Effects of ginseng total saponins from ginseng stem leaf on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR2B-CaMKII signal in rat hippocampus

Effects of ginseng total saponins from ginseng stem leaf on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR2B-CaMKII signal in rat hippocampus | 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 Effects of ginseng total saponins from ginseng stem leaf on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR2B-CaMKII signal in rat hippocampus Chungen Guo, Wenli WANG, Meiju ZHU, Hongzhu ZHU This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4275142/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 This study is to investigate ginseng total saponins from ginseng stem leaf on the learning and memory of fatigue rats and the mechanism of action. Sixty Sprague-Dawley male rats were randomly divided into six groups: normal group, normal + ginseng total saponins (200 mg/kg) group, exercise group, exercise + ginseng total saponins (50, 100, 200 mg/kg)–treated groups. The learning and memory was tested by Morris water maze experiment. After 7 days of exhaustive exercise, we measured hippocampal morphology by electron microscopy. The protein expression levels of synaptophysin ( SYP ), and postsynaptic density (PSD) protein 95 (PSD 95), N-methyl-D-aspartic acid receptor 2B (NR2B), calcium / calmodulin - dependent protein kinase II ༈CaMKII༉, phospho - NR2B ( p-NR2B ) and phospho - CaMKII ( p - CaMKII ) were measured by western blot analysis. The results demonstrated that ginseng total saponins (100, 200 mg/kg) treatment significantly decreased the latency to find the platform, increased dwell time in the target quadrant and the number of platform crossings of fatigued rats. ginseng total saponins (100, 200 mg/kg) treatment also increased the number of synapses and postsynaptic density (PSD) thickness, shrink the synaptic cleft of synapses in hippocampus of fatigue rats, significantly up-regulated NR2B -CaMKII signal, increased the levels of SYP and PSD 95 protein expression. It suggests that ginseng total saponins could improve the learning and memory of fatigue rats, relating to protecting the morphology of hippocampus, up-regulating NR2B-CaMKII signal in the hippocampus of fatigued rats. ginseng saponin exercise fatigue learning and memory synapse NR2B-CaMKII signal Figures Figure 1 Figure 2 Figure 3 Figure 4 1. Introduction Moderate exercise is beneficial to learning and memory. However, excessive exercise without adequate rest may cause fatigue, which not only affects the output of motor skills (Aune, et al, 2008 ), but also impairs brain cognition, such as attention, information handing and decision processing (Moore, et al, 2012 ; Thomson, et al, 2009). Recent animal studies have also pointed that fatigue induced by exercise weakens brain function (Ma, et al, 2018 ; Wang, et al, 2019 ; Zhu, et al, 2020 ). The ability of memory and learning is an important basis for daily work life and competitive sports. But there are few methods that can validly facilitate the memory and learning of fatigued body. Ginseng radix is the root of panax ginseng C.A. MEYER (Araliaceae, ginseng radix ). It is used as an important nootropic herb for many years in traditional Chinese medicine (Gao,et al., 2018 ). Hydrolyzed red ginseng extract improves learning and memory capability of scopolamine-treated C57BL/6J mice ( Ju et al., 2021 ). Total ginsenoside from ginseng root can improve the learning and memory impairment of rats induced by hindlimb suspension through inhibiting body inflammation and regulating hypothalamus-pituitary-adrenocortical axis imbalance (Bao et al., 2021 ). But there are few reports about the effects of ginseng saponin on the learning and memory of fatigue body. Furthermore, Ginseng's leaf stems are more readily available at a lower cost than its root. Extracts from ginseng leaf-stem also contain similar active ingredients with pharmacological functions of ginseng root (Wang et al., 2009 ). It is well known that the hippocampus is the key center of memory and learning, and it is also the most sensitive part of the brain damaged by fatigue stress (Xuan, et al, 2012 ). N-methyl-D-aspartic acid receptor (NR) is a kind of excitatory amino acid receptor that is closely related to memory and learning (Yang et al., 2022; Sahin, et al, 2023). NR2B can lead to changes in memory and learning and motion in training with different intensity (Ren, et al, 2017 ). Calcium / calmodulin - dependent protein kinase II (CaMKII) is a crucial protein kinase in neuroplasticity and memory (Zalcman, et al, 2018 ). CaMKII can be activated by calcium influx through NR (Nicoll & Schulman, 2023 ). Furthermore, inhibition of NR2B-CaMKII signalling pathway also reduces the expression of synaptic protein markers: postsynaptic density 95 (PSD95) and synaptophysin (SYP) (Iyaswamy, et al, 2018 ), which damaged the neurons. In the process of spatial memory formation, PSD95 can be recruited to the correspondent synaptic membrane surface, which improves the efficiency of synaptic transmission during synaptic plasticity (Delint-Ramírez, et al, 2008 ). The protein and mRNA expression of PSD95 in hippocampus of rats were significantly decreased by intensive training (Ren, et al., 2017 ). SYP is also participated in the last step of hippocampal neuron exocytosis and synapse formation (Tarsa & Goda, 2002 ). Intense exercise significantly decreased the expression level of SYP protein in hippocampus of rats (Ding, et al, 2014 ). This study explored the effects of ginseng total saponins on memory and learning, synaptic morphology and synaptic protein expression levels and NR2B-CaMKII signal in hippocampus of rats with fatigue induced by exhaustive exercise. 2. Materials and methods 2.1 Total ginsenoside from ginseng stem leaf Ginseng total saponins were obtained from Shaanxi Jin KangTai Biotech Co.,Ltd (Shanxi, China) (purity > 80%), which was aqueous extractions of ginseng stem leaf. Batch number is JKT20220623. The certificate of analysis for the test material is provided as a Supplementary document. 2.2 Animals The experiment used grade SPF, 8-week-old adult Sprague-Dawley male rats weighing 216 ± 12.52 g. Rats were purchased from Hunan Lake King of laboratory animal Co. Ltd, Changsha, Hunan, China. Each poly-propylene cage (41 cm × 34 cm × 16 cm (height)) was kept with 5 animals with the controlled conditions of temperature (20℃-24℃), humidity (40%), a light/dark cycle (12 h/12 h) with free access to food and water. Let the animals adapt to the laboratory environment before the experiment begins. The rodent license of the laboratory is no.SCXK (Xiang) 2016-0002 and laboratory animal use certificate is no. SYXK(Gan)2017-0003. All animal experiments comply with the ARRIVE guidelines and are carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, the Guidelines for Care and Use of Laboratory Animals of Jinggangshan University and approved by the Guidance Suggestions for the Hunan Province Laboratory Animal Care and Use Committee and the Medical Ethics Committee of Jinggangshan University. 2.3 Experimental design Sixty rats were randomly divided into six groups (n = 10 in each group): normal group (A), normal + ginseng total saponins (200 mg/ kg) (B), exercise group (C), exercise + ginseng total saponins (50 mg/ kg)-treated group (D), exercise + ginseng total saponins (100 mg/ kg)-treated group (E) and exercise + ginseng total saponins (200 mg/ kg)-treated group (F). The rats of ginseng total saponins treated groups (B, D, E, F) were injected by the intragastric gavage (ig) once per day with ginseng total saponins at the respective one time dose. Rats of group A and C were administrated with the same volume of saline by ig. The volume of each ig was 2 ml per rat and performed at 2 h before the start of treadmill running for 7 days. Rats in the exercised groups (C, D, E and F) had been doing exercises through running on a treadmill with 0° of inclination according to the exercise scheme in the exhaustive exercise-induced fatigue model for 7 days. All rats were conducted by Morris water maze experiment at 2 h after the end of the treadmill exercise every day for 6 days. On the 7th day, after spatial probe test, brain samples were harvested for transmission electron microscopy and western blotting (3 rats for each examination). 2.4 The model of exhaustive exercise-induced fatigue According to the method described in the literature (Wang, et al., 2019 ), the rats were placed on the treadmill for 30 min to familiarize themselves with the running environment, and then the treadmill running began. The exercise loading was classifified into three stages: I: 10 m/min, 15min; II: 15 m/ min, 15min; III: 20 m/min, run till exhaustion. Criteria of exhaustion was as follows: the running posture changed from stomp-style into prostrate-style, and it remained stationary in the rear part of the treadmill, sound/light/electric stimulation could not keep it running. 2.5 Morris water maze According to the method described in the literature (Li, et al, 2017 ), all rats were trained to search for a under warm (23–25°C) water platform for six days, one training session per day, consisting of four training trials, and cues around the pool were consistent. In each trial, put each rat into water of each of the four quadrants in succession and the longest time to discover the platform is 120 seconds. If animals can’t find the platform in this limited time, it is guided to the platform, and stayed there for 15 seconds. After the training of six days, the positioning navigation experiment and space exploration experiment were carried out. A digital video camera connected to a computer (Anhui Zhenghua Biological Instrument Equipment Co., Ltd, China) recorded escape latency, the time of staying in the previous target quadrant, the times of crossing the target platform and the average swimming speed of the rats. 2.6 Preparation of electron microscopy specimen Preparation of electron microscopy specimen was performed according to the method described in the literature (Wang, et al., 2019 ). Rats were anesthetized with 1% pentobarbital (40 mg/kg body weight) by intraperitoneal injection and then were fixed by perfusing 4% paraformaldehyde solution through heart. One cubic millimeter of hippocampus was taken and fixed in 4% glutaraldehyde for 3 h. After immersion in 0.1 mol/L phosphate buffered solution (PBS) for 10 min × 3 times, the hippocampal tissue was placed in 1% osmic acid (prepared with 0.1 mol/L PBS) for 40min. After dehydration and resin embedding, ultrathin sections of 70 nm thickness were cut and then counterstained with acetyl uranium and lead acetate. 