Abcam (Cambridge, UK) provided antibodies for A$\beta$-1 (cat. no. ab201060), BCL-2 (cat. no. ab182858), Bax (cat. no. ab32503), HO1 (cat. no. ab1346) and Nrf2 (cat. no. ab62352), while cell signaling tech., (Danvers, MA, USA) supplied antibodies for p-tau (cat. no. 12885), Akt (cat. no. 9272), P-Akt (cat. no. 4060), GSK3$\beta$ (cat. no. 12456), P-GSK3$\beta$ (cat. no. 5558), LaminB1 (cat. no. 17416), caspase-3 (cat. no. 9662S), cleave caspase-3 (cat. no. 9664S), GAPDH (cat. no. 5174), and goat anti-rabbit (cat. no. 14708) and mouse (cat. no. 14709) IgG (H+L) HRP. Affinity (Melbourne, FL, USA) provided ECL reagent (cat. no. KF8003), while Sigma-Aldrich (St. Louis, MO, USA) supplied Dulbecco’s modified-Eagle medium (DMEM) and fetal bovine serum (FBS). Also, we obtained trypsin, multicolor protein marker, sodium-dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) kit and 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide (MTT) dye from Solarbio (Beijing, China). Millipore (Millipore, Bedford, MA, USA) supplied polyvinylidene fluoride membranes (PVDF). The BCA protein concentration assay (Enhanced) (cat. no. P0010) and One-step TUNEL cell apoptosis detection kit (cat. no. C1089) were provided by Beyotime (Shanghai, China). Malondialdehyde (MDA) (cat. no. ml094962), Superoxide dismutase (SOD) (cat. no. ml092620), and reduced glutathione (GSH) (cat. no. ml092952) content assay kits were supplied by Elisa (Shanghai, China). Macklin Biochemical Co., Ltd. (Shanghai, China) provided ECR (purity 98%) (cat. no. H811108) and Donepezil (cat. no. D849374).

Changzhou Cavins Laboratory Animal Co., Ltd. (Changzhou, China) provided the male mice, namely senescence accelerated-resistant mouse (SAMR1) and Senescence-accelerated mouse prone 8 (SAMP8), which were without any specific pathogen, 4 months old and weight 30–35 g. All the animals were fed in cages in the same quiet environment with a light (12 L)/dark (12 D) cycle, respectively fed and drank sterilized feed and water. The experiment was carried out after 7 days of adaptation. The Jiangsu University (UJS IACUC) institutional committee for the care and use of laboratory animals reviewed and approved (approval number: UJS-IACUC-2022031401) for studies involving animals.

One week later, 48 qualified animals were selected, and 40 SAMP8 mice except for 8 SAMR1 mice in the control group were randomly divided into 5 groups. All the animals were divided into six groups: SAMR1 blank group (Control), SAMP8 Model group (Model), SAMP8 model + ecdysterone Low dose group (Low), SAMP8 model + ecdysterone Medium dose group (Medium), SAMP8 model + ecdysterone High dose group (High), SAMP8 model + Donepezil group (Positive). Mice in the administration group were given ECR intragastric administration (high, medium and low doses were 5, 10, and 20 mg/kg/day, respectively) [24]. The control group and model group were intragastrically given 0.9% sodium chloride solution of the same volume, and the positive group was intragastrically given Donepezil (1 mg/kg/day). The drug treatment group and the control group were fed the same way in different cages. And continued administration for 4 weeks. Animals were sacrificed after the treatment, and tissues were collected and kept in a –80 °C freezer until further analysis.

