Protective Effects of Curcumin/Magnesium Oxide Nanoparticles Against Ketamine- induced Neurotoxicity

Protective Effects of Curcumin/Magnesium Oxide Nanoparticles Against Ketamine- induced Neurotoxicity | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Protective Effects of Curcumin/Magnesium Oxide Nanoparticles Against Ketamine- induced Neurotoxicity Mahsa Salehirad, A. Wallace Hayes, Majid Motaghinejad, Mina Gholami This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4008048/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract A curcumin-conjugated MgO nanostructure (Cur/MgO NPs) was synthesized, and its composition was verified. XRD and a particle size analyzer were used to determine the average crystalline and particle sizes. Morphological studies were conducted using FE-SEM. UV-Vis was also employed to examine absorption patterns, and FT-IR spectroscopy analyzed the functional groups involved in the reaction. The following protocol evaluated the effectiveness of Cur/MgO NPs in ketamine-treated male BALB/c mice. Group 1 received 0.2 mL of normal saline. Group 2 animals received Ket (25 mg/kg). Group 3 animals received 40 mg/kg Cur and 25 mg/kg Ket. Groups 4–6 received Ket (25 mg/kg) and Cur/MgO N.P.s (10, 20, or 40 mg/kg). Group 7 received 5 mg/kg MgO and Ket (25 mg/kg). Mice were injected ip daily for two weeks. The hippocampal tissue was analyzed for oxidative stress, inflammation, apoptotic markers, and mitochondrial quadruple complex enzymes. The Cur/MgO N.P.s were neuroprotective against the inflammation, apoptosis, and oxidative stress induced by Ket. Ketamine Curcumin/MgO N.P.s neurodegeneration Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 1. Introduction Nanotechnology can improve drug delivery while, at the same time, reducing adverse events and potential costs. Additional improvements include cell selectivity, release at specific target sites, and improved bioavailability from increased drug solubility [ 1 , 2 ]. Magnesium is the second most abundant intracellular cation after potassium and is a noncompetitive antagonist for the N-methyl-D-aspartate receptor (NMDA). The NMDA receptor is blocked and downregulated by excess magnesium [ 3 ]. MgO N.P.s have been shown to influence the regulation of neuronal cell activity [ 4 ]. Because of their size, formation, and structure, MgO N.P.s have the potential to be functional drugs [ 5 ]. MgO N.P.s reduced pain and inflammation in mice through central and peripheral pathways [ 6 ]. Additionally, Moeini-Nodeh et al. showed that MgO N.P.s have both antiapoptotic and anti-oxidative effects [ 7 ]. Curcumin (Cur) is a traditionally used medicinal ingredient with centuries of use as a safe and effective natural product [ 8 ]. Cur is the principal curcuminoid in turmeric, a popular Indian spice derived from the rhizome of Curcuma longa [ 9 ]. The antioxidant, anti-inflammatory, anti-proliferative, antiseptic, analgesic, anti-malarial, antitumoral, apoptosis-inducing, and anti-angiogenesis properties of Cur have been reported [ 10 ]. All appear to involve the inhibition of NFk B signaling, which lowers pro-inflammatory cytokines IL-1, IL-6, and TNF-α [ 11 ]. In an alcoholic animal neuropathy model, Cur antagonized the cellular consequences of oxidative stress by preventing DNA damage [ 12 ]. Curcumin may be a promising remedy for certain NDDs [ 11 , 13 , 14 ]. In obstetric and pediatric patients, ketamine (Ket), an NMDA receptor antagonist, is used for its analgesic and anesthetic effects [ 15 ]. Ket, however, has the potential for abuse due to its hallucinogenic and other reinforcing characteristics [ 16 , 17 ]. Ket has been reported to promote neuronal cell death and neurodegeneration, but the mechanism underlying its harmful effects remains elusive [ 18 , 19 ]. The frequencies with which practitioners prescribe Ket and the frequency of its illicit trade for recreational use have contributed to a significant increase in use. Ketamine's impact on the adult mouse brain has not been resolved, especially when used over an extended time. We evaluated whether Ket-induced neurotoxicity in the mouse hippocampus would respond to Cur/MgO N.P.s 2. Materials and Methods 2.1. Materials: Sigma-Aldrich provided the ketamine, lithium, potassium hydroxide, and magnesium nitrate hexahydrate (Mg (NO3) 2 6H 2 O), while Merck supplied the Cur. All reagents were analytical grade. The reagents were provided by DNA Biotech Co (Tehran, Iran). ddH 2 O was the solvent except for Ket and lithium, which were dissolved in normal saline. 2.2. Nanoparticle Synthesis: Cur/MgO nanoparticles were synthesized as follows. Fifty mg of Cur was dissolved in 50 mL of ddH 2 O at 80°C. The solubilized Cur was added to a magnesium nitrate solution (Mg (NO 3 ) 2 50 mL, 0.1 M), resulting in a yellowish-colored solution. The solution was refluxed at 85–90°C for two hours. When the refluxed solution cooled to 40°C, 5 mL of KOH (0.2 M) was gradually added, leading to an orange-yellow gel-like suspension. This suspension was centrifuged at 10,000 rpm, and the precipitate was washed with acetone and water until no yellow color (curcumin) was observed. The resulting Cur/MgO nanoparticle residue was vacuum-dried at ambient temperature. The nanoparticles were dissolved in normal saline in an ultrasonic bath for 15 min. 2.3. Nanoparticle Characterization FE-SEM, XRD, FT-IR spectral analysis, and UV-Vis spectra assessed the nanostructure's particle size, morphology, and chemical composition. FT-IR spectra were measured using a PerkinElmer instrument, and a Philips powder diffractometer was used to determine the X-ray diffraction records. The scanning rate was in the range of 10–80°. To confirm chemical formation, a double-beam spectrophotometer measured the UV-visible spectrum. FE-SEM analyzed the morphology and particle dispersion of the nanoparticles, and chemical composition was evaluated using Energy Dispersive X-ray Spectroscopy. Uncorrected melting points were determined using a capillary tube and a B510K melting point instrument. These techniques provided a comprehensive understanding of the characteristics of the synthesized nanostructure. 2.4. Materials and Methodology 2.4.1. Animals Fifty-six male BALB/c mice weighing 25–30 g were obtained from the University of Medical Sciences, Iran. After a week of acclimatization in the animal unit laboratory, they were randomly divided into experimental and control groups and housed in groups of eight. The mice had access to pellet feed (Parsfeed Co, Tehran, Iran) and water in a controlled environment (room temperature: 22 ± 0.5°C; relative humidity: 6–40%; 12-hour light/dark cycles). Animals were monitored for signs of toxicity post-treatment and continuously during the study. 2.4.2. Ethics Statement The research followed the Shahid Beheshti University of Medical Sciences Animal Care and Use Committee's guidelines, approved under I.R.SBMU.NRITLD.REC.1402.93. The investigators also adhered to the Animal Ethics and Welfare Guidelines and the ARRIVE procedure to guarantee the ethical treatment of the animals [ 20 ]. 2.4.3. Experimental Procedure The mice were divided into groups: Group 1 received 0.2 mL of normal saline. Group 2 animals received Ket (25 mg/kg). Group 3 animals received 40 mg/kg Cur and 25 mg/kg Ket. Groups 4–6 received Ket (25 mg/kg) and Cur/MgO N.P.s (10, 20, or 40 mg/kg). Group 7 received 5 mg/kg MgO and Ket (25 mg/kg). Mice were injected ip daily for two weeks. 2.4.5: Molecular and Biochemical Assessment The experimental design is represented in Fig. 1, while Fig. 2 presents an overview of the experimental procedure and timeline. 2.4.5.1. Total Protein On day 15, mice were anesthetized with Trapanal (50 mg/kg, i.p.), and the brains were removed. The hippocampus was carefully separated and evaluated for oxidative stress, inflammation, apoptosis, and mitochondrial respiratory chain enzyme alterations. The hippocampus was homogenized in a cold buffer and then centrifuged at 450 g for 10 min. The buffer contained MOPS (25 mM), sucrose (400 mM), magnesium chloride (4 mM), EGTA (0.05 mM), and the pH of the solution was 7.3. Subsequently, the samples underwent a second 10-min centrifugation at 12,000 g. The resulting sediment was re-suspended in the buffer and stored at 0°C. Bio-Rad CA DC protein assay kits (Providence, RI, USA) were employed to measure the concentration of BDNF, CREB, TNF-α, IL-1, Bax, Bcl-2, SOD, GPx, and G.R. Total protein concentration was determined using the Bradford method (Bio-Rad, Providence, RI, USA). A serial dilution of BSA, ranging from 0.1 to 1.0 mg/mL, was prepared in the homogenization buffer. A standard curve was created. Protein extracts of 10, 15, 20, 25, and 30 µL were added to individual wells, along with the Bradford reagent. Absorbance was measured at 630 nm utilizing a plate reader (Hiperion Microplate Reader, MPR4+, Rayto Company, China) [ 21 – 23 ]. 2.4.5.2. Oxidative Stress 2.4.5.2.1. Lipid Peroxidation The primary byproduct of cellular lipid peroxidation is malondialdehyde (MDA). One hundred uL of the MDA standard (DNA Biotech Co, Tehran, Iran, 100 uL) or 100 uL of the tissue homogenate was added to wells of the 96 microwell plate. SDS Lysis Solution (100 Ul) was added to each well. TBA reagent (250 uL) (DNA Biotech Co, Tehran, Iran) was added, and the mixture was gently shaken and incubated at 95°C for 45–60 min. The samples were then centrifuged at 1000 g for 15 min. n-Butanol (DNA Biotech Co, Tehran, Iran) (300 ul) was added to stop the reaction. Samples were centrifuged at 10,000 g for 7 min, and the absorbance was read at 532 nm. Results are reported as nmol/mg of protein [ 24 – 29 ]. 2.4.5.2.2. GSH and GSSG Twenty-five uL of the glutathione reductase solution (1X) (DNA Biotech Co, Tehran, Iran) and 25 uL of the NADPH solution (1X) (DNA Biotech Co, Tehran, Iran) were added, respectively, to 96-well plates to measure GSH and GSSG content. A standard glutathione solution (DNA Biotech Co, Tehran, Iran) or 25 uL homogenized tissue sample was added. Chromogen (1X) (50 uL, DNA Biotech Co, Tehran, Iran) was added, and the mixture was vigorously blended. Absorbance was measured at 405 nm. The results are reported as nmol/mg of protein [ 29 , 30 ]. 