IJMM International Journal of Molecular Medicine 1107-3756 1791-244X D.A. Spandidos 10.3892/ijmm.2018.4005 ijmm-43-02-0717 Articles Ginsenoside Rg1 protects against H2O2-induced neuronal damage due to inhibition of the NLRP1 inflammasome signalling pathway in hippocampal neurons in vitro XuTan-Zhen1* ShenXiao-Yan1* SunLing-Ling1* ChenYa-Li1* ZhangBi-Qiong1 HuangDa-Ke2 LiWei-Zu1 Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education Synthetic Laboratory of Basic Medicine College, Anhui Medical University, Hefei, Anhui 230032, P.R. China Correspondence to: Dr Wei-Zu Li, Department of Pharmacology, Key Laboratory of Anti-inflammatory and Immunopharmacology, Ministry of Education, Anhui Medical University, 81 Meishan Road, Hefei, Anhui 230032, P.R. China, E-mail: liweizu@126.com

Contributed equally

 02 2019 30 11 2018 43 2 717 726 23 08 2018 21 11 2018 Copyright: © Xu et al. 2019 This is an open access article distributed under the terms of the Creative Commons Attribution-NonCommercial-NoDerivs License, which permits use and distribution in any medium, provided the original work is properly cited, the use is non-commercial and no modifications or adaptations are made.

Oxidative stress and neuroinflammation are important in the pathogenesis of ageing and age-related neurodegenerative diseases, including Alzheimer’s disease. NADPH oxidase 2 (NOX2) is a major source of reactive oxygen species (ROS) in the brain. The nucleotide-binding oligomerisation domain (NOD)-like receptor protein 1 (NLRP1) inflammasome is responsible for the formation of pro-inflammatory molecules in neurons. Whether the NOX2-NLRP1 inflammasome signalling pathway is involved in neuronal ageing and age-related damage remains to be elucidated. Ginsenoside Rg1 (Rg1) is a steroidal saponin found in ginseng. In the present study, the primary hippocampal neurons were treated with H2O2 (200 µM) and Rg1 (1, 5 and 10 µM) for 24 h to investigate the protective effects and mechanisms of Rg1 on H2O2-induced hippocampal neuron damage, which mimics age-related damage. The results showed that H2O2 treatment significantly increased ROS production and upregulated the expression of NOX2 and the NLRP1 inflammasome, and led to neuronal senescence and damage to hippocampal neurons. Rg1 decreased ROS production, reducing the expression of NOX2 and the NLRP1 inflammasome in H2O2-treated hippocampal neurons. Furthermore, Rg1 and tempol treatment significantly decreased neuronal apoptosis and the expression of β-galactosidase, and alleviated the neuronal senescence and damage induced by H2O2. The present study indicates that Rg1 may reduce NOX2-mediated ROS generation, inhibit NLRP1 inflammasome activation, and inhibit neuronal senescence and damage.

 reactive oxygen species oxidative stress NADPH oxidase 2 nucleotide-binding oligomerisation domain-like receptor protein 1 inflammasome hippocampal neurons ginsenoside Rg1 Introduction

Brain ageing is a progressive and gradual accumulation of harmful changes in the brain tissue with age, including oxidative stress injury and neuronal inflammation, which increase the risk of age-related learning and memory impairment (1,2). At present, the oxidative stress theory is recognised as the most feasible hypothesis to explain the ageing process, particularly age-related neuronal degeneration (3-5). Increasing evidence has suggested that reactive oxygen species (ROS) may cause neuronal oxidative stress, inflammation, and neurodegeneration with age (3,6-7). There are several enzymes that can generate intracellular ROS. NADPH oxidase (NOX) is a major source of ROS generation (4,5). It has been found that NOX2 (gp91phox) is constitutively expressed in neurons and significantly increased in the brain of ageing mice (4). The expression of NOX2 subunits p47phox and p67phox are also increased in the cortex of patients with mild cognitive disorders (5). Inflammasomes are NOD-like receptor (NLR) family multiprotein complexes that are responsible for the formation of pro-inflammatory molecules. Neuroinflammation mediated by inflammasomes is important in several age-related neurodegenerative diseases (8,9). NOD-like receptor protein 1 (NLRP1) was the first reported inflammasome (10). The NLRP1 inflammasome is widely expressed in the body and is the main inflammasome in neurons. An increasing number of studies have shown that the NLRP1 inflammasome is associated with age-related neuronal damage and cognitive impairment (8,11). NADPH oxidase-derived ROS accumulation is one of the main pathways for NLRP3 inflammasome activation (12,13). However, whether NOX2-derived ROS can activate the NLRP1 inflammasome, and whether it is involved in neuronal senescence and damage, remain to be elucidated.

Ginsenoside Rg1 (Rg1) is one of the active ingredients of ginseng, which has been used to improve health conditions and delay senescence in China (14,15). It has been reported that Rg1 has significant antioxidant, anti-ageing and neuroprotective effects (16), and Rg1 can improve learning and memory impairment in ageing mice (17,18). Our previous studies showed that Rg1 significantly decreased ROS generation and attenuated the neuronal oxidative stress damage induced by chronic restrain stress in mice (19), and Rg1 protected against neuroinflammation and neuronal injury induced by chronic glucocorticoid exposure (20). These data suggest that Rg1 has a protective effect on neuronal damage due to decreasing ROS generation. Therefore, it was hypothesised that Rg1 may downregulate NOX2 and reduce the production of ROS, thereby inhibiting activation of the NLRP1 inflammasome in hippocampal neurons and protecting against age-related neuronal damage.

