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Background: Chemokine C–C motif ligand 2 (CCL2) is one of the most widely recognised proinflammatory chemokines in cognitive disorders. Currently, CCL2‑targeting drugs are extremely limited. Thus, this study aimed to explore the neuroprotection afforded by naringin in CCL2‑induced cognitive impairment in rats. Methods: Before the CCL2 intra‑hippocampal injection, rats were treated with naringin for 3 consecutive days via intraperitoneal injection. Two days post‑surgery, the Morris water maze (MWM) and novel object recognition (NORT ) tests were performed to detect spatial learning and memory and object cognition, respectively. Nissl staining and dUTP nick‑ end labelling ( TUNEL) staining were performed to assess histopathological changes in the hippocampus. Commercial kits were used to measure the activity of superoxide dismutase (SOD) and glutathione peroxidase (GSH‑ Px) and the content of malondialdehyde (MDA). Quantitative real‑time polymerase chain reaction (qRT ‑PCR) was performed to examine the relative mRNA expression of interleukin 1β, (IL‑1β), interleukin 6 (IL ‑6), glutamate/aspartate transporter (GLAST ), glutamate transporter‑1 (GLT ‑1), phosphate ‑activated glutaminase (PAG), cysteine aspartic acid‑ specific protease 8 (caspase ‑8), cysteine aspartic acid‑specific protease 3 (caspase ‑3), cell lymphoma/leukaemia‑2 (Bcl‑2), and Bcl‑2 associated X protein (Bax). Results: In the MWM, the average escape latency and average swimming distance were significantly reduced and the crossing times were increased in the naringin‑treated groups, compared with the CCL2 group. The NORT results revealed that, compared with the CCL2 rats, the discrimination index in the naringin‑treated rats increased signifi‑ cantly. Nissl and TUNEL staining revealed that naringin protected the structure and survival of the neurons in the CA1 zone of the hippocampus. In the naringin‑treated groups, the SOD and GSH‑Px activities were increased, whereas the MDA levels were decreased. Furthermore, in the naringin‑treated groups, the relative mRNA expression of IL ‑1β and IL‑6 was significantly decreased; GLAST and GLT ‑1 mRNA expression levels were increased, whereas PAG was decreased. In the naringin‑treated groups, the relative mRNA expression levels of caspase ‑8, caspase ‑3, and Bax were decreased, whereas that of Bcl‑2 was increased. *Correspondence: firstname.lastname@example.org Jiang‑ yi Long—The first author Department of Pharmacology, Guangxi Medical University, Nanning 53002, Guangxi, China Full list of author information is available at the end of the article © The Author(s) 2020. 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The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/publi cdoma in/ zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data. Long et al. Behav Brain Funct (2020) 16:4 Page 2 of 13 Conclusion: Collectively, these data indicated that naringin alleviated the CCL2‑induced cognitive impairment. The underlying mechanisms could be associated with the inhibition of neuroinflammation, oxidative stress, apoptosis, and the regulation of glutamate metabolism. Keywords: Naringin, Cognitive impairment, Anti‑inflammation, Antioxidants, Apoptosis, Glutamate metabolism Background investigated the effect of naringin on CCL2-induced The chemokine C–C motif ligand 2 (CCL2), also known cognitive impairment and elucidated the possible as the monocyte chemoattractant protein-1(MCP-1) underlying mechanisms. [1, 2], belongs to the C–C chemokine family . It has the potent ability to activate and recruit mononuclear Materials and methods phagocytes and activate T cells and B cells; functions Reagents and instruments that are well-characterised in the immune system [4, 5]. CCL2 (R&D system) and naringin (purity > 98%) were However, recent studies have revealed that CCL2 is also purchased from Sigma Chemicals (USA). RT-PCR prim- involved in several central nervous system (CNS) dis- ers were obtained from Generay Biotech (Japan). The eases, such as epilepsy, Alzheimer’s disease, and ischae- TRIzol reagent for RT-PCR was purchased from TaKaRa mic brain injury . Elevated CCL2 has been detected Bio Inc (Japan). The commercial kits were purchased in the cerebrospinal fluid (CSF) of patients with Alzhei - from Nanjing Jiancheng Bio-engineering Institute (Nan- mer’s disease (AD) and HIV-associated neurocognitive jing, China). TUNEL and the Nissl kit were procured disorder (HAND) [6–9]. In the brain, CCL2 is pro- from Beyotime Institute of Biotechnology (Haimen, duced mainly by macrophages and microglia, which, in China). turn, activate microglia and release numerous inflam - matory cytokines [10, 11], such as interleukin 6 (IL-6) Experimental animal grouping and interleukin 1β (IL-1β), exacerbating the extent of All animal experiments were performed in accord- inflammation and resulting in neuronal injury. Thus, as ance with the guidelines of the Animal Ethical Com- a potential proinflammatory mediator, we postulated mittee of Guangxi University. Seventy-seven SPF male that CCL2 is closely associated with neuroinflamma - Sprague–Dawley rats (4–6 weeks old, weighing 180– tion and cognitive impairment. 