity, CUR alone and in the presence of EVG increases antioxidant capacity and perhaps decreases oxidative stress.

Effect of CUR on modulation of EVG-induced cytokine pro le in U1 macrophages
In this study, the effect of combining CUR with EVG on the release of proin ammatory cytokines (IL-1β, TNFα, IL-6, IL-8, IL-18), anti-in ammatory cytokines (IL-10, IL-1RA), and chemokines (RANTES, MCP-1) was evaluated in the media released from U1 macrophages. The results indicated that the addition of CUR to EVG treatment signi cantly reduced the level of the proin ammatory cytokine TNFα (Fig. 6a, *p ≤ 0.05) when compared to the control group. Furthermore, the results showed that the level of IL-1β was elevated in EVG-only (Fig. 6a, *p ≤ 0.05) treatment but signi cantly decreased in the EVG + CUR (Fig. 6a, #p ≤ 0.05) treatment group. Additionally, CUR signi cantly lowered the level of IL-18 (Fig. 6a, *p ≤ 0.05, #p ≤ 0.05) when compared to both the control group and the EVG-only treatment group. However, we noticed no statistical difference in the levels of IL-6 and IL-8 in all treatment groups when compared to control. The level of anti-in ammatory cytokine IL-10 ( Fig. 6a, *p ≤ 0.05) was increased in EVG + CUR treatment group compared to control. However, IL-1ra showed no signi cant changes in any treatment groups compared to control. With respect to the chemokines, RANTES and MCP-1, both showed increased levels by EVG-only treatment (Fig. 6a, *p ≤ 0.05 for MCP-1). However, CUR alone (Fig. 6a, #p ≤ 0.05 for MCP-1) or combination (Fig. 6a, #p ≤ 0.05 for RANTES) showed decreased levels of chemokines. Taken together, relatively decreased levels of proin ammatory cytokines and chemokines and increased levels of anti-in ammatory cytokines by EVG + CUR compared to control and/or EVG-only suggest that the combination therapy can decrease systemic in ammatory response in brain macrophages.
Furthermore, to validate the results obtained on cytokines released in the media (systemic in ammation), we also measured the effect of CUR + EVG treatment on cellular cytokines (IL1-β and TNF-α) using Western blot. TNF-α and IL-1β play a major role in HIV pathogenesis. Our ndings demonstrated that treatment with EVG alone signi cantly increased IL-1β levels compared to the control group (Fig. 6b, **p ≤ 0.01). However, the addition of CUR to the EVG treatment effectively reversed this increase in IL-1β expression (Fig. 6b, ##p ≤ 0.01). Regarding TNF-α, both CUR treatment alone and EVG + CUR (Fig. 6c, *p ≤ 0.05) (Fig. 6c, *p ≤ 0.05) resulted in decreased levels compared to the control treatment. These results further suggest that CUR alone or combination reduces cellular in ammatory response.