One of the most popular cell lines for neuroscience research is PC12, which is employed in investigations on synaptogenesis, neurotoxicity, neuroprotection, neurosecretion, and neuroinflammation [25]. STR profiling was used to confirm the PC12 Cell Line, and mycoplasma testing came out negative. Every cell was cultivated at 37 °C and 5% CO${}_2$ in a humidified incubator. In order to cultivate PC12 cells, DMEM with 10% FBS, 100 U/mL penicillin, and 0.1 mg/mL streptomycin (C0222, Beyotime) was used. Later on, we categorized the cells into five groups, viz., control, model, low-dose ECR (25 µM), medium-dose ECR (50 µM) and high-dose ECR (100 µM). Only normal media of equal volume was used to cultivate the cells in the control category. The model group was treated with A$\beta$25-35 in oligomer form at 25 µM (A4559, Sigma) for 24 h to establish an in vitro AD model. Likewise, we employed A$\beta$25-35 (25 µM) to treat the cells in low, medium and high dose groups in normal medium for 24 h, before replacement with medium containing ECR (25, 50 and 100 µM) for another 24 h.

Paraffin sections of mouse hippocampal tissue were dewaxed and immersed in water, dyed with 1% toluidine blue for 10 min, and rinsed with distilled water. The colour is then separated in 70% alcohol for seconds to minutes. Then in anhydrous ethanol dehydration, xylene is transparent. Finally, seal it with a neutral glue and observe the cornu ammonis 1 (CA1) and CA3 regions of the hippocampus under a microscope.

After administration, hippocampal tissue was taken from mice and placed in 4% (w/v) paraformaldehyde (PFA) solution overnight. Before cutting the tissue into slices (4 µm), we embedded them in paraffin. Dewaxing of paraffin sections to water was carried out, before antigen repairing, blocking and sealing as well as overnight incubation at 4 °C with primary antibodies of A$\beta$ and P-Tau and afterwards with secondary antibodies. Later, we visualized the tissue with 3, 3${}^{\prime }$-diamino-benzidine tetra-hydrochloride before counterstaining with hematoxylin, dehydration, mounting and imaging using a high-power microscope. Image J software (V1.8.0; LOCI, University of Wisconsin, Madison, WI, USA) was used for quantitative analysis of staining results.

Measurement of ROS levels in tissues: At the end of the last administration, the hippocampus tissues of mice were extracted and cut into pieces, and then completely soaked in 2 mL digestive fluid (DMEM containing 1 mg/mL collagenase Ⅳ and 1 mg/mL DNA enzyme Ⅰ). The above mixture was then placed in a 37 °C water bath for digestion. Ice was added after 45 min to stop digestion. The digested brain tissue solution was run through a 40 µm cell filter in order to exclude cell masses and tissue masses that had not been adequately digested. To obtain a single-cell suspension, the filtrate was centrifuged at 1000 rpm for 5 min while the cell precipitation was resuspended in PBS. Wash with PBS twice, centrifuge at 1000 r/min for 5 min, remove the supernatant, add an appropriate volume of diluted DCFH-DA working liquid, and incubate at 37 °C for 30 min in the dark. Following incubation, the cells underwent two PBS washes in order to eliminate any remaining DCFH-DA from the cells. In 500 µL of PBS, the cells were suspended. The ROS positive rate was found using flow cytometry.

Measurement of ROS levels in cells: PC12 cells were placed in a 6-well plate, 3.5 $\times$ 10${}^6$ cells/well, and adherent cultured for 24 h. 24 h after the preparation of the model in vitro, the medium containing different concentrations of ecdysterone was replaced. Following a 24-hour period, the original supernatant was disposed of, the cells in the 6-well plate underwent two PBS washes, the diluted DCFH-DA probe was applied, and the mixture was incubated for 15–30 min in a dark environment. After incubation, the supernatant was discarded and washed twice with PBS to remove the DCFH-DA probe that did not enter the cell. Add 1 mL of 0.02% pancreatic enzyme without EDTA, and when the cells become round, add PBS to terminate digestion, and gently blow the cells with a pipette to suspend the cells. Collect in EP tube, centrifuge at 3500 rpm for 5 min, and discard supernatant. After adding 1 mL of pre-cooled PBS at 4 °C to the fully suspended cells, they were centrifuged for 5 min at 3500 rpm, and the supernatant was disposed of. In 500 µL of PBS, the cells were suspended. Using flow cytometry, the ROS positivity rate was discovered.