220 g) were provided by the Guangxi Medical Univer- In addition to neuroinflammation, our previous study sity. The rats were maintained in an air-conditioned has indicated a novel role of CCL2 in mediating exci- room (22 ± 2 °C, 12-h light/dark cycle) with free access totoxicity in an in vitro study. The administration of to water. The rats were randomly divided into seven CCL2 enhances N-methyl-d-aspartic acid (NMDA) groups (n = 11), including the control group, sham group, receptor-mediated excitatory postsynaptic currents model group (5 ng CCL2), positive drug treatment group (EPSCs) and ultimately impairs neuronal dendrites in (CCL2 + 10 mg/kg memantine), naringin low dose group the hippocampal CA1 region, inducing neuronal death (CCL2 + 25 mg/kg naringin), naringin middle dose group in the hippocampus . Based on these findings, we (CCL2 + 50 mg/kg naringin), and naringin high dose hypothesised that CCL2 impairs neurons via multiple group (CCL2 + 100 mg/kg naringin), as shown in Fig. 1. pathways and could be a potential therapeutic target in several CNS diseases. Stereotaxic surgery and drugs treatment −1 However, drugs currently available against CCL2 Firstly, CCL2 was dissolved in 100 ng μL using sterile −1 are extremely limited. Naringin, a flavonoid naturally saline and then diluted to 1 ng μL before the experi- existing in grapefruit and other citrus fruits, pos- ment. With the exception of the control group, each sesses numerous biological benefits. Preclinical evi - group underwent a bilateral hippocampal injection. dence has suggested the protective role of naringin in Briefly, rats were anaesthetised using an intraperitoneal −1 the prevention of cardiovascular disease, diabetes, and (i.p.) injection of 1% sodium pentobarbital (45 mg kg ). neurodegeneration via antioxidant and anti-inflam - The fur on the rat’s head was removed following anaes - mation properties [13–17]. Another study has dem- thesia. According to the stereotaxic map of rat brain, a 26 onstrated that naringin can alleviate the progression GS micro-syringe was used to inject the test drugs into of atherosclerosis by downregulating CCL2 expres- the hippocampus following the coordinate positions of sion . However, the potential protective effects of AP = − 3.7 mm, ML = ± 3.0 mm, and DV = − 3.0 mm. naringin against CCL2-induced neuronal impairment The injection volume was 2.5 μL per side, with a constant have not been investigated. Therefore, in this study, we speed of 0.3 μL/min; the sham group received an equal L ong et al. Behav Brain Funct (2020) 16:4 Page 3 of 13 volume of sterile saline. After administration, the needle box was wiped using 75% alcohol to prevent the odour was left in place for another 5 min to avoid leakage of the of the preceding rat to influence the next rat. Twenty- drug and ensure complete absorption. Next, we sutured four hours later, we first placed two identical object As −1 the skin and administered penicillin (300,000 units kg , at the two adjacent corners of the box. The rat was given i.p.) to prevent the development of any infection. Before 10 min to freely explore the environment. One hour the administration of CCL2, the rats in the treatment later, one object A was replaced with object B and the rat groups were treated with naringin and memantine was allowed to explore for 5 min. We recorded the total repeatedly for 3 consecutive days via i.p. injection. The time spent at A (time for the familiar; TF) and B (time rats in the control, sham, and model (5 ng CCL2) groups for novel; TN), respectively. The discrimination index were administered equal volumes of normal saline for 3 (DI) was evaluated according to the following formula: consecutive days. On the third day, drug administration DI = TN/(TN + TF) * 100%. was performed 30 min before the bilateral hippocampal injection. Samples preparation Following the performance of NORT, the rats were Morris water maze (MWM) anaesthetised with 10% chloral hydrate and decapitated. In rats, spatial learning and memory assess began on the The brain was quickly removed on a culture dish filled third day following