Protective effects of ginsenoside Rg1 on neuronal senescence due to inhibition of NOX2 and NLRP1 inflammasome activation in SAMP8 mice

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Protective effects of ginsenoside Rg1 on neuronal senescence due to inhibition of NOX2 and NLRP1 inflammasome activation in SAMP8 mice Yali Chena,1, Shixin Dinga,1, Han Zhanga,1, Zhenghao Suna,1, Xiaoyan Shena, Lingling Suna, ⁎ ⁎ Yanyan Yina, Sen Qunb, , Weizu Lia, a Department of Pharmacology, Basic Medicine College, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, Hefei 230032, China b Department of Neurology, The First Affiliated Hospital of USTC, University of Science and Technology of China, Hefei 230001, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Ginsenoside Rg1 ROS oxidative stress NADPH oxidase 2 NLRP1 inflammasome SAMP8

With aging of population, aging-related neurodegenerative diseases have become a major public health concern. Oxidative stress and neuroinflammation have been reported to play important roles in the aging process. Ginsenoside Rg1 (Rg1) is the main active ingredient in ginseng, and may be a potential agent for neurodegenerative diseases. In the present study, we investigated the effects of Rg1, tempol (ROS scavenger) and apocynin (NOX inhibitor) treatment for 9 weeks on cognitive performance, neuronal damage, and NOX2 and NLRP1 inflammasome expressions in 8-month old SAMP8 mice. The results showed that Rg1 alleviated learning and memory impairment and neuronal damage in SAMP8 mice. Meanwhile, Rg1 could reduce production of ROS and decreased expressions of NOX2 and NLRP1-related proteins in brain cortex and hippocampus tissues in SAMP8 mice. The findings suggest that Rg1 can alleviate aging-related neuronal damage, the mechanisms may be involved in reducing NOX2-mediated ROS generation and inhibiting NLRP1 inflammasome activation.

1. Introduction Aging is complex and inevitable in nearly all species and all organs. Brain exhibits high energy metabolism and is one of the most susceptible target organ in the course of aging (Zhu & Chen, 2018). Brain aging has been reported to been an important risk factor in a wide range of human diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD) (Cenini, Lloret, & Cascella, 2019). With increasing of age population, the brain pathological aging and aging-related neurodegenerative diseases have become a major public health concern (Youssef et al., 2016). Accordingly, it is very important to study brain aging and its mechanisms for prevention of aging-related neurodegenerative diseases. At present, the mechanism of brain aging is still not entirely clear. Oxidative stress and neuroinflammation have been reported to play an important role in the aging process (Fan et al., 2017; Joseph, ShukittHale, Casadesus, & Fisher, 2005). Oxidative stress is harmful to cells due to the excessive generation of endogenous reactive oxygen species (ROS) resulting in disturbance of prooxidant and antioxidant balance (Casciaro et al., 2018). Growing evidence showed that accumulation of ROS plays an important role in aging-associated neuronal oxidative

stress and inflammation resulting in neuronal damage (Calabrese et al., 2010). NADPH oxidases (NOX) are important family of enzymes dedicated to generation of ROS that can damage cellular components and induce functional abnormalities in many cell types (Altenhofer et al., 2012; Halliwell, 2006). The NADPH oxidase 2 (NOX2) is constitutively expressed in many cells in the brain, especially in neurons. NOX2 is a major source of excessive ROS generation and closely related to the cause and progression of aging-related neuronal damage and neurodegenerative diseases, such as AD and PD (d'Avila et al., 2018; Fan et al., 2017). Our previous study found that the expressions of NOX2 and its subunits, Rac1, p47phox and p22phox were significantly increased in prolonged cultured hippocampal neurons (Xu, Sun, et al., 2019). These data suggest that NOX2-derived ROS oxidative stress plays a critical role in the development of brain aging. Additionally, inflammation is reported to be an underlying cause of many human diseases, and also plays an important role in aging-related neurodegenerative diseases such as AD (Zotova et al., 2013). The initial phase of the inflammatory response involves the interaction of inflammasome multiple complexes in the cells. As a critical component of the inflammasome, nucleotide-binding oligomerization domain (NOD)like receptor protein 1 (NLRP1) was the first member of the NOD-like

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Corresponding authors at: Department of Pharmacology, Basic Medicine College, Anhui Medical University, No. 81 Meishan Road, Hefei, Anhui 230032, China. E-mail addresses: [email protected] (S. Qun), [email protected] (W. Li). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.jff.2019.103713 Received 6 August 2019; Received in revised form 26 November 2019; Accepted 27 November 2019 1756-4646/ © 2019 Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/BY-NC-ND/4.0/).

Please cite this article as: Yali Chen, et al., Journal of Functional Foods, https://doi.org/10.1016/j.jff.2019.103713

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(5 mg/kg) and Rg1 (10 mg/kg) treatment groups (n = 9 in each group). The tempol (Merck Millipore), apocynin (Merck Millipore) and Rg1(content of Rg1 > 98%; Chengdu Desite Biotechnology Co., China) were dissolved in distilled water and were treated intragastrically (i.g., 0.1 ml/10 g) for 9 weeks. The SAMR1 control group and SAMP8 model group received equivalent volumes of distilled water for 9 weeks. The handling of mice and experimental procedures were approved by the Animal Care and Use Committee of Anhui Medical University.