In the present study, primary cultured hippocampal neurons were treated with H2O2 (200 µM) for 24 h to mimic age-related neuronal damage. The study evaluated the protective effects of ROS scavenger tempol and Rg1 on H2O2-induced neuronal damage and assessed whether Rg1 regulated the activation of the NLRP1 inflammasome via the inhibition of NOX2 in hippocampal neurons.

 Materials and methods Culture and treatment of hippocampal neurons

Primary hippocampal neurons were prepared from postnatal Sprague-Dawley rats (within 24 h; 6-8 neonates/experiment) obtained from the Center of Laboratory Animals of Anhui Medical University (Hefei, China) and placed in plates coated with poly-L-lysine (10 µg/ml). Neurobasal medium with B-27 supplement (Thermo Fisher Scientific, Inc., Waltham, MA, USA) was used to culture the neurons at 37°C with 5% CO2 as described previously (21). The neurons were cultured for 7 days and divided into six groups: Control group, H2O2 (200 µM) group, H2O2 (200 µM) + tempol (100 µM) group, and H2O2 (200 µM) + Rg1 (1, 5 and 10 µM) groups. With the exception of the control group, the neurons in the groups were treated with H2O2 (200 µM) and tempol (100 µM) or Rg1 (1, 5 or 10 µM) for 24 h. Tempol (Santa Cruz Technology, Inc., Dallas, TX, USA) and Rg1 (content of Rg1 >98%; Chengdu Desite Biotechnology Co., Chengdu, China) were dissolved in distilled water and stored at −80°C. All experimental procedures were performed in accordance with the approved protocol of the Ethics Committee of Anhui Medical University (Anhui, China).

 Neuronal apoptosis assay (Hoechst 33258 staining)

Hoechst 33258 can stain the nucleus and is often used to assess the apoptotic rate of neurons (22). First, the neurons were fixed with 4% paraformaldehyde and incubated with Hoechst 33258 (5 µg/ml, Zhongshan Golden Bridge Biotechnology Co., Beijing, China) for 15 min. Following washing with PBS, the neurons were sealed onto slides with anti-fade mounting medium. Fluorescence microscopy was then used to examine the apoptosis of neurons (Olympus IX71; Olympus Corporation, Tokyo, Japan). Morphologically, the apoptotic neurons appeared smaller and bright blue, the nucleus was condensed and deeply stained (23). The apoptotic neurons were counted and the relative neuronal apoptotic rate was examined in each culture and compared with the control.

 Examination of ROS production with dihydroethidium (DHE) staining

DHE is the most commonly used ROS fluorescent probe and is often used for labelling living cells to detect the production of ROS (24). For the ROS examination, DHE (10 µM) was added to the medium and incubated for 30 min at 37°C. The production of ROS was then detected using a fluorescence microscope (Olympus IX71, Olympus Corporation). The red mean optical density was performed using Image-Pro Plus 6.0 software (Media Cybernetics, Inc., Rockville, MD, USA), to indicate the ROS production in the hippocampal neurons.

 Immunofluorescence

Caspase-3 is the common pathway and the executor of apoptosis (25). To confirm the effect of Rg1 on neuronal apoptosis in H2O2-damaged hippocampal neurons, the expression of caspase-3 was detected by immunofluorescence. First, the hippocampal neurons were fixed with 4% paraformaldehyde for 30 min and washed thoroughly with PBS. Following permeabilisation with Triton X-100 (0.25%) for 30 min, and blocking with 1% BSA (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in PBS for 1 h, the neurons were incubated with caspase-3 primary antibody (cat. no. BS7003; lot no. CJ32131; 1:200; Bioworld Technology, Inc., St. Louis Park, MN, USA) overnight at 4°C. Subsequently, the neurons were incubated in secondary antibody conjugated to FITC (cat. no. ZF-0311; lot no. 127805; 1:200; ZSGB-BIO; OriGene Technologies, Inc., Beijing, China) for 1 h at room temperature. The slides were then mounted and examined with a fluorescence microscope (Olympus IX71; Olympus Corporation). The relative fluorescent intensity was quantified from three random fields per slide using the Image-Pro Plus 6.0 analysis system to indicate the expression of caspase-3.