hippocampal injection. The method - with ice-cold saline (~ 4 °C). Three samples of the whole- ology was as described by Vorhees et al. . The MWM brain were used for Nissl and TUNEL staining (n = 3), paradigm consisted of a circular pool, with a diameter of four samples of one side of the hippocampus were used 120 cm and a height of 110 cm, a video capture system, for RT-PCR (n = 4), and eight samples of the other side of and a software analysis system. The pool was divided into the hippocampus were used for oxidative stress detection four equal quadrants, including NW, SW, SE, and NE. (n = 8). Near the wall of each quadrant, a distinct marker of simi- lar size and different shape was placed. The water tem - Nissl staining and TUNEL staining perature was controlled at 22 ± 1 °C. In the centre of the Nissl staining was conducted according to the kit proto- SW quadrant, a platform was submerged 2 cm below the cols. Images were photographed using an Olympus BX53 water surface. The experiment consisted of three phases: fluorescence microscope at 400×. TUNEL staining was (1) habituation phase: To enable environmental adap- performed in accordance with the manufacturer’s pro- tation and avoid stress, all rats were allowed to swim in tocol. An Olympus BX53 fluorescence microscope was the pool for 60 s before performing the experiment; (2) used to capture the images under 400×. The apoptotic spatial navigation phase: This phase was performed for cells were quantitatively assessed, with three animals five consecutive days. On each day, the rats were placed examined per group and three slices per hippocampal in the water from different quadrants and administered sample. four trails each day. We recorded the time from the start to find the platform, termed the escape latency. Addition - Oxidative stress determination ally, swimming speed and swimming distance were meas- To examine the effects of naringin in CCL2-induced hip - ured. Each trial was performed for 90 s. If the rat failed to pocampal oxidative stress, we measured the expression reach the platform within 90 s, we guided the animal to levels of glutathione peroxidase (GSH-PX), malondialde- the platform for 30 s and recorded the escape latency as hyde (MDA), and superoxide dismutase (SOD). Briefly, 90 s. (3) Probe trial: This phase was performed 24 h after the hippocampus was prepared as a 10% homogenate the end of spatial navigation. We removed the platform using iced saline. The total protein concentration of each and introduced the rats at a random quadrant for 90 s. sample was determined by the BCA method. SOD, GSH- The crossing times to reach the position of the platform PX activity, and MDA content were detected according were recorded as the evaluated index. to the specific kit instructions. New object recognition test (NORT) qRT‑PCR experiment This experiment was conducted after the MWM for 2 Briefly, total RNA extracted from the hippocampus consecutive days. The methods were as described by was evaluated according to the RNA extraction kit Leger et al. . The apparatus consisted of a transpar - instructions. RNA was reverse transcribed into cDNA ent plastic box (60 cm × 40 cm × 80 cm), two identical as directed by the reverse transcription kit. The PCR objects A, a different object B, and a video recording sys - reaction was quantified using the SYBE Green reagent tem. On the first day, the rat was habituated to the box and analysed by the StepOnePlus Real-Time PCR for 5 min. Then, the rat was returned to the cage and the System. GAPDH was used as the reference gene. The Long et al. Behav Brain Funct (2020) 16:4 Page 4 of 13 latency, swimming speed, and swimming distance were relative mRNA expression was calculated using the 2 ΔΔ analysed using the two-way ANOVA of repeated meas- Ct method. Table 1 presents the primer sequence. ures, whereas one-way ANOVA was performed on the rest of the measured data analysis. Significance was Statistical analysis defined at P < 0.05. Data analyses were performed using SPSS version 20.0 (IBM Corp., Armonk, NY, USA). All data are expressed as the mean ± SEM. In the case of the MWM, escape Results Naringin improves the spatial learning and memory of CCL2‑administered rats Table 1 The primer sequences of target genes To evaluate the protective role of naringin in CCL2- Gene Primer sequence induced spatial learning and memory impairment, we performed the MWM test. In spatial navigation train- GAPDH F: 5′‑GAC ATG CCG CCT GGA GAA AC‑3′ R: 5′‑AGC CCA GGA TGC CCT TTA GT‑3′ ing, the swimming speed for all groups demonstrated Caspase‑3 F: 5′‑GCA GCA GCC TCA AAT TGT TGAC‑3′ no significant differences (Fig. 2a). The escape latency R: 5′‑TGC TCC GGC TCA AAC CAT C‑3′ of each group gradually decreased across the training Caspase‑8 F: 5′‑CCT GTT CTA AGC CTG TCT C‑3′ days (Fig. 2d). Compared to the sham group, the escape R: 5′‑TGG GAA GGA AGC CTC TAT ‑3′ latency of the CCL2 group was significantly increased; Bax F: 5′‑GAG ACA CTC GCT CAG CTT CTTG‑3′ naringin treatment significantly decreased the escape R: 5′‑TTG CTA CAG GGT TTC ATC CAGG‑3′ latency (F = 5.448, P < 0.001, Fig . 