2.4.5.2.3. SOD A kit from DNA Biotech Co (Persequor Park Pretoria South Africa) was used to assess SOD activity. The first and third blank wells were filled with ddH 2 O, and the second blank well was filled with homogenized tissue solution with a reagent volume of 20 L. The process included adding a working solution to each well, gently mixing it, and then adding a dilution buffer and an enzyme working solution to specific wells. For 20 min, the solutions were incubated at 37°C after being thoroughly mixed. Subsequently, the absorbance was measured at 450 nm, and SOD was quantified as units/mL/mg protein. 2.4.5.2.4. GPx The GPx function in the hippocampal tissue was assessed using a commercial kit from DNA Biotech Company. The process included the addition of precise volumes of the homogenized tissue solution, assay buffer, reaction solution, and peroxidase substrate solution to the wells, followed by the measurement of absorbance at 340 nm over an 8-min period at 25°C. The results are in mU/mg protein. 2.4.5.2.5. GR The assessment of glutathione reductase activity was carried out using a kit from DNA Biotech Co. In the presence of NADPH, the reaction involved the transformation of GSSG to GSH. The reaction mixture was thoroughly mixed before measuring the absorbance at 340 nm for 120 seconds. The outputs are reported as mU/mg protein. [ 29 – 31 ]. 2.4.5.2.6. Mitochondrial Complex Enzymes The activities of mitochondrial complexes I, II, III, and IV were assessed using commercial kits from Abcam, Co (Boston, MA, USA). Measurement of NADH oxidation to NAD + at 450 nm estimated the function of mitochondrial complex I. For mitochondrial complex II, the assay involved the Electron-Transfer Catalysis of succinate to ubiquinone by measuring absorption at 550 nm. The activity of mitochondrial complex III was evaluated by determining the rate of conversion of CYCS to its reduced form at 600 nm. The function of mitochondrial complex IV was monitored by quantifying the oxidation of the reduced form of CYCS at 550 nm. The results are activity per milligram of protein per minute. 2.4.5.2.7. Protein Expression TNF-α, IL-1β, Bax, Bcl-2, caspase-3, and caspase-7 were estimated using commercial ELISA kits (Genzyme Diagnostics, Cambridge, USA). Sheep anti-mouse IL-1β, TNF-α Bax, Bcl-2, caspase-3, and caspase-7 polyclonal antibodies (Sigma Chemical Co., Poole, Dorset, U.K.)) were washed three times (0.5M sodium chloride (NaCl), 2.5mM sodium dihydrogen phosphate (NaH 2 PO 4 ), 7.5mM Na2HPO4, 0.1% Tween 20, pH 7.2). One hundred mL of 1% (w/v) ovalbumin solution (Sigma Chemical Co., Poole, Dorset, U.K.) was added to each well and incubated at 37° C for 1 hour. After washing three times, 100 mL of sample or standard was added to each well and incubated at 48°C for 20 hours. After three washes, 100 mL of biotinylated sheep anti-mouse IL-1β or TNF-α antibody (1:1000 dilutions in wash buffer containing 1% sheep serum, Sigma Chemical Co., Poole and Dorset, U.K.) was added to each well. Following 1 hour of incubation and three washes, 100 mL of Avidin-HRP (Dako Ltd, U.K.) (1:5000 dilutions in wash buffer) was added to each well, and the plates were incubated for 15 min. After washing three times, 100 mL of TMB substrate solution (Dako Ltd., U.K.) was added to each well and incubated for 10 min at room temperature. Finally, 100 mL of 1M H 2 SO 4 was added to stop the reaction, and the absorbance was read at 450 nm. The results are expressed as ng /mL for TNF-α and IL-1β and as pg /mL for Bax, Bcl-2, caspase-3, and caspase-7 (32–35). 2.5. Statistical data Analysis method The statistical analyses of the data were carried out using GraphPad Prism v.6 software by GraphPad Company, San Diego, USA. The Kolmogorov-Smirnov test was used to assess the normal distribution of the molecular parameters, indicating that all variables were evenly distributed. The data were then presented as mean ± SEM. A statistically significant P < 0.001 was found by performing one-way ANOVA with Bonferroni's post-hoc test for comparing the treatment groups. 3. Results 3.1. Nanoparticle Synthesis 3.1.1. Analysis of MgO N.P.s In the UV-visible spectroscopy, the MgO-NPs displayed absorbance at 266 nm, attributable to the magnesium oxide (MgO) nanoparticles, which are specific to MgO N.P.s and have a wavelength range between 260 and 280 nm (Fig-3). Utilizing XRD pattern recognition, a confirmation analysis of the crystal structure was performed. XRD patterns were obtained by calcining the precursor at 500°C. The particle size of the MgO N.P. (0.9λ/ (B*cosθ) was 20 to 25 nm using Debye Scherer's formula (Fig-4). Employing the KBr pellet approach, FT-IR spectra in the solid phase were recorded between 400 and 4000 cm -1 . The sample underwent calcination at 500°C for four hours, and Fig-5 depicts the I.R. spectra. According to the spectrum, the broad-band stretching vibration mode of the Mg-O moiety occurs between 438 and 769 cm -1 . The bending vibrations of the absorbed water molecules and the surface hydroxyl group (-O.H.) are attributed to two distinct bands observed at the wave ranges of 1014–1074 cm -1 and 1590–1641 cm -1 , respectively. The presence of an aromatic ring was confirmed by the band near 1387 cm -1 , caused by the C = C stretching frequency. The O-H stretching vibrations of the absorbed water molecule and the surface hydroxyl group caused a broad vibration band within the wave range of 3325–3553 cm -1 . The asymmetric stretching of the carbonate ion, CO3 2- species, is attributed to the FT-IR absorption peak visible at 1501 cm -1 . The morphology of the MgO N.P.s was studied using a FE-SEM, which determined the average size of the N.P.s to be 164 nm with a modest amount of agglomeration (Fig-6). The nanoparticles had a somewhat round shape. As a result of the coordination with Cur, the MgO N.P.s may also have been stabilized. 3.2. Results of pharmacological assay 3.2.1 Glutathione/Glutathione Disulfide Ket (25 mg/kg) represented a significant cutback in GSH and a boost of GSSG levels compared to controls (P < 0.05). In contrast, compared to mice treated with Ket alone (P < 0.05), the group treated with the ketamine-curcumin combination (40 mg/kg) had elevated GSH levels and lower GSSG levels (Table-1). Cur/MgO nanoparticles reversed the Ket-induced decline in GSH and increased GSSG (P < 0.05) (Table-1). Table -1: Effects of Cur/MgO N.P.s on mitochondrial GSH and GSSG content in ketamine treated rats Group Mean± SEM GSH (nmol/mg protein) GSSG (nmol/mg protein) GSH/GSSG Control group 112.9±8.6 0.82±0.6 140 Ketamine (25mg/kg) 21.4±6.9 a 19.1±0.3 a 1.1 a Ketamine + Curcumin (40 mg/kg) 90.2±9.3 b 4.1±0.5 b 21 b Ketamine+ Cur/MgO N.P.s (10 mg/kg) 79.6±5.9 b 8.1±0.4 b 9.8 b Ketamine+ Cur/MgO N.P.s (20 mg/kg) 95.2±8.2 b 4.2±0.3 b 23 b Ketamine+ Cur/MgO N.P.s 40 mg/kg) 110.6±11.9 b 1.2±0.1 b 91 b MgO (5 mg/kg) 37.4±8.9 10.3±7.2 a 3.7 All data are presented as mean ±SEM, n=8. a Showed significant level with P< 0.001 vs. control group . b Showed significant level with P<0.001 vs. Ketamine (25mg/kg). 3.2.2. Oxidative Stress Animals treated with Ket decreased SOD, GPx, and G.R. enzyme activity with higher MDA levels than controls (P < 0.001) (Fig-7A, B, C, and D). Ketamine- curcumin combination (40 mg/kg) induced higher levels of SOD, GPx, and G.R. activity but lower MDA levels (P < 0.001) (Fig-7A, B, C, and D). When compared to subjects that received Ket, Cur/MgO N.P.s reversed the effect of Ket-induced oxidative stress (P < 0.001) (Fig-7 A, B, C, and D). 3.2.3. Inflammation Compared to control mice, Ket (25 mg/kg) significantly elevated both IL-1β and TNF-α levels (P < 0.001) (Fig-8 A and B). Ket plus Cur (40 mg/kg) lowered the levels of IL-1β and TNF-α compared to ketamine-treated mice (P < 0.001) (Fig-8A and B). The levels of IL-1β and TNF-α (P < 0.001) were lower in those animals given the N.P. compared to Ket alone, indicating the Ket-induced inflammation was suppressed (Fig-8A and B). 3.2.4. Apoptosis Compared to controls, Ket administered (25 mg/kg) significantly elevated Bcl-2, caspase-3, and caspase-7 while significantly lowering Bax levels (P < 0.001)(Fig-9A, B, C, and D). Compared to Ket treatment, mice receiving Ket combined with Cur exhibited enhanced Bax levels and decreased Bcl-2, caspase-3, and caspase-7 levels (P < 0.001) (Fig-9A, B, C, and D). Additionally, when compared to Ket treatment mice, Cur/MgO N.P.s significantly reduced the apoptotic effects of Ket, as seen by a rise in Bax levels and a decrease in Bcl-2, caspase-3, and caspase-7 levels (P < 0.001) (Fig-9A, B, C, and D). 3.2.5. Mitochondrial Chain Enzyme Compared to control mice, Ket (25 mg/kg) significantly altered the function of the mitochondrial complex I, II, III, and IV enzymes (P < 0.001) (Fig-10 A, B, C, and D). However, compared to the group that received only Ket, the mitochondrial complex I, II, III, and IV enzymes increased (P < 0.001) in the groups treated with Ket combined with Cur (40 mg/kg) (Fig-10A, B, C, and D). Additionally, when compared to subjects treated with Ket, Cur/MgO N.P.s partially reversed the activity of the mitochondrial complex I, II, III, and IV enzymes, suppressing Ket-induced mitochondrial abnormalities (P < 0.001) (Fig-10A, B, C, and D). 