 Immunoblotting assay

Radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Haimen, China) were used to extract the total protein. The protein concentration was determined by BCA Protein Assay kit (Beyotime Institute of Biotechnology). Equal samples of protein (30 µg) were separated by 12% SDS-PAGE and transferred onto a PVDF membrane. The membrane was blocked with 5% skim milk for 1 h at room temperature. The membrane was then incubated with primary antibodies targeting β-galactosidase (β-Gal), NOX2, p22phox, p47phox, NLRP1, apoptosis-associated speck-like protein containing a carboxy-terminal CARD (ASC), caspase-1 and β-actin (1:1,000) overnight at 4°C. The primary antibodies for β-Gal (cat. no. ab9361; lot no. GR27325-12), NOX2 (cat. no. ab31092; lot no. GR91755-40), NLRP1 (cat. no. ab3683; lot no. GR281560-21) and caspase-1 (cat. no. ab1872; lot no. GR615230-38) were from Abcam (Cambridge, UK). The p22phox (cat. no. BS60290; lot no. CN21141) and p47phox (cat. no. BS4852; lot no. CN33161) antibodies were from Bioworld Technologies, Inc. ASC antibody (cat. no. SC-514414; lot no. G1916) was from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) The dilutions of β-Gal, NOX2, NLRP1 and p47phox were 1:1,000. The dilutions of ASC, caspase-1 and p22phox were 1:500. The membranes were then washed with TBS-Tween 20 and incubated with anti-rabbit IgG antibody conjugated to HRP (cat. no. ZF-2301; lot no. 128615; 1:10,000; ZSGB-BIO; OriGene Technologies, Inc.) at room temperature for 1 h. The protein bands were visualised by using a Chemi Q4800 mini imaging system (Shanghai Bioshine Technology, Shanghai, China). The protein bands were measured with ImageJ 1.44 software (National Institutes of Health, Bethesda, MD, USA) and normalised to the corresponding β-actin bands. The relative density of each target protein over the control was used to represent the changes in expression of target proteins.

 Enzyme-linked immunosorbent assay (ELISA)

Following incubation with H2O2 and tempol or Rg1, the supernatants were collected from each group. ELISA kits were used to detect the levels of interleukin (IL)-1β and IL-18 (Cloud-Clone Corp., Houston, TX, USA). Briefly, the samples and the standards of IL-1β and IL-18 were placed into the assay plate and incubated for 1 h at 37°C. Following incubation for 1 h, the HRP-conjugated reagent (100 µl) was added to the wells and incubated for 1 h at 37°C. Subsequently, the chromogen solution (100 µl) was added to the wells. The plate was mixed and incubated for 15 min at 37°C. Stop solution (50 µl) was then added to the wells and the absorbance at 450 nm was detected with a microplate reader (Thermo Fisher Scientific, Inc.) within 15 min.

 Statistical analysis

Data are presented as the mean ± standard deviation. Statistical differences were analysed by one-way analysis of variance and the between-group comparisons were subjected to the Bonferroni post hoc test using SPSS 17.0 software (SPSS, Inc., Chicago, IL, USA. P<0.05 was considered to indicate a statistically significant difference.

 Results Rg1 inhibits H<sub>2</sub>O<sub>2</sub>-induced neuronal apoptosis and the expression of caspase-3

The present study first examined the effects of Rg1 treatment on H2O2-induced neuronal apoptosis by staining with Hoechst 33258, which can bind to chromatin allowing the visualisation of normal and condensed chromosomes with a fluorescence microscope (22). The results showed that there were few apoptotic neurons in the control group. Compared with the control group, H2O2 exposure significantly increased neuronal apoptosis (Fig. 1A and B; P<0.05). Compared with the H2O2-treated group, Rg1 (5 and 10 µM) treatment significantly reduced neuronal apoptosis (Fig. 1A and B; P<0.05). Treatment with the ROS scavenger tempol (100 µM) significantly decreased the apoptosis of hippocampal neurons induced by H2O2 treatment (Fig. 1A and B; P<0.05).

To confirm the anti-apoptotic effect of Rg1, the effects of Rg1 (5 µM) on the expression of caspase-3 in H2O2-damaged hippocampal neurons was further examined by immunofluorescence. The results showed that, compared with the control group, H2O2 treatment significantly increased the expression of caspase-3. Compared with the H2O2-treated model group, tempol and Rg1 treatments significantly decreased the expression of caspase-3 in H2O2-treated hippocampal neurons (Fig. 2A and B; P<0.05).

 Rg1 decreases the expression of senescence-associated β-gal in H<sub>2</sub>O<sub>2</sub>-treated hippocampal neurons

Senescent cells exhibit increased senescence-associated β-Gal activity (26). In order to observe the effect of Rg1 on H2O2-induced senescence in hippocampal neurons, the expression of β-Gal was detected by western blot analysis. The results showed that H2O2 (200 µM) treatment significantly increased the expression of β-Gal in the hippocampal neurons compared with the control group (Fig. 3A and B; P<0.01). Compared with the H2O2-treated group, tempol (100 µM) and Rg1 (5 and 10 µM) treatments significantly decreased the expression of β-Gal in the hippocampal neurons (Fig. 3A and B; P<0.05 and P<0.01, respectively).

 Rg1 reduces ROS generation in H<sub>2</sub>O<sub>2</sub>-treated hippocampal neurons

H2O2 exposure induces cell redox balance disorder, which may cause neuronal senescence and damage. Therefore, ROS production was detected using DHE staining. Compared with the control group, H2O2 treatment significantly increased the production of ROS in the hippocampal neurons (Fig. 4A and B; P<0.01). Compared with the H2O2-treated group, tempol (100 µM) and ginsenoside Rg1 (1, 5 and 10 µM) treatments significantly reduced the production of ROS in hippocampal neurons (Fig. 4A and B; P<0.05).