receptor (NLR) family to be discovered (Kummer et al., 2007; Tan et al., 2015). NLRP1 inflammasome was widely expressed in the central nervous system, especially in neurons (Abulafia et al., 2009). Recent studies have shown that NLRP1 inflammasome is involved in the pathogenesis of a variety of neurological diseases, especially the age-related neuronal damage and cognitive impairment (Mawhinney, de Rivero Vaccari, Dale, Keane, & Bramlett, 2011). Activated NLRP1 can generate a functional caspase-1-containing inflammasome to cause inflammatory response and pyroptotic cell death (Kaushal et al., 2015; Masters et al., 2012). Moreover, inhibition of the NLRP1 inflammasome can reduce the innate immune response and ameliorate age-related cognitive deficits (de Rivero Vaccari et al., 2009; Mawhinney et al., 2011). Our latest study showed that NOX2 and NLRP1 inflammasome acitvation could exacerbate age-related damage in prolonged primary cultured hippocampal neurons (Xu, Sun, et al., 2019). However, it remains unclear whether NOX2-derived ROS can activate NLRP1 inflammasome and induce aging-associated neuronal damage in vivo. At present, there are still no effective drugs for preventing brain aging and aging-related neurodegenerative diseases. Ginseng, as a famous traditional Chinese medicine, has been used as a nourishing drug for improving health conditions, and delaying senescence since ancient times (Y. Wang et al., 2014). Ginsenoside Rg1 (Rg1) is the main active ingredient in ginseng. Previous studies suggested that Rg1 has the functions of improving learning and memory impairment, and may be a potential agent for neurodegenerative diseases such as AD (Bae, Chung, Lee, & Kang, 2014; Zhou et al., 2012; Zhu, Wang, Li, & Wang, 2015). Our previous studies demonstrated that Rg1 could decrease ROS production and protect against neuronal damage caused by chronic glucocorticoids exposure (Zhang et al., 2017). Our latest study found that Rg1 treatment significantly reduced the expressions of NOX2 and p47phox in H2O2-treated hippocampal neurons (Xu, Shen, et al., 2019). Nevertheless, whether the protective effect of Rg1 on senescence-associated neuronal damage is related to inhibition of NOX2 has not yet been fully elucidated. The senescence-accelerated mouse prone 8 strain (SAMP8) is one of the most widely accepted murine models for the study of late-onset or age-related AD etiopathogenesis (Wang et al., 2019). In this study, we hypothesize that Rg1 may ameliorate neuronal damage and cognitive deterioration via inhibition of NOX2-mediated ROS generation and NLRP1 inflammasome activation in SAMP8 mice. Tempol is a ROS scavenger that promotes the metabolism of many ROS. A recent study reported that the effectiveness of tempol in catalyzing the metabolism of cellular O2− was similar to native superoxide dismutase (SOD) (Wilcox, 2010). Studies have found that tempol was effective in many models of neurodegeneration due to oxidative stress (Kumar & Singh, 2015). Apocynin is a naturally methoxy-substituted catechol. As a NOX inhibitor, APO attenuates the activity of NADPH oxidase by interfering with the intracellular translocation of two cytosolic components, p47phox and p67phox (Stolk, Hiltermann, Dijkman, & Verhoeven, 1994). Increasing studies have shown the beneficial antioxidant effects of apocynin on neurological disorders (Li et al., 2013; Qiu et al., 2016). To study the effects of Rg1 on NOX2-mediated oxidative stress and NLRP1 activation during aging, we examined the effects of Rg1, tempol and apocynin on cognitive performance, neuronal damage, and NOX2 and NLRP1 inflammasome expressions in SAMP8 mice.

2.2. Open field test (OFT) The open field test apparatus (Shanghai Biowill Co., Ltd., China) consisted of a computer-tracking cage (60 × 60 × 50 cm) which was separated as 9 squares by two vertical lines and transverse lines as previously described (Hu et al., 2016). The OFT test was performed on the 5th day of the 9th week. The mouse was placed in the cage for 2 min in each testing period. Then the ANY-maze behavioral tracking software (Stoeling Co., USA) was used to record the paths of its motor activity and exploration for 3 min. The number of lines crossing, the moving distance (m), the mean moving speed (m/s) and the number of standing up events (indicating exploratory behavior) were recorded by the software to represent the motor and exploratory behaviors (Frye, Paris, & Rhodes, 2007).

2.3. Novel object recognition (NOR) test NOR test is an indicative memory task that is based on the behavior of animals which prefer a novel object to a familiar object and spend more time in searching the novel object (Ennaceur & Delacour, 1988). The NOR test consists of the open field apparatus and the ANY-maze behavioral tracking system. For NOR test, the animals were individually habituated to the open field with no object for 5 min (that is also the OFT) before the task. After 24 h of the OFT, the mice were placed in the open field with two identical objects (blue cubes) in A and B position, and the animals were allowed to explore them freely for 5 min. The next day, a new object (red cone) replaced the original position of B, and the animals were allowed to explore them freely for 5 min. The discrimination index (DI) was recorded to evaluate the effects of Rg1 on recognition and memory impairments. The DI was calculated as the time spent exploring the new object divided by the total time exploring the both objects multiplied by 100. A higher DI was considered to reflect greater memory retention (Acar, Akkoc, & Erdinc, 2019).

2.4. ROS detection (DHE staining) To assess the generation of ROS in neurons during the course of aging, the dihydroethidine (DHE) fluorescence staining was performed to detect the ROS levels in brain (Wang et al., 2014). For DHE staining, the animals (n = 3) were injected intravenously with DHE (100 μM, 0.1 ml/10 g, Beyotime Biotechnology, China) for 30 min. Then the brains were removed and half of the brains embedded in OCT (Sakura, Torrance, CA) at −20 °C and remaining brains were stored at −80 °C for immunoblot analysis. The brains were sectioned into 5 μm slices with a freezing microtome (Leica CM3050, Germany). Sections were washed three times with PBS, then incubated with Hoechst 33258 (Sigma, 5 mg/L) for 5 min and washed three times with PBS. The sections were examined by a fluorescence microscope (Olympus IX71, Japan) and photographed for Hoechst 33258 (excitation: 360 nm, emission: 450 nm) and DHE (excitation: 480 nm, emission: 590 nm). The mean fluorescence density of DHE staining was quantified from 3 random fields (400×) in brain cortex and hippicampal CA1 and CA3 areas by using an Image Pro Plus 6.0 analysis software to indicate the ROS production.