The isolated hippocampal tissue was homogenized in cold phosphate buffer (pH 7.4) and centrifuged at 4 °C at 10,000 rpm for 15 min. The centrifuged supernatant (serum, cell culture supernatant) was collected and 100 µL supernatant was added to the plate. The levels of MDA, SOD, and GSH in supernatant, serum, and cell culture supernatant were measured using specific kits according to the manufacturer’s instructions.

Extraction of total protein from hippocampus and determination of protein content were carried out respectively with RIPA lysis and BCA protein assay kit. Later on, we performed electrophoresis of protein (40 µg) for 1 h on SDS-PAGE (10%) to PVDF membrane with 120 V of transmembrane step. After being blocked with 5% BSA, membranes were incubated with the primary antibodies (BCL-2, Bax, HO-1, P-Akt, Akt, GSK3$\beta$, P-GSK3$\beta$, LaminB1, Cleave-caspase 3, caspase 3 and Nrf2) for a whole night at 4 °C. Before using goat anti-mouse antibody or goat anti-rabbit antibody that had been HRP-labeled for 1 h at room temperature, the membrane was subjected to three Tris Buffered Saline with Tween (TBST) washes. After three washes of the membrane with TBST, we quantified peroxidase-labelled protein bands with an ECL kit before assessment of protein intensity with Image J.

Tissue or cell samples that have been fixed in PFA (4%) were subjected to drying, paraffin embedding, slicing, dewaxing, hydrating, antigen extraction, blocking, and all-night incubation at 4 °C with primary antibody. Afterwards, sections were subjected to three PBS washes before the FITC conjugate secondary antibody was used for their incubation for 1 h at 37 °C. Later on, an anti-fade mounting medium with 4, 6-diamidino-2-phenyl-indole (DAPI) was used to mount the slices after washing. Ultimately, we evaluated the sections with a fluorescent microscope.

Following the manufacturer’s instructions, TUNEL labeling was used to identify neuronal cell death in the hippocampal regions of mice. Under a fluorescent microscope, the sections were taken in pictures. To count the cells that were TUNEL positive, Image J was employed. Data was expressed as a ratio of the number of TUNEL positive cells to the square millimeter.

Assessment of the effects of various dosage forms on cell viability was accomplished with the MTT assay. Growing of PC12 cells was carried out in 96-well plates for 24 h with 8000 cells/well. Twenty-four (24) h after an in vitro cell model preparation, we exposed the cells to various amounts of drug-containing serum. Following a 24 h treatment period, we applied MTT (5 mg/mL, 20 µL) to each well, before the removal of supernatant after 4 h. Later on, we added DMSO (150 µL) to each well, while complete dissolution of dirty crystals was accomplished with 10 min of oscillations at low-speed. At a wavelength of 570 nm, we used an enzymatic-labeled meter to determine the absorbance OD values.

According to the manufacturer’s instructions, we detected cell apoptosis with Annexin V-FITC/propyl iodide (PI) apoptotic kit. To put it simply, we inoculated PC12 cells into 6-well plates with 3.5 $\times$ 10${}^6$ cells/well density, before culturing for 24 h. After 24 h of preparation, the cultured medium containing different concentrations of ECR was changed and treated for 24 h. Afterwards, we used a binding buffer to digest, collect, centrifuge and resuspend the cells. At ambient temperature without any light exposure, we incubated the mixture after the addition of Annexin V-FITC (5 µL) and PI (5 µL). Later on, we examined the apoptotic cells using flow cytometry and the program FlowJo (V7.1.0; TreeStar, Ashland, OR, USA).

To do the statistical analysis, GraphPad Prism 8.0 (GraphPad Software, Inc., San Diego, CA, USA) was used. We expressed the data with mean $\pm$ SD. One-way or two-way analyses of variance (ANOVA) were used to assess all of the data. Statistically, the accepted significant level was p 0.05, while acceptance at p 0.01 or p 0.001 was regarded as being very significant.