Discussion:
The ndings of our study address the challenges associated with drug delivery across the BBB in context to HIV neuropathogenesis. The limited penetration of ART drugs into the CNS leads to persistent viral replication and neuroin ammation, contributing to neuronal damage 4 . To overcome this barrier, prior research has explored innovative strategies for enhancing CNS drug delivery. One approach is the In our previous study, we investigated the IN delivery of darunavir (DRV) to improve brain drug concentration in mice 26 . We compared the biodistribution of DRV at high (25 mg/kg) and low (2.5 mg/kg) concentrations using IV and IN routes in the brain, liver, lungs, and plasma. Our results showed that IN administration signi cantly increased DRV penetration in the brain compared to IV administration, at both low (5 mg/kg) and high (25 mg/kg) concentrations. Furthermore, IN administration resulted in lower DRV concentrations in plasma and liver compared to IV administration. These ndings indicate that the IN route can enhance the concentration of DRV in the brain, potentially suppressing HIV in brain reservoirs, while reducing off-target effects in peripheral organs 26 .
In the present study, we investigated the potential of CUR as an adjuvant to improve the delivery of the ART drug EVG across the BBB and reduce pathogenesis associated with HIV such as oxidative stress and in ammatory response in macrophages, an important CNS viral reservoir. Our results demonstrated that the addition of CUR increased the intracellular concentration of EVG in U1 macrophages, suggesting improved e cacy. Moreover, in vivo experiments using mouse models showed that CUR enhances the brain delivery of EVG, particularly when administered through IN route. The IN route provides a noninvasive mode of drug delivery to the brain by bypassing the BBB 26 . This route takes advantage of the direct nose-to-brain pathway, resulting in faster onset of action and improved drug delivery to speci c brain regions. The nding is consistent with our previous ndings with DRV delivery via IN route. Thus, our ndings support the potential of IN administration of CUR-adjuvanted EVG as an effective approach for enhancing CNS drug delivery.
The ine ciency of current ART regimens in treating HAND can be attributed to their limited ability to target the in ammatory cascades associated with neuroHIV 27 . HIV-infected individuals receiving ART have been reported to exhibit higher levels of free radical species compared to untreated HIV-positive individuals or healthy subjects 28 . This suggests that HIV infection itself, along with the introduction of ART, may induce oxidative stress and exacerbate HIV pathogenesis. Various intracellular antioxidant defenses play a crucial role in detoxifying ROS and protecting cells. Enzymes such as SOD1 and catalase contribute to the quenching and conversion of ROS into harmless byproducts 29 . However, the balance between ROS production and antioxidant defenses can be disrupted in HIV-infected individuals 28 . CUR, with its well-documented anti-in ammatory and antioxidant properties, is a promising candidate for adjuvant therapy in HIV treatment 30 . It inhibits key enzymes involved in HIV replication, such as protease and integrase, and targets the NF-κB pathway, which is essential for HIV gene expression 30,31 . CUR acts as a bifunctional antioxidant by directly scavenging ROS and inducing an antioxidant response 32 . These properties make CUR an attractive option for enhancing the e cacy of HIV treatment.
Oxidative stress and in ammation are closely linked processes. The accumulation of ROS triggers oxidative stress, which in turn enhances in ammation by activating transcription factors associated with in ammation. CUR can reduce ROS production by affecting NADPH oxidase and increasing the activity of antioxidant enzymes 29 . Additionally, CUR's modulation of the Nrf2-Keap1 pathway contributes to its antiin ammatory and antioxidant effects 33 . In our study, we observed that the combination of CUR and EVG had bene cial effects on modulating oxidative stress and antioxidant capacity in U1 macrophages. While EVG alone showed no signi cant changes, CUR alone and in combination with EVG reduced oxidative stress and increased antioxidant capacity. These ndings underscore the potential neuroprotective effects of CUR and its ability to modulate oxidative capacity in the context of HIV infection. In our study, the combination of CUR and EVG has demonstrated promising results. The addition of CUR to EVG treatment led to a signi cant decrease in proin ammatory cytokines (IL-1β, TNFα, IL-18) and chemokines (RANTES, MCP-1) in cell-based experiments. In HAND, key events involve neuronal apoptosis, dysregulation of neuronal support cells, and dendritic arbor loss. To explore neural markers, we examined synaptophysin, L1CAM, NeuN, and GFAP in mouse brains. NeuN, a splicing regulator, shows increased cytoplasmic localization in HIV-associated neurocognitive disorders. GFAP, an astrocyte marker, is upregulated in reactive astrocytes. Synaptophysin, a synaptic vesicle regulator, is abundant in synaptic transmission. L1CAM plays a role in neural development and regeneration. Our study found that CUR addition to EVG treatment did not signi cantly alter the expression of these neuronal markers, indicating preserved neural homeostasis.
In conclusion, the ndings of our study, along with the additional evidence presented, highlight the potential bene ts of incorporating CUR as an adjuvant therapy in the treatment of HIV neuropathogenesis. The combination of CUR with the antiretroviral drug EVG has shown improvements in total antioxidant activity and a reduction in in ammation, as demonstrated by the modulation of cytokine pro les in an in vitro HIV setting. Further investigation using HIV-infected primary macrophages and HIV mouse models would provide valuable insights into the implications of CUR on HIV neuropathogenesis. Additionally, development of EVG nanoparticles using poloxamer-PLGA by our research group has demonstrated signi cant increase in brain EVG concentrations and subsequent HIV suppression. Therefore, formulating CUR along with EVG in PLGA nanoparticles can further enhance its adjuvant pro le and improve the overall outcomes in the treatment of HIV neuropathogenesis. Animals: Male and female Balb/c mice, aged 10 to 12 weeks, were obtained from Jackson Laboratory (Bar Harbor, MA) and allowed to acclimate in the animal facility for a minimum of 7 days before the start of the study. The mice were housed in groups of ve per cage in a sterile room with a 12/12-hour lightdark cycle. The room maintained a constant temperature and humidity, and the mice had ad libitum access to food and water throughout the study. All animal procedures were approved by the institutional animal care and use committee of the University of Tennessee Health Science Center (UTHSC-IACUC protocol #20-0165) and were performed in accordance with the Guide for the Care and Use of Laboratory Animals from the National Institutes of Health. All methods with animal studies were reported in accordance with ARRIVE guidelines. For the EVG biodistribution study, a total of 96 mice were randomly divided into two groups for IP and IN administration. Each group was further divided into four subgroups corresponding to four time points (1, 3, 6, and 12 hours), with three females and three males in each subgroup for both EVG and EVG + CUR treatment. In the low-dose study, mice were administered EVG (5 mg/kg) and CUR (4 mg/kg), while in the high-dose study, EVG (25 mg/kg) and CUR (20 mg/kg) were administered. These concentrations were determined based on previous studies and literature sources 26, 39 . The drugs were dissolved in a solution containing 5% DMSO, 80% PEG400, and 15% PBS. For IN administration, the concentration of EVG in the nal solution was adjusted to 0.5 ml/kg in mice.