2. Materials and methods 2.1. Animals and treatment Adult male SAMP8 mice and male senescence-accelerated resistant mouse 1 (SAMR1) mice (6 months old, weight 30–40 g) were obtained from Department of Laboratory Animal Science, Peking University Health Science Center. The SAMR1 mice (n = 9) were used as normal age control. The SAMP8 mice were randomly divided into 5 groups: SAMP8 model group, tempol (50 mg/kg), apocynin (50 mg/kg), Rg1 2

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Japan).

2.5. Histological examination The hematoxylin and eosin (H&E) staining is commonly used to observe the neuronal histopathology (Wang, Liu, Lu, et al., 2016). After behavior test, the mice (n = 4) were perfused transcardially with 4% paraformaldehyde (PFA) to fix the brains. Then the brains were carefully removed and immersed into 4% paraformaldehyde for further fixation. Paraffin-embedded brain tissues were cut into 5 µm sections. The paraffin sections were deparaffinized by xylene and hydrated by gradient ethanol. Then the sections were stained with H&E reagents and the neuronal morphology in the cortex and hippocampus CA1 and CA3 was captured by using an optical microscope (Olympus IX71, Japan).

All data were presented as mean ± SD. Statistical differences were performed by SPSS 17.0 statistical software. The results were analyzed by one-way ANOVA and then subjected to between-group comparisons using the Tukey's test. Statistical significance was defined as P < 0.05.

2.6. Nissl dyeing

3.1. Effect of Rg1 treatment for 9 weeks on changes of body weight in SAMP8 mice

2.9. Statistical analysis

3. Results

Nissl staining is also an important method to study the neuronal pathological alterations (Peng, Gao, Huo, Liu, & Yan, 2015). For Nissl staining, the paraffin sections were deparaffinized with xylene and hydrated with gradient ethanol and distilled water. Then the sections were treated with Nissl staining solution (Beyotime Biotechnology, China) for 10 min. After washing with distilled water, the sections were differentiated with 95% alcohol and cleared with xylene. The sections were observed and photographed by a light microscope (Olympus IX71, Japan).

In this study, the body weight was measured every week before and after treatment with tempol, apocynin and Rg1. The results showed that there were no significant differences in body weight before administration among the groups, the average body weight was less than 2 g between SAMR1 and SAMP8 groups. The mice were treated with distilled water or drugs for 9 weeks (W). The body weight of SAMR1 mice increased from 32.02 ± 1.87 g to 34.39 ± 2.30 g. While the body weight of SAMP8 mice decreased from 33.39 ± 2.73 g to 29.29 ± 1.61 g, and the body weight was significantly different during 7–9 W as compared with SAMR1 group (Fig. 1, P < 0.05 or P < 0.01). Similar to SAMP8 group mice, the mice body weight in the tempol, apocynin and Rg1 (5 mg/kg) groups also had a decreased trend from 1 W to 7 W after treatment with drugs. However, from 7 W to 9 W, the body weight in SAMP8 group still has a decreased trend, while the body weight in drugs-treated groups were no longer decrease. And in the 9 W, the body weight in apocynin and Rg1 (5 mg/kg) treated groups was significantly higher than the SAMP8 model group (Fig. 1, P < 0.05 or P < 0.01). While in Rg1 (10 mg/kg) group, the body weight has no significant decrease from 1 W to7W, and the body weight was significantly higher in 7 W, 8 W and 9 W than that of SAMP8 mice (Fig. 1, P < 0.05 or P < 0.01). The results indicated that Rg1 treatment significantly improved the body weight in SAMP8 mice during the course of aging.

2.7. Immunoblot analysis Immunoblot analysis was performed as our previously described (Xu, Shen, et al., 2019). The total protein of cortex and hippocampus was extracted. The protein concentration was determined by BCA Protein Assay Kit (Shanghai Sangon Bio-Tech, China). Equal amounts of proteins were separated electrophoretically by SDS-PAGE gels and then transferred to PVDF membranes (Millipore, Bedford, MA, USA). The membranes were blocked in 5% skimmed milk for 1 h and then incubated with primary antibodies of NOX2 (1:1000, Abcam, ab31092), p22phox (1:500, Bioworld Technology, BS60290), p47phox (1:1000, Bioworld Technology, BS4852), NLRP1 (1:1000, Abcam, ab3683), ASC (1:500, Santa Cruz Technology, SC-514414), caspase-1 (1:1000, Abcam, ab1872), IL-1β (1:1000, Abcam, ab9722) and β-actin (1:1000; ZSGB-BIO, TA-09) overnight at 4 °C. The blots were then incubated with horseradish peroxidase-conjugated goat anti-rabbit (ZSGB-BIO, ZB-2301) secondary antibodies (1:10,000) for 1 h at room temperature. The blots were performed with ECL substrate kit (Bio-Rad Laboratories, CA, USA). The protein bands were visualized by using a Bioshine Chemi Q4600 Mini Imaging System (Shanghai Bioshine Technology, China). The density of protein bands was performed with Image J software and normalized to the corresponding β-actin bands. The relative density of each target protein over SAMR1 control was used to represent the changes of target proteins.