 Effects of Rg1 on the expression of NOX2, p22phox and p47phox in H<sub>2</sub>O<sub>2</sub>-treated hippocampal neurons

To confirm whether NADPH oxidase is involved in H2O2-induced neuronal senescence and damage in primary hippocampal neurons, the expression levels of NOX2, p22phox and p47phox in hippocampal neurons were measured by immunoblotting. The results showed that, compared with the control group, the expression levels of NOX2, p22phox and p47phox were significantly increased in the H2O2-treated group (Fig. 5A-C; P<0.05). Compared with the model group, Rg1 (5 and 10 µM) treatment significantly reduced the expression levels of NOX2 and p47phox in the H2O2-treated hippocampal neurons, whereas tempol (100 µM) treatment had no significant influence on their expression. The results showed that Rg1 had no significant influence on the expression of p22phox, whereas tempol treatment increased the expression of p22phox in the H2O2-treated hippocampal neurons (Fig. 5B; P<0.05).

 Effect of Rg1 on the expression levels of NLRP-1, ASC and caspase-1 in H<sub>2</sub>O<sub>2</sub>-treated hippocampal neurons

To confirm whether Rg1 regulated NLRP1 inflammasome activation, the expression levels of NLRP1, ASC and caspase-1 in primary hippocampal neurons were detected by immunoblotting. The results showed that, compared with the control group, the expression levels of NLRP-1, ASC and caspase-1 were significantly increased in the H2O2-treated group. Compared with the model group, tempol (100 µM) and Rg1 (5 and 10 µM) treatments significantly decreased the expression levels of NLRP1, ASC and caspase-1 in the H2O2-treated hippocampal neurons (Fig. 6A-C; P<0.05).

 Rg1 decreases the release of IL-1β and IL-18 in H<sub>2</sub>O<sub>2</sub>-treated hippocampal neurons

To confirm the effect of Rg1 on downregulation of the NLRP1 inflammasome, the levels of IL-1β and IL-18 released in supernatants were examined by ELISA. The results showed that, compared with the control group, the release of IL-1β and IL-18 was significantly increased in the H2O2-treated group (Fig. 7A and B; P<0.05). Compared with the model group, tempol (100 µM) and Rg1 (5 and 10 µM) treatments significantly reduced the release of IL-1β and IL-18 in supernatants from the H2O2-treated hippocampal neurons (Fig. 7A and B; P<0.05).

 Discussion

It has been reported that oxidative stress and neuroinflammation are involved in neuronal senescence and neurodegenerative diseases, including Alzheimer’s disease (27). The effect of Rg1 is largely associated with its antioxidant and other scavenging properties, for example, inhibiting the expression of inducible nitric oxide synthase and the overgeneration of nitric oxide. However, whether Rg1 can alleviate H2O2-induced senescence and neuronal damage, and its mechanism, warrants further investigation. In the present study, it was demonstrated that H2O2 treatment significantly induced oxidative stress damage and apoptosis in primary hippocampal neurons, and the NOX2-NLRP1 inflammasome pathway was important in H2O2-induced neuronal senescence and damage. In addition, Rg1 significantly reduced the production of ROS and the expression of NOX2 and the NLRP1 inflammasome, and inhibited neuronal senescence and damage in the H2O2-treated hippocampal neurons.

Oxidative stress is caused by the imbalance of redox in the body, resulting in excessive ROS generation. The accumulation of ROS can induce neuronal oxidative stress damage and is involved in the development of ageing-related neurodegenerative diseases (28). In the process of ageing, excessive ROS can lead to destruction of neuronal structure and impairment of learning and memory (29,30). H2O2 treatment is often used to simulate ROS accumulation-induced neuronal damage (31). In the present study, the results showed that H2O2 treatment significantly increased the generation of ROS, promoted neuronal apoptosis and increased the expression of senescence-associated β-Gal in hippocampal neurons.

Ginseng has been used as an anti-ageing drug for thousands of years in China. Numerous studies have shown that Rg1, a main active ingredient in ginseng, has antioxidant and anti-ageing effects, in addition to promoting cognitive function and improving immunity (32,33). Rg1 protects against neuronal damage induced by various mechanisms, including oxidative stress and inflammatory responses (20,34). Tempol is an effective antioxidant and can attenuate the damage caused by excessive accumulation of ROS (35). In the present study, the results showed that both Rg1 and tempol significantly decreased ROS generation and inhibited the expression of β-Gal in H2O2-treated hippocampal neurons. In addition, the Hoechst 33258 staining showed that Rg1 (5 and 10 µM) had a significant effect on H2O2-induced hippocampal neuron apoptosis, whereas Rg1 (1 µM) had no effect on the neuronal apoptosis. Caspase-3 is the most important terminal shear enzyme in the process of apoptosis (33). To confirm the anti-apoptotic effect of Rg1, the effects of Rg1 (5 µM) on the expression of caspase-3 were examined by immunofluorescence. The results showed that Rg1 (5 µM) significantly decreased the expression of caspase-3 in H2O2-induced hippocampal neurons. These results indicate that Rg1 may decrease ROS generation and inhibit neuronal senescence and apoptosis. However, the mechanism underlying the effect of Rg1 on reducing the production of ROS remains to be fully elucidated.