Materials and methods
The nal volume of DMSO used was 0.025 ml/kg, which was below the reported non-toxic dose. Mice were euthanized under deep iso urane anesthesia followed by cervical dislocation, and blood samples were collected at the designated time points (1, 3, 6, and 12 hours) via cardiac puncture using EDTAcontaining blood-collection tubes. The blood samples were allowed to settle at room temperature and then centrifuged at 6000 rpm for 10 minutes at 4°C to obtain plasma. Tissues, including the brain, liver, and lungs, were collected at the terminal time point of 12 hours. Plasma and brain samples were stored in tubes and frozen at -80°C until further analysis using LC-MS/MS. Tissue samples were homogenized in 1X phosphate-buffered saline (PBS) at a ratio of 1:4 (wt/vol). Fifty µL of each plasma and tissue sample was used for LC-MS/MS analysis, which was performed following established protocols using appropriate LC-MS/MS equipment and methodologies.
Cell culture and treatment U1 cells, a chronically HIV-1-infected U937 cell line, were obtained from the NIH AIDS Reagent Program (Germantown, MD). U1 cells are the major HIV model cells to study in vitro HIV-associated pathogenesis including oxidative stress and in ammatory response 40,41 . The data obtained with U1 macrophages are correlated with human primary monocyte-derived macrophages 42 . The U1 cells were cultured in RPMI 1640 media supplemented with 10% fetal bovine serum (FBS) and 1% L-glutamine. To differentiate the U1 cells into macrophages, 0.3 million cells in 0.4 ml of media containing 100 nM phorbol 12-myristate 13-acetate (PMA) were seeded in each well of a 12-well plate. After 3 days of differentiation, the media was aspirated, and the cells were washed with PBS before adding fresh media to the differentiated cells. The cells were then incubated for 3-4 hours before starting the treatment. The differentiated U1 macrophages were subjected to different treatment conditions. This included a control group treated with DMSO, as well as experimental groups treated with EVG (1 µM), CUR (5 µM). These EVG and CUR concentrations, which are near physiological, were chosen based on our previous study and studies from other group 26,39,43 . The cells were exposed to the respective treatments for a de ned period as per the treatment protocol of each assay. After the treatment duration, the U1 macrophages were harvested for further analysis. The cells were collected and processed for downstream experiments as per the speci c requirements of each assay.