2b). Addition- (6,63) Bcl‑2 F: 5′‑TGC AGA TGC CGG TTC AGG TAC‑3′ ally, the results of swimming distances were compara- R: 5′‑GGG AGC GTC AAC AGG GAG ATG‑3′ ble to those of escape latency (F = 6.280, P < 0.001, GLAST F: 5′‑CAT CTT GGT TTC GCT GTC T‑3′ (6,63) R: 5′‑GGG GAA CTC CGT GAT TGA ‑3′ Fig. 2c). The swimming path on training day 5 is shown GLT‑1 F: 5′‑AAG CAG CCC GCC ACA TAC ‑3′ in Fig. 2e. In the probe trial, the crossing times in the R: 5′‑AAC CGA GGG TGC CAA CAA ‑3′ CCL2 group decreased significantly compared to the PAG F: 5′‑GCG TTC TCA GGC GGG ATT ‑3′ sham group; in the naringin-treated group, crossing R: 5′‑TCA GCC ATT CAG CGA CCA G‑3′ times were significantly increased compared to the IL‑1β F: 5′‑AGG AGA GAC AAG CAA CGA CA‑3′ CCL2 group (F = 4.794, P < 0.001, Fig . 2f ). R: 5′‑CTT TTC CAT CTT CTT CTT TGG TAT ‑3′ (6, 63) IL‑6 F: 5′‑ATG GGC CTT CTT GGG ACT GATGT‑3′ R: 5′‑GGT CTG TTG TGG GTG GTA TCCTC‑3′ SD rats control sham CCL2 CCL2+memantine CCL2+Nar(L) CCL2+Nar(M) CCL2+Nar(H) Behavioral test mRNA expression of Nissl TUNEL caspase-3, caspase-8, Bax, SOD, MDA, staining Morris water Novel object staining Bcl-2, IL-1β, IL-6, PAG, GSH-PX maze recognition test GLAST, GLT-1 Data staistical analysis Fig. 1 Experimental designs L ong et al. Behav Brain Funct (2020) 16:4 Page 5 of 13 control sham CCL2 CCL2+Nar 50 CCL2+Memantine CCL2+Nar 25 CCL2+Nar 100 Fig. 2 Naringin improves the learning and memory functions of CCL2 administered rats. Eec ff t of Naringin on spatial learning in CCL2‑treated rat were evaluated using the Morris water maze for five consecutive days. a The average swimming speeds among groups. There were no significance among groups. b The escape latency among groups. CCL2 group spent more time to found hidden platform compared with the Naringin treatment groups. c The average swimming distances among groups. The CCL2 treatment rats showed a significantly longer courses to arrived platform compaerd with the Naringin treatment groups. d The escape latency tendency during 5‑ day training. The time to found platform is shorten across days. e The typical track of searching for the hidden platform on training day 5. f The number of crossings of the target quadrant. The number of crossings of the target quadrant in the Naringin treatment groups significantly incresed compared with CCL2 group was observed. Values are expressed as mean ± SEM, control group, sham group, memantine group, Naringin low dose group, Naringin middle dose group, # ## * ** Naringin high dose group: n = 10;CCL2 group: n = 9; Positive drug group: n = 11 ( P < 0.05, P < 0.01 vs sham, P < 0.05, P < 0.01 vs CCL2) Long et al. Behav Brain Funct (2020) 16:4 Page 6 of 13 Naringin ameliorates recognition memory activity of SOD (F = 11.736, P < 0.001, Fig. 5a) and (6, 49) in CCL2‑administered rats GSH-Px (F = 6.603, P < 0.001, Fig. 5c) while decreas- (6, 49) To further evaluate cognitive function, NORT was per- ing the MDA content (F = 48.557, P < 0.001, Fig. 5b). (6, 49) formed following the MWM assay. In the CCL2 group, the DIs was significantly reduced compared to the sham Naringin decreases inflammatory‑associated mRNA group. Compared to the CCL2 group, the naringin- expression treated groups demonstrated a significant increase in the Here, we detected the mRNA expression of IL-6 and DI in a dose-dependent manner (F = 4.899, P < 0.001, IL-1β to evaluate whether naringin could decrease the (6, 48) Fig. 3). extent of CCL2-induced inflammation. In the CCL2 group, the mRNA expression levels of IL-6 and IL-1β Naringin protects neurons in the hippocampal CA1 zone were significantly higher than the sham group. Compared Nissl staining revealed the morphological changes to the CCL2 group, naringin significantly decreased the induced in the CA1 structure of the hippocampus. Com- mRNA expression of IL-6 (F = 3.087, P < 0.05, Fig. 6a) (6, 17) pared to the sham group, the CCL2 group exhibited and IL-1β (F = 2.541, P < 0.05, Fig. 6b). (6, 17) numerous damaged neurons in the hippocampal CA1 region, presenting indistinct cell boundaries, with small Naringin regulates glutamate metabolism‑associated darkened and shrunken nuclei. In contrast, the naringin- mRNA expression treated groups