NOX is the most important enzyme system involved in the generation of ROS and has been shown to be important in various neurological diseases (28). NOX2 is a major source of ROS involved in the development of age-related cognitive dysfunction (28). The expression of NOX2 has been found to be increased in the brains of ageing mice (36). NOX2 consists of a membrane-bound catalytic core, including NOX2 (gp91phox) and p22phox), and several cytosolic subunits, including p40phox, p47phox, p67phox and rac1 (37). NOX2 is activated when the cytosolic subunits translocate to the membrane and combine with gp91phox (37,38). There is a linear relationship between the activity of NOX2 and cognitive decline in aged mice (39). In addition, NOX contributes to oxidative stress and neuronal apoptosis. NOX-deficient mice exhibit reduced injury following stroke (40). It is not entirely clear whether Rg1 can reduce NOX2 and, thus, reduce the production of ROS in H2O2-treated hippocampal neurons. In the present study, the results showed that the expression levels of NOX2, p22phox and p47phox were significantly increased in H2O2-treated hippocampal neurons. Rg1 treatment significantly reduced the expression levels of NOX2 and p47phox in H2O2-treated hippocampal neurons, whereas tempol (100 µM) treatment had no significant influence on their expression. The results also showed that Rg1 had no significant influence on the expression of p22phox, whereas tempol treatment increased the expression of p22phox in H2O2-treated hippocampal neurons. Therefore, Rg1 may protect against neuronal ageing and damage by scavenging ROS and inhibiting the formation of ROS derived from NOX2.

Neuroinflammation can accelerate brain ageing, causing neuronal damage and cognitive deficits (41). The chronic, progressive pro-inflammatory response is an important feature of the ageing process and the leading cause of neuronal apoptosis (42). Inflammasomes are multimeric proteins in the cytoplasm of a wide variety of cells (43). Among them, the NLRP1 inflammasome was the first characterised and is expressed widely in the body, particularly in neurons (44,45). The NLRP1 inflammasome consists mainly of NLRP1, caspase-1 and ASC proteins. NLRP1 can activate the key regulatory agent caspase-1 through its own oligomerization. Activated caspase-1 can further cleave pro-IL-18 and pro-IL-1β into active IL-18 and IL-1β, which in turn promote inflammatory responses and apoptosis (46). It has been reported that the NLRP3 inflammasome can be activated by excessive ROS production (47). Our previous study showed that Rg1 protected against chronic dexamethasone-induced neuronal degeneration by inhibiting NLRP-1 inflammasomes in mice (19). It has also been reported that Rg1 can protect against H2O2-induced neuronal apoptosis (33). These studies suggest that Rg1 has an anti-oxidative effect and can inhibit the NLRP1 inflammasome in hippocampal neurons. However, whether Rg1 can down-regulate the NOX2-mediated generation of ROS and thereby inhibit the NLRP1 inflammasome in H2O2-treated hippocampal neurons remained to be elucidated. The present study found that H2O2 treatment significantly increased the expression levels of NLRP-1, ASC and caspase-1 in hippocampal neurons and the levels of IL-1β and IL-18 released into the supernatant. These results suggest that the accumulation of ROS can activate the NLRP1 inflammasome, which may be involved in senescence and damage in hippocampal neurons. The results also showed that tempol and Rg1 treatments significantly decreased the expression levels of NLRP1, ASC and caspase-1, and reduced the levels of IL-1β and IL-18 released into the supernatant. A previous study showed that Rg1 attenuated H2O2-induced neuronal oxidative stress and apoptosis via the downregulation of caspase-3, Rho-associated kinase1 activation and myosin light chain (Ser-19) phosphorylation (33), whereas the present study suggested that Rg1 alleviated H2O2-induced neuronal oxidative stress and apoptosis via the inhibition of NOX2-derived ROS generation and downregulation of the NLRP1 inflammasome.

Overall, the present study demonstrated that Rg1 has antioxidant effects and can reduce the expression of NOX2 and the production of ROS, which in turn inhibits the activation of the NLRP1 inflammasome in H2O2-treated hippocampal neurons. These findings provide support for the hypothesis that Rg1 reduces NOX2-mediated ROS generation and inhibits NLRP1 inflammasome activation. However, whether the overexpression of NOX2 can activate the NLRP1 inflammasome and accelerate neuronal senescence remains to be fully elucidated. Other associated mechanisms of Rg1 on neuronal senescence associated with the NOX2-NLRP1 inflammasome signalling pathway warrant further investigation.

 Funding

This study was supported by the National Natural Science Foundation of China (grant nos. 81671384 and 81371329) and the Natural Science Foundation of Anhui Province Education Department (grant no. KJ2016A357).

 Availability of data and materials

The datasets used and analysed during the present study are available from the corresponding author on reasonable request.

 Authors’ contributions

TZX analysed data, performed the experiments, and was a major contributor in writing the manuscript. XYS, LLS and YLC collated the data, BQZ contributed to the immunoblot analysis and interpretation of the results. DKH was mainly responsible for the immunofluorescence detection. WZL designed the study, critically revised the manuscript for intellectually important content, supervised the study and wrote the manuscript. All authors read and approved the final submitted manuscript.

 Ethics approval and consent to participate

All experiments involving animals were approved by the Ethics Committee of Laboratory Animals of Anhui Medical University.

 Patient consent for publication

Not applicable.

 Competing interests

The authors declare that they have no competing interests.

 Acknowledgments

The authors would like to thank Mr. Bao Li (Synthetic Laboratory of Basic Medicine College, Anhui Medical University) for their technical assistance.