4. Discussion Ket caused neurodegeneration accompanied by apoptosis, oxidative stress, inflammation, and increased mitochondrial respiratory chain enzymes in male mice. Cur or Cur/MgO N.P.s counteracted the Ket-induced neurodegeneration. Ket (25 mg/kg) increased lipid peroxidation and GSSG status while decreasing GSH and anti-oxidant enzymes such as GPx, GR, and SOD. Ket treatment boosted IL-1β, TNF-α, and Bcl 2 levels while decreasing Bax levels. Importantly, Ket significantly reduced the function of mitochondrial respiratory chain enzymes, such as mitochondrial complex I, II, III, and IV. Furthermore, in Ket-dependent mice, Cur/MgO N.P.s reduced neuroinflammation and oxidative stress in a dose-dependent way. In addition, in Ket-treated animals, Cur/MgO N.P.s improved mitochondrial complex I, II, III, and IV enzyme function and decreased apoptosis. In obstetric and pediatric treatment, Ket, an NMDA receptor antagonist, is frequently used for anesthesia and analgesia [ 15 , 19 , 32 ], even though it has a high-risk profile for producing hallucinations and delusions [ 33 ] and is thus potentially a drug of abuse [ 17 ]. According to recent studies [ 19 , 34 , 35 ], Ket appears to cause neuronal cell death and neurodegeneration, but the mechanism behind these pathological alterations is not well characterized. In the current study, Ket treatment increased lipid peroxidation and oxidative stress, as shown by a marked increase in MDA levels followed by an increase in mitochondrial GSSG status and a decline in GSH content. Others have reported that Ket negatively affected hippocampus cells in adult mice [ 19 , 34 , 35 ] and lowered lipid peroxidation [ 28 , 36 ]. In the Ket-treated group, Cur/MgO N.P.s (10, 20, and 40) prevented increased MDA levels, thereby reducing lipid peroxidation. In addition, it decreased GSSG levels and enhanced GSH content in the Ket-treated mice. In mice treated with Ket, Cur (40 mg/kg) decreased MDA levels while increasing GSH but decreasing GSSG levels. Cur has been reported to confer neuroprotection, partly through scavenging free radicals. [ 37 ]. Due to its ability to stimulate GSH production, Cur has been suggested as a potential treatment for neurodegenerative illnesses [ 38 ]. We found that the antioxidant enzyme activity, including SOD, GR, and GPx, was significantly increased by Cur/MgO N.P.s. Ketamine's negative effects were also reduced by Cur (40 mg/kg), which enhanced several antioxidant enzymes. By enhancing GPx and GR activity, Cur/MgO N.P.s, even at low dosages and Cur at intermediate doses (40 mg/kg), may have accelerated the production of GSH from GSSG and adjusted protection of the brain from Ket-induced oxidative stress. Numerous studies [ 24 , 26 , 39 ] have noted Cur's potential oxidant-antioxidant modification system in drug users. The anti-oxidative characteristics of Cur have been verified by in vivo and in vitro experiments, but the anti-oxidative effects of Cur/MgO N.P.s were not assessed in these experiments [ 39 ]. Our conclusions align with earlier research that showed increased pro-inflammatory cytokines and apoptosis after persistent Ket use or abuse [ 40 – 42 ]. The neurodegenerative effects of this hazardous substance have been attributed to an increase in pro-inflammatory cytokines and apoptosis following Ket [ 40 – 42 ]. Cur can prevent apoptosis and neuro-inflammation caused by Ket. This aligns with earlier research [ 24 , 26 , 39 ], which demonstrated that Cur may inhibit various locations of the TNF-α, TGF-β, and apoptosis signaling cascades. Among cell types, hippocampal cells seem particularly sensitive to Ket [ 40 – 43 ]. In mouse brain granular and glial cells, Ket can cause a persistent stress reaction, which can cause neuronal injury and dysfunction [ 43 , 44 ]. Ket treatment drastically lowers neuronal cell survival rates in a time- and dose-dependent way [ 45 , 46 ]. Oxidative stress and decreased antioxidant levels in the rodent brain have been linked to Ket-induced toxicity [ 40 , 47 ]. Hydroxyl radicals, such as ROS, are formed during oxidative stress and can induce DNA oxidation, resulting in errors and DNA replication mutations [ 45 , 48 ]. Ket enhances the production of ROS, which, in turn, causes inflammation. DNA damage and cellular death may follow [ 48 ]. There are few comprehensive, systematic studies on managing Ket toxicity. Since Cur is known for its immunomodulatory properties and activity against oxidation, inflammation, and apoptosis, [ 49 ] it may have therapeutic promise for NDD like A.D. and P.D. [ 26 , 50 – 52 ]. By lowering lipid peroxidation and boosting the function of antioxidant enzymes such as SOD, CAT, and GSH-related antioxidants, Cur may be able to combat oxidative stress [ 53 , 54 ]. Additionally, long-term use of Cur lowers the increase in TNF-α, IL-1β, and TGF-β1levels brought on by alcohol [ 55 – 57 ]. Cur has potential as a treatment for Ket-induced neuronal dysfunction [ 8 , 58 , 59 ]. The precise distribution of drugs to particular cells and tissues, such as the brain, is an active area of research in nanomedicine, and, as such, the technology can potentially enhance the effects of Cur [ 60 , 61 ]. Targeted delivery can improve the effectiveness of Cur [ 62 , 63 ]. Novel curcuminoid formulations complexed with N.P.s may overcome some of Cur’s pharmacokinetic restrictions [ 8 , 58 , 61 , 63 , 64 ]. 5. Conclusion A curcumin-conjugated MgO nanostructure was synthesized. The Cur/MgO N.P.s were neuroprotective against the inflammation, apoptosis, and oxidative stress induced by Ket (Fig-11). For the treatment of Ket-induced neurodegeneration and neurotoxicity, Cur/MgO N.P.s may have promise as a therapeutic candidate. Abbreviations A.D.: Alzheimer’s disease , ANOVA : Analysis of Variance, BAX: Bcl-2 Associated X-protein, Bcl-2: B-cell lymphoma 2, BSA: Bovine serum albumin, CAT: catalase, CYCS: cytochrome c, somatic, Cur: curcumin , Cur/MgO N.P.s : Curcumin/ Magnesium Oxide nanoparticles, ddH 2 O: Double Distilled water , EDS: Energy Dispersive X-ray Spectroscopy, EGTA: Ethylene glycol tetra acetic acid, FESEM: field emission scanning electron microscope , ELISA: Enzyme-linked immunosorbent assay , FTIR: Fourier transform infrared, GPx: Glutathione peroxidase, GR: glutathione reductase, GSH: Reduced form of Glutathione, GSSG: Oxidized form of Glutathione, IL-1β: Interleukin-1 beta, KET: ketamine , KOH: Potassium Hydroxide, MDA: Malondialdehyde, MgO: Magnesium Oxide, MOPS: 4-Morpholinepropanesulfonic acid , NaCl: Sodium Chloride, NADPH: Nicotinamide adenine dinucleotide phosphate, NADH /NAD + : Nicotinamide adenine dinucleotide, Na 2 EDTA: Disodium ethylenediaminetetraacetate dehydrate, NDD: neurodegenerative diseases , NFk B: nuclear factor kappa B , Na 2 HPO 4 : Sodium phosphate dibasic, NMDA: N-methyl-D-aspartate, NS: normal saline , O.D.: optical density, P.D.: Parkinson’s disease, PSA: particle size analyzer, ROS: Reactive oxygen species , SEM: standard error of the mean, SDS: Sodium dodecyl sulfate, SOD: Super oxide dismutase, TBA: Thiobarbituric acid, TMB: 3,3',5,5' tetramethylbenzidine , TNF-α: Tumor Necrosis Factor alpha, Trapanal: Sodium thiopental , UV-Vis: ultraviolet-visible spectroscopy , XRD: X-ray diffractometer. Declarations Conflict of interest We hereby certify that there is not any actual or potential conflict of interest Acknowledgments We sincerely thank the Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Chemistry, Tehran Medical Sciences, Islamic Azad University, and Tehran, Iran for their support of this work. Data availability declaration: The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. Funding: None declared. Author Declarations section Ethics approval: The research followed the Shahid Beheshti University of Medical Sciences Animal Care and Use Committee's guidelines, approved under I.R.SBMU.NRITLD.REC.1402.93. The investigators also adhered to the Animal Ethics and Welfare Guidelines and the ARRIVE procedure to guarantee the ethical treatment of the animals. Consent to participate: None applicable. Consent for publication: None applicable. 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Life Sci. 2017;179:37–53. Motaghinejad M, et al. Topiramate via NMDA, AMPA/kainate, GABAA and Alpha2 receptors and by modulation of CREB/BDNF and Akt/GSK3 signaling pathway exerts neuroprotective effects against methylphenidate-induced neurotoxicity in rats. J Neural Transm. 2017;124(11):1369–87. Motaghinejad M, et al. Neuroprotective effects of various doses of topiramate against methylphenidate-induced oxidative stress and inflammation in isolated rat amygdala: the possible role of CREB/BDNF signaling pathway. J Neural Transm. 2016;123(12):1463–77. Motaghinejad M, et al. Possible involvement of CREB/BDNF signaling pathway in neuroprotective effects of topiramate against methylphenidate induced apoptosis, oxidative stress and inflammation in isolated hippocampus of rats: molecular, biochemical and histological evidences. Brain Research Bulletin; 2017. Jørum E, Warncke T, Stubhaug A. 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Pharmacological and Molecular Evidence of Neuroprotective Curcumin Effects Against Biochemical and Behavioral Sequels Caused by Methamphetamine: Possible Function of CREB-BDNF Signaling Pathway. Basic and Clinical Neuroscience: p. 0–0. Kandezi N, et al. Novel Insight to Neuroprotective Potential of Curcumin: A Mechanistic Review of Possible Involvement of Mitochondrial Biogenesis and PI3/Akt/GSK3 or PI3/Akt/CREB/BDNF Signaling Pathways. Int J Mol Cell Med. 2020;9(1):1. Menon VP, Sudheer AR. Antioxidant and anti-inflammatory properties of curcumin , in The molecular targets and therapeutic uses of curcumin in health and disease . 2007, Springer. p. 105–125. Onaolapo A, et al. Subchronic ketamine alters behaviour, metabolic indices and brain morphology in adolescent rats: Involvement of oxidative stress, glutamate toxicity and caspase-3-mediated apoptosis. J Chem Neuroanat. 2019;96:22–33. Li Y, et al. Effects of ketamine on levels of inflammatory cytokines IL-6, IL-1β, and TNF-α in the hippocampus of mice following acute or chronic administration. Front Pharmacol. 2017;8:139. Sun L, et al. Chronic ketamine exposure induces permanent impairment of brain functions in adolescent cynomolgus monkeys. Addict Biol. 2014;19(2):185–94. Ding R, et al. Changes in hippocampal AMPA receptors and cognitive impairments in chronic ketamine addiction models: another understanding of ketamine CNS toxicity. Sci Rep. 2016;6:38771. Sinner B, et al. The toxic effects of s (+)-ketamine on differentiating neurons in vitro as a consequence of suppressed neuronal Ca2 + oscillations. Volume 113. Anesthesia & Analgesia; 2011. pp. 1161–9. 5. Bosnjak J. Ketamine induces toxicity in human neurons differentiated from embryonic stem cells via mitochondrial apoptosis pathway. Curr Drug Saf. 2012;7(2):106–19. Slikker W, et al. Ketamine-induced toxicity in neurons differentiated from neural stem cells. Mol Neurobiol. 2015;52(2):959–69. Félix LM, et al. Ketamine induction of p53-dependent apoptosis and oxidative stress in zebrafish (Danio rerio) embryos. Chemosphere. 2018;201:730–9. Bai X, et al. Ketamine enhances human neural stem cell proliferation and induces neuronal apoptosis via reactive oxygen species-mediated mitochondrial pathway. Anesth Analg. 2013;116(4):869. Aggarwal BB, Harikumar KB. Potential therapeutic effects of curcumin, the anti-inflammatory agent, against neurodegenerative, cardiovascular, pulmonary, metabolic, autoimmune and neoplastic diseases. Int J Biochem Cell Biol. 2009;41(1):40–59. Cole GM, Teter B, Frautschy SA. Neuroprotective effects of curcumin. The Molecular Targets and Therapeutic Uses of Curcumin in Health and Disease. Springer; 2007. pp. 197–212. Darvesh AS, et al. Curcumin and neurodegenerative diseases: a perspective. Expert Opin Investig Drugs. 