The aging process of SAMP8 mice is accompanied by complex physiological changes related to cognitive dysfunction, such as brain A$\beta$ deposition, increased oxidative stress, Tau hyperphosphorylation and neuroinflammation, which is currently recognized as a natural senescence dementia model [26]. In this regard, we assessed the potential of ECR to improve cognitive impairment in vivo using SAMP8 and SAMR1 as the respective mice models and controls. Usually, MWM which includes experiments such as navigation and exploration of space exploration by laboratory animals, is a well-known approach for testing spatial learning and memory in trials of these animals [27]. We first employed MWM to assess the learning and memory capacity of mice to determine the impact of ECR on cognitive deficits in SAMP8 mice. According to Fig. 1A, the escape latency of the model group was much higher during the first 5 days compared to that of the control group, and the learning impairment of SAMP8 mice was improved after ECR or positive drug administration, and ECR was dose-dependent. On the last day of the space exploration experiment, mice in the ECR and positive groups increased the number of crossings in the platform region (Fig. 1B) and the time spent in the target quadrant region (Fig. 1C) compared to the model group. Additionally, we tested SAMR1 mice using MWM but found no significant effect on their behavior due to ECR treatment (Supplementary Fig. 1). Nissl staining was performed to evaluate histopathological abnormalities in the hippocampus, which are frequently linked to the course of the disease. As shown in Fig. 2A–C, SMAP8 mice displayed considerably fewer intact neurons in hippocampal regions (CA1 and CA3) compared with SAMR1 mice. Nonetheless, we observed a reversal of the above alteration after ECR and positive drug treatment, wherein amid ECR effect was in dose dependent fashion. In addition, immunohistochemical staining showed that A$\beta$ and P-tau expressions in the hippocampus of model mice were increased markedly compared to control mice, but we observed a reversal of this increase after ECR and positive drug treatments (Fig. 2D–F). These data indicate that ECR effectively alleviated memory loss and learning disabilities in SAMP8 mice.

Fig. 1.

ECR improves cognitively deficient behavior in SAMP8 mice. Effects of ECR treatment on (A) escape latency, (B) number of times crossed the target platform position and (C) time spent in the target quadrant in Morris water maze (MWM). (A) Data were analyzed using two-way ANOVA and a Bonferroni test or (B,C) using a one-way ANOVA and Tukey’s post hoc test and presented as the mean $\pm$ standard deviation (SD), n = 8 in each group. The groups were compared as follows; ${}^{*}$p 0.05 and ${}^{**}$p 0.01 compared to model mice; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared to control mice. ECR, ecdysterone; SAMP8, senescence-accelerated mouse prone 8; ANOVA, analyses of variance.

Fig. 2.

ECR improves cognitive deficits in SAMP8 mice. (A) Illustration of images of Nissl staining of hippocampal regions (CA1 and CA3). Surviving cells per 1 mm of (B) CA1 and (C) CA3 were analyzed quantitatively. In each group, n = 3, while the scale bar = 100 µm. (D) By using immunohistochemical staining, A$\beta$ and P-Tau expressions in the hippocampus were discovered. (E,F) Quantitative analysis of immunohistochemical staining. In each group, n = 3, while the scale bar = 100 µm. The data were presented as the mean $\pm$ SD after being subjected to one-way ANOVA and Tukey’s post hoc test analysis. The groups were compared as follows; ${}^{*}$p 0.05 and ${}^{**}$p 0.01 compared to model mice; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared to control mice.

Scientists have shown that oxidative stress has a significant influence on the development of AD [7]. Based on this assertion, we evaluated oxidative stress-linked biomarkers to ascertain the involvement of this process in the ability of ECR to ameliorate cognitive deficits in SAMP8 mice. As shown in Fig. 3A,B, ROS levels considerably increased in the hippocampus of SAMP8 mice compared to the control group but decreased significantly after treatment with ECR or Donepezil, and ECR showed a dose-dependent manner. Important indexes that reflect oxidative system imbalance are GSH, MDA and SOD. As can be seen from Fig. 3C–E, SOD (Fig. 3D) and GSH (Fig. 3E) activities in the hippocampus of the model group were significantly decreased, while MDA (Fig. 3C) content was increased. These changes were significantly reversed by ECR or Donepezil, with a stronger reversal effect observed at higher doses of ECR. Similarly, the detection of MDA, SOD and GSH levels in the serum of mice was consistent with that in the hippocampus (Fig. 3F–H). Thus, inhibition of oxidative stress by ECR may facilitate its ameliorating effect on cognitive deficits in SAMP8.