demonstrated only minimal morpho- We detected the major regulators involved in glutamate logical changes (Fig. 4a). Furthermore, TUNEL staining metabolism, including glutamate transporter-1 (GLT-1), was used to quantify the number of apoptotic neurons glutamate/aspartate transporter (GLAST), and phos- in the CA1 zone. In the CCL2 group, the number of the phate-activated glutaminase (PAG). In the CCL2 group, apoptotic neurons was significantly higher than that in the mRNA expression of PAG was higher than that in the the sham group; naringin treatment inhibited neuronal sham group, whereas the GLAST and GLT-1 expression apoptosis (F = 124.862, P < 0.001, Fig. 4b, c). levels were lower. Compared to the CCL2 group, nar- (6, 14) ingin significantly decreased the mRNA expression of Naringin reduces oxidative stress induced by CCL2 in ratsPAG (F = 2.582, P > 0.05, Fig. 7a) and increased the (6, 17) In the hippocampus, the MDA content and enzymatic mRNA expression of GLAST (F = 2.421, P > 0.05, (6, 17) activity of SOD and GSH-Px were measured using com- Fig. 7b) and GLT-1(F = 2.723, P < 0.05, Fig. 7c). (6, 17) mercial kits. Compared to the sham group, GSH-Px and SOD activities were significantly decreased in the hip - Naringin decreases apoptosis‑associated mRNA expression pocampus of the CCL2 group, whereas the MDA con- In the CCL2 group, the mRNA expression of cysteine tent was significantly increased. Compared to the CCL2 aspartic acid-specific protease 3 (caspase-3), cysteine group, naringin treatment significantly increased the aspartic acid-specific protease 8 (caspase-8), and Bcl-2 Fig. 3 Naringin ameliorates recognition memory of CCL2–administered rats in novel object recognition. Eec ff ts of Naringin on cognitive function in CCL2‑Induced rats was evaluate by novel object recognition test. a The discrimination index among group. CCL2 group rats showed significant lower discrimination index compared with the Naringin treatment groups. b The discrimination index of individual of each group. Naringin treatment groups rats spent more time to explore the novel object when compared to CCL2 group. Values are expressed as mean ± SEM, control group, sham group, Positive drug group, memantine group, Naringin low dose group, Naringin middle dose group, Naringin high dose group: n = 8; # ## * ** CCL2 group: n = 7 ( P < 0.05, P < 0.01 vs sham, P < 0.05, P < 0.01 vs CCL2) L ong et al. Behav Brain Funct (2020) 16:4 Page 7 of 13 control sham CCL2 CCL2+memantine CCL2+Nar 25 CCL2+Nar 50 CCL2+Nar 100 control sham CCL2 CCL2+memantine CCL2+Nar 25 CCL2+Nar 50 CCL2+Nar 100 Fig. 4 The effects of Naringin on neuroprotection. a Represents photomicrographs of Nissl staining of brain tissue sections across hippocampus CA1 region (magnification 400×). b Represents photomicrographs of TUNEL staining of brain tissue sections across hippocampus CA1 region,TUNEL staining to detect neuronal apoptosis in the brain tissues of rats, arrows indicate apoptotic cells (magnification 400×). c The # ## * ** TUNEL‑positive cells. Values are expressed as mean ± SEM, n = 3 ( P < 0.05, P < 0.01 vs sham, P < 0.05, P < 0.01 vs CCL2) Long et al. Behav Brain Funct (2020) 16:4 Page 8 of 13 Fig. 5 Eec ff t of different dose of Naringin 25 mg/kg, Naringin 50 mg/kg, and Naringin 100 mg/kg on the oxidative stress status in CCL2 treated rats. The supernatant of hippocampus homogenate was used for the assay of SOD, GSH‑PX activity and MDA levels. A significant increase in (a) SOD and (c) GSH‑PX activity in Naringin treatment groups compared with model group. b A significant reduction in MDA levels in Naringin treatment groups # ## * ** compared with model group. Values are expressed as mean ± SEM, n = 8 ( P < 0.05, P < 0.01, P < 0.05, P < 0.01 vs CCL2) Fig. 6 Eec ff ts of Naringin on IL ‑1β,IL ‑6 mRNA expression in the hippocampus of CCL2‑treated rats. Quantitative real‑time PCR analysis of messenger (m)RNA levels. Relative expression of: a IL‑1βmRNA and b IL ‑6mRNA. qPCR analysis showed increased expression of IL ‑1β,IL ‑6 mRNA in Hippocampus of CCL2 rats. This result showing that Naringin significantly decreased expression of IL ‑1β, IL ‑6 mRNA in CCL2group rat.The # ## * ** experiments were repeated four times independently. Values are expressed as mean ± SEM, n = 4 ( P < 0.05, P < 0.01 vs sham, P < 0.05, P < 0.01 vs CCL2) associated X protein (Bax) were significantly upregu - Discussion lated, whereas cell lymphoma/leukaemia-2 (Bcl-2) was In addition to its well-characterised immune response downregulated. Compared to the CCL2 group, naringin function, CCL2 has recently demonstrated a patho- significantly decreased the mRNA expression levels of physiological