2012;21(8):1123–40. Huang H-C, et al. Protective effects of curcumin on amyloid-β-induced neuronal oxidative damage. Neurochem Res. 2012;37(7):1584–97. Huang H-C, Xu K, Jiang Z-F. Curcumin-mediated neuroprotection against amyloid-β-induced mitochondrial dysfunction involves the inhibition of GSK-3β. J Alzheimers Dis. 2012;32(4):981–96. Liu L, et al. Curcumin prevents cerebral ischemia reperfusion injury via increase of mitochondrial biogenesis. Neurochem Res. 2014;39(7):1322–31. Perez-Torres I et al. Hibiscus Sabdariffa Linnaeus (Malvaceae), curcumin and resveratrol as alternative medicinal agents against metabolic syndrome. Cardiovascular & Hematological Agents in Medicinal Chemistry (Formerly Current Medicinal Chemistry-Cardiovascular & Hematological Agents), 2013. 11(1): p. 25–37. Tiwari V, Chopra K. Attenuation of oxidative stress, neuroinflammation, and apoptosis by curcumin prevents cognitive deficits in rats postnatally exposed to ethanol. Psychopharmacology. 2012;224(4):519–35. Lu H-F, et al. Curcumin-induced DNA damage and inhibited DNA repair genes expressions in mouse–rat hybrid retina ganglion cells (N18). Neurochem Res. 2009;34(8):1491. Kaufmann FN, et al. Curcumin in depressive disorders: an overview of potential mechanisms, preclinical and clinical findings. Eur J Pharmacol. 2016;784:192–8. Lopresti AL. Curcumin for neuropsychiatric disorders: a review of in vitro, animal and human studies. J Psychopharmacol. 2017;31(3):287–302. Di Martino P, et al. Nano-medicine improving the bioavailability of small molecules for the prevention of neurodegenerative diseases. Curr Pharm Design. 2017;23(13):1897–908. Rakotoarisoa M, Angelova A. Amphiphilic nanocarrier systems for curcumin delivery in neurodegenerative disorders. Medicines. 2018;5(4):126. Gera M, et al. Nanoformulations of curcumin: an emerging paradigm for improved remedial application. Oncotarget. 2017;8(39):66680. Ghalandarlaki N, Alizadeh AM, Ashkani-Esfahani S. Nanotechnology-applied curcumin for different diseases therapy. BioMed research international, 2014. 2014. Sun M, et al. Advances in nanotechnology-based delivery systems for curcumin. Nanomedicine. 2012;7(7):1085–100. Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4008048","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":282547484,"identity":"ebee9652-5886-48a3-9c4d-e8cfa64c525d","order_by":0,"name":"Mahsa Salehirad","email":"","orcid":"","institution":"Tehran Medical Sciences, Islamic Azad University","correspondingAuthor":false,"prefix":"","firstName":"Mahsa","middleName":"","lastName":"Salehirad","suffix":""},{"id":282547485,"identity":"81e86a4f-3362-4141-a244-fd4f2ed2fc43","order_by":1,"name":"A. Wallace Hayes","email":"","orcid":"","institution":"Michigan State University","correspondingAuthor":false,"prefix":"","firstName":"A.","middleName":"Wallace","lastName":"Hayes","suffix":""},{"id":282547486,"identity":"d4a63121-3467-48ac-859a-592d8b3fc425","order_by":2,"name":"Majid Motaghinejad","email":"data:image/png;base64,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","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":true,"prefix":"","firstName":"Majid","middleName":"","lastName":"Motaghinejad","suffix":""},{"id":282547487,"identity":"8f42f6ae-00c7-4bec-bcff-eb942028baa2","order_by":3,"name":"Mina Gholami","email":"","orcid":"","institution":"Shahid Beheshti University of Medical Sciences","correspondingAuthor":false,"prefix":"","firstName":"Mina","middleName":"","lastName":"Gholami","suffix":""}],"badges":[],"createdAt":"2024-03-03 09:02:51","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4008048/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4008048/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":53454635,"identity":"d5b83c26-ced8-44cc-9d30-bd766d4cbc25","added_by":"auto","created_at":"2024-03-26 07:31:07","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":197128,"visible":true,"origin":"","legend":"\u003cp\u003eSchematic illustration of experimental grouping\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/8a4a5d99ebf13969fa71f54d.png"},{"id":53454634,"identity":"5ddd1d80-80b1-4ce9-a724-e5975429c3a2","added_by":"auto","created_at":"2024-03-26 07:31:07","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":171626,"visible":true,"origin":"","legend":"\u003cp\u003eTimeline for experimental procedure and molecular evaluation\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/cf51208047e78dfda167f146.png"},{"id":53453941,"identity":"ed7f11cb-2af9-4f57-9eeb-128602f41870","added_by":"auto","created_at":"2024-03-26 07:23:07","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":41405,"visible":true,"origin":"","legend":"\u003cp\u003eUV SPECTRA OF Cur/MgO NPs\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/ed6153a64e9e7b4db890b3b2.png"},{"id":53453940,"identity":"3786d731-79b7-4af0-8636-6ab3b5014516","added_by":"auto","created_at":"2024-03-26 07:23:07","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":56630,"visible":true,"origin":"","legend":"\u003cp\u003eXRD PATTERN OF Cur/MgO NPs\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/0eb986eba21f1a50ec41a3ef.png"},{"id":53453939,"identity":"9f992a42-e4bd-454e-b135-44bccd793fe5","added_by":"auto","created_at":"2024-03-26 07:23:07","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":94660,"visible":true,"origin":"","legend":"\u003cp\u003eFT-IR SPECTRA OF Cur/MgO NPs\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/be81fbe17fd3f3e5b56a1883.png"},{"id":53453942,"identity":"2e7d3717-07a9-42b7-b365-fa2944d35508","added_by":"auto","created_at":"2024-03-26 07:23:07","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":784487,"visible":true,"origin":"","legend":"\u003cp\u003eSEM image of Cur/MgO NPs\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/b19a740585400454fd6acb8f.png"},{"id":53455111,"identity":"e148a34b-2cb2-4b6d-b7b3-49d3515162b4","added_by":"auto","created_at":"2024-03-26 07:39:08","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":482822,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Cur/MgO N.P.s on Ketamine induced\u003csub\u003e \u003c/sub\u003ealteration in lipid peroxidation (A), SOD activity (B), GPx activity (C) and G.R. activity (D) in rat isolated hippocampus.\u003c/p\u003e\n\u003cp\u003eAll data are expressed as Mean ± SEM (n=8).\u003c/p\u003e\n\u003cp\u003e*** P\u0026lt; 0.001 vs. control\u003c/p\u003e\n\u003cp\u003e### P\u0026lt; 0.001 vs. Ketamine (25mg/kg).\u003c/p\u003e","description":"","filename":"Figure7final.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/4f1c580efb8eee115cb5959c.png"},{"id":53453944,"identity":"08adf9ef-bb03-4ae7-b4c2-391836ede2b0","added_by":"auto","created_at":"2024-03-26 07:23:07","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":343503,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Cur/MgO N.P.s on Ketamine induced\u003csub\u003e \u003c/sub\u003ealteration in TNF-α (A) and IL-1β (B) level in rat isolated hippocampus.\u003c/p\u003e\n\u003cp\u003eAll data are expressed as Mean ± SEM (n=8).\u003c/p\u003e\n\u003cp\u003e*** P\u0026lt; 0.001 vs. control\u003c/p\u003e\n\u003cp\u003e### P\u0026lt; 0.001 vs. Ketamine (25mg/kg).\u003c/p\u003e","description":"","filename":"Figure8final.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/5cd9ed68c67e87903d7acee9.png"},{"id":53453947,"identity":"81ea243c-1e12-4d59-b480-d93c16788cf4","added_by":"auto","created_at":"2024-03-26 07:23:08","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":476861,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Cur/MgO N.P.s on Ketamine induced in protein expression of Bax (A), Bcl-2 (B), Caspase-3(C) and Caspase-7(D) in rat isolated hippocampus.\u003c/p\u003e\n\u003cp\u003eAll data are expressed as Mean ± SEM (n=8).\u003c/p\u003e\n\u003cp\u003e*** P\u0026lt; 0.001 vs. control\u003c/p\u003e\n\u003cp\u003e### P\u0026lt; 0.001 vs. Ketamine (25mg/kg).\u003c/p\u003e","description":"","filename":"Figure9final.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/31bb75c2ab13a72a8df315ea.png"},{"id":53454637,"identity":"61be0849-a4d2-4f3d-86c9-87aa07a99c02","added_by":"auto","created_at":"2024-03-26 07:31:08","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":497678,"visible":true,"origin":"","legend":"\u003cp\u003eEffects of Cur/MgO N.P.s on Ketamine induced in mitochondrial complex enzymes activity, complex I (A), complex II (B), complex III (C) and complex III (D) in rat isolated hippocampus.\u003c/p\u003e\n\u003cp\u003eAll data are expressed as Mean ± SEM (n=8).\u003c/p\u003e\n\u003cp\u003e*** P\u0026lt; 0.001 vs. control\u003c/p\u003e\n\u003cp\u003e### P\u0026lt; 0.001 vs. Ketamine (25mg/kg).\u003c/p\u003e","description":"","filename":"Figure10final.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/45261aac36155f920f32432d.png"},{"id":53453949,"identity":"42498c2d-83d1-41f7-8d0a-f7c2ff492cfe","added_by":"auto","created_at":"2024-03-26 07:23:08","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":220258,"visible":true,"origin":"","legend":"\u003cp\u003eCurcumin/MgO NPs are an effective neuroprotective agent against oxidative stress, inflammation, and apoptosis caused by ketamine.\u003c/p\u003e","description":"","filename":"Figure11.png","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/f82df4d4280f7163eb900b98.png"},{"id":65838811,"identity":"6a7ad164-a0f9-4353-a261-381aa11a518f","added_by":"auto","created_at":"2024-10-03 11:31:53","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4078612,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4008048/v1/bf932a37-b566-434f-b16a-e5eb37fb77fb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Protective Effects of Curcumin/Magnesium Oxide Nanoparticles Against Ketamine- induced Neurotoxicity","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNanotechnology can improve drug delivery while, at the same time, reducing adverse events and potential costs. Additional improvements include cell selectivity, release at specific target sites, and improved bioavailability from increased drug solubility [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. Magnesium is the second most abundant intracellular cation after potassium and is a noncompetitive antagonist for the N-methyl-D-aspartate receptor (NMDA).