2.7 Transmission electron microscopy and image analysis Three consecutive slices were cut at the same position of each rat's CA1 region of hippocampus. Tissue was examined under a Hitachi H-7650 Transmission Electron Microscope operated at 100 kV. The number of synapses under the same field of vision, the length of synaptic active area and the width of synaptic cleft and the thickness of postsynaptic density (PSD) in each synapse were measured by Leica-200 image analyzer. The length of synaptic active zone and the thickness of PSD were measured following published method (Güldner and Ingham, 1980 ), and the width of synaptic cleft was measured by multi-point averaging method (Wang, et al., 2019 ). 2.8 Western blot analysis Western blot analysis was performed as previously described (Zhu, et al., 2020 ). Hippocampal fragments were isolated after behavioral test and stored in a − 80°C freezer until further use. The hippocampal tissues homogenate was centrifuged at 13 000 g for 15 min at 4 ◦ C and the supernatant was collected. Protein concentration was measured by Bio-Rad Protein Assay (BioRad, Hercules, CA, USA). Sample proteins were separated on SDS-polyacrylamide gels, and then transferred to a polyvinylidine diflfluoride membrane, blocked by 5% non-fat milk and incubated overnight with the primary antibodies: SYP ( 1:1000; 2644T; CST ), PSD95 (1:1000; A7889; Abclonal), NR2B (1:1000; A3056; Abclonal), CaMKII (1:1000; A3231; Abclonal), p-NR2B ( 1:5000; ab81271; Abcam ), p-CaMKII (1:1000; 12716T;CST). The membrane was washed twice and then incubated for 1 h with secondary antibodies (anti-rabbit IgG for SYN, PSD95, NR2B, CaMKII, p-NR2B and p-CaMKII)(1:10 000; ab97060, Abcam,Cambridge, UK), and the bound antibody was detected by using an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK). For the gel loading control, membranes were re-probed with a monoclonal anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz, sc-25778, 1:1000; CA, USA). Protein levels in the groups were expressed as a percentage of respective control values. 2.9 Statistical analysis The first author used SPSS 17.0 software (SPSS, Chicago, IL, USA) for statistical analysis. The results were expressed as mean ± standard (SD). All the data were analyzed by one - way analysis of variance (ANOVA) and then tested by Tukey’s post hoc test, p < 0.05 was statistically significant. 3. Results 3.1 Effect of ginseng total saponins on the learning and memory of rats with exhaustive exercise-induced fatigue Figure 1 and Table 1 showed the influence of ginseng total saponins on the learning and memory of rats. The results showed that fatigue exercise (the exercise group, C) prolonged the latency to find the platform, decreased the dwell time in the target quadrant and the number of platform crossings compared to the normal group (A) ( p < 0.01). The administration of 100 and 200 mg/kg of ginseng total saponins (groups E and F) shortened the latency to find the platform, increased the dwell time in the target quadrant and the number of platform crossings compared to the exercise group (C) ( p < 0.01), and the administration of 200 mg/kg of ginseng total saponins (group F) is more active than 100 mg/kg of ginseng total saponins (group E) ( p < 0.01). No significant difference was found in the latency to find the platform, dwell time in the target quadrant and umber of platform crossings among groups F (the exercise + ginseng total saponins (200 mg/ kg)-treated group), A (the normal group) and B (the normal + ginseng total saponins (200 mg/ kg)- treated group), between groups C (the exercise group) and D (exercise + ginseng total saponins 50 mg/ kg)-treated group), p > 0.05. The average speed of reaching the platform in each group has no significant difference (p > 0.05). Table 1 Effect of ginseng total saponins on learning and memory ability of rats with exhaustive exercise-induced fatigue(x̄ ± s, n = 10) Group Index A B C D E F escape latency(s) 15.90 ± 1.79 16.00 ± 1.56 ** 21.50 ± 1.78 †† 20.80 ± 2.97 ††§§ 18.30 ± 1.58 ††**§ 16.30 ± 1.89 ** crossing platform times 7.50 ± 0.97 8.10 ± 0.74 ** 5.20 ± 1.23 ††§ 5.30 ± 1.49 ††§§ 6.70 ± 0.95 ††**§ 8.40 ± 0.84 ** Target quadrant residence time (s) 12.50 ± 1.84 13.30 ± 2.21 ** 8.80 ± 0.79 †† 8.90 ± 1.29 ††§§ 11.00 ± 1.89 †**§§ 13.40 ± 1.35 ** Average swimming speed(cm/s) 29.00 ± 1.44 28.70 ± 1.29 28.18 ± 1.82 28.49 ± 2.02 29.05 ± 2.09 29.57 ± 2.13 Note: A: normal group, B: normal + ginseng total saponins (200 mg/kg) group, C: exercise group, D: exercise + ginseng total saponins (50 mg/kg)-treated group, E: exercise + ginseng total saponins (100 mg/ kg)-treated group, F: exercise + ginseng total saponins (200 mg/kg)-treated group. †† p < 0.01 , compared with group A, ** p < 0.01 , compared with group C, § § p < 0.01 , compared with group F. 3.2 Effect of ginseng total saponins on the morphology of synapse in hippocampus of rats with exhaustive exercise-induced fatigue The effects of ginseng total saponins on the number of synapses in hippocampal CA1 area of rats are shown in Fig. 2-a and Table 1 . The results showed that the number of synapses in hippocampal CA1 area were significantly less in the exercise group (C) compared to the other groups except the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) ( P < 0.01). The exercise + ginseng total saponins (200 mg/ kg)-treated group (F) had more synapses of hippocampal CA1 area compared to both the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) and exercise + ginseng total saponins (100 mg/ kg)-treated group (E) ( P < 0.01 ). There were no significant difference in the number of synapses among the exercise + ginseng total saponins (200 mg/ kg)-treated group (F), the normal group (A) and normal + ginseng total saponins (200 mg/ kg) (B) ( P > 0.05). The effects of ginseng total saponins on the length of synaptic active area, synaptic cleft width and PSD thickness of synapses in hippocampal CA1 area of rats are shown in Fig. 2-b and Table 1 .The results showed that the width of the synaptic cleft in the exercise group (C) were wider compared to the other groups except the exercise + ginseng total saponins (50 mg/ kg)-treated group (D), while the thickness of PSD were opposite ( P < 0.01). The width of the synaptic cleft in exercise + ginseng total saponins (200 mg/ kg)-treated group (F) were narrower compared to both the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) and exercise + ginseng total saponins (100 mg/ kg)-treated group (E), and the thickness of PSD were opposite ( P < 0.01 or P < 0.05 ). There were no significant difference in the width of synaptic cleft and thickness of PSD among the normal group (A), normal + ginseng total saponins (200 mg/ kg) (B) and exercise + ginseng total saponins (200 mg/ kg)-treated group (F)( P > 0.05 ). There were no significant difference in the length of the synaptic active area in hippocampal CA1 area in each group ( P > 0.05). Table 2 Comparison of structural parameters of synapses in hippocampal CA1 area of rats (x̄ ± s, n = 5) Group Index A B C D E F The number of synapses 3.20 ± 0.84 3.00 ± 0.71 ** 1.00 ± 0.71 †† 1.20 ± 0.84 ††,§§ 2.00 ± 0.00 †,**,§ 3.40 ± 0.55 ** The width of synaptic cleft (nm) 16.40 ± 1.82 16.60 ± 1.95 ** 19.20 ± 1.92 ††,§ 20.00 ± 1.58 ††,§§ 18.80 ± 1.64 ††,**,§ 16.40 ± 1.67 ** The thickness of post-synaptic density (nm) 71.20 ± 10.83 71.40 ± 12.01 ** 41.00 ± 6.78 ††,§§ 43.20 ± 9.76 ††,§§ 59.20 ± 5.72 ††,**,§ 72.40 ± 10.92 ** The length of synaptic active zone (nm) 349.80 ± 84.30 351.20 ± 69.11 275.20 ± 36.31 280.60 ± 63.82 307.40 ± 66.62 337.20 ± 65.73 Note: A: normal group, B: normal + ginseng total saponins (200 mg/kg/day) group, C: exercise group, D: exercise + ginseng total saponins (50 mg/kg/day)-treated group, E: exercise + ginseng total saponins (100 mg/kg/day)-treated group, F: exercise + ginseng total saponins (200 mg/kg/day)-treated group. Values are mean ± SD. †† p < 0.01 , compared with group A, ** p < 0.01 , compared with group C, § § p < 0.01 , compared with group F. 3.3 Effect of ginseng total saponins on synaptic protein levels in hippocampus of rats with exhaustive exercise-induced fatigue SYP and PSD are synaptic proteins closely related to learning and memory and morphology of synapse. Therefore, we measured the effects of ginseng total saponins on SYP and PSD95 protein expression in the hippocampus. We observed that the levels of SYP and PSD95 protein expression in the hippocampus of rats in the exercise group (C) were significantly lower compared to the other groups except the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) ( p < 0.01). The protein expression levels of SYP and PSD95 in the hippocampus of rats in exercise + ginseng total saponins (200 mg/ kg)-treated group (F) were higher compared to both the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) and exercise + ginseng total saponins (100 mg/ kg)-treated group (E) ( p < 0.01). There were no significant difference in the protein expression levels of SYP and PSD95 in the hippocampus of rats in the normal group (A), normal + ginseng total saponins (200 mg/ kg) (B) and exercise + ginseng total saponins (200 mg / kg)-treated group (F) ( p > 0.05). See Fig. 3 3.4 Effect of ginseng total saponins on NMDAR-CaMKII signalling pathway in hippocampus of rats with exhaustive exercise-induced fatigue The effects of ginseng total saponins on NMDAR-CaMKII signalling pathway in hippocampus of rats were presented in Fig. 4. The protein expression levels of hippocampal NR2B, p-NR2B, CaMKⅡ and p-CaMKⅡ in the exercise group (C) were significantly lower compared to the other groups except the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) ( p < 0.01). The protein expression levels of NR2B, p-NR2B, CaMKⅡ and p-CaMKⅡ in the hippocampus of rats in exercise + ginseng total saponins (200 mg/ kg)-treated group (F) were higher compared to both the exercise + ginseng total saponins (50 mg/ kg)-treated group (D) and exercise + ginseng total saponins (100 mg/ kg)-treated group (E) ( p < 0.01). The protein expression levels of hippocampus NR2B, p-NR2B, CaMKⅡ and p-CaMKⅡ in the normal group (A), normal + ginseng total saponins (200 mg/ kg) (B) and exercise + ginseng total saponins (200 mg/ kg)-treated group (F) were not significantly different, ( p > 0.05). 