3.2. Effects of Rg1 treatment on exploration behavior in SAMP8 mice In the study, the open field test (OFT) and novel object recognition (NOR) test were used to observe the effects of Rg1 treatment on exploratory behavior in SAMP8 mice. The OFT results showed that the mean moving distance (m), the mean moving speed (m/s), the number of lines crossing and standing up were significantly decreased in SAMP8 mice compared with SAMR1 control mice (Table1, P < 0.05 or P < 0.01). The results suggested that the SAMP8 mice showed obvious aging phenomenon at the age of 8 months, the activity and exploration behavior have a significant decline. While compared with the SAMP8 model group, the tempol, apocynin and Rg1 (5, 10 mg/kg) treatment for 9 weeks significantly increased the number of lines crossing and standing up, but had no significant effect on moving distance and mean speed. The results demonstrating that tempol, apocynin and Rg1 treatment had no influence on motor activity but significantly improved the exploratory behavior in the SAMP8 mice (Table 1, P < 0.05 or P < 0.01). The further NOR test results also showed that the discrimination index was significantly decreased in the SAMP8 mice compared to the SAMR1 control group mice (Fig. 2, P < 0.05). Meanwhile, the results showed that treatment with tempol, apocynin and Rg1 (5 and 10 mg/ kg) significantly increased the discrimination index as compared with SAMP8 model group (Fig. 2, P < 0.05 or P < 0.01). The results indicate that the tempol, apocynin and Rg1 treatment can improve the learning and exploration ability in SAMP8 mice.

2.8. Immunofluorescence We further detected the co-expression of NLRP1 and NEUN to confirm whether the NLRP1 expresses in cortex and hippocampal neurons in SAMP8 mice by immunofluorescence staining. The paraffin sections were deparaffinized and hydrated. Subsequently, the sections were permeabilized with 0.25% Triton X-100 for 30 min, followed by blocking with 1% BSA for 60 min. Then, the sections were incubated with primary antibodies of mouse monoclonal neuronal specific nuclear protein (NEUN) antibody (1:200, Abcam, ab104224) and rabbit polyclonal NLRP1 antibody (1:200, Abcam, ab3683) overnight at 4 °C. Secondary antibodies against mouse and rabbit were conjugated respectively to Rhodamine (1:200, ZSGB-BIO) and FITC (1:200, ZSGBBIO). The sections were then washed and stained with Hoechst 33258 for 10 min. Then the sections were mounted by using anti-fade medium and examined with the fluorescence microscope (Olympus IX71, 3

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Fig. 1. Effects of Rg1 treatment on body weight in SAMP8 mice. Data are expressed as mean ± SD, n = 9. *P < 0.05, **P < 0.01 compared with SAMR1 control group; #P < 0.05, ##P < 0.01 compared with SAMP8 model group.

group mice (Fig. 3). The results of Nissl staining revealed that there were abundant Nissl bodies in the cortex and hippocampal neurons in the SAMR1 mice. Meanwhile, the pyknotic and hyperchromatic neurons were less in the SAMR1 mice. Compared with the SAMR1 group mice, the Nissl bodies were significantly decreased, the pyknotic and hyperchromatic neurons were significantly increased in the cortex and hippocampal CA1 and CA3 regions in the SAMP8 group mice. And the boundaries between the nucleus and cytoplasm were unclear (Fig. 4). While compared with the SAMP8 model group, tempol, apocynin and Rg1 (5, 10 mg/kg) treatment for 9 weeks significantly alleviated the neuronal damage, showing that the Nissl bodies and the pyknotic and hyperchromatic neurons were decreased (Fig. 4). The results suggest that Rg1 treatment can effectively alleviate the aging-related neuronal damages in SAMP8 mice.

3.3. Effects of Rg1 treatment on neuronal histopathological changes in SAMP8 mice Aging has been reported to cause atrophy or loss of neurons in the brain (Padurariu, Ciobica, Mavroudis, Fotiou, & Baloyannis, 2012). To investigate the protective effects of Rg1 treatment on morphological changes of neurons in SAMP8 mice, the H&E staining and Nissl staining were used to observe the histopathological changes in the cortex and hippocampal CA1 and CA3. As shown in the results of H&E staining, there were no abnormal changes in the brain cortex and hippocampal CA1 and CA3 neurons in the SAMR1 control group. The neurons were clear, intact structure and good arrangement. But compared with the SAMR1 control mice, there were significant neuronal damages in brain cortex and hippocampal CA1 and CA3 in SAMP8 mice. There were more neurons showed unclear nucleoli, karyopyknosis, hyperchromatism, and obvious cytoplasmic eosinophilic changes, indicating that there were obvious aging-related neuronal damages in cortex and hippocampus CA1 and CA3 in the SAMP8 mice (Fig. 3). However, the tempol, apocynin, and Rg1 (5, 10 mg/kg) treatment for 9 weeks significantly alleviated the aging-related neuronal damages compared with the SAMP8 model group mice, especially in Rg1 (10 mg/kg) treatment

3.4. Effects of Rg1 treatment on the generation of ROS in brain tissues of SAMP8 mice To further identify the effect of Rg1 treatment on the generation of ROS, the DHE fluorescence staining was performed to examine the levels of ROS in brain cortex and hippocampal CA1 and CA3 areas. The

Table 1 Protective effects of Rg1 on motor activity and exploratory behavior in SAMP8 mail mice (open field test). Groups

Dose (mg/kg)

Moving distance (m)

Mean speed (m/s)