role in several CNS diseases such as stroke, caspase-3 (F = 2.269, P > 0.05, Fig. 8a), caspase-8 (F epilepsy, ischaemic brain injury , and neurodegenera- (6, 17) (6, = 3.545, P < 0.05, Fig. 8b), Bax (F = 1.679, P > 0.05, tive diseases [21, 22]. Previously, studies have reported 17) (6, 18) Fig. 8c), and increased Bcl-2 mRNA expression (F increased CCL2 expression in the cerebrospinal fluid (6, = 3.677, P < 0.05, Fig. 8d). of patients with AD and HAND [6–9] in association 17) with cognitive decline [6, 10]. Our previous study has L ong et al. Behav Brain Funct (2020) 16:4 Page 9 of 13 Fig. 7 Eec ff ts of Naringin on PAG, GLAST, and GLT ‑1mRNA expression in the hippocampus of CCL2‑treated rats. Quantitative real‑time PCR analysis of messenger (m)RNA levels. Relative expression of: a The PAG mRNA. b The GLAST mRNA. c The GLT‑1 mRNA. qPCR analysis showed increased expression of PAG mRNA while decreased expression of GLAST, GLT‑1 mRNA in hippocampus of CCL2 rats, This result showing that Naringin significantly decreased expression of PAG mRNA while significantly increased GLAST, GLT ‑1 mRNA in CCL2 group rat.The experiments were repeated # ## * ** four times independently. Values are expressed as mean ± SEM, n = 4 ( P < 0.05, P < 0.01 vs sham, P < 0.05, P < 0.01 vs CCL2) Fig. 8 Eec ff ts of Naringin oncaspase ‑3, caspase ‑8,Bax,Bcl‑2mRNA expression in the hippocampus of CCL2‑treated rats. Quantitative real‑time PCR analysis of messenger (m)RNA levels. Relative expression of: a The caspase‑3 mRNA. b The caspase ‑8 mRNA. c The Bax mRNA expression. d The Bcl‑2 mRNA. qPCR analysis showed increased expression of caspase ‑3, caspase ‑8, Bax mRNA while decreased expression of Bcl‑2 in hippocampus of CCL2 rats, This result showing that Naringin significantly decreased expression of caspase ‑3, caspase ‑8, Bax mRNA while significantly increased Bcl‑2 # ## mRNA in CCL2 group rat.The experiments were repeated four times independently. Values are expressed as mean ± SEM, n = 4 ( P < 0.05, P < 0.01 * ** vs sham, P < 0.05, P < 0.01 vs CCL2) Long et al. Behav Brain Funct (2020) 16:4 Page 10 of 13 demonstrated that CCL2 dose-dependently impairs spa- Notably, a few studies have demonstrated that naringin tial memory and object recognition in rats . Further- improves learning and memory impairments induced by more, in-depth investigations have elucidated that the gp120, a crucial pathogenic factor of HAND pathogen- potential mechanisms are related to inflammation, oxida - esis . tive stress, excitotoxicity, and neuronal apoptosis [6, 10]. In the brain, CCL2 is mainly produced by macrophages u Th s, CCL2, a multi-potent pathological factor, could and microglia, the key mediators of neuroinflammation be established as a therapeutic target to prevent or treat . During inflammatory progression, CCL2 not only neurodegenerative diseases. However, currently available attracts immune cells to specific sites but also promotes anti-CCL2 drugs remain limited. Thus, in the present the release of other inflammatory factors, including IL-6 study, we explored the neuroprotective effects of naringin and IL-1β, exacerbating the extent of neuroinflammation against CCL2 induced damage. [10, 11]. Researchers have revealed that naringin demon- The MWM and NORT are common behavioural para - strates anti-inflammatory activity. For instance, naringin digms used to evaluate spatial learning and memory and can attenuate increased tumor necrosis factor-α (TNF- object cognition in rodents. In the MWM, swimming α) levels in a kainic acid-induced animal model  and speeds did not significantly differ among groups, indicat - reduce periplaque-activated microglia and astrocytes in ing that the surgery and drug treatment did not impair APP/PS1 transgenic mice . Therefore, in this study, locomotor function in the experimental animals. In the we examined the hippocampal expression of IL-1β and CCL2 group, the escape latency, as well as the swim- IL-6 mRNA to elucidate whether naringin could alleviate ming distances, were significantly longer compared to CCL2-induced neuroinflammation. In the CCL2 group, the sham group; the crossing times were significantly IL-1β and IL-6 mRNA expression significantly increased decreased, confirming the detrimental effects of CCL2 in compared to the sham group; naringin treatment sig- spatial learning and memory. Additionally, in the CCL2- nificantly decreased the mRNA expression of both inter - treated group, NORT exhibited poor object recognition leukins, confirming the anti-inflammatory effects of behaviour. However, naringin significantly improved naringin against CCL2-mediated neuroinflammation. cognition, as indicated by