 References FloydRAHensleyKOxidative stress in brain aging. Implications for therapeutics of neurodegenerative diseasesNeurobiol Aging23795807200210.1016/S0197-4580(02)00019-212392783 JosephJAShukitt-HaleBCasadesusGFisherDOxidative stress and inflammation in brain aging: Nutritional considerationsNeurochem Res30927935200510.1007/s11064-005-6967-416187227 CalabreseVCorneliusCMancusoCLentileRStellaAMButterfieldDARedox homeostasis and cellular stress response in aging and neurodegenerationMethods Mol Biol610285308201010.1007/978-1-60327-029-8_17 FanLMCahill-SmithSGengLDuJBrooksGLiJMAging-associated metabolic disorder induces Nox2 activation and oxidative damage of endothelial functionFree Radic Biol Med108940951201710.1016/j.freeradbiomed.2017.05.008284999115489050 AnsariMAScheffSWNADPH-oxidase activation and cognition in Alzheimer disease progressionFree Radic Biol Med51171178201110.1016/j.freeradbiomed.2011.03.025214577773109185 RehmanSUShahSAAliTChungJIKimMOAnthocyanins reversed d-galactose-induced oxidative stress and neuroinflammation mediated cognitive impairment in adult ratsMol Neurobiol54255271201710.1007/s12035-015-9604-5 ZotovaEBharambeVCheaveauMMorganWHolmesCHarrisSNealJWLoveSNicollJABocheDInflammatory components in human Alzheimer’s disease and after active amyloid-β42 immunizationBrain13626772696201310.1093/brain/awt21023943781 KaushalVDyeRPakavathkumarPFoveauBFloresJHymanBGhettiBKollerBHLeBlancACNeuronal NLRP1 inflammasome activation of Caspase-1 coordinately regulates inflammatory interleukin-1-beta production and axonal degeneration-associated Caspase-6 activationCell Death Differ2216761686201510.1038/cdd.2015.16257440234563782 ZhangBZhangYXuTYinYHuangRWangYZhangJHuangDLiWChronic dexamethasone treatment results in hippocampal neurons injury due to activate NLRP1 inflam-masome in vitroInt Immunopharmacol49222230201710.1016/j.intimp.2017.05.03928605710 FaustinBLartigueLBrueyJMLucianoFSergienkoEBailly-MaitreBVolkmannNHaneinDRouillerIReedJCReconstituted NALP1 inflammasome reveals two-step mechanism of caspase-1 activationMol Cell25713724200710.1016/j.molcel.2007.01.03217349957 MawhinneyLJde Rivero VaccariJPDaleGAKeaneRWBramlettHMHeightened inflammasome activation is linked to age-related cognitive impairment in fischer 344 ratsBMC Neurosci12123201110.1186/1471-2202-12-123221332033259063 ChoiAJRyterSWInflammasomes: Molecular regulation and implications for metabolic and cognitive diseasesMol Cells37441448201410.14348/molcells.2014.0104248501494086337 MoonJSNakahiraKChungKPDeNicolaGMKooMJPabónMARooneyKTYoonJHRyterSWStout-DelgadoHChoiAMNOX4-dependent fatty acid oxidation promotes NLRP3 inflammasome activation in macrophagesNat Med2210021012201610.1038/nm.4153274555105204248 YangLZhangJZhengKShenHChenXLong-term ginsenoside Rg1 supplementation improves age-related cognitive decline by promoting synaptic plasticity associated protein expression in C57BL/6J miceJ Gerontol A Biol Sci Med Sci69282294201410.1093/gerona/glt091 SunXCRenXFChenLGaoXQXieJXChenWFGlucocorticoid receptor is involved in the neuroprotective effect of ginsenoside Rg1 against inflammation-induced dopaminergic neuronal degeneration in substantia nigraJ Steroid Biochem Mol Biol15594103201610.1016/j.jsbmb.2015.09.040 AtteleASWuJAYuanCSGinseng pharmacology: Multiple constituents and multiple actionsBiochem Pharmacol5816851693199910.1016/S0006-2952(99)00212-910571242 ZhuJMuXZengJXuCLiuJZhangMLiCChenJLiTWangYGinsenoside Rg1 prevents cognitive impairment and hippocampus senescence in a rat model of D-galactose-induced agingPLoS One9e101291201410.1371/journal.pone.0101291249797474076296 ZhuGWangYLiJWangJChronic treatment with ginsenoside Rg1 promotes memory and hippocampal long-term potentiation in middle-aged miceNeuroscience2928189201510.1016/j.neuroscience.2015.02.03125724866 WangYKanHYinYWuWHuWWangMLiWLiWProtective effects of ginsenoside Rg1 on chronic restraint stress induced learning and memory impairments in male micePharmacol Biochem Behav1207381201410.1016/j.pbb.2014.02.01224560910 ZhangYHuWZhangBYinYZhangJHuangDHuangRLiWLiWGinsenoside Rg1 protects against neuronal degeneration induced by chronic dexamethasone treatment by inhibiting NLRP-1 inflammasomes in miceInt J Mol Med4011341142201710.3892/ijmm.2017.3092288491715593467 ZhangBZhangYWuWXuTYinYZhangJHuangDLiWChronic glucocorticoid exposure activates BK-NLRP1 signal involving in hippocampal neuron damageJ Neuroinflammation14139201710.1186/s12974-017-0911-9287325025521122 CaoGXiaoMSunFXiaoXPeiWLiJGrahamSHSimonRPChenJCloning of a novel Apaf-1-interacting protein: A potent suppressor of apoptosis and ischemic neuronal cell deathJ Neurosci2461896201200410.1523/JNEUROSCI.1426-04.200415240811 HuWZhangYWuWYinYHuangDWangYLiWLiWChronic glucocorticoids exposure enhances neurodegeneration in the frontal cortex and hippocampus via NLRP-1 inflammasome activation in male miceBrain Behav