\u003c/p\u003e \u003cp\u003eThe NMDA receptor is blocked and downregulated by excess magnesium [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. MgO N.P.s have been shown to influence the regulation of neuronal cell activity [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Because of their size, formation, and structure, MgO N.P.s have the potential to be functional drugs [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]. MgO N.P.s reduced pain and inflammation in mice through central and peripheral pathways [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e]. Additionally, Moeini-Nodeh et al. showed that MgO N.P.s have both antiapoptotic and anti-oxidative effects [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Curcumin (Cur) is a traditionally used medicinal ingredient with centuries of use as a safe and effective natural product [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. Cur is the principal curcuminoid in turmeric, a popular Indian spice derived from the rhizome of \u003cem\u003eCurcuma longa\u003c/em\u003e [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. The antioxidant, anti-inflammatory, anti-proliferative, antiseptic, analgesic, anti-malarial, antitumoral, apoptosis-inducing, and anti-angiogenesis properties of Cur have been reported [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. All appear to involve the inhibition of NFk B signaling, which lowers pro-inflammatory cytokines IL-1, IL-6, and TNF-α [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e]. In an alcoholic animal neuropathy model, Cur antagonized the cellular consequences of oxidative stress by preventing DNA damage [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. Curcumin may be a promising remedy for certain NDDs [\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn obstetric and pediatric patients, ketamine (Ket), an NMDA receptor antagonist, is used for its analgesic and anesthetic effects [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Ket, however, has the potential for abuse due to its hallucinogenic and other reinforcing characteristics [\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. Ket has been reported to promote neuronal cell death and neurodegeneration, but the mechanism underlying its harmful effects remains elusive [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. The frequencies with which practitioners prescribe Ket and the frequency of its illicit trade for recreational use have contributed to a significant increase in use.\u003c/p\u003e \u003cp\u003eKetamine's impact on the adult mouse brain has not been resolved, especially when used over an extended time. We evaluated whether Ket-induced neurotoxicity in the mouse hippocampus would respond to Cur/MgO N.P.s\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Materials:\u003c/h2\u003e \u003cp\u003eSigma-Aldrich provided the ketamine, lithium, potassium hydroxide, and magnesium nitrate hexahydrate (Mg (NO3)\u003csub\u003e2\u003c/sub\u003e 6H\u003csub\u003e2\u003c/sub\u003eO), while Merck supplied the Cur. All reagents were analytical grade. The reagents were provided by DNA Biotech Co (Tehran, Iran). ddH\u003csub\u003e2\u003c/sub\u003eO was the solvent except for Ket and lithium, which were dissolved in normal saline.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Nanoparticle Synthesis:\u003c/h2\u003e \u003cp\u003eCur/MgO nanoparticles were synthesized as follows. Fifty mg of Cur was dissolved in 50 mL of ddH\u003csub\u003e2\u003c/sub\u003eO at 80\u0026deg;C. The solubilized Cur was added to a magnesium nitrate solution (Mg (NO\u003csub\u003e3\u003c/sub\u003e)\u003csub\u003e2\u003c/sub\u003e 50 mL, 0.1 M), resulting in a yellowish-colored solution. The solution was refluxed at 85\u0026ndash;90\u0026deg;C for two hours. When the refluxed solution cooled to 40\u0026deg;C, 5 mL of KOH (0.2 M) was gradually added, leading to an orange-yellow gel-like suspension. This suspension was centrifuged at 10,000 rpm, and the precipitate was washed with acetone and water until no yellow color (curcumin) was observed. The resulting Cur/MgO nanoparticle residue was vacuum-dried at ambient temperature. The nanoparticles were dissolved in normal saline in an ultrasonic bath for 15 min.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Nanoparticle Characterization\u003c/h2\u003e \u003cp\u003eFE-SEM, XRD, FT-IR spectral analysis, and UV-Vis spectra assessed the nanostructure's particle size, morphology, and chemical composition. FT-IR spectra were measured using a PerkinElmer instrument, and a Philips powder diffractometer was used to determine the X-ray diffraction records. The scanning rate was in the range of 10\u0026ndash;80\u0026deg;. To confirm chemical formation, a double-beam spectrophotometer measured the UV-visible spectrum. FE-SEM analyzed the morphology and particle dispersion of the nanoparticles, and chemical composition was evaluated using Energy Dispersive X-ray Spectroscopy. Uncorrected melting points were determined using a capillary tube and a B510K melting point instrument. These techniques provided a comprehensive understanding of the characteristics of the synthesized nanostructure.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Materials and Methodology\u003c/h2\u003e \u003cdiv id=\"Sec7\" class=\"Section3\"\u003e \u003ch2\u003e2.4.1. Animals\u003c/h2\u003e \u003cp\u003eFifty-six male BALB/c mice weighing 25\u0026ndash;30 g were obtained from the University of Medical Sciences, Iran. After a week of acclimatization in the animal unit laboratory, they were randomly divided into experimental and control groups and housed in groups of eight. The mice had access to pellet feed (Parsfeed Co, Tehran, Iran) and water in a controlled environment (room temperature: 22\u0026thinsp;\u0026plusmn;\u0026thinsp;0.5\u0026deg;C; relative humidity: 6\u0026ndash;40%; 12-hour light/dark cycles). Animals were monitored for signs of toxicity post-treatment and continuously during the study.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section3\"\u003e \u003ch2\u003e2.4.2. Ethics Statement\u003c/h2\u003e \u003cp\u003e The research followed the Shahid Beheshti University of Medical Sciences Animal Care and Use Committee's guidelines, approved under I.R.SBMU.NRITLD.REC.1402.93. The investigators also adhered to the Animal Ethics and Welfare Guidelines and the ARRIVE procedure to guarantee the ethical treatment of the animals [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section3\"\u003e \u003ch2\u003e2.4.3. Experimental Procedure\u003c/h2\u003e \u003cp\u003eThe mice were divided into groups: Group 1 received 0.2 mL of normal saline. Group 2 animals received Ket (25 mg/kg). Group 3 animals received 40 mg/kg Cur and 25 mg/kg Ket. Groups 4\u0026ndash;6 received Ket (25 mg/kg) and Cur/MgO N.P.s (10, 20, or 40 mg/kg). Group 7 received 5 mg/kg MgO and Ket (25 mg/kg). Mice were injected ip daily for two weeks.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section3\"\u003e \u003ch2\u003e2.4.5: Molecular and Biochemical Assessment\u003c/h2\u003e \u003cp\u003eThe experimental design is represented in Fig.\u0026nbsp;1, while Fig.\u0026nbsp;2 presents an overview of the experimental procedure and timeline.\u003c/p\u003e \u003cdiv id=\"Sec11\" class=\"Section4\"\u003e \u003ch2\u003e2.4.5.1. Total Protein\u003c/h2\u003e \u003cp\u003eOn day 15, mice were anesthetized with Trapanal (50 mg/kg, i.p.), and the brains were removed. The hippocampus was carefully separated and evaluated for oxidative stress, inflammation, apoptosis, and mitochondrial respiratory chain enzyme alterations.\u003c/p\u003e \u003cp\u003eThe hippocampus was homogenized in a cold buffer and then centrifuged at 450 g for 10 min. The buffer contained MOPS (25 mM), sucrose (400 mM), magnesium chloride (4 mM), EGTA (0.05 mM), and the pH of the solution was 7.3. Subsequently, the samples underwent a second 10-min centrifugation at 12,000 g. The resulting sediment was re-suspended in the buffer and stored at 0\u0026deg;C.\u003c/p\u003e \u003cp\u003eBio-Rad CA DC protein assay kits (Providence, RI, USA) were employed to measure the concentration of BDNF, CREB, TNF-α, IL-1, Bax, Bcl-2, SOD, GPx, and G.R. Total protein concentration was determined using the Bradford method (Bio-Rad, Providence, RI, USA).\u003c/p\u003e \u003cp\u003eA serial dilution of BSA, ranging from 0.1 to 1.0 mg/mL, was prepared in the homogenization buffer. A standard curve was created. Protein extracts of 10, 15, 20, 25, and 30 \u0026micro;L were added to individual wells, along with the Bradford reagent. Absorbance was measured at 630 nm utilizing a plate reader (Hiperion Microplate Reader, MPR4+, Rayto Company, China) [\u003cspan additionalcitationids=\"CR22\" citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section4\"\u003e \u003ch2\u003e2.4.5.2. Oxidative Stress\u003c/h2\u003e \u003cdiv id=\"Sec13\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.1. Lipid Peroxidation\u003c/h2\u003e \u003cp\u003eThe primary byproduct of cellular lipid peroxidation is malondialdehyde (MDA). One hundred uL of the MDA standard (DNA Biotech Co, Tehran, Iran, 100 uL) or 100 uL of the tissue homogenate was added to wells of the 96 microwell plate. SDS Lysis Solution (100 Ul) was added to each well. TBA reagent (250 uL) (DNA Biotech Co, Tehran, Iran) was added, and the mixture was gently shaken and incubated at 95\u0026deg;C for 45\u0026ndash;60 min. The samples were then centrifuged at 1000 g for 15 min. n-Butanol (DNA Biotech Co, Tehran, Iran) (300 ul) was added to stop the reaction. Samples were centrifuged at 10,000 g for 7 min, and the absorbance was read at 532 nm. Results are reported as nmol/mg of protein [\u003cspan additionalcitationids=\"CR25 CR26 CR27 CR28\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.2. GSH and GSSG\u003c/h2\u003e \u003cp\u003eTwenty-five uL of the glutathione reductase solution (1X) (DNA Biotech Co, Tehran, Iran) and 25 uL of the NADPH solution (1X) (DNA Biotech Co, Tehran, Iran) were added, respectively, to 96-well plates to measure GSH and GSSG content. A standard glutathione solution (DNA Biotech Co, Tehran, Iran) or 25 uL homogenized tissue sample was added. Chromogen (1X) (50 uL, DNA Biotech Co, Tehran, Iran) was added, and the mixture was vigorously blended. Absorbance was measured at 405 nm. The results are reported as nmol/mg of protein [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e, \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.3. SOD\u003c/h2\u003e \u003cp\u003eA kit from DNA Biotech Co (Persequor Park Pretoria South Africa) was used to assess SOD activity. The first and third blank wells were filled with ddH\u003csub\u003e2\u003c/sub\u003eO, and the second blank well was filled with homogenized tissue solution with a reagent volume of 20 L. The process included adding a working solution to each well, gently mixing it, and then adding a dilution buffer and an enzyme working solution to specific wells. For 20 min, the solutions were incubated at 37\u0026deg;C after being thoroughly mixed. Subsequently, the absorbance was measured at 450 nm, and SOD was quantified as units/mL/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.4. GPx\u003c/h2\u003e \u003cp\u003eThe GPx function in the hippocampal tissue was assessed using a commercial kit from DNA Biotech Company. The process included the addition of precise volumes of the homogenized tissue solution, assay buffer, reaction solution, and peroxidase substrate solution to the wells, followed by the measurement of absorbance at 340 nm over an 8-min period at 25\u0026deg;C. The results are in mU/mg protein.