Fig. 3.

Antioxidative effect of ECR against oxidative stress in SAMP8 mouse model. (A,B) Determination of levels of ROS in the hippocampus with flow cytometric technique after administration of different dosage forms (n = 3). (C) MDA, (D) SOD, and (E) GSH in the hippocampus were detected by biochemical kits (n = 6). (F) MDA, (G) SOD, and (H) GSH in serum were detected by biochemical kits (n = 6). The data were presented as the mean $\pm$ SD after being subjected to one-way ANOVA and Tukey’s post hoc test analysis. The groups were compared as follows; ${}^{*}$p 0.05 and ${}^{**}$p 0.01 compared to model mice; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared to control mice. ROS, reactive oxygen species; MDA, Malondialdehyde; SOD, superoxide dismutase; GSH, glutathione; DCF, 2${}^{\prime }$,7${}^{\prime }$-dichlorofluorescein.

Much evidence suggests that peroxidation of lipids and proteins is the consequence of oxidative system imbalance, which ultimately culminates in apoptosis in cells [28]. To investigate whether inhibition of oxidative stress by ECR may further result in reduced cell apoptosis, we evaluated cell apoptosis and expression levels of apoptotic-linked protein in the hippocampus of the mice. To achieve this, we employed TUNEL staining to observe the ECR effect on the apoptosis of neurons in the hippocampus. In terms of results, we discovered a substantially increased number of apoptostic-positive cells in model mice compared to control, with the cells displaying distinctive morphological features of cellular apoptosis. Meanwhile, we observed a significantly decreased number of TUNEL positive cells in the hippocampus of mice that received ECR compared to model mice (Fig. 4A,B). Following that, we ascertained the alterations in proteins that have been linked to apoptosis with the western blotting technique. Regarding the findings, we saw that model mice expressed less Bcl-2 than control mice did, while the former group expressed more Bax and cleaved caspase-3 (Fig. 4D,E) . Nevertheless, ECR treatment could significantly reverse these changes, wherein the reversal was more obvious with an increase in ECR dose (Fig. 4C,F). These data suggest that, in the hippocampus of SAMP8 mice, ECR has a protective impact against cell apoptosis, and that this protective effect increased with increasing ECR concentration within a specific range.

Fig. 4.

ECR inhibits neuron apoptosis in SAMP8 mice. (A) Representative images of TUNEL staining in each group. (B) Apoptotic cells were quantitatively analyzed. In each group, n = 3, while the scale bar = 50 µm. (C) Detection of apoptosis-linked proteins, namely Bax, Bcl-2, cleave caspase-3, and caspase-3 in the hippocampus with western blotting technique. (D,E) Normalization of quantified levels of protein to GAPDH (n = 3). (F) Normalization of the quantified level of protein to caspase-3 (n = 3). The data were presented as the mean $\pm$ SD after being subjected to one-way ANOVA and Tukey’s post hoc test analysis. The groups were compared as follows; ${}^{*}$p 0.05 and ${}^{**}$p 0.01 compared to model mice; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared to control mice. TUNEL, terminal deoxynucleotidyl transferase dUTP nick end labeling.