4. Discussion This study shows that ginseng total saponins can significantly improve the learning and memory, resist the changes in the morphology of synapse in hippocampus, increase the expression levels of synaptic-related proteins SYP and PSD95 and up-regulate the NR2B-CaMKII signal in hippocampus of rats with exhaustive exercise-induced fatigue in a dose-dependent manner. Appropriate exercise is an important part of a healthy life style because of its beneficial effects on brain functions. However, excessively intense exercise that results in fatigue can induce brain dysfunction(Baker, et al., 2004 ). High-intensity treadmill running impairs the spatial memory ability of mice (Sun, et al., 2017 ). The Morris water maze test is a method of testing spatial learning and memory. In this study, according to behavioral test results, rats have weaker spatial learning and memory after 7 days of exhaustive exercise, which is consistent with previous studies (Wang, et al., 2019 ; Ma, et al., 2018 ). Ginseng total saponins treatment significantly improved the learning and memory of rats with exhaustive exercise-induced fatigue. The most potent effects were observed at the dose of 200 mg/kg of ginseng total saponins. The hippocampus is one of the key brain regions for learning and memory. Synapses are the structural basis of functional connections between neurons. Learning and memory are closely related to synapses of the hippocampus (Kempf, et al., 2016 ). Learning disabilities may not only be caused by neural regeneration, neurodevelopmental abnormalities, neuron migration, and neuronal apoptosis, but also by synapses (Lunardi, et al., 2010 ). Intense exercise can cause synapse plasticity damage in the hippocampus (Ding, et al., 2015 ). High- intensity treadmill exercise can impair synaptic functional plasticity in the hippocampus (Sun, et al., 2017 ). Exercise-induced fatigue could make the PSD thickness thinner and the width of the synaptic cleft wider in the striatum synapses (Hou, et al., 2017 ). In this study, we found that ginseng total saponins could resist the decrease of the number and PSD thickness of synapses and the increase of the width of synaptic cleft of synapses in the CA1 region of hippocampus of rats with exhaustive exercise-induced fatigue. It is suggested that the effects of ginseng total saponins on improving the learning and memory of rats were related to protecting from the damage of hippocampal synapses caused by exhaustive exercise-induced fatigue. Intense exercise significantly reduced the protein expression levels of SYP in the hippocampus of rats (Ding, et al., 2014 ). High-intensity training significantly reduced the expression of protein and mRNA of PSD95 in hippocampus of rats (Ren, et al., 2017 ). In this study, we found the level of the protein expression of SYP and PSD95 significantly decreased after 7 days of exhaustive exercise. Reduced the levels of synaptic protein markers (PSD95, SYN) were coincided with the decrease of the number and PSD thickness of synapses (Pan, et al., 2016 ). We also found that ginseng total saponins could resisted the decrease of the protein expression levels of SYP and PSD95 in hippocampus of rats with exhaustive exercise-induced fatigue in a dose-dependent manner. The most potent effects were observed at the dose of 200 mg/kg of ginseng total saponins. It is suggested that ginseng total saponins could improve their learning and memory through increasing the protein expression levels of SYP and PSD95 in the hippocampus of rats with exhaustive exercise-induced fatigue. The enhanced presynaptic glutamate release and downregulated postsynaptic NR function lead to the impaired corticostriatal plasticity in mice with exercise-induced fatigue (Ma, et al., 2018 ). High-intensity platform training decreased the the protein expression levels of NR1 and NR2A and NR2B in hippocampus of rats (Ren,et al., 2017 ). Our previous research showed that the level of NR1 and NR2B mRNA expression in hippocampus of rats decreased after exercise-induced fatigue (Zhu, et al., 2006 ). In this study, we found that ginseng total saponins could not only resist the decrease of the protein expression levels of NR2B and CAMKII, but also significantly increase the phosphorylation levels of NR2B, CAMKII in hippocampus of rats with exhaustive exercise-induced fatigue. The phosphorylation status of NR is an important factor that reflects the activation status of NR (Sanderson, et al., 2016 ). CaMKII is a key protein kinase in neural plasticity and memory (Zalcman, et al., 2018 ). CaMKII directly binds to the NR subunits NR1 and NR2B (Leonard, et al., 1999 ). CaMKII can be activated by calcium influx through NR (Nicoll & Schulman, 2023 ). When exhaustive exercise-induced fatigue inhibited the expression and phosphorylation levels of NR2B, it inhibited the phosphorylation of CaMKII which leads to the memory decline. Furthermore, inhibition of this signalling pathway also reduces the expression of PSD95 and SYN (Iyaswamy, et al., 2018 ), which damaged the neurons. So resistance to the inhibition of NR2B-CaMKII signal caused by exhaustive exercise-induced fatigue may be an important mechanism for ginseng total saponins to improve learning and memory of rats with exhaustive exercise-induced fatigue. Ginseng is used as an important nootropic herb for many years in traditional Chinese medicine (Gao,et al., 2018 ). Ginseng saponin is the main active ingredient of ginseng. Promoting learning and memory is one of the main pharmacological effects of ginseng saponin (Wang, et al., 2014 ). The researches have demonstated that total ginsenoside from ginseng root can improve the learning and memory impairment of rats induced by hindlimb suspension through inhibiting body inflammation and regulating HPA axis imbalance (Bao et al., 2021 ). Ginsenoside Rg1 alleviates learning and memory impairments and Aβ disposition through inhibiting NLRP1 inflammasome and autophagy dysfunction in APP/PS1 mice (Li et al, 2023 ). This study clearly demonstrate that ginseng total saponins from ginseng stem leaf can also significantly improve the impairment of learning and memory of rats with exhaustive exercise-induced fatigue. This effect was associated with ginseng total saponins protecting from the damage of hippocampal synapses, increasing the expression levels of SYP and PSD95 and up-regulating NR2B-CaMKII signalling pathway in the hippocampus of rats with exhaustive exercise-induced fatigue. Ginseng total saponins from ginseng stem leaf has no obvious effects on learning and memory in normal rats, which is inconsistent with the literature (Yang, et al., 1994 ). Conclusion Ginseng total saponins from ginseng stem leaf at 200 mg/kg has shown a good effect on improving the learning and memory of rats with exhaustive exercise-induced fatigue. The mechanism of ginseng total saponins’s improving learning and memory might be related to its protecting from the damage of hippocampal synapses, increasing the protein expression levels of SYN and PSD95 and up-regulating NR2B-CaMKII signal in the hippocampus of rats with exhaustive exercise-induced fatigue. Results from this study could provide partial experimental basis for the use of ginseng total saponins and ginseng stem leaf in sports medicine. Declarations Competing Interests The authors have no relevant financial or non-financial interests to disclose. Funding Declaration This study was supported by Natural Science Foundation of Jiangxi Province, China ( No. 20202BABL206124 ), National Natural Science Foundation of China ( No. 31660291), Natural Science Foundation of Education Department of Jiangxi Province（No.GJJ2201646） Author Contribution Meiju Zhu is responsible for the design of the article and writing, data statistics and the research of morphology of synapses in hippocampus and WB detection. Chungen Guo is in charge of the animal treadmills, the intraperitoneal injection and Morris water maze test. Hongzhu Zhu and Wenli Wang are in charge of the feeding of animals and Morris water maze test and the animal treadmills. All authors reviewed the manuscript. 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Gao Q., Zhang F.S., Zhang D.D., Wang Y.H., Liu G.L., Lin J.F. , Wang Y.H., Chang Z., Tian D.F., Han Z.Y., 2018. Research progress of TCM pathogenesis of vascular dementia and the nootropic mechanism of ginsenosides. CJTCMP, 2018,33:5508-5510. Güldner, F.H., Ingham, C.A., 1980. Increase in postsynaptic density material in optic target neurons of the rat suprachiasmatic nucleus after bilateral enucleation. Neurosci Lett. 17, 27–31. Hou, L.J.，Chen, J.L., Wang, X.X., Zhang, S.，Liu, X.L.，Qiao, D.C., 2017. Exercise-induced fatigue influenced striatal neuron’s synaptic ultrastructure and D2DR intervention role in rat. China Sport Scince (Chinese). 37, 62-68. Ju, S.H., Seo, J.Y., Lee, S.K., Oh, J.S., Kim, J.S., 2021. Oral administration of hydrolyzed red ginseng extract improves learning and memory capability of scopolamine-treated C57BL/6J mice via upregulation of Nrf2-mediated antioxidant mechanism. J Ginseng Res. 45(1):108-118. Kempf, S.J., Metaxas, A., Ibanez-Vea, M., Darvesh, S., Finsen, B., Larsen, M. R., 2016. An integrated proteomics approach shows synaptic plasticity changes in an APP/PS1 Alzheimer’s mouse model. Oncotarget. 7:33627–33648. Kwon, S.E., Chapman, E.R., 2011. Synaptophysin regulates the kinetics of synaptic vesicle endocytosis in central neurons . Neuron. 70, 847-854. Leonard, A.S., Lim, I.A., Hemsworth, D.E., Horne, M.C., Hell, J.W., 1999. Calcium/calmodulin-dependent protein kinase II is associated with the N-methyl-Daspartate receptor. Proc Natl Acad Sci USA. 96, 3239–3244. Li, X.W., Lei, H., Kong, L.L., Su, Y., Zhou, H.M., Ji, P.M., Sun, R., Wang, C., Li, W.P., Li, W.Z., 2023. Ginsenoside Rg1 alleviates learning and memory impairments and Aβ disposition through inhibiting NLRP1 inflammasome and autophagy dysfunction in APP/PS1 mice.Mol Med Rep. 27:6. Li,Y.N., Li, X.R.,Guo, C., Li, L., Wang, Y.X., Zhang, Y.M., Chen, Y., Liu, W.H., Gao, L., 2017. Long-term neurocognitive dysfunction in offspring via NGF/ERK/CREB signaling pathway caused by ketamine exposure during the second trimester of pregnancy in rats. Oncotarget. 8, 30956-30970. Lunardi, N., Ori, C., Erisir, A., Jevtovic-Todorovic, V., 2010. General anesthesia causes long-lasting disturbances in the ultrastructural properties of developing synapses in young rats. Neurotox Res. 17,179–188. Iyaswamy, A., Kammella, A.K., Thavasimuthu, C., Wankupar, W., Dapkupar, W., Shanmugam, S., Rajan, R., Rathinasamy, S., 2018. Oxidative stress evoked damages leading to attenuated memory and inhibition of NMDAR-CaMKII-ERK/CREB signalling on consumption of aspartame in rat model. J Food Drug Anal. 26, 903-916. Ma, J., Chen, H.M., Liu, X.L., Zhang, L.T., Qiao, D.C., 2018. Exercise-induced fatigue impairs bidirectional corticostriatal synaptic plasticity. Front Cell Neurosci. 