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec17\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.5. GR\u003c/h2\u003e \u003cp\u003eThe assessment of glutathione reductase activity was carried out using a kit from DNA Biotech Co. In the presence of NADPH, the reaction involved the transformation of GSSG to GSH. The reaction mixture was thoroughly mixed before measuring the absorbance at 340 nm for 120 seconds. The outputs are reported as mU/mg protein. [\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec18\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.6. Mitochondrial Complex Enzymes\u003c/h2\u003e \u003cp\u003eThe activities of mitochondrial complexes I, II, III, and IV were assessed using commercial kits from Abcam, Co (Boston, MA, USA). Measurement of NADH oxidation to NAD\u003csup\u003e+\u003c/sup\u003e at 450 nm estimated the function of mitochondrial complex I. For mitochondrial complex II, the assay involved the Electron-Transfer Catalysis of succinate to ubiquinone by measuring absorption at 550 nm. The activity of mitochondrial complex III was evaluated by determining the rate of conversion of CYCS to its reduced form at 600 nm. The function of mitochondrial complex IV was monitored by quantifying the oxidation of the reduced form of CYCS at 550 nm. The results are activity per milligram of protein per minute.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section5\"\u003e \u003ch2\u003e2.4.5.2.7. Protein Expression\u003c/h2\u003e \u003cp\u003eTNF-α, IL-1β, Bax, Bcl-2, caspase-3, and caspase-7 were estimated using commercial ELISA kits (Genzyme Diagnostics, Cambridge, USA). Sheep anti-mouse IL-1β, TNF-α Bax, Bcl-2, caspase-3, and caspase-7 polyclonal antibodies (Sigma Chemical Co., Poole, Dorset, U.K.)) were washed three times (0.5M sodium chloride (NaCl), 2.5mM sodium dihydrogen phosphate (NaH\u003csub\u003e2\u003c/sub\u003ePO\u003csub\u003e4\u003c/sub\u003e), 7.5mM Na2HPO4, 0.1% Tween 20, pH 7.2). One hundred mL of 1% (w/v) ovalbumin solution (Sigma Chemical Co., Poole, Dorset, U.K.) was added to each well and incubated at 37\u0026deg; C for 1 hour. After washing three times, 100 mL of sample or standard was added to each well and incubated at 48\u0026deg;C for 20 hours. After three washes, 100 mL of biotinylated sheep anti-mouse IL-1β or TNF-α antibody (1:1000 dilutions in wash buffer containing 1% sheep serum, Sigma Chemical Co., Poole and Dorset, U.K.) was added to each well. Following 1 hour of incubation and three washes, 100 mL of Avidin-HRP (Dako Ltd, U.K.) (1:5000 dilutions in wash buffer) was added to each well, and the plates were incubated for 15 min. After washing three times, 100 mL of TMB substrate solution (Dako Ltd., U.K.) was added to each well and incubated for 10 min at room temperature. Finally, 100 mL of 1M H\u003csub\u003e2\u003c/sub\u003eSO\u003csub\u003e4\u003c/sub\u003e was added to stop the reaction, and the absorbance was read at 450 nm. The results are expressed as ng /mL for TNF-α and IL-1β and as pg /mL for Bax, Bcl-2, caspase-3, and caspase-7 (32\u0026ndash;35).\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Statistical data Analysis method\u003c/h2\u003e \u003cp\u003eThe statistical analyses of the data were carried out using GraphPad Prism v.6 software by GraphPad Company, San Diego, USA. The Kolmogorov-Smirnov test was used to assess the normal distribution of the molecular parameters, indicating that all variables were evenly distributed. The data were then presented as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM. A statistically significant P\u0026thinsp;\u0026lt;\u0026thinsp;0.001 was found by performing one-way ANOVA with Bonferroni's post-hoc test for comparing the treatment groups.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec22\" class=\"Section2\"\u003e\n \u003ch2\u003e3.1. Nanoparticle Synthesis\u003c/h2\u003e\n \u003cdiv id=\"Sec23\" class=\"Section3\"\u003e\n \u003ch2\u003e3.1.1. Analysis of MgO N.P.s\u003c/h2\u003e\n \u003cp\u003eIn the UV-visible spectroscopy, the MgO-NPs displayed absorbance at 266 nm, attributable to the magnesium oxide (MgO) nanoparticles, which are specific to MgO N.P.s and have a wavelength range between 260 and 280 nm (Fig-3). Utilizing XRD pattern recognition, a confirmation analysis of the crystal structure was performed. XRD patterns were obtained by calcining the precursor at 500\u0026deg;C. The particle size of the MgO N.P. (0.9\u0026lambda;/ (B*cos\u0026theta;) was 20 to 25 nm using Debye Scherer\u0026apos;s formula (Fig-4).\u003c/p\u003e\n \u003cp\u003eEmploying the KBr pellet approach, FT-IR spectra in the solid phase were recorded between 400 and 4000 cm\u003csup\u003e-1\u003c/sup\u003e. The sample underwent calcination at 500\u0026deg;C for four hours, and Fig-5 depicts the I.R. spectra. According to the spectrum, the broad-band stretching vibration mode of the Mg-O moiety occurs between 438 and 769 cm\u003csup\u003e-1\u003c/sup\u003e. The bending vibrations of the absorbed water molecules and the surface hydroxyl group (-O.H.) are attributed to two distinct bands observed at the wave ranges of 1014\u0026ndash;1074 cm\u003csup\u003e-1\u003c/sup\u003e and 1590\u0026ndash;1641 cm\u003csup\u003e-1\u003c/sup\u003e, respectively. The presence of an aromatic ring was confirmed by the band near 1387 cm\u003csup\u003e-1\u003c/sup\u003e, caused by the C\u0026thinsp;=\u0026thinsp;C stretching frequency. The O-H stretching vibrations of the absorbed water molecule and the surface hydroxyl group caused a broad vibration band within the wave range of 3325\u0026ndash;3553 cm\u003csup\u003e-1\u003c/sup\u003e. The asymmetric stretching of the carbonate ion, CO3\u003csup\u003e2-\u003c/sup\u003e species, is attributed to the FT-IR absorption peak visible at 1501 cm\u003csup\u003e-1\u003c/sup\u003e. The morphology of the MgO N.P.s was studied using a FE-SEM, which determined the average size of the N.P.s to be 164 nm with a modest amount of agglomeration (Fig-6). The nanoparticles had a somewhat round shape. As a result of the coordination with Cur, the MgO N.P.s may also have been stabilized.\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec24\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Results of pharmacological assay\u003c/h2\u003e\n \u003cdiv id=\"Sec25\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.1 Glutathione/Glutathione Disulfide\u003c/h2\u003e\n \u003cp\u003eKet (25 mg/kg) represented a significant cutback in GSH and a boost of GSSG levels compared to controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05). In contrast, compared to mice treated with Ket alone (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05), the group treated with the ketamine-curcumin combination (40 mg/kg) had elevated GSH levels and lower GSSG levels (Table-1). Cur/MgO nanoparticles reversed the Ket-induced decline in GSH and increased GSSG (P\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Table-1).\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eTable -1:\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eEffects of\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eCur/MgO N.P.s\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eon\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003emitochondrial GSH and GSSG content\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003ein\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eketamine\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003etreated rats\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003cdiv align=\"\"\u003e\n \u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\" width=\"687\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGroup \u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMean\u0026plusmn; SEM\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003eGSH (nmol/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eGSSG (nmol/mg protein)\u003c/strong\u003e\u003c/p\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; GSH/GSSG\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eControl group\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e112.9\u0026plusmn;8.