Line crossings

Number of standing up

SAMR1 SAMP8 Tempol Apocynin Rg1

– – 50 50 5 10

19.12 14.65 16.23 17.06 19.35 17.07

0.11 0.08 0.09 0.09 0.11 0.09

64.00 45.63 62.88 63.89 65.38 66.56

41.90 16.88 25.13 31.22 36.63 46.44

± ± ± ± ± ±

2.69 5.55* 5.21 2.96 6.56 6.32

± ± ± ± ± ±

0.02 0.03* 0.03 0.02 0.04 0.04

± ± ± ± ± ±

8.73 17.86* 13.10# 16.04# 6.77# 16.49#

± ± ± ± ± ±

7.91 1.81** 3.87## 2.82## 6.12## 7.20##

Results are expressed as mean ± SD. n = 9. *P < 0.05, **P < 0.01 compared with SAMR1 control group; #P < 0.05, ##P < 0.01 compared with SAMP8 model group. 4

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caspase-1 and IL-1β in the cortex and hippocampus tissues in SAMP8 mice (Fig. 7B–E; P < 0.05 or P < 0.01). These data suggest that Rg1 treatment can inhibit the NLRP1 inflammasome in the cortex and hippocampus in SAMP8 mice. 3.7. NLRP1 mainly expresses in cortex and hippocampal neurons in SAMP8 mice To confirm whether the NLRP1 expresses in cortex and hippocampal neurons in SAMP8 mice, we further examined the co-expression of NLRP1 and NEUN by using immunofluorescence staining in SAMP8 model mice. The results showed that the NEUN expressed in the neuronal nucleus and the NLRP1expressed in the cytoplasm. There were more cells co-expressed NEUN and NLRP1 indicating that the NLRP1 mainly expresses in the cortex and hippocampal neurons in the SAMP8 mice (Fig. 8). 4. Discussion

Fig. 2. Effects of Rg1 on memory and exploration in SAMP8 male mice (new object recognition test). Results are expressed as mean ± SD. n = 9. *P < 0.05 compared with SAMR1 control group; #P < 0.05, ##P < 0.01 compared with SAMP8 model group.

Pathological aging and age-related cognitive decline have become a major health problem for our society with the aging population (Fjell et al., 2014). Aging is a major risk factor for many neurological diseases, including Alzheimer’s disease (AD) (Verheijen, Vermulst, & van Leeuwen, 2018). At present, there were no fully effective medicines and methods for delaying brain aging (Snell et al., 2016). Therefore, understanding the molecular mechanisms of brain aging may provide new insights into the etiology of such dysfunctions and promote the development of treatments for delaying or preventing brain aging. The SAMP8 mouse, which has been described as a nature, non-transgenic animal for accelerated brain senescence, displays multiple features known to occur early in the pathogenesis of AD including increased oxidative stress, neuronal loss, amyloid-alterations, tau phosphorylation, and severe deficits of learning and cognition (Butterfield & Poon, 2005; Cheng, Zhou, & Zhang, 2014; Del Valle et al., 2010; Yanai & Endo, 2016; Zhang et al., 2009). The development of new therapeutic methods will be a great significance in alleviating senescence-related neuronal damage and reducing the morbidity rate of aging-related neurodegenerative diseases. Our previous studies suggested that Rg1 could decrease ROS generation by inhibiting NOX2 and attenuate the neuronal damage in chronic restrain stress-induced mice (Wang et al., 2014). Additionally, Rg1 could also down-regulate the NLRP1 inflammasome and protect against the neuronal damage induced by chronic DEX exposure (Zhang et al., 2017). The present study was designed to investigate the effects and mechanisms of the Rg1 treatment for 9 weeks on senescence-associated neuronal damage in SAMP8 mice. The current results demonstrated that NOX2 and NLRP1 inflammasome activation closely correlated with oxidative stress, inflammation and senescence-related neuronal damage in brain cortex and hippocampus in the SAMP8 mice. The results also showed that Rg1 significantly improved the learning and memory, and reduced the production of ROS and the expressions of NOX2 and NLRP1 inflammasome in SAMP8 mice. Our results suggest that treatment with Rg1 in the early stages of aging may be a potential strategy for delaying the progression of brain aging. Brain aging is characterized by progressive cognitive impairment and neurodegeneration and is a basis of many aging-related neurodegenerative diseases in the elderly, such as AD. Clinical studies have shown that the majority of individuals over the age of 65 will experience cognitive decline that interferes with their quality of life and ability to maintain independence (Banuelos et al., 2014; Hernandez et al., 2015). Behavioral tests are often used to evaluate the recognition function of animal models with cognitive dysfunction. The open field test and novel object recognition test are generally used to assess motor activity and exploratory behavior in a novel environment (Prut & Belzung, 2003). Ginseng has been reported as an anti-aging herb for thousands of years. The ginsenoside Rg1 (Rg1) is one of active