Immunofluorescent staining and western blotting techniques were utilized to detect the expression of proteins linked to the Akt/GSK3$\beta$ signaling pathway and Nrf2 to confirm that the potentiality of ECR to improve cognitive deficits in SAMP8 involves inhibition of oxidative stress and neuronal apoptosis. The immunofluorescence results (Fig. 5A–C) imply that Akt and P-GSK3$\beta$ expression levels in model mice reduced considerably compared to control mice, but ECR treatment could partially reverse this effect. Additionally, there was no discernible change in the level of Nrf2 expression between the model group and the control group, however, ECR therapy may dramatically raise the Nrf2 expression level. As shown in Fig. 5D–H, western blot analysis revealed that ECR treatment could not only significantly increase P-Akt and P-GSK3$\beta$ expression levels in the hippocampus of SAMP8 mice, but also markedly increased the hippocampal expression levels of Nucleus-Nrf2 and the Nrf2-related protein HO1. However, compared with the model group, ECR treatment significantly reduced the expression level of Cytosolic-Nrf2 (Fig. 5I). These findings suggest that ECR may improve cognitive impairments in SAMP8 mice via activating Akt/GSK3$\beta$to regulate Nrf2 and prevent oxidative damage.

Fig. 5.

ECR improves cognitive deficits in SAMP8 mice by activating Akt/GSK3$\beta$ to regulate Nrf2. Detection of (A) Akt, (B) Nrf2 and (C) P-GSK3$\beta$ expressions in the hippocampus with immunofluorescence staining. In each group, n = 3, while scale bar = 50 µm. (D) Analysis of Akt, P-Akt, P-GSK3$\beta$, GSK3$\beta$, nuclear-Nrf2, Cytosolic-Nrf2 and HO1 in the hippocampus with western blotting. (E) Normalization of P-Akt protein level to Akt (n = 3). (F) Normalization of P-GSK3$\beta$ protein level to GSK3$\beta$ (n = 3). (G) Normalize HO1 protein level to GAPDH (n = 3). (H) Normalize Cytosolic-Nrf2 protein level to GAPDH (n = 3). (I) Normalize Nuclear-Nrf2 protein level to LaminB (n = 3). The data were presented as the mean $\pm$ SD after being subjected to one-way ANOVA and Tukey’s post hoc test analysis. The groups were compared as follows; ${}^{*}$p 0.05 and ${}^{**}$p 0.01 compared to model mice; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared to control mice. Akt, protein kinase B; Nrf2, nuclear factor erythroid-2-related factor-2; GSK3$\beta$, glycogen synthase kinase 3$\beta$; HO-1, heme oxygenase-1.

After demonstrating the potential of ECR to alleviate cognitive impairment via prevention of oxidative stress and death of neurons in vivo, we conducted several in vitro experiments to investigate the probable mechanistic action of ECR in cognitive impairment improvement. First, we screened the cell use concentrations of A$\beta$25-35 and ECR by MTT assay (Supplementary Fig. 2). We cultured PC12 cells and established an AD model treated with 25 µM A$\beta$25-35. Then we observed the effect of ECR on cell vitality by MTT, and the results showed that compared with the control group, the cell vitality decreased significantly after A$\beta$25-35 stimulation, but the cell vitality was reversed after ECR administration (Fig. 6C). Excessive ROS mediated antioxidant stress system imbalance is thought to be related to the AD process. Therefore, we investigated the ECR effect on oxidative stress in the A$\beta$25-35-induced AD model in PC12 cells with flow cytometric technique. Findings (Fig. 6A,B) of the above experiment showed that levels of ROS in model mice increased markedly compared to control, but their levels dose dependently decreased in mice that received ECR. Additionally, PC12 cells stimulated with A$\beta$25-35 showed alterations in SOD and GSH levels as well as an increase in MDA levels, with ECR reversing these modifications in the same manner as stated above (Fig. 6D–F). The expression levels of proteins associated with the Nrf2 antioxidant system and the levels of Cytosolic-Nrf2 and Nucleus-Nrf2 were detected using western blot to determine if the suppression of ECR on oxidative stress in A$\beta$25-35-induced PC12 cells in vitro is connected to the Nrf2 antioxidant system. Fig. 6G–M showed the results of western blot and cellular immunofluorescence analysis. We discovered that ECR activated the Nrf2 system, which substantially increased the expression of Nrf2 at the protein level in the nucleus.