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e0.82\u0026plusmn;0.6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e140\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKetamine\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;(25mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e21.4\u0026plusmn;6.9\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e19.1\u0026plusmn;0.3\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e1.1\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKetamine + Curcumin (40 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e90.2\u0026plusmn;9.3\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e4.1\u0026plusmn;0.5\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u0026nbsp;21\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKetamine+ Cur/MgO N.P.s (10 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e79.6\u0026plusmn;5.9\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e8.1\u0026plusmn;0.4\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e9.8\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKetamine+ Cur/MgO N.P.s (20 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e95.2\u0026plusmn;8.2\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e4.2\u0026plusmn;0.3\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e23\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eKetamine+ Cur/MgO N.P.s 40 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e\u0026nbsp; \u0026nbsp; \u0026nbsp;110.6\u0026plusmn;11.9\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e1.2\u0026plusmn;0.1\u003csup\u003e\u0026nbsp;b\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e91\u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"37.88098693759071%\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eMgO\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003e(5 mg/kg)\u003c/strong\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"18.57764876632801%\" valign=\"top\"\u003e\n \u003cp\u003e37.4\u0026plusmn;8.9\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e10.3\u0026plusmn;7.2\u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"21.77068214804064%\" valign=\"top\"\u003e\n \u003cp\u003e3.7\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003c/table\u003e\n \u003c/div\u003e\n \u003cp\u003eAll data are presented as mean \u0026plusmn;SEM, n=8.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003ea\u0026nbsp;\u003c/sup\u003eShowed\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003esignificant level with\u003csup\u003e\u0026nbsp;\u003c/sup\u003eP\u0026lt; 0.001\u0026nbsp;vs.\u0026nbsp;control\u0026nbsp;group\u003csup\u003e\u0026nbsp;\u003c/sup\u003e.\u003c/p\u003e\n \u003cp\u003e\u003csup\u003eb\u0026nbsp;\u003c/sup\u003eShowed\u0026nbsp;\u003csup\u003e\u0026nbsp;\u003c/sup\u003esignificant level with\u003csup\u003e\u0026nbsp;\u003c/sup\u003e P\u0026lt;0.001 vs. Ketamine (25mg/kg).\u0026nbsp;\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec26\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.2. Oxidative Stress\u003c/h2\u003e\n \u003cp\u003eAnimals treated with Ket decreased SOD, GPx, and G.R. enzyme activity with higher MDA levels than controls (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-7A, B, C, and D). Ketamine- curcumin combination (40 mg/kg) induced higher levels of SOD, GPx, and G.R. activity but lower MDA levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-7A, B, C, and D). When compared to subjects that received Ket, Cur/MgO N.P.s reversed the effect of Ket-induced oxidative stress (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-7 A, B, C, and D).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec27\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.3. Inflammation\u003c/h2\u003e\n \u003cp\u003eCompared to control mice, Ket (25 mg/kg) significantly elevated both IL-1\u0026beta; and TNF-\u0026alpha; levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-8 A and B). Ket plus Cur (40 mg/kg) lowered the levels of IL-1\u0026beta; and TNF-\u0026alpha; compared to ketamine-treated mice (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-8A and B). The levels of IL-1\u0026beta; and TNF-\u0026alpha; (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) were lower in those animals given the N.P. compared to Ket alone, indicating the Ket-induced inflammation was suppressed (Fig-8A and B).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec28\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.4. Apoptosis\u003c/h2\u003e\n \u003cp\u003eCompared to controls, Ket administered (25 mg/kg) significantly elevated Bcl-2, caspase-3, and caspase-7 while significantly lowering Bax levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001)(Fig-9A, B, C, and D). Compared to Ket treatment, mice receiving Ket combined with Cur exhibited enhanced Bax levels and decreased Bcl-2, caspase-3, and caspase-7 levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-9A, B, C, and D). Additionally, when compared to Ket treatment mice, Cur/MgO N.P.s significantly reduced the apoptotic effects of Ket, as seen by a rise in Bax levels and a decrease in Bcl-2, caspase-3, and caspase-7 levels (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-9A, B, C, and D).\u003c/p\u003e\n \u003c/div\u003e\n \u003cdiv id=\"Sec29\" class=\"Section3\"\u003e\n \u003ch2\u003e3.2.5. Mitochondrial Chain Enzyme\u003c/h2\u003e\n \u003cp\u003eCompared to control mice, Ket (25 mg/kg) significantly altered the function of the mitochondrial complex I, II, III, and IV enzymes (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-10 A, B, C, and D). However, compared to the group that received only Ket, the mitochondrial complex I, II, III, and IV enzymes increased (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) in the groups treated with Ket combined with Cur (40 mg/kg) (Fig-10A, B, C, and D). Additionally, when compared to subjects treated with Ket, Cur/MgO N.P.s partially reversed the activity of the mitochondrial complex I, II, III, and IV enzymes, suppressing Ket-induced mitochondrial abnormalities (P\u0026thinsp;\u0026lt;\u0026thinsp;0.001) (Fig-10A, B, C, and D).\u003c/p\u003e\n \u003c/div\u003e\n\u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eKet caused neurodegeneration accompanied by apoptosis, oxidative stress, inflammation, and increased mitochondrial respiratory chain enzymes in male mice. Cur or Cur/MgO N.P.s counteracted the Ket-induced neurodegeneration. Ket (25 mg/kg) increased lipid peroxidation and GSSG status while decreasing GSH and anti-oxidant enzymes such as GPx, GR, and SOD. Ket treatment boosted IL-1β, TNF-α, and Bcl 2 levels while decreasing Bax levels. Importantly, Ket significantly reduced the function of mitochondrial respiratory chain enzymes, such as mitochondrial complex I, II, III, and IV. Furthermore, in Ket-dependent mice, Cur/MgO N.P.s reduced neuroinflammation and oxidative stress in a dose-dependent way. In addition, in Ket-treated animals, Cur/MgO N.P.s improved mitochondrial complex I, II, III, and IV enzyme function and decreased apoptosis.\u003c/p\u003e \u003cp\u003eIn obstetric and pediatric treatment, Ket, an NMDA receptor antagonist, is frequently used for anesthesia and analgesia [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e], even though it has a high-risk profile for producing hallucinations and delusions [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e] and is thus potentially a drug of abuse [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. According to recent studies [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e], Ket appears to cause neuronal cell death and neurodegeneration, but the mechanism behind these pathological alterations is not well characterized. In the current study, Ket treatment increased lipid peroxidation and oxidative stress, as shown by a marked increase in MDA levels followed by an increase in mitochondrial GSSG status and a decline in GSH content. Others have reported that Ket negatively affected hippocampus cells in adult mice [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e] and lowered lipid peroxidation [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e, \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eIn the Ket-treated group, Cur/MgO N.P.s (10, 20, and 40) prevented increased MDA levels, thereby reducing lipid peroxidation. In addition, it decreased GSSG levels and enhanced GSH content in the Ket-treated mice. In mice treated with Ket, Cur (40 mg/kg) decreased MDA levels while increasing GSH but decreasing GSSG levels. Cur has been reported to confer neuroprotection, partly through scavenging free radicals. [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e]. Due to its ability to stimulate GSH production, Cur has been suggested as a potential treatment for neurodegenerative illnesses [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. We found that the antioxidant enzyme activity, including SOD, GR, and GPx, was significantly increased by Cur/MgO N.P.s. Ketamine's negative effects were also reduced by Cur (40 mg/kg), which enhanced several antioxidant enzymes. By enhancing GPx and GR activity, Cur/MgO N.P.s, even at low dosages and Cur at intermediate doses (40 mg/kg), may have accelerated the production of GSH from GSSG and adjusted protection of the brain from Ket-induced oxidative stress. Numerous studies [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] have noted Cur's potential oxidant-antioxidant modification system in drug users. The anti-oxidative characteristics of Cur have been verified by \u003cem\u003ein vivo\u003c/em\u003e and \u003cem\u003ein vitro\u003c/em\u003e experiments, but the anti-oxidative effects of Cur/MgO N.P.s were not assessed in these experiments [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eOur conclusions align with earlier research that showed increased pro-inflammatory cytokines and apoptosis after persistent Ket use or abuse [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. The neurodegenerative effects of this hazardous substance have been attributed to an increase in pro-inflammatory cytokines and apoptosis following Ket [\u003cspan additionalcitationids=\"CR41\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Cur can prevent apoptosis and neuro-inflammation caused by Ket. This aligns with earlier research [\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e, \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e], which demonstrated that Cur may inhibit various locations of the TNF-α, TGF-β, and apoptosis signaling cascades.