Immun525870201610.1016/j.bbi.2015.09.019 KazamaKAnratherJZhouPGirouardHFrysKMilnerTAIadecolaCAngiotensin II impairs neurovascular coupling in neocortex through NADPH oxidase-derived radicalsCirc Res9510191026200410.1161/01.RES.0000148637.85595.c515499027 ChengXLZhangJJEffect of edaravone on apoptosis of hippocampus neuron in seizures rats kindled by pentylenetetrazoleEur Rev Med Pharmacol Sci18769774201424706298 DongWChengSHuangFFanWChenYShiHHeHMitochondrial dysfunction in long-term neuronal cultures mimics changes with agingMed Sci Monit17BR91BR96201110.12659/MSM.881706214551013539510 QuintanillaRAOrellanaJAvon BernhardiRUnderstanding risk factors for Alzheimer’s disease: Interplay of neuroinflammation, connexin-based communication and oxidative stressArch Med Res43632644201210.1016/j.arcmed.2012.10.01623142264 Cahill-SmithSLiJMOxidative stress, redox signalling and endothelial dysfunction in ageing-related neurodegenerative diseases: A role of NADPH oxidase 2Br J Clin Pharmacol78441453201410.1111/bcp.12357252794044243896 LeeKWWooJMImJYParkESHeLIchijoHJunnEMouradianMMApoptosis signal-regulating kinase 1 modulates the phenotype of α-synuclein transgenic miceNeurobiol Aging36519526201510.1016/j.neurobiolaging.2014.07.034 VerriMPastorisODossenaMAquilaniRGuerrieroFCuzzoniGVenturiniLRicevutiGBongiornoAIMitochondrial alterations, oxidative stress and neuroinflammation in Alzheimer’s diseaseInt J Immunopathol Pharmacol25345353201210.1177/03946320120250020422697066 ZhouXSuCFZhangZWangCYLuoJQZhouXWCaiLYanLZhangWLuoHMNeuroprotective effects of methyl 3,4-dihydroxybenzoate against H(2)O(2)-induced apoptosis in RGC-5 cellsJ Pharmacol Sci1255158201410.1254/jphs.13055FP OngWYFarooquiTKohHLFarooquiAALingEAProtective effects of ginseng on neurological disordersFront Aging Neurosci7129201510.3389/fnagi.2015.00129262362314503934 WangYLiuQXuYZhangYLvYTanYJiangNCaoGMaXWangJGinsenoside rg1 protects against oxidative stress-induced neuronal apoptosis through myosin IIA-actin related cytoskeletal reorganizationInt J Biol Sci1213411356201610.7150/ijbs.15992278770865118780 LiuQKouJPYuBYGinsenoside Rg1 protects against hydrogen peroxide-induced cell death in PC12 cells via inhibiting NF-kB activationNeurochem Int58119125201110.1016/j.neuint.2010.11.004 AksuUYanarKTerziogluDErkolTEceEAydinSUsluEÇakatayUEffect of tempol on redox homeostasis and stress tolerance in mimetically aged drosophilaArch Insect Biochem Physiol871325201410.1002/arch.2117625044374 DuganLLAliSSShekhtmanGRobertsAJLuceroJQuickKLBehrensMMIL-6 mediated degeneration of forebrain GABAergic interneurons and cognitive impairment in aged mice through activation of neuronal NADPH oxidasePLoS One4e5518200910.1371/journal.pone.0005518194367572678193 TammarielloSPQuinnMTEstusSNADPH oxidase contributes directly to oxidative stress and apoptosis in nerve growth factor-deprived sympathetic neuronsJ Neurosci20RC53200010.1523/JNEUROSCI.20-01-j0006.200010627630 DeLeoFRQuinnMTAssembly of the phagocyte NADPH oxidase: Molecular interaction of oxidase proteinsJ Leukoc Biol60677691199610.1002/jlb.60.6.6778975869 AliSSYoungJWWallaceCKGresackJJesteDVGeyerMADuganLLRisbroughVBInitial evidence linking synaptic superoxide production with poor short-term memory in aged miceBrain Res13686570201110.1016/j.brainres.2010.11.0093018762 WalderCEGreenSPDarbonneWCMathiasJRaeJDinauerMCCurnutteJTThomasGRIschemic stroke injury is reduced in mice lacking a functional NADPH oxidaseStroke2822522258199710.1161/01.STR.28.11.22529368573 XuYZNygardMKristenssonKBentivoglioMRegulation of cytokine signaling and T-cell recruitment in the aging mouse brain in response to central inflammatory challengeBrain Behav Immun24138152201010.1016/j.bbi.2009.09.006 GlassCKSaijoKWinnerBMarchettoMCGageFHMechanisms underlying inflammation in neurodegenerationCell140918934201010.1016/j.cell.2010.02.016203038802873093 ZouPLiuXLiGWangYResveratrol pretreatment attenuates traumatic brain injury in rats by suppressing NLRP3 inflammasome activation via SIRT1Mol Med Rep17321232172018 YinYYanYJiangXMaiJChenNCWangHYangXFInflammasomes are differentially expressed in cardiovascular and other tissuesInt J Immunopathol Pharmacol22311322200910.1177/039463200902200208195053852847797 TanCCZhangJGTanMSChenHMengDWJiangTMengXFLiYSunZLiMMNLRP1 inflammasome is activated in patients with medial temporal lobe epilepsy and contributes to neuronal pyroptosis in amygdala kindling-induced rat modelJ Neuroinflammation1218201510.1186/s12974-014-0233-0256263614314732 FannDYLeeSYManzaneroSTangSCGelderblomMChunduriPBernreutherCGlatzelMChengYLThundyilJIntravenous immunoglobulin suppresses NLRP1 and NLRP3 inflammasome-mediated neuronal death in ischemic strokeCell Death Dis4e790201310.1038/cddis.2013.326240087343789184 AminzadehMRoghaniMSarfallahARiaziGHTRPM2 dependence of ROS-induced NLRP3 activation in Alzheimer’s diseaseInt Immunopharmacol547885201810.1016/j.intimp.2017.10.024