12:14. Moore, R.D., Romine, M.W., O'connor, P.J., Tomporowski, P.D., 2012. The influence of exercise-induced fatigue on cognitive function. J Sports Sci. 30, 841-850. Nicoll R.A., Schulman H., (2023). Synaptic memory and CaMKII. Physiol Rev., 103(4):2877-2925. Pan, W., Han, S., Kang, L., Li, S., Du, J., Cui, H.X., 2016. Effects of dihydrotestosterone on synaptic plasticity of the hippocampus in mildcognitive impairment male SAMP8 mice. Exp Ther Med. 12, 1455-1463. Ren, H.,Yu, X.,Yu, L., Zhang, Y.G., Xie, H., Shi, N., Chen, L.J., 2017. Effects of different training loads on emotional state and mRNA and protein expressions of N-methyl-D-aspartate receptor subunits, postsynaptic density 95, and kinesin family member 17 in hippocampus of rats. Med Sci Monit. 23,4954-4960. Sanderson, J.L., Gorski, J.A., Dell'Acqua, M.L., 2016. NMDA receptor-dependent LTD requires transient synaptic incorporation of Ca2+-permeable AMPARs mediated by AKAP150-anchored PKA and calcineurin. Neuron. 89, 1000-1015. Sun, L.N., Li, X.l., Wang, F., Zhang, J., Wang, D.D., Yuan, L., Wu, M.N.,Wang, Z.J., Qi, J.S., 2017. High-intensity treadmill running impairs cognitive behavior and hippocampal synaptic plasticity of rats via activation of inflammatory response. J Neurosci Res. 95,1611–1620. Tarsa, L., Goda, Y., 2002. Synaptophysin regulates activity dependent synapse formation in cultured hippocampal neurons. Proc Natl Acad Sci U S A. 99, 1012–1016. Thomson, K., Watt, A.P., Liukkonen, J., 2009. Differences in ball sports athletes speed discrimination skills before and after exercise induced fatigue. J Sports Sci Med. 8, 259-264. Wang, H.W., Peng, D.C., Xie., J.T., 2009. Ginseng leaf-stem: bioactive constituents and pharmacological functions. Chinese Medicine. 4: 20-28. Wang, Q., Wang Y., Han C.Y., Liu X.M., 2014. Nootropic effect of gensenosides Rg1 and Rb1 and their metabolites. Chinese Traditional and Herbal Drugs. 45:1960-1965. Wang, Z.F., Hou, L.J., Wang, D.M., 2019. Effects of exercise-induced fatigue on the morphology of asymmetric synapse and synaptic protein levels in rat striatum. Neurochem Int. 129, 104476-104488. Xuan, A., Long, D., Li J., Ji, W.D., Zhang, M., Hong, L.P., Liu, J.H., 2012. Hydrogen sulfide attenuates spatial memory impairment and hippocampal neuroinflammation in beta-amyloid rat model of Alzheimer’s disease. J Neuroinflammation. 9, 202. Yang, Y., Zhang J.T., Shi C. Z., Qu Z.W., Liu Y., 1994. Study on the nootropic mechanism of gensenoside Rb1 and Rg1- influence on mouse brain development. Acta Pharmaceutic Sinica. 29: 241-245. Zalcman, G., Federman, N., Romano, A., 2018. CaMKII Isoforms in Learning and Memory: Localization and Function. Front Mol Neurosci. 11:445. Zhu, M.J., Li, S.C., Chen, L.G., 2006. The expression and effect of NR1, NR2A and NR2B in hippocampus tissue of rats with exercise fatigue. China Sport Science. 26, 71-74. Zhu, M.J., Zhu, H.Z., Ding, X.M., Liu, S.S., Zou, Y.H., 2020. Analysis of the anti-fatigue activity of polysaccharides from Spirulina platensis: role of central 5-hydroxytryptamine mechanisms. Food Funct. 11: 1826-1834. Additional Declarations No competing interests reported. Supplementary Files Seethe7WBimagesontheleft.tif 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-4275142","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":307982534,"identity":"3f49c69c-31a5-405d-b504-4247928d970a","order_by":0,"name":"Chungen Guo","email":"","orcid":"","institution":"Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"Chungen","middleName":"","lastName":"Guo","suffix":""},{"id":307982538,"identity":"94cc101a-9943-4aa3-a1de-f4549523f11a","order_by":1,"name":"Wenli WANG","email":"","orcid":"","institution":"Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"Wenli","middleName":"","lastName":"WANG","suffix":""},{"id":307982539,"identity":"838965d4-692b-4a02-b548-c3519fc291b8","order_by":2,"name":"Meiju ZHU","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAAqUlEQVRIiWNgGAWjYDACCRDBY8PDz99AmpY0GckZB0jSwnDYxqAhgUgd8rN7DJhuyJznMWA4wPjhYw4RWhjnnDFgzuG5zWPO3MAsOXMbEVqYJXIgWiwbDrAx8xKjhQ2i5RyPwYEEIrXwQLQcIEGLhERaAVBLMo/kjIPNxPlFfkbyBubcHjt7fv7mgx8+EqMFCNh/MPaAaMYG4tRDwA9SFI+CUTAKRsGIAwAaVC0oCvs/HwAAAABJRU5ErkJggg==","orcid":"","institution":"Jinggangshan University","correspondingAuthor":true,"prefix":"","firstName":"Meiju","middleName":"","lastName":"ZHU","suffix":""},{"id":307982540,"identity":"43b15560-2838-449d-ba3c-577a7024be81","order_by":3,"name":"Hongzhu ZHU","email":"","orcid":"","institution":"Jinggangshan University","correspondingAuthor":false,"prefix":"","firstName":"Hongzhu","middleName":"","lastName":"ZHU","suffix":""}],"badges":[],"createdAt":"2024-04-16 10:12:13","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4275142/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4275142/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":58097490,"identity":"bc51b2b5-f2fc-40cc-8696-33b5cc7eb190","added_by":"auto","created_at":"2024-06-11 06:02:38","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":385603,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative swimming paths of rats in each group in Morris water maze task\u003c/p\u003e","description":"","filename":"1.png","url":"https://assets-eu.researchsquare.com/files/rs-4275142/v1/12b7763d408b22b3236e7827.png"},{"id":58097492,"identity":"ec0dd581-4d07-4ab2-8080-b2133deb0e9e","added_by":"auto","created_at":"2024-06-11 06:02:38","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":671673,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ginseng total saponins on the morphology of synapse in hippocampus of rats with exhaustive exercise-induced fatigue. (a) Representative synapses in CA1 area of hippocampus of rats in each group（Scale bar, 0.5μm) and the black arrows point to the synapses. (b) Representative synapses in CA1 area of hippocampus of rats in each group（Scale bar, 200 nm）and the black arrows point to the thickened part of the postsynaptic membranes. A: normal group，B: normal + ginseng total saponins (200 mg/kg/day) group, C: exercise group, D: exercise + ginseng total saponins (50 mg/kg/day)-treated group, E: exercise + ginseng total saponins (100 mg/kg/day)-treated group, F: exercise + ginseng total saponins (200 mg/kg/day)-treated group.\u003c/p\u003e","description":"","filename":"2.png","url":"https://assets-eu.researchsquare.com/files/rs-4275142/v1/04951009805e27c1fc8b5669.png"},{"id":58097489,"identity":"63e2bb93-029d-4699-96b7-4b7714df9805","added_by":"auto","created_at":"2024-06-11 06:02:38","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":187606,"visible":true,"origin":"","legend":"\u003cp\u003eThe effects of ginseng total saponins on synaptic protein levels in hippocampus of rats with exhaustive exercise-induced fatigue. (a) Representative immunoblots for expression levels of the corresponding proteins. (b) Comparison of expression levels of the corresponding proteins. A: normal group，B: normal + ginseng total saponins (200 mg/kg/day) group, C: exercise group, D: exercise + ginseng total saponins (50 mg/kg/day)-treated group, E: exercise + ginseng total saponins (100 mg/kg/day)-treated group, F: exercise + ginseng total saponins (200 mg/kg/day)-treated group. Values are mean ± SD. \u003csup\u003e††\u003c/sup\u003e \u003cem\u003ep\u0026lt;0.01\u003c/em\u003e, compared with group A, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u0026lt;0.01\u003c/em\u003e, compared with group C, \u003csup\u003e§ §\u003c/sup\u003e \u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e, compared with group F.\u003c/p\u003e","description":"","filename":"3.png","url":"https://assets-eu.researchsquare.com/files/rs-4275142/v1/311bbc9a79472c3062f0a1a7.png"},{"id":58097879,"identity":"ad60d778-49ee-41df-be27-e6919e26afc1","added_by":"auto","created_at":"2024-06-11 06:10:38","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":228051,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of ginseng total saponins on the NR2B-CaMKII signalling pathway in hippocampus of rats with exhaustive exercise-induced fatigue. (a) Representative immunoblots for expression levels of the corresponding proteins. (b) Comparison of expression levels of the corresponding proteins. A: normal group，B: normal +\u0026nbsp; ginseng total saponins (200 mg/kg/day) group, C: exercise group, D: exercise +\u0026nbsp; ginseng total saponins (50 mg/kg/day)-treated group, E: exercise + ginseng total saponins (100 mg/kg/day)-treated group, F: exercise + ginseng total saponins (200 mg/kg/day)-treated group. Values are mean±SD. \u003csup\u003e††\u003c/sup\u003e \u003cem\u003ep\u0026lt;0.01\u003c/em\u003e, compared with group A, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u0026lt;0.01\u003c/em\u003e, compared with group C, \u003csup\u003e§ §\u003c/sup\u003e \u003cem\u003ep \u0026lt; 0.01\u003c/em\u003e, compared with group F.\u003c/p\u003e","description":"","filename":"4.png","url":"https://assets-eu.researchsquare.com/files/rs-4275142/v1/67e912a6a62dae1802b96291.png"},{"id":78867719,"identity":"2f80d1a9-f57d-496c-a5df-ebd77764180f","added_by":"auto","created_at":"2025-03-20 04:46:37","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2410716,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4275142/v1/8715e882-0098-47b0-ac65-18f33039f67d.pdf"},{"id":58097493,"identity":"d25b2e51-3cf1-4852-90d9-fea3296fac3c","added_by":"auto","created_at":"2024-06-11 06:02:39","extension":"tif","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":24830582,"visible":true,"origin":"","legend":"","description":"","filename":"Seethe7WBimagesontheleft.tif","url":"https://assets-eu.researchsquare.com/files/rs-4275142/v1/d6164d8bd91be673682d0ea8.tif"}],"financialInterests":"No competing interests reported.","formattedTitle":"Effects of ginseng total saponins from ginseng stem leaf on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR2B-CaMKII signal in rat hippocampus","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eModerate exercise is beneficial to learning and memory. However, excessive exercise without adequate rest may cause fatigue, which not only affects the output of motor skills (Aune, et al, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), but also impairs brain cognition, such as attention, information handing and decision processing (Moore, et al, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2012\u003c/span\u003e; Thomson, et al, 2009). Recent animal studies have also pointed that fatigue induced by exercise weakens brain function (Ma, et al, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e; Wang, et al, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e; Zhu, et al, \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). The ability of memory and learning is an important basis for daily work life and competitive sports. But there are few methods that can validly facilitate the memory and learning of fatigued body.