\u003c/p\u003e \u003cp\u003eAmong cell types, hippocampal cells seem particularly sensitive to Ket [\u003cspan additionalcitationids=\"CR41 CR42\" citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. In mouse brain granular and glial cells, Ket can cause a persistent stress reaction, which can cause neuronal injury and dysfunction [\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e, \u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Ket treatment drastically lowers neuronal cell survival rates in a time- and dose-dependent way [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e]. Oxidative stress and decreased antioxidant levels in the rodent brain have been linked to Ket-induced toxicity [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Hydroxyl radicals, such as ROS, are formed during oxidative stress and can induce DNA oxidation, resulting in errors and DNA replication mutations [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e, \u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Ket enhances the production of ROS, which, in turn, causes inflammation. DNA damage and cellular death may follow [\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThere are few comprehensive, systematic studies on managing Ket toxicity. Since Cur is known for its immunomodulatory properties and activity against oxidation, inflammation, and apoptosis, [\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e] it may have therapeutic promise for NDD like A.D. and P.D. [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e, \u003cspan additionalcitationids=\"CR51\" citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. By lowering lipid peroxidation and boosting the function of antioxidant enzymes such as SOD, CAT, and GSH-related antioxidants, Cur may be able to combat oxidative stress [\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e, \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. Additionally, long-term use of Cur lowers the increase in TNF-α, IL-1β, and TGF-β1levels brought on by alcohol [\u003cspan additionalcitationids=\"CR56\" citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. Cur has potential as a treatment for Ket-induced neuronal dysfunction [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe precise distribution of drugs to particular cells and tissues, such as the brain, is an active area of research in nanomedicine, and, as such, the technology can potentially enhance the effects of Cur [\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e]. Targeted delivery can improve the effectiveness of Cur [\u003cspan citationid=\"CR62\" class=\"CitationRef\"\u003e62\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e]. Novel curcuminoid formulations complexed with N.P.s may overcome some of Cur\u0026rsquo;s pharmacokinetic restrictions [\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e, \u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e, \u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e, \u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e].\u003c/p\u003e"},{"header":"5. Conclusion","content":"\u003cp\u003eA curcumin-conjugated MgO nanostructure was synthesized. The Cur/MgO N.P.s were neuroprotective against the inflammation, apoptosis, and oxidative stress induced by Ket (Fig-11). For the treatment of Ket-induced neurodegeneration and neurotoxicity, Cur/MgO N.P.s may have promise as a therapeutic candidate.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003e\u003cstrong\u003eA.D.:\u003c/strong\u003e Alzheimer\u0026rsquo;s disease\u003cstrong\u003e\u0026nbsp;, ANOVA :\u003c/strong\u003e Analysis of Variance,\u003cstrong\u003e\u0026nbsp;BAX:\u003c/strong\u003e Bcl-2 Associated X-protein,\u003cstrong\u003e\u0026nbsp;Bcl-2:\u003c/strong\u003e B-cell lymphoma 2, \u003cstrong\u003eBSA:\u003c/strong\u003e Bovine serum albumin, \u003cstrong\u003eCAT:\u003c/strong\u003e catalase, \u003cstrong\u003eCYCS:\u003c/strong\u003e cytochrome c, somatic, \u003cstrong\u003eCur:\u003c/strong\u003e curcumin ,\u003cstrong\u003eCur/MgO\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eN.P.s\u003c/strong\u003e: Curcumin/ Magnesium Oxide nanoparticles, \u0026nbsp; \u003cstrong\u003eddH\u003csub\u003e2\u003c/sub\u003eO:\u0026nbsp;\u003c/strong\u003eDouble Distilled water\u003cstrong\u003e,\u003c/strong\u003e\u003cstrong\u003eEDS:\u003c/strong\u003e Energy Dispersive X-ray Spectroscopy, \u003cstrong\u003eEGTA:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eEthylene glycol tetra acetic acid, \u003cstrong\u003eFESEM:\u003c/strong\u003e field emission scanning electron microscope , \u003cstrong\u003eELISA:\u003c/strong\u003e Enzyme-linked immunosorbent assay\u003cstrong\u003e, FTIR:\u003c/strong\u003e Fourier transform infrared, \u003cstrong\u003eGPx:\u003c/strong\u003e Glutathione peroxidase, \u003cstrong\u003eGR:\u0026nbsp;\u003c/strong\u003eglutathione reductase, \u003cstrong\u003eGSH:\u003c/strong\u003e Reduced form of Glutathione, \u003cstrong\u003eGSSG:\u0026nbsp;\u003c/strong\u003eOxidized form of Glutathione, \u003cstrong\u003eIL-1\u0026beta;:\u003c/strong\u003e Interleukin-1 beta, \u003cstrong\u003eKET:\u003c/strong\u003e ketamine , \u003cstrong\u003eKOH:\u0026nbsp;\u003c/strong\u003ePotassium Hydroxide, \u003cstrong\u003eMDA:\u0026nbsp;\u003c/strong\u003eMalondialdehyde,\u003cstrong\u003e\u0026nbsp;MgO:\u003c/strong\u003e Magnesium Oxide, \u003cstrong\u003eMOPS:\u003c/strong\u003e 4-Morpholinepropanesulfonic acid ,\u003cstrong\u003eNaCl:\u003c/strong\u003e Sodium Chloride,\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003cstrong\u003eNADPH:\u003c/strong\u003e Nicotinamide adenine dinucleotide phosphate, \u003cstrong\u003eNADH /NAD\u003csup\u003e+\u003c/sup\u003e:\u003c/strong\u003e Nicotinamide adenine dinucleotide, \u003cstrong\u003eNa\u003csub\u003e2\u003c/sub\u003eEDTA:\u003c/strong\u003e Disodium ethylenediaminetetraacetate dehydrate, \u003cstrong\u003eNDD:\u0026nbsp;\u003c/strong\u003eneurodegenerative diseases \u0026nbsp;,\u003cstrong\u003eNFk B:\u003c/strong\u003e nuclear factor kappa B , \u003cstrong\u003eNa\u003csub\u003e2\u003c/sub\u003eHPO\u003csub\u003e4\u003c/sub\u003e:\u003c/strong\u003e Sodium phosphate dibasic, \u003cstrong\u003eNMDA:\u003c/strong\u003e\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003eN-methyl-D-aspartate, \u003cstrong\u003eNS:\u003c/strong\u003e normal saline ,\u003cstrong\u003e\u0026nbsp;O.D.:\u003c/strong\u003e optical density, \u003cstrong\u003eP.D.:\u003c/strong\u003e Parkinson\u0026rsquo;s disease,\u003cstrong\u003ePSA:\u003c/strong\u003e particle size analyzer, \u003cstrong\u003eROS:\u003c/strong\u003e Reactive oxygen species\u003cem\u003e,\u003c/em\u003e\u003cstrong\u003eSEM:\u003c/strong\u003e standard error of the mean, \u003cstrong\u003eSDS:\u003c/strong\u003e Sodium dodecyl sulfate, \u003cstrong\u003eSOD:\u003c/strong\u003e Super oxide dismutase, \u003cstrong\u003eTBA:\u003c/strong\u003e Thiobarbituric acid, \u003cstrong\u003eTMB:\u003c/strong\u003e 3,3\u0026apos;,5,5\u0026apos; tetramethylbenzidine\u003cstrong\u003e, TNF-\u0026alpha;:\u003c/strong\u003e Tumor Necrosis Factor alpha, \u003cstrong\u003eTrapanal:\u003c/strong\u003e Sodium thiopental , \u003cstrong\u003eUV-Vis:\u003c/strong\u003e ultraviolet-visible spectroscopy , \u003cstrong\u003eXRD:\u003c/strong\u003e X-ray diffractometer.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eConflict of interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe hereby certify that there is not any actual or potential conflict of interest\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe sincerely thank the Department of Pharmaceutical Chemistry, Faculty of Pharmaceutical Chemistry, Tehran Medical Sciences, Islamic Azad University, and Tehran, Iran for their support of this work. \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability declaration:\u0026nbsp;\u003c/strong\u003eThe datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e None declared.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Declarations section\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval:\u003c/strong\u003e The research followed the Shahid Beheshti University of Medical Sciences Animal Care and Use Committee\u0026apos;s guidelines, approved under I.R.SBMU.NRITLD.REC.1402.93. The investigators also adhered to the Animal Ethics and Welfare Guidelines and the ARRIVE procedure to guarantee the ethical treatment of the animals.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate:\u003c/strong\u003e None applicable. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication:\u003c/strong\u003e None applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026apos; contributions:\u003c/strong\u003e Contributions to the conception and design of the work was\u003cstrong\u003e\u0026nbsp;done by Majid Motaghinejad\u003c/strong\u003e, Data acquisition and analysis was done by Mahsa Salehirad, interpretations of data were done by A.Wallace Hayes and Mina Gholami. Drafted the work or substantively revised it was done by Majid Motaghinejad and A.Wallace Hayes.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eCouvreur P. 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Nanomedicine. 2012;7(7):1085\u0026ndash;100.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ketamine, Curcumin/MgO N.P.s, neurodegeneration","lastPublishedDoi":"10.21203/rs.3.rs-4008048/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4008048/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eA curcumin-conjugated MgO nanostructure (Cur/MgO NPs) was synthesized, and its composition was verified. XRD and a particle size analyzer were used to determine the average crystalline and particle sizes. Morphological studies were conducted using FE-SEM. UV-Vis was also employed to examine absorption patterns, and FT-IR spectroscopy analyzed the functional groups involved in the reaction. The following protocol evaluated the effectiveness of Cur/MgO NPs in ketamine-treated male BALB/c mice. Group 1 received 0.2 mL of normal saline. Group 2 animals received Ket (25 mg/kg). Group 3 animals received 40 mg/kg Cur and 25 mg/kg Ket. Groups 4\u0026ndash;6 received Ket (25 mg/kg) and Cur/MgO N.P.s (10, 20, or 40 mg/kg). Group 7 received 5 mg/kg MgO and Ket (25 mg/kg). Mice were injected ip daily for two weeks. The hippocampal tissue was analyzed for oxidative stress, inflammation, apoptotic markers, and mitochondrial quadruple complex enzymes. The Cur/MgO N.P.s were neuroprotective against the inflammation, apoptosis, and oxidative stress induced by Ket.\u003c/p\u003e","manuscriptTitle":"Protective Effects of Curcumin/Magnesium Oxide Nanoparticles Against Ketamine- induced Neurotoxicity","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-26 07:23:02","doi":"10.21203/rs.3.rs-4008048/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"72a786ad-5f2e-4601-9270-72d64175e066","owner":[],"postedDate":"March 26th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-10-03T11:23:34+00:00","versionOfRecord":[],"versionCreatedAt":"2024-03-26 07:23:02","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4008048","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4008048","identity":"rs-4008048","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}