the shorter escape latency and In general, neuroinflammation is accompanied by oxi - swimming distances, increased crossing times, and DIs. dative stress. Oxidative stress has been known to par- Collectively, these results revealed the protective role of ticipate in the pathogenesis of several neurodegenerative naringin against CCL2-induced cognitive impairment. diseases [27, 28]. A large number of reactive oxygen Fig. 9 Naringin improves CCL2‑induced cognition impairment mechanism illustration. Naringin treatment inhibited the oxidation, inflammation, reducing excitotoxicity and ultimately alleviating neurons apotosis and damage in rats with learning and memory impairment L ong et al. Behav Brain Funct (2020) 16:4 Page 11 of 13 species (ROS) produced by oxidative stress can cause demonstrated that compared to the sham group, lipid peroxidation and DNA damage, provoking second- PAG mRNA expression levels increased, whereas the ary neuronal damage, and ultimately damaging cogni- GLAST and GLT-1 mRNA expression levels decreased tion . Under normal circumstances, antioxidant in the CCL2 group. In contrast, naringin treatment defence systems, including antioxidative enzyme systems significantly reduced the PAG mRNA expression and such as SOD and non-enzyme systems such as GSH-Px, increased the mRNA expression levels of GLAST and could maintain equilibrium between the oxidative and GLT-1, demonstrating that naringin has a protective antioxidative stress levels. For example, SOD scavenges effect on CCL2-induced excitotoxicity via the regula - free radicals and prevents lipid peroxidation in vivo to tion of glutamate metabolism. prevent oxidative damage. GSH-Px specifically catalyses Apoptosis is a type of programmed cell death that hydrogen peroxide (H O ) into water (H O) to decrease clears the aging and necrotic organelles to maintain 2 2 2 the expression of H O [16, 29]. Therefore, elevated SOD the normal physiological function in the body [38–40]. 2 2 and GSH-Px activities could directly reflect a powerful However, abnormal activation of apoptosis has been antioxidative ability. In addition, the increased expression known to play a role in the pathophysiological pro- of MDA, as a major metabolite of lipid oxidation, reflects cesses of several diseases, including neurodegenera- the oxidative degree . Here, we observed that the tive diseases [12, 32]. In the CNS, neuroinflammation, expression levels of GSH-Px and SOD were significantly oxidative stress, and excitotoxicity are the main fac- reduced in the CCL2 group, whereas the expression of tors inducing excessive neuronal apoptosis and cogni- MDA was increased. This further confirmed the role of tion decline [29, 41–44]. The present results, combined CCL2 in mediating oxidative stress. Naringin treatment with our previous research, demonstrated that CCL2 markedly increased the expression of GSH-Px and SOD impaired cognitive function; the underlying mecha- in the hippocampus, and significantly reduced MDA lev - nisms may associate with neuroinflammation, oxida - els. These results revealed the antioxidative stress effects tive stress, and excitotoxicity. Thus, we proposed that of naringin. In fact, a few reports have revealed that nar- the CL2 administration can induce hippocampal neu- ingin could ameliorate cognitive deficits by enhancing ronal apoptosis. First, we observed the morphological antioxidative stress [29, 31], as demonstrated by our cur- changes in the hippocampal CA1 zones using Nissl and rent results. TUNEL staining. An obvious impairment of CA1 struc- In addition, our previous studies have observed that ture was observed in the CCL2 group via Nissl staining. CCL2 can enhance NMDA receptor-mediated EPSC Additionally, in the CCL2 treatment group, TUNEL 2+ and mediate C a influx [12, 32]. This can impair the staining demonstrated a significant increase in apop - structure of neuronal dendrites in the hippocampal totic hippocampal neurons in the CA1 zone, validat- CA1 region and induce neuronal death, indicating ing our hypothesis. Reportedly, naringin demonstrates that CCL2 can provoke excitotoxicity via a presynaptic anti-apoptotic effects in a cerebral infarction model mechanism and increase the release of glutamate [12,  and quinolinic acid (QA)-induced neurotoxicity 32]. As the major excitatory neurotransmitter in the rat mode . In our results, naringin treatment sig- CNS, glutamate is involved in normal synaptic trans- nificantly protected the hippocampal neurons, consist - mission and the process of long-term potentiation ent with the other observed outcomes. As apoptosis is (LTP). However, the abnormal and excessive accumula- regulated