results showed that there was slight production of ROS in SAMR1 brain tissues. While compared with SAMR1 mice, the production of ROS was significantly elevated in the cortex and hippocampal CA1 and CA3 in SAMP8 mice (Fig. 5A–F, P < 0.01). Compared with SAMP8 group mice, tempol, apocynin and Rg1 (5, 10 mg/kg) treatment significantly decreased the ROS levels in brain tissues, especially in the Rg1 (10 mg/ kg) treatment group (Fig. 5A–F, P < 0.05 or P < 0.01). These results indicate that ROS oxidative stress plays an important role in brain senescence and Rg1 treatment may ameliorate senescence-associated neuronal oxidative stress damage. 3.5. Effects of Rg1 treatment on expressions of NOX2, p22phox and p47phox in the cortex and hippocampus tissues in SAMP8mice To confirm whether NADPH oxidase is involved in ROS generation during the course of aging, the expressions of NOX2, p22phox and p47phox were measured in the cortex and hippocampus by immunoblot (Fig. 6A). The results showed that, compared with the SAMR1 control group, the expressions of NOX2, p22phox and p47phox were significantly increased in SAMP8 mice. Compared with the SAMP8 model group, Rg1 (5 and 10 mg/kg) treatment significantly reduced the expressions of NOX2, p22phox and p47phox in the cortex and hippocampus tissues (Fig. 6B–D, P < 0.05 or P < 0.01). Tempol (50 mg/ kg) treatment can decrease p22phox expression (Fig. 6B–D, P < 0.05), while had no significant influence on NOX2 and p47phox expression. Apocynin (50 mg/kg) treatment can decrease p22phox and p47phox expression (Fig. 6B–D, P < 0.05) and had no significant influence on NOX2 expression. These data suggest that Rg1treatment can inhibit the NOX2-mediated ROS generation in the cortex and hippocampus in SAMP8 mice. 3.6. Effects of Rg1 treatment on expressions of NLRP1, ASC, caspase-1 and IL-1β in the cortex and hippocampus tissues in SAMP8 mice To confirm whether Rg1 can modulate NLRP1 inflammasome activation, we further detected the expression of NLRP1, ASC, caspase-1 and IL-1β in the cortex and hippocampus brain tissues by immunoblot (Fig. 7A). The results revealed that, compared with the SAMR1 control group, the expressions of NLRP1, ASC, caspase-1 and IL-1β were significantly increased in the cortex and hippocampus in SAMP8 mice (Fig. 7B–E; P < 0.05). Compared with the model group, tempol (50 mg/kg), apocynin (50 mg/kg) and Rg1 (5 and 10 mg/kg) treatment for 9 weeks significantly decreased the expressions of NLRP1, ASC, 5

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Fig. 3. Effects of Rg1 treatment on neuronal histopathological changes in SAMP8 mice (H&E staining, 400×, n = 4). Black arrows represent normal neurons and red arrows represent damage neurons.

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Fig. 4. Effects of Rg1 treatment on neuronal morphology changes in SAMP8 mice (Nissl staining, 400×, n = 4). Black arrows represent normal neurons and red arrows represent damage neurons.

significant impairment in motor activity and exploration behavior, suggesting that the 8-month-old SAMP8 mice have shown obvious aging characteristics. Meanwhile, treatment with tempol (a ROS scavenger), apocynin (a NOX inhibiter) and Rg1 for 9 weeks significantly

ingredient in ginseng. It has been reported that Rg1 has antioxidant and anti-aging effects, as well as promoting intelligence (Ong, Farooqui, Koh, Farooqui, & Ling, 2015; Wang, Liu, Xu, et al., 2016). In this study, the data demonstrated that 8-month-old SAMP8 mice showed 7

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Fig. 5. Effects of Rg1 treatment on ROS generation in SAMP8 mice (DHE staining, 400×). (A) The production of ROS in the cortex. (B) The production of ROS in the hippocampal CA1. (C) The production of ROS in the hippocampal CA3. (D) Quantitative analysis of the ROS production in cortex. (E) Quantitative analysis of the ROS production in hippocampal CA1. (F) Quantitative analysis of the ROS production in hippocampal CA3. Data are expressed as mean ± SD, n = 3. **P < 0.01 compared with SAMR1 control group; #P < 0.05, ##P < 0.01 compared with SAMP8 model group.

revealed that the body weight showed a significant decreasing trend from 6-month-old to 8-month-old in SAMP8 mice compared to the SAMR1control mice. While the Rg1 (10 mg/kg) treatment for 9 weeks significantly inhibited weight loss in SAMP8 mice. These data suggest that Rg1 treatment has protective effects on brain senescence in SAMP8 mice. Ageing-related neurodegenerative diseases are characterized by the

ameliorated the behavior impairment in the SAMP8 mice. In addition, studies have shown that neurological diseases were associated with a reduction in body weight (Bradford et al., 2009; de Waard et al., 2010). And aging-related weight loss is also an important characteristic, which primarily associated with decreased physiological function caused by cells loss and metabolic changes (Haigis & Yankner, 2010; Lopez-Otin, Blasco, Partridge, Serrano, & Kroemer, 2013). The present study 8

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Fig. 6. Effects of Rg1 treatment on the expressions of NOX2, p22phox and p47phox in the cortex and hippocampus tissues in SAMP8 mice (immunoblot). (A) The bands of NOX2, p22phox, p47phox and β-actin examined by immunoblot. (B) Quantitative analysis of the relative expression of NOX2 over SAMR1 control. (C) Quantitative analysis of the relative expression of p22phox over SAMR1 control. (D) Quantitative analysis of the relative expression of p47phox over SAMR1 control. Results are expressed as the mean ± SD, n = 3. *P < 0.05 compared with the SAMR1 control group; #P < 0.05 and ##P < 0.01 compared with the SAMP8 model group.