\u003c/p\u003e \u003cp\u003eGinseng radix is the root of \u003cem\u003epanax ginseng\u003c/em\u003e C.A. MEYER (Araliaceae, \u003cem\u003eginseng radix\u003c/em\u003e). It is used as an important nootropic herb for many years in traditional Chinese medicine (Gao,et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Hydrolyzed red ginseng extract improves learning and memory capability of scopolamine-treated C57BL/6J mice ( Ju et al., \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Total ginsenoside from ginseng root can improve the learning and memory impairment of rats induced by hindlimb suspension through inhibiting body inflammation and regulating hypothalamus-pituitary-adrenocortical axis imbalance (Bao et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). But there are few reports about the effects of ginseng saponin on the learning and memory of fatigue body. Furthermore, Ginseng's leaf stems are more readily available at a lower cost than its root. Extracts from ginseng leaf-stem also contain similar active ingredients with pharmacological functions of ginseng root (Wang et al., \u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e2009\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIt is well known that the hippocampus is the key center of memory and learning, and it is also the most sensitive part of the brain damaged by fatigue stress (Xuan, et al, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e2012\u003c/span\u003e). N-methyl-D-aspartic acid receptor (NR) is a kind of excitatory amino acid receptor that is closely related to memory and learning (Yang et al., 2022; Sahin, et al, 2023). NR2B can lead to changes in memory and learning and motion in training with different intensity (Ren, et al, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Calcium / calmodulin - dependent protein kinase II (CaMKII) is a crucial protein kinase in neuroplasticity and memory (Zalcman, et al, \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). CaMKII can be activated by calcium influx through NR (Nicoll \u0026amp; Schulman, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). Furthermore, inhibition of NR2B-CaMKII signalling pathway also reduces the expression of synaptic protein markers: postsynaptic density 95 (PSD95) and synaptophysin (SYP) (Iyaswamy, et al, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which damaged the neurons. In the process of spatial memory formation, PSD95 can be recruited to the correspondent synaptic membrane surface, which improves the efficiency of synaptic transmission during synaptic plasticity (Delint-Ram\u0026iacute;rez, et al, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2008\u003c/span\u003e). The protein and mRNA expression of PSD95 in hippocampus of rats were significantly decreased by intensive training (Ren, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). SYP is also participated in the last step of hippocampal neuron exocytosis and synapse formation (Tarsa \u0026amp; Goda, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e2002\u003c/span\u003e). Intense exercise significantly decreased the expression level of SYP protein in hippocampus of rats (Ding, et al, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). This study explored the effects of ginseng total saponins on memory and learning, synaptic morphology and synaptic protein expression levels and NR2B-CaMKII signal in hippocampus of rats with fatigue induced by exhaustive exercise.\u003c/p\u003e"},{"header":"2. Materials and methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Total ginsenoside from ginseng stem leaf\u003c/h2\u003e \u003cp\u003eGinseng total saponins were obtained from Shaanxi Jin KangTai Biotech Co.,Ltd (Shanxi, China) (purity\u0026thinsp;\u0026gt;\u0026thinsp;80%), which was aqueous extractions of ginseng stem leaf. Batch number is JKT20220623. The certificate of analysis for the test material is provided as a Supplementary document.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals\u003c/h2\u003e \u003cp\u003eThe experiment used grade SPF, 8-week-old adult Sprague-Dawley male rats weighing 216\u0026thinsp;\u0026plusmn;\u0026thinsp;12.52 g. Rats were purchased from Hunan Lake King of laboratory animal Co. Ltd, Changsha, Hunan, China. Each poly-propylene cage (41 cm \u0026times; 34 cm \u0026times; 16 cm (height)) was kept with 5 animals with the controlled conditions of temperature (20℃-24℃), humidity (40%), a light/dark cycle (12 h/12 h) with free access to food and water. Let the animals adapt to the laboratory environment before the experiment begins. The rodent license of the laboratory is no.SCXK (Xiang) 2016-0002 and laboratory animal use certificate is no. SYXK(Gan)2017-0003. All animal experiments comply with the ARRIVE guidelines and are carried out in accordance with the U.K. Animals (Scientific Procedures) Act, 1986 and associated guidelines, EU Directive 2010/63/EU for animal experiments, the Guidelines for Care and Use of Laboratory Animals of Jinggangshan University and approved by the Guidance Suggestions for the Hunan Province Laboratory Animal Care and Use Committee and the Medical Ethics Committee of Jinggangshan University.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3 Experimental design\u003c/h2\u003e \u003cp\u003eSixty rats were randomly divided into six groups (n\u0026thinsp;=\u0026thinsp;10 in each group): normal group (A), normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg) (B), exercise group (C), exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D), exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/ kg)-treated group (E) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F). The rats of ginseng total saponins treated groups (B, D, E, F) were injected by the intragastric gavage (ig) once per day with ginseng total saponins at the respective one time dose. Rats of group A and C were administrated with the same volume of saline by ig. The volume of each ig was 2 ml per rat and performed at 2 h before the start of treadmill running for 7 days. Rats in the exercised groups (C, D, E and F) had been doing exercises through running on a treadmill with 0\u0026deg; of inclination according to the exercise scheme in the exhaustive exercise-induced fatigue model for 7 days. All rats were conducted by Morris water maze experiment at 2 h after the end of the treadmill exercise every day for 6 days. On the 7th day, after spatial probe test, brain samples were harvested for transmission electron microscopy and western blotting (3 rats for each examination).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4 The model of exhaustive exercise-induced fatigue\u003c/h2\u003e \u003cp\u003eAccording to the method described in the literature (Wang, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), the rats were placed on the treadmill for 30 min to familiarize themselves with the running environment, and then the treadmill running began. The exercise loading was classifified into three stages: I: 10 m/min, 15min; II: 15 m/ min, 15min; III: 20 m/min, run till exhaustion. Criteria of exhaustion was as follows: the running posture changed from stomp-style into prostrate-style, and it remained stationary in the rear part of the treadmill, sound/light/electric stimulation could not keep it running.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Morris water maze\u003c/h2\u003e \u003cp\u003eAccording to the method described in the literature (Li, et al, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e2017\u003c/span\u003e), all rats were trained to search for a under warm (23\u0026ndash;25\u0026deg;C) water platform for six days, one training session per day, consisting of four training trials, and cues around the pool were consistent. In each trial, put each rat into water of each of the four quadrants in succession and the longest time to discover the platform is 120 seconds. If animals can\u0026rsquo;t find the platform in this limited time, it is guided to the platform, and stayed there for 15 seconds. After the training of six days, the positioning navigation experiment and space exploration experiment were carried out. A digital video camera connected to a computer (Anhui Zhenghua Biological Instrument Equipment Co., Ltd, China) recorded escape latency, the time of staying in the previous target quadrant, the times of crossing the target platform and the average swimming speed of the rats.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6 Preparation of electron microscopy specimen\u003c/h2\u003e \u003cp\u003ePreparation of electron microscopy specimen was performed according to the method described in the literature (Wang, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). Rats were anesthetized with 1% pentobarbital (40 mg/kg body weight) by intraperitoneal injection and then were fixed by perfusing 4% paraformaldehyde solution through heart. One cubic millimeter of hippocampus was taken and fixed in 4% glutaraldehyde for 3 h. After immersion in 0.1 mol/L phosphate buffered solution (PBS) for 10 min \u0026times; 3 times, the hippocampal tissue was placed in 1% osmic acid (prepared with 0.1 mol/L PBS) for 40min. After dehydration and resin embedding, ultrathin sections of 70 nm thickness were cut and then counterstained with acetyl uranium and lead acetate.