by a cascade of genes, we further examined tion of glutamate in the synaptic cleft can trigger neu- the mRNA expression of caspase-8, caspase-3, Bax, and ronal damage, termed as excitotoxicity. Physiologically, Bcl-2, to explore the apoptotic pathway. Caspase-8 is there is a glutamate-glutamine cycle between neurons the upstream molecule that further activates caspase-3, and glial cells, mainly regulated by GLT-1, GLAST, and a key apoptosis executive molecular [47, 48]. Further- PAG. GLT-1 and GLAST are located in astrocytes; they more, Bax and Bcl-2 are important mediators for apop- take in the excessive glutamate and maintain normal totic regulation. Bax is released from the mitochondrial neurotransmission. PAG is an enzyme located in the inter-membrane space and amplifies the apoptotic sig - presynaptic terminal and catalyses glutamine to gluta- nal. Conversely, Bcl-2 possesses anti-apoptotic effects mate, which could enhance the level of glutamate [33, [49–51]. In the model group, the mRNA expression lev- 34]. Therefore, the dysregulation of these regulators els of caspase-3,caspase-8, and Bax were upregulated, could lead to the excessive accumulation of glutamate whereas the mRNA expression of Bcl-2 was down- in the synaptic cleft and eventually induce excitotox- regulated. Naringin treatment significantly decreased icity [35–37]. Here, we tested the mRNA expression the mRNA expression of caspase-3,caspase-8,and Bax, of PAG, GLAST, GLT-1 to elucidate whether nar- and significantly increased Bcl-2 expression, suggesting ingin influenced glutamate metabolism. The results anti-apoptotic properties. Long et al. Behav Brain Funct (2020) 16:4 Page 12 of 13 6. Carvallo L, Lopez L, Che FY, Lim J, Eugenin EA, Williams DW, et al. Conclusion Buprenorphine decreases the CCL2‑mediated chemotactic response of In our study, we observed that naringin can afford pro - monocytes. J Immunol. 2015;194(7):3246–58. tection against CCL2-induced cognition impairment; 7. Lee WJ, Liao YC, Wang YF, Lin IF, Wang SJ, Fuh JL. Plasma MCP‑1 and cognitive decline in patients with Alzheimer’s disease and mild cognitive moreover, the underlying mechanisms were related to impairment: a two‑ year follow‑up study. Sci Rep. 2018;8(1):1280. reduced inflammation, antioxidative stress, anti-apopto - 8. Dhillon NK, Williams R, Callen S, Zien C, Narayan O, Buch S. Roles of MCP‑1 sis, and glutamate metabolism, indicating the potential in development of HIV‑ dementia. Front Biosci. 2008;13(10):3913–8. 9. Thames AD, Briones MS, Magpantay LI, Martinez‑Maza O, Singer EJ, Hinkin neuronal protective effects of naringin as shown in Fig. 9. CH, Morgello S, et al. The role of chemokine C‑ C motif ligand 2 genotype and cerebrospinal fluid chemokine C‑ C motif ligand 2 in neurocognition Acknowledgements among HIV‑infected patients. AIDS. 2015;29(12):1483–91. The authors would like to thank the National Natural Science Foundation 10. Xu J, Dong H, Qian Q, Zhang X, Wang Y, Jin W, Qian Y. Astrocyte‑ derived of China and the National Foundation of Guangxi for financial support. The CCL2 participates in surgery‑induced cognitive dysfunction and authors are also thankful to the Departmental Facility and Central Facility at neuroinflammation via evoking microglia activation. Behav Brain Res. the Guangxi Medical University Life Science Research Institute. 2017;332:145–53. 11. Persidsky Y, Hill J, Zhang M, Dykstra H, Winfield M, Reichenbach NL, et al. Authors’ contributions Dysfunction of brain pericytes in chronic neuroinflammation. J Cereb JL and YZ were involved in designing this study JC conducted the behavioural Blood Flow Metab. 2016;36(4):794–807. analysis and data acquisition. All authors were involved in the experimentation 12. Zhou Y, Tang H, Xiong H. Chemokine CCL2 enhances NMDA receptor‑ and analysis of data and provided their intellectual input. All authors read and mediated excitatory postsynaptic current in rat hippocampal slices‑a approved the final manuscript. potential mechanism for HIV‑1‑associated neuropathy? J Neuroimmune Pharmacol. 2016;11(2):306–15. Funding 13. Hsueh TP, Sheen JM, Pang JH, Bi KW, Huang CC, Wu HT, Huang ST. This work was supported by the National Natural Foundation of China (No The anti‑atherosclerotic effect of naringin is associated with reduced 81660213, 81360192, 81660706), the National Foundation of Guangxi (No expressions of cell adhesion molecules and chemokines through NF‑κB 2017GXNSFAA198187, 2018JJA140536), and the Guangxi First‑ class Discipline pathway. 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Behavioral and Brain Functions – Springer Journals
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