accordingly when gp91phox expression was up-regulated (Zeng, Zhao, Ma, Jiang, & Tu, 2012). Our previous results had found that Rg1 treatment significantly reduced the expressions of NOX2 and p47phox in H2O2-treated hippocampal neurons (Xu, Shen, et al., 2019). The present study showed that the expressions of NOX2, p22phox and p47phox in the brain cortex and hippocampus were significantly increased in SAMP8 mice, suggesting that NOX2-derived ROS accumulation closely involved in aging-related neuronal damage. Moreover, Rg1 treatment significantly decreased the expressions of NOX2, p22phox and p47phox in the brain cortex and hippocampus in SAMP8 mice. Tempol is similar to catalase or superoxide dismutase and is often used as a radical scavenger (Sunkaria, Sharma, Wani, & Gill, 2014; Youn et al., 2016). Tempol was effective in some models of neurodegeneration due to oxidative stress (Wilcox, 2010). The present results showed that tempol (50 mg/kg) treatment could significantly decrease ROS production in brain cortex and hippocampus in SAMP8 mice, whereas only had a decreased trend on NOX2 and p47phox expressions. But tempol treatment could down-regulate p22phox expression, which may be a result of ROS scavenging and alleviating neuronal oxidative stress damage. Apocynin, as an inhibitor of NADPH oxidase, can prevent the generation of ROS (Stolk et al., 1994). The apocynin can inhibit the NOX2 with an IC50 about 10 µM, while for NOX4, the IC50 more than 200 µM (Serrander et al., 2007). Our results showed that apocynin (50 mg/kg) treatment could significantly decrease ROS production and down-regulate p22phox and p47phox expressions in brain cortex and hippocampus in SAMP8 mice. It has been reported that the molecular structure of Rg1 contains (carbon-3, -6 -20) hydroxyl groups, and shows strong hydroxyl radical scavenging activity (Lu, Yao, & Chen, 2009). Our results suggested that Rg1 may decrease ROS production and alleviate aging-related neuronal damage not only by scavenging ROS, but also inhibiting NOX2-mediated ROS generation. Neuroinflammation also plays a detrimental role in the development of aging-related neurodegenerative diseases, such as Alzheimer's

damage and loss of neurons in specific regions of the brain such as the cortex and hippocampus, which is particularly susceptible to ROS oxidative stress (Neal & Richardson, 2018). Ample evidence has shown that ROS played an important role in brain aging (Beckman & Ames, 1998; Fan et al., 2019; Wyss-Coray, 2016). In the process of aging, over production of ROS can induce a series of oxidative stress events, not only destroying the function of mitochondria, but also further inducing the occurrence and progression of neurodegeneration (Lee et al., 2015; Venkat, Chopp, & Chen, 2015; Zhang, Wu, Nie, Jia, & Yu, 2014). In the present study, it was demonstrated that the ROS levels in brain cortex and hippocampus were significantly increased in SAMP8 mice. And treatment with tempol, apocynin and Rg1 significantly decreased the ROS levels in brain cortex and hippocampus. Consistent with the elevation of ROS levels, there were significant neuronal damages in brain cortex and hippocampus in SAMP8 mice detected by H&E and Nissl staining. Moreover, treatment with tempol, apocynin and Rg1 for 9 weeks also significantly alleviated the neuronal damage in SAMP8 mice. Our data suggest that over production of ROS plays an important role in senescence-associated neuronal damage, Rg1 treatment may delay brain aging by decreasing ROS accumulation in brain cortex and hippocampus. NADPH oxidase (NOX) is a major source of ROS generation in vivo, and involved in the development of age-related cognitive dysfunction (Cahill-Smith & Li, 2014). Accumulating evidence indicated that the NADPH oxidase 2 (NOX2) played an important role in ROS generation in brain and closely involved in the development of age-associated cognitive dysfunction (Maejima, Kuroda, Matsushima, Ago, & Sadoshima, 2011). The latest study suggested a crucial role for NOX2derived ROS in aging-related loss of brain function (Fan et al., 2019). The NOX2 consists of two membrane subunits p22phox and catalyzed gp91phox (NOX2), and several cytoplasmic subunits, such as p47phox, p67phox, p40phox and rac1 (Lambeth, Kawahara, & Diebold, 2007). Responding to many factors, the activity of NADPH oxidase increased 9

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Fig. 7. Effects of Rg1 treatment on the expressions of NLRP1, ASC, caspase-1 and IL1β in the cortex and hippocampus in SAMP8 mice (immunoblot). (A) The bands of NLRP1, ASC, caspase-1, IL-1β and β-actin measured by immunoblot. (B) Quantitative analysis of the relative expression of NLRP1 over SAMR1 control. (C) Quantitative analysis of the relative expression of ASC over SAMR1 control. (D) Quantitative analysis of the relative expression of caspase-1 over SAMR1 control. (E) Quantitative analysis of the relative expression of IL-1β over SAMR1 control. Results are expressed as the mean ± SD, n = 3. * P < 0.05 compared with the SAMR1 control group; #P < 0.05 and ##P < 0.01 compared with the SAMP8 model group.

procaspase-1 activation to caspase-1. Activated caspase-1 cleaves proIL-1β to generate the mature, secreted form of IL-1β (Artlett & Thacker, 2015; de Rivero Vaccari, Dietrich, & Keane, 2016). It has been reported that ROS accumulation activated NLRP1 inflammasome and induced macrophages apoptosis in vitro and in vivo, which were significantly inhibited by pretreatment with ROS inhibitors (Gan, Gao, Zhao, & Qi, 2016). Our previous study also showed that NOX2 activation and excessive ROS production play an important role in activating the NLRP1 inflammasome and closely involves in neuronal senescence (Xu, Sun, et al., 2019). In addition, our previous study showed that Rg1 protected against chronic dexamethasone-induced neuronal degeneration by inhibiting NLRP1 inflammasomes in mice (Zhang et al., 2017). And Rg1 could alleviate H2O2-induced neuronal oxidative stress and apoptosis via inhibition of NOX2-derived ROS

disease and Parkinson’s disease (Liu & Chan, 2014; Xu, Nygard, Kristensson, & Bentivoglio, 2010). The chronic inflammatory response is an important feature of the aging process and the leading cause of aging-related neuronal damage (Glass, Saijo, Winner, Marchetto, & Gage, 2010). Inflammasomes are intracellular multiprotein complexes, which play a pivotal role in pro-inflammation. Among them, the NLRP1 inflammasome is expressed widely in the body, particularly in neurons (Tan et al., 2015; Yin et al., 2009). The NLRP1 inflammasome activation is closely related to the neurodegenerative diseases and patients with epilepsy (Tan et al., 2015). The NLRP1 inflammasome consists of NLRP1, apoptosis-associated speck-like protein containing a caspase recruitment domain (ASC), and caspase-1, an inflammatory cysteineaspartic protease (Jorgensen & Miao, 2015; Martinon, Burns, & Tschopp, 2002). ASC can interact with procaspase-1 and induce

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Fig. 8. The expressions of NEUN and NLRP1 in the cortex and hippocampus CA1 and CA3 in SAMP8 mice (immunofluorescence, 400×).