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Transmission electron microscopy and image analysis\u003c/h2\u003e \u003cp\u003eThree consecutive slices were cut at the same position of each rat's CA1 region of hippocampus. Tissue was examined under a Hitachi H-7650 Transmission Electron Microscope operated at 100 kV. The number of synapses under the same field of vision, the length of synaptic active area and the width of synaptic cleft and the thickness of postsynaptic density (PSD) in each synapse were measured by Leica-200 image analyzer. The length of synaptic active zone and the thickness of PSD were measured following published method (G\u0026uuml;ldner and Ingham, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e1980\u003c/span\u003e), and the width of synaptic cleft was measured by multi-point averaging method (Wang, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Western blot analysis\u003c/h2\u003e \u003cp\u003eWestern blot analysis was performed as previously described (Zhu, et al., \u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Hippocampal fragments were isolated after behavioral test and stored in a \u0026minus;\u0026thinsp;80\u0026deg;C freezer until further use. The hippocampal tissues homogenate was centrifuged at 13 000 g for 15 min at 4 \u003csup\u003e◦\u003c/sup\u003eC and the supernatant was collected. Protein concentration was measured by Bio-Rad Protein Assay (BioRad, Hercules, CA, USA). Sample proteins were separated on SDS-polyacrylamide gels, and then transferred to a polyvinylidine diflfluoride membrane, blocked by 5% non-fat milk and incubated overnight with the primary antibodies: SYP ( 1:1000; 2644T; CST ), PSD95 (1:1000; A7889; Abclonal), NR2B (1:1000; A3056; Abclonal), CaMKII (1:1000; A3231; Abclonal), p-NR2B ( 1:5000; ab81271; Abcam ), p-CaMKII (1:1000; 12716T;CST). The membrane was washed twice and then incubated for 1 h with secondary antibodies (anti-rabbit IgG for SYN, PSD95, NR2B, CaMKII, p-NR2B and p-CaMKII)(1:10 000; ab97060, Abcam,Cambridge, UK), and the bound antibody was detected by using an enhanced chemiluminescence detection kit (Amersham, Buckinghamshire, UK). For the gel loading control, membranes were re-probed with a monoclonal anti-Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (Santa Cruz, sc-25778, 1:1000; CA, USA). Protein levels in the groups were expressed as a percentage of respective control values.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9 Statistical analysis\u003c/h2\u003e \u003cp\u003eThe first author used SPSS 17.0 software (SPSS, Chicago, IL, USA) for statistical analysis. The results were expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard (SD). All the data were analyzed by one - way analysis of variance (ANOVA) and then tested by Tukey\u0026rsquo;s post hoc test, \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 was statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cp\u003e\u003cstrong\u003e3.1 Effect of ginseng total saponins on the learning and memory of rats with exhaustive exercise-induced fatigue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFigure \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e showed the influence of ginseng total saponins on the learning and memory of rats. The results showed that fatigue exercise (the exercise group, C) prolonged the latency to find the platform, decreased the dwell time in the target quadrant and the number of platform crossings compared to the normal group (A) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). The administration of 100 and 200 mg/kg of ginseng total saponins (groups E and F) shortened the latency to find the platform, increased the dwell time in the target quadrant and the number of platform crossings compared to the exercise group (C) (\u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01), and the administration of 200 mg/kg of ginseng total saponins (group F) is more active than 100 mg/kg of ginseng total saponins (group E) ( \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.01). No significant difference was found in the latency to find the platform, dwell time in the target quadrant and umber of platform crossings among groups F (the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group), A (the normal group) and B (the normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)- treated group), between groups C (the exercise group) and D (exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins 50 mg/ kg)-treated group), \u003cem\u003ep\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05. The average speed of reaching the platform in each group has no significant difference (p\u0026thinsp;\u0026gt;\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab1\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eEffect of ginseng total saponins on learning and memory ability of rats with exhaustive exercise-induced fatigue(x̄ \u0026plusmn; s, n\u0026thinsp;=\u0026thinsp;10)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003cp\u003eIndex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eescape latency(s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e15.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.79\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.56\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e21.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.78\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e20.80\u0026thinsp;\u0026plusmn;\u0026thinsp;2.97\u003csup\u003e\u0026dagger;\u0026dagger;\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003csup\u003e\u0026dagger;\u0026dagger;**\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003ecrossing platform times\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e7.50\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.10\u0026thinsp;\u0026plusmn;\u0026thinsp;0.74\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e5.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.23\u003csup\u003e\u0026dagger;\u0026dagger;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e5.30\u0026thinsp;\u0026plusmn;\u0026thinsp;1.49\u003csup\u003e\u0026dagger;\u0026dagger;\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e6.70\u0026thinsp;\u0026plusmn;\u0026thinsp;0.95\u003csup\u003e\u0026dagger;\u0026dagger;**\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eTarget quadrant residence time (s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e12.50\u0026thinsp;\u0026plusmn;\u0026thinsp;1.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.30\u0026thinsp;\u0026plusmn;\u0026thinsp;2.21\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.80\u0026thinsp;\u0026plusmn;\u0026thinsp;0.79\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e8.90\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003csup\u003e\u0026dagger;\u0026dagger;\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e11.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.89\u003csup\u003e\u0026dagger;**\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e13.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.35\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eAverage swimming speed(cm/s)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.44\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.70\u0026thinsp;\u0026plusmn;\u0026thinsp;1.29\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.18\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e28.49\u0026thinsp;\u0026plusmn;\u0026thinsp;2.02\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.05\u0026thinsp;\u0026plusmn;\u0026thinsp;2.09\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e29.57\u0026thinsp;\u0026plusmn;\u0026thinsp;2.13\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote: A: normal group, B: normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/kg) group, C: exercise group, D: exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/kg)-treated group, E: exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/ kg)-treated group, F: exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/kg)-treated group. \u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, compared with group A, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, compared with group C, \u003csup\u003e\u0026sect; \u0026sect;\u003c/sup\u003e \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, compared with group F.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.2 Effect of ginseng total saponins on the morphology of synapse in hippocampus of rats with exhaustive exercise-induced fatigue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of ginseng total saponins on the number of synapses in hippocampal CA1 area of rats are shown in Fig. 2-a and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e. The results showed that the number of synapses in hippocampal CA1 area were significantly less in the exercise group (C) compared to the other groups except the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). The exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F) had more synapses of hippocampal CA1 area compared to both the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/ kg)-treated group (E) (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e). There were no significant difference in the number of synapses among the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F), the normal group (A) and normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg) (B) (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e\n\u003cp\u003eThe effects of ginseng total saponins on the length of synaptic active area, synaptic cleft width and PSD thickness of synapses in hippocampal CA1 area of rats are shown in Fig. 2-b and Table \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e.The results showed that the width of the synaptic cleft in the exercise group (C) were wider compared to the other groups except the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D), while the thickness of PSD were opposite (\u003cem\u003eP\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). The width of the synaptic cleft in exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F) were narrower compared to both the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/ kg)-treated group (E), and the thickness of PSD were opposite (\u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e or \u003cem\u003eP\u0026thinsp;\u0026lt;\u0026thinsp;0.05\u003c/em\u003e). There were no significant difference in the width of synaptic cleft and thickness of PSD among the normal group (A), normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg) (B) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F)(\u003cem\u003eP\u0026thinsp;\u0026gt;\u0026thinsp;0.05\u003c/em\u003e). There were no significant difference in the length of the synaptic active area in hippocampal CA1 area in each group (\u003cem\u003eP\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05).