Quanti cation of intracellular ROS with uorescence-based assay
To quantify the ROS level, we used ow cytometry analysis along with the uorescence dye CM-H2DCFDA (ThermoFisher Scienti c) as described before 44 . After thoroughly washing the treated cells with PBS, they were resuspended in 5 µM of CM-H2DCFDA in PBS and incubated in the dark at room temperature for 45 minutes. Following the incubation, the cells were washed and resuspended in 300 µL of PBS. The ROS produced in the cells was then detected and analyzed using the built-in ow cytometer software (Agilent NovoCyte).

Total Antioxidant Capacity
The antioxidant capacity of U1 cells treated with EVG + CUR was determined using the Total Antioxidant Capacity Assay (TCA) Kit (Cell Biolabs, San Diego, CA, USA) according to the manufacturer's instructions. The assay quanti es the antioxidant capacity by measuring the copper reducing equivalents (CRE) in the samples. The results are reported as µM CRE, which is indicative of the total antioxidant capacity of the samples.

Cytokine Analysis
The levels of various cytokines and chemokines, including pro-in ammatory cytokines IL-1β, TNF-α, IL-8, IL-6, IL-18; anti-in ammatory cytokines IL-1RA, IL-10; and chemokines MCP-1 and RANTES, were measured from the culture media of differentiated U1 macrophages and mice plasma. The measurements were performed using Human Custom Procartaplex 9-plex and Mouse Custom Procartaplex 6-plex (Invitrogen, ThermoFisher Scienti c, Grand Island, NY, USA), following the manufacturer's protocol as previously described 40 . Samples, standards, and magnetic beads were added to a 96-well ELISA plate and mixed thoroughly on a plate shaker for 1 hour at room temperature, followed by overnight incubation at 4°C. The beads were then washed, and the detection antibody, streptavidin-PE, and reading buffer were added, with subsequent washing steps after each addition. The concentration of cytokines and chemokines (pg/mL) was measured using a Magpix system, and the data were analyzed using the xPONENT® software.
Western Blotting: Protein expression in the treated cells and mouse brain was determined using Western blotting. For the evaluation of IL-1β, TNF-α, catalase, and SOD1 expression in EVG + CUR treated U1 cells, an equal amount of protein (15 µg Quanti cation of EVG using LC-MS/MS: The concentration of EVG in mouse plasma, tissue samples, and cell lysate samples was analyzed using our standardized LC-MS/MS method, as previously described 39 .

Statistical analysis
The data obtained from the experiments were presented as mean ± standard error of the mean (SEM) and were derived from a minimum of three independent experiments. Statistical analyses were performed using GraphPad Prism version 9.5.1 for Windows (GraphPad Software, San Diego, California, USA, www.graphpad.com). The signi cance of differences between groups was determined using analysis of variance (ANOVA) with multiple comparisons or t-tests for comparisons between two groups, as appropriate. The level of signi cance was set at p ≤ 0.05.

Declarations:
Funding This study is supported by funding from the NIH grants AG081140, and MH125670 to SK.  Biodistribution of EVG (5 mg/kg) in presence of CUR (4 mg/kg) administered via IP and IN in Balb/c mice. The concentration of EVG was measured in plasma (a, c), brain (e), lungs (f), and liver (g) and expressed as ng/g in tissues and ng/ml in plasma. Statistical analysis was performed using ANOVA (multiple comparisons) and t-test (two groups). Results are expressed as means ± S.E.M (n=6). $$$ represents p ≤0.001 when compared to EVG+CUR (IP).   Effects of EVG and CUR on reactive oxygen species (ROS), antioxidant capacity and anti-oxidant enzymes in U1 macrophages. U1 macrophages were treated with EVG (1 µM) and CUR (5 µM) for one