Effect of Rg1 on neuronal apoptosis in hippocampal neurons induced by H2O2 exposure (Hoechst 33258 staining, magnification, ×400). (A) Representative images of neuronal apoptosis in hippocampal neurons. The apoptotic cells appeared smaller than normal and were more deeply stained (yellow arrow). (B) Quantitative analysis of the relative apoptotic rate of hippocampal neurons over the control. (C) The level of LDH released in the supernatant. Results are expressed as the mean ± standard deviation, n=3, *P<0.05 and **P<0.01 compared with the control group; #P<0.05 and ##P<0.01 compared with the model group. Rg1, ginsenoside Rg1.

Effect of Rg1 on the expression of caspase-3 in H2O2-treated hippocampal neurons (immunofluorescence, magnification, ×400). (A) Representative images of the expression of caspase-3 in hippocampal neurons. (B) Quantitative analysis of the relative expression of caspase-3 over the control. Results are expressed as the mean ± standard deviation, n=3, *P<0.05 compared with the control group; ##P<0.01 compared with the model group. Rg1, ginsenoside Rg1.

Effect of Rg1 treatment on the expression of senescence-associated β-Gal in H2O2-treated hippocampal neurons (immunoblot). (A) Representative blots of β-Gal in hippocampal neurons. (B) Analysis of the relative expression of β-Gal over the control. Results are expressed as the mean ± standard deviation, n=3, **P<0.01 compared with the control group; #P<0.05 and ##P<0.01 compared with the model group. Rg1, ginsenoside Rg1; β-Gal, β-galactosidase.

Effect of Rg1 treatment on ROS production in H2O2-treated hippocampal neurons (dihydroethidium staining). (A) ROS production was detected by fluorescence microscopy in hippocampal neurons (magnification, ×400). Left-panel images are bright-field microscopy, representing cell morphology. Right-panel images are fluorescent microscopy, indicating the formation of ROS. (B) Relative production of ROS over the control group. Results are expressed as the mean ± standard deviation, n=3. **P<0.01 compared with the control group; #P<0.05 compared with the H2O2-treated model group. Rg1, ginsenoside Rg1; ROS, reactive oxygen species.

Effect of Rg1 treatment on the expression of NOX2, p22phox, and p47phox in H2O2-treated hippocampal neurons (immunoblot). (A) Relative expression of NOX2 over the control. (B) Relative expression of p22phox over the control. (C) Relative expression of p47phox over the control. Results are expressed as the mean ± standard deviation, n=3, *P<0.05 compared with the control group; #P<0.05 and ##P<0.01 compared with the model group. Rg1, ginsenoside Rg1; NOX2, NADPH oxidase 2.

Effect of Rg1 treatment on the expression of NLRP-1, ASC and caspase-1 in H2O2-treated hippocampal neurons (immunoblot). (A) Relative expression of NLRP-1 over the control. (B) Relative expression of ASC over the control. (C) Relative expression of caspase-1 over the control. Results are expressed as the mean ± standard deviation, n=3, *P<0.05 and **P<0.01 compared with the control group; #P<0.05 and ##P<0.01 compared with the model group. Rg1, ginsenoside Rg1; NLRP-1, nucleotide-binding oligomerisation domain-like receptor protein 1; ASC, apoptosis-associated speck-like protein containing a carboxy-terminal CARD.

Effect of Rg1 on the levels of IL-1β and IL-18 released into supernatants in H2O2-treated hippocampal neurons (enzyme-linked immunosorbent assay). (A) Release of IL-1β into supernatants. (B) Release of IL-18 into supernatants. Results are expressed as the mean ± standard deviation, n=3,*P<0.05 compared with the control group; #P<0.05 compared with the model group. Rg1, ginsenoside Rg1; IL, interleukin.