\u0026nbsp;\u003c/p\u003e\n\u003ctable id=\"Tab2\" border=\"1\"\u003e\n \u003ccaption language=\"En\"\u003e\n \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e\n \u003cdiv class=\"CaptionContent\"\u003e\n \u003cp\u003eComparison of structural parameters of synapses in hippocampal CA1 area of rats (x̄ \u0026plusmn; s, n\u0026thinsp;=\u0026thinsp;5)\u003c/p\u003e\n \u003c/div\u003e\n \u003c/caption\u003e\n \u003cthead\u003e\n \u003ctr\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n \u003cp\u003eGroup\u003c/p\u003e\n \u003cp\u003eIndex\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eA\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eB\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eC\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eD\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eE\u003c/p\u003e\n \u003c/th\u003e\n \u003cth align=\"left\"\u003e\n \u003cp\u003eF\u003c/p\u003e\n \u003c/th\u003e\n \u003c/tr\u003e\n \u003c/thead\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe\u0026nbsp;number\u003c/p\u003e\n \u003cp\u003eof \u0026nbsp;synapses\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.71\u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e1.20\u0026thinsp;\u0026plusmn;\u0026thinsp;0.84\u003csup\u003e\u0026dagger;\u0026dagger;,\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e2.00\u0026thinsp;\u0026plusmn;\u0026thinsp;0.00\u003csup\u003e\u0026dagger;,**,\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e3.40\u0026thinsp;\u0026plusmn;\u0026thinsp;0.55\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe width of synaptic cleft (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.60\u0026thinsp;\u0026plusmn;\u0026thinsp;1.95\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e19.20\u0026thinsp;\u0026plusmn;\u0026thinsp;1.92\u003csup\u003e\u0026dagger;\u0026dagger;,\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e20.00\u0026thinsp;\u0026plusmn;\u0026thinsp;1.58\u003csup\u003e\u0026dagger;\u0026dagger;,\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e18.80\u0026thinsp;\u0026plusmn;\u0026thinsp;1.64\u003csup\u003e\u0026dagger;\u0026dagger;,**,\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e16.40\u0026thinsp;\u0026plusmn;\u0026thinsp;1.67\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe thickness of post-synaptic density (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.20\u0026thinsp;\u0026plusmn;\u0026thinsp;10.83\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e71.40\u0026thinsp;\u0026plusmn;\u0026thinsp;12.01\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e41.00\u0026thinsp;\u0026plusmn;\u0026thinsp;6.78\u003csup\u003e\u0026dagger;\u0026dagger;,\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e43.20\u0026thinsp;\u0026plusmn;\u0026thinsp;9.76\u003csup\u003e\u0026dagger;\u0026dagger;,\u0026sect;\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e59.20\u0026thinsp;\u0026plusmn;\u0026thinsp;5.72\u003csup\u003e\u0026dagger;\u0026dagger;,**,\u0026sect;\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e72.40\u0026thinsp;\u0026plusmn;\u0026thinsp;10.92\u003csup\u003e**\u003c/sup\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003eThe length of synaptic active zone (nm)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e349.80\u0026thinsp;\u0026plusmn;\u0026thinsp;84.30\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e351.20\u0026thinsp;\u0026plusmn;\u0026thinsp;69.11\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e275.20\u0026thinsp;\u0026plusmn;\u0026thinsp;36.31\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e280.60\u0026thinsp;\u0026plusmn;\u0026thinsp;63.82\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"char\"\u003e\n \u003cp\u003e307.40\u0026thinsp;\u0026plusmn;\u0026thinsp;66.62\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd align=\"left\"\u003e\n \u003cp\u003e337.20\u0026thinsp;\u0026plusmn;\u0026thinsp;65.73\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n \u003ctfoot\u003e\n \u003ctr\u003e\n \u003ctd colspan=\"7\"\u003eNote: A: normal group, B: normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/kg/day) group, C: exercise group, D: exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/kg/day)-treated group, E: exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/kg/day)-treated group, F: exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/kg/day)-treated group. Values are mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. \u003csup\u003e\u0026dagger;\u0026dagger;\u003c/sup\u003e \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, compared with group A, \u003csup\u003e**\u003c/sup\u003e \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, compared with group C, \u003csup\u003e\u0026sect; \u0026sect;\u003c/sup\u003e \u003cem\u003ep\u0026thinsp;\u0026lt;\u0026thinsp;0.01\u003c/em\u003e, compared with group F.\u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tfoot\u003e\n\u003c/table\u003e\n\u003cp\u003e\u003cbr\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3 Effect of ginseng total saponins on synaptic protein levels in hippocampus of rats with exhaustive exercise-induced fatigue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSYP and PSD are synaptic proteins closely related to learning and memory and morphology of synapse. Therefore, we measured the effects of ginseng total saponins on SYP and PSD95 protein expression in the hippocampus. We observed that the levels of SYP and PSD95 protein expression in the hippocampus of rats in the exercise group (C) were significantly lower compared to the other groups except the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). The protein expression levels of SYP and PSD95 in the hippocampus of rats in exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F) were higher compared to both the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/ kg)-treated group (E) (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). There were no significant difference in the protein expression levels of SYP and PSD95 in the hippocampus of rats in the normal group (A), normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg) (B) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg / kg)-treated group (F) (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05). See Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.4 Effect of ginseng total saponins on NMDAR-CaMKII signalling pathway in hippocampus of rats with exhaustive exercise-induced fatigue\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe effects of ginseng total saponins on NMDAR-CaMKII signalling pathway in hippocampus of rats were presented in Fig.\u0026nbsp;4. The protein expression levels of hippocampal NR2B, p-NR2B, CaMKⅡ and p-CaMKⅡ in the exercise group (C) were significantly lower compared to the other groups except the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). The protein expression levels of NR2B, p-NR2B, CaMKⅡ and p-CaMKⅡ in the hippocampus of rats in exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F) were higher compared to both the exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50 mg/ kg)-treated group (D) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (100 mg/ kg)-treated group (E) (\u003cem\u003ep\u0026thinsp;\u0026lt;\u003c/em\u003e\u0026thinsp;0.01). The protein expression levels of hippocampus NR2B, p-NR2B, CaMKⅡ and p-CaMKⅡ in the normal group (A), normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg) (B) and exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/ kg)-treated group (F) were not significantly different, (\u003cem\u003ep\u0026thinsp;\u0026gt;\u003c/em\u003e\u0026thinsp;0.05).\u003c/p\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eThis study shows that ginseng total saponins can significantly improve the learning and memory, resist the changes in the morphology of synapse in hippocampus, increase the expression levels of synaptic-related proteins SYP and PSD95 and up-regulate the NR2B-CaMKII signal in hippocampus of rats with exhaustive exercise-induced fatigue in a dose-dependent manner.\u003c/p\u003e \u003cp\u003eAppropriate exercise is an important part of a healthy life style because of its beneficial effects on brain functions. However, excessively intense exercise that results in fatigue can induce brain dysfunction(Baker, et al., \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e2004\u003c/span\u003e). High-intensity treadmill running impairs the spatial memory ability of mice (Sun, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). The Morris water maze test is a method of testing spatial learning and memory. In this study, according to behavioral test results, rats have weaker spatial learning and memory after 7 days of exhaustive exercise, which is consistent with previous studies (Wang, et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e ; Ma, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Ginseng total saponins treatment significantly improved the learning and memory of rats with exhaustive exercise-induced fatigue. The most potent effects were observed at the dose of 200 mg/kg of ginseng total saponins.\u003c/p\u003e \u003cp\u003eThe hippocampus is one of the key brain regions for learning and memory. Synapses are the structural basis of functional connections between neurons. Learning and memory are closely related to synapses of the hippocampus (Kempf, et al., \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). Learning disabilities may not only be caused by neural regeneration, neurodevelopmental abnormalities, neuron migration, and neuronal apoptosis, but also by synapses (Lunardi, et al., \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). Intense exercise can cause synapse plasticity damage in the hippocampus (Ding, et al., \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e2015\u003c/span\u003e). High- intensity treadmill exercise can impair synaptic functional plasticity in the hippocampus (Sun, et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Exercise-induced fatigue could make the PSD thickness thinner and the width of the synaptic cleft wider in the striatum synapses (Hou, et al., \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, we found that ginseng total saponins could resist the decrease of the number and PSD thickness of synapses and the increase of the width of synaptic cleft of synapses in the CA1 region of hippocampus of rats with exhaustive exercise-induced fatigue. It is suggested that the effects of ginseng total saponins on improving the learning and memory of rats were related to protecting from the damage of hippocampal synapses caused by exhaustive exercise-induced fatigue.