Declaration of Competing Interest

generation and down-regulation of the NLRP1 inflammasome (Xu. Shen, et al., 2019). These studies suggest that Rg1 has an anti-oxidative effect and can inhibit the NLRP1 inflammasome in cortex and hippocampal neurons in SAMP8 mice. In this study, the results showed that the expressions of NLRP1, ASC, caspase-1 and IL-1β were significantly increased in the brain cortex and hippocampus tissues of SAMP8 mice. Treatment with Rg1, tempol and apocynin could significantly reduce the expressions of NLRP1, ASC, caspase-1 and IL-1β. Additionally, the immunofluorescence results showed that the NLRP1 mainly co-expressed with NEUN in the cortex and hippocampus neurons. These data suggest that Rg1 may alleviate senescence-related neuronal damage via down-regulation of the NLRP1 inflammasome in SAMP8 mice. However, whether Rg1 can inhibit the NLRP1 inflammasome by downregulating the NOX2-mediated ROS generation in brain aging remains further elucidate.

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper. Acknowledgements This study was supported by the National Natural Science Foundation of China (81671384, 81371329). The authors would like to thank Dake Huang and Bao Li from the Synthetic Laboratory of Basic Medicine College, Anhui Medical University for their technical assistance. References Abulafia, D. P., de Rivero Vaccari, J. P., Lozano, J. D., Lotocki, G., Keane, R. W., & Dietrich, W. D. (2009). Inhibition of the inflammasome complex reduces the inflammatory response after thromboembolic stroke in mice. Journal of Cerebral Blood Flow & Metabolism, 29(3), 534–544. https://doi.org/10.1038/jcbfm.2008. 143jcbfm2008143. Acar, A., Akkoc, H., & Erdinc, M. (2019). The effects of spinosad on antioxidant system and cognitive performance of mice. Archives of Physiology and Biochemistry, 1–5. https://doi.org/10.1080/13813455.2019.1623264. Altenhofer, S., Kleikers, P. W., Radermacher, K. A., Scheurer, P., Rob Hermans, J. J., Schiffers, P., ... Schmidt, H. H. (2012). The NOX toolbox: Validating the role of NADPH oxidases in physiology and disease. Cellular and Molecular Life Sciences, 69(14), 2327–2343. https://doi.org/10.1007/s00018-012-1010-9. Artlett, C. M., & Thacker, J. D. (2015). Molecular activation of the NLRP3 Inflammasome in fibrosis: Common threads linking divergent fibrogenic diseases. Antioxidants & Redox Signaling, 22(13), 1162–1175. https://doi.org/10.1089/ars.2014.6148. Bae, H. J., Chung, S. I., Lee, S. C., & Kang, M. Y. (2014). Influence of aging process on the bioactive components and antioxidant activity of ginseng (Panax ginseng L.). Journal of Food Science, 79(10), H2127–H2131. https://doi.org/10.1111/1750-3841.12640. Banuelos, C., Beas, B. S., McQuail, J. A., Gilbert, R. J., Frazier, C. J., Setlow, B., & Bizon, J. L. (2014). Prefrontal cortical GABAergic dysfunction contributes to age-related working memory impairment. Journal of Neuroscience, 34(10), 3457–3466. https:// doi.org/10.1523/JNEUROSCI.5192-13.201434/10/3457. Beckman, K. B., & Ames, B. N. (1998). The free radical theory of aging matures. Physiological Reviews, 78(2), 547–581. https://doi.org/10.1152/physrev.1998.78.2. 547. Bradford, J., Shin, J. Y., Roberts, M., Wang, C. E., Li, X. J., & Li, S. (2009). Expression of mutant huntingtin in mouse brain astrocytes causes age-dependent neurological symptoms. Proceedings of the National Academy of Sciences USA, 106(52), 22480–22485. https://doi.org/10.1073/pnas.09115031060911503106. Butterfield, D. A., & Poon, H. F. (2005). The senescence-accelerated prone mouse (SAMP8): A model of age-related cognitive decline with relevance to alterations of

5. Conclusion Overall, the present study suggested that Rg1 can alleviate learning and memory impairment and aging-related neuronal damage in SAMP8 mice. Meanwhile, we further found that Rg1 had obvious antioxidant effects and reduced ROS production and NOX2 expression. Furthermore, Rg1 significantly inhibited the activation of NLRP1 inflammasome in brain cortex and hippocampus tissues in SAMP8 mice. These findings provide support that Rg1 can delay brain aging and alleviate aging-related neuronal damage, and the mechanisms may be related to inhibition of NOX2, ROS generation and NLRP1 inflammasome activation. However, this study only provided a basic experiment for Rg1 in delaying brain senescence, and the precise mechanisms of Rg1 on regulation of the NOX2-NLRP1 signaling pathway warrant further investigation.

6. Ethics statement All animals were housed and handled according to Anhui Medical University Institutional Animal Care and Use Committee guidelines. All experiments were performed according to institutional guidelines.

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