\u003c/p\u003e \u003cp\u003eIntense exercise significantly reduced the protein expression levels of SYP in the hippocampus of rats (Ding, et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). High-intensity training significantly reduced the expression of protein and mRNA of PSD95 in hippocampus of rats (Ren, et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). In this study, we found the level of the protein expression of SYP and PSD95 significantly decreased after 7 days of exhaustive exercise. Reduced the levels of synaptic protein markers (PSD95, SYN) were coincided with the decrease of the number and PSD thickness of synapses (Pan, et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). We also found that ginseng total saponins could resisted the decrease of the protein expression levels of SYP and PSD95 in hippocampus of rats with exhaustive exercise-induced fatigue in a dose-dependent manner. The most potent effects were observed at the dose of 200 mg/kg of ginseng total saponins. It is suggested that ginseng total saponins could improve their learning and memory through increasing the protein expression levels of SYP and PSD95 in the hippocampus of rats with exhaustive exercise-induced fatigue.\u003c/p\u003e \u003cp\u003eThe enhanced presynaptic glutamate release and downregulated postsynaptic NR function lead to the impaired corticostriatal plasticity in mice with exercise-induced fatigue (Ma, et al., \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). High-intensity platform training decreased the the protein expression levels of NR1 and NR2A and NR2B in hippocampus of rats (Ren,et al., \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e2017\u003c/span\u003e). Our previous research showed that the level of NR1 and NR2B mRNA expression in hippocampus of rats decreased after exercise-induced fatigue (Zhu, et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2006\u003c/span\u003e). In this study, we found that ginseng total saponins could not only resist the decrease of the protein expression levels of NR2B and CAMKII, but also significantly increase the phosphorylation levels of NR2B, CAMKII in hippocampus of rats with exhaustive exercise-induced fatigue. The phosphorylation status of NR is an important factor that reflects the activation status of NR (Sanderson, et al., \u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e2016\u003c/span\u003e). CaMKII is a key protein kinase in neural plasticity and memory (Zalcman, et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). CaMKII directly binds to the NR subunits NR1 and NR2B (Leonard, et al., \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e1999\u003c/span\u003e). CaMKII can be activated by calcium influx through NR (Nicoll \u0026amp; Schulman, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). When exhaustive exercise-induced fatigue inhibited the expression and phosphorylation levels of NR2B, it inhibited the phosphorylation of CaMKII which leads to the memory decline. Furthermore, inhibition of this signalling pathway also reduces the expression of PSD95 and SYN (Iyaswamy, et al., \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e2018\u003c/span\u003e), which damaged the neurons. So resistance to the inhibition of NR2B-CaMKII signal caused by exhaustive exercise-induced fatigue may be an important mechanism for ginseng total saponins to improve learning and memory of rats with exhaustive exercise-induced fatigue.\u003c/p\u003e \u003cp\u003eGinseng is used as an important nootropic herb for many years in traditional Chinese medicine (Gao,et al., \u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e2018\u003c/span\u003e). Ginseng saponin is the main active ingredient of ginseng. Promoting learning and memory is one of the main pharmacological effects of ginseng saponin (Wang, et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2014\u003c/span\u003e). The researches have demonstated that total ginsenoside from ginseng root can improve the learning and memory impairment of rats induced by hindlimb suspension through inhibiting body inflammation and regulating HPA axis imbalance (Bao et al., \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e2021\u003c/span\u003e). Ginsenoside Rg1 alleviates learning and memory impairments and Aβ disposition through inhibiting NLRP1 inflammasome and autophagy dysfunction in APP/PS1 mice (Li et al, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e2023\u003c/span\u003e). This study clearly demonstrate that ginseng total saponins from ginseng stem leaf can also significantly improve the impairment of learning and memory of rats with exhaustive exercise-induced fatigue. This effect was associated with ginseng total saponins protecting from the damage of hippocampal synapses, increasing the expression levels of SYP and PSD95 and up-regulating NR2B-CaMKII signalling pathway in the hippocampus of rats with exhaustive exercise-induced fatigue. Ginseng total saponins from ginseng stem leaf has no obvious effects on learning and memory in normal rats, which is inconsistent with the literature (Yang, et al., \u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e1994\u003c/span\u003e).\u003c/p\u003e "},{"header":"Conclusion","content":"\u003cp\u003eGinseng total saponins from ginseng stem leaf at 200 mg/kg has shown a good effect on improving the learning and memory of rats with exhaustive exercise-induced fatigue. The mechanism of ginseng total saponins\u0026rsquo;s improving learning and memory might be related to its protecting from the damage of hippocampal synapses, increasing the protein expression levels of SYN and PSD95 and up-regulating NR2B-CaMKII signal in the hippocampus of rats with exhaustive exercise-induced fatigue. Results from this study could provide partial experimental basis for the use of ginseng total saponins and ginseng stem leaf in sports medicine.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors have no relevant financial or non-financial interests to disclose.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis study was supported by Natural Science Foundation of Jiangxi Province, China ( No. 20202BABL206124 ), National Natural Science Foundation of China ( No. 31660291), Natural Science Foundation of Education Department of Jiangxi Province（No.GJJ2201646）\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMeiju Zhu is responsible for the design of the article and writing, data statistics and the research of morphology of synapses in hippocampus and WB detection. Chungen Guo is in charge of the animal treadmills, the intraperitoneal injection and Morris water maze test. Hongzhu Zhu and Wenli Wang are in charge of the feeding of animals and Morris water maze test and the animal treadmills. All authors reviewed the manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAdams, D.J., Arthur, C.P., Stowell, M.H., 2015. Architecture of the synaptophysin / synaptobrevincomplex: structural evidence for an entropic clustering function at the synapse. Sci Rep. 5, 1-9. \u003c/li\u003e\n\u003cli\u003eAune,\u0026ensp;T.K., Ingvaldsen, R.P., Ettema, G.J., 2008. Effect of physical fatigue on motor control at different skill levels. Percept Mot Skills.106, 371-386. \u003c/li\u003e\n\u003cli\u003eBaker, J.S., Bailey, D.M., Hullin, D., Young, Ian., Davies, Bruce.,2004. Metabolic implications of resistive force selection for oxidative stress and markers of muscle damage during 30 s of high-intensity exercise. 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The expression and effect of NR1, NR2A and NR2B in hippocampus tissue of rats with exercise fatigue. China Sport Science. 26, 71-74.\u003c/li\u003e\n\u003cli\u003eZhu, M.J., Zhu, H.Z., Ding, X.M., Liu, S.S., Zou, Y.H., 2020. Analysis of the anti-fatigue activity of polysaccharides from Spirulina platensis: role of central 5-hydroxytryptamine mechanisms. Food Funct. 11: 1826-1834.\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"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":"ginseng saponin, exercise, fatigue, learning and memory, synapse, NR2B-CaMKII signal","lastPublishedDoi":"10.21203/rs.3.rs-4275142/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4275142/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study is to investigate ginseng total saponins from ginseng stem leaf on the learning and memory of fatigue rats and the mechanism of action. Sixty Sprague-Dawley male rats were randomly divided into six groups: normal group, normal\u0026thinsp;+\u0026thinsp;ginseng total saponins (200 mg/kg) group, exercise group, exercise\u0026thinsp;+\u0026thinsp;ginseng total saponins (50, 100, 200 mg/kg)\u0026ndash;treated groups. The learning and memory was tested by Morris water maze experiment. After 7 days of exhaustive exercise, we measured hippocampal morphology by electron microscopy. The protein expression levels of synaptophysin ( SYP ), and postsynaptic density (PSD) protein 95 (PSD 95), N-methyl-D-aspartic acid receptor 2B (NR2B), calcium / calmodulin - dependent protein kinase II ༈CaMKII༉, phospho - NR2B ( p-NR2B ) and phospho - CaMKII ( p - CaMKII ) were measured by western blot analysis. The results demonstrated that ginseng total saponins (100, 200 mg/kg) treatment significantly decreased the latency to find the platform, increased dwell time in the target quadrant and the number of platform crossings of fatigued rats. ginseng total saponins (100, 200 mg/kg) treatment also increased the number of synapses and postsynaptic density (PSD) thickness, shrink the synaptic cleft of synapses in hippocampus of fatigue rats, significantly up-regulated NR2B -CaMKII signal, increased the levels of SYP and PSD 95 protein expression. It suggests that ginseng total saponins could improve the learning and memory of fatigue rats, relating to protecting the morphology of hippocampus, up-regulating NR2B-CaMKII signal in the hippocampus of fatigued rats.\u003c/p\u003e","manuscriptTitle":"Effects of ginseng total saponins from ginseng stem leaf on spatial learning and memory impairment by exhaustive exercise-induced fatigue: Role of NR2B-CaMKII signal in rat hippocampus","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-06-11 06:02:33","doi":"10.21203/rs.3.rs-4275142/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":"4a3f8dac-70c1-4a77-ab68-e5dbf2ee83f1","owner":[],"postedDate":"June 11th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2025-03-20T04:38:29+00:00","versionOfRecord":[],"versionCreatedAt":"2024-06-11 06:02:33","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4275142","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4275142","identity":"rs-4275142","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}