In addition, we observed that PC12 cells treated with A$\beta$25-35 had an increased apoptotic rate and an increased expression of apoptosis-related proteins. In light of the flow cytometry result, the proportion of apoptotic cells in PC12 increased under the intervention of A$\beta$25-35 compared to control, while different doses of ECR significantly decreased apoptotic cells proportion in the above-mentioned cells (Fig. 6N,O). Furthermore, we evaluated the potential of ECR to prevent apoptosis in PC12 cells with western blot. Protein expression of Bcl-2 was found to be downregulated in PC12 cells stimulated with A$\beta$25-35, whereas the levels of caspase-3 and Bax were upregulated. ECR treatment ably upregulated Bcl-2 expression at protein level but downregulated Bax and caspase-3 protein expressions compared to PC12 cells in the model group (Fig. 6P–S).

Fig. 6.

Protective effect of ECR on oxidative stress of neurons in A$\beta$25-35-induced PC12 cells. (A) Determination of ROS levels in PC12 cells treatment with different dosage forms with flow cytometry. (B) Levels of ROS quantifications (n = 3). (C) Assessment of cell viability with MTT assay and treatment of A$\beta$25-35 induced PC12 cells with various doses of ECR (n = 6). Detection of biomarkers of oxidative stress (D) MDA, (E) SOD and (F) GSH in PC12 cells after treatment with different dosage forms (n = 6). (G) The expression levels of Nrf2 antioxidant system-related proteins HO1, NQO1, Cytosolic-Nrf2 and Nuclear-Nrf2 were analyzed by western blotting. (H) Normalize Nuclear- Nrf2 protein level to LaminB (n = 3). (I–K) Normalize HO1, NQO1 and Cytosolic-Nrf2 protein levels to GAPDH (n = 3). (L,M) Immunofluorescence staining analysis of Nrf2 expression in PC12 cells after treatment with different dosage forms. In each group, n = 3, while scale bar = 50 µm. (N,O) Determination of ECR effect on the percentage of apoptotic cells in PC12 cells induced by A$\beta$25-35 with flow cytometric technique (n = 3). (P) Western blotting analysis of Bax, Bcl-2, cleave caspase-3 and caspase-3 protein expressions in PC12 cells after different treatments. (Q,R) Normalization of levels of Bax and Bcl-2 protein to GAPDH (n = 3). (S) Normalization of the level of cleaved caspase-3 to caspase-3 (n = 3). The data were presented as the mean $\pm$ SD after being subjected to one-way ANOVA and Tukey’s post hoc test analysis. The groups were compared as follows; ${}^{*}$p 0.05 and ${}^{**}$p 0.01 compared to model mice; ${}^{\mathrm{\#}\mathrm{\#}}$p 0.01 compared to control mice. MTT, 3-(4,5-dimethyl-thiazol-2-yl)-2, 5-diphenyl-2H-tetrazolium bromide; PI, propidium iodide; NQO1, NADH dehydrogenase (quinone 1).

Collectively, ECR demonstrated a beneficial effect on oxidative stress and cell apoptosis accordingly induced in PC12 cells by A$\beta$25-35.

During this experiment, we employed Akt inhibitor MK2206 to further clarify the possibility that ECR protected PC12 against oxidative stress and apoptosis induced by A$\beta$25-35 via regulation of Nrf2 by the Akt/GSK3$\beta$ pathway. On the basis of the finding (Fig. 7A), we identified that the declined cell viability in A$\beta$25-35-induced cells was ameliorated by ECR, but inhibition of Akt caused this effect to disappear. Similarly, ECR treatment alleviated intracellular ROS levels in A$\beta$25-35-induced PC12 cells, but this phenomenon could also be eliminated by inhibiting Akt (Fig. 7B). Additionally, as depicted in the western blot results presented in Fig. 7C, ECR treatment increased the expression levels of P-GSK3$\beta$ and Nucleus-Nrf2, while decreasing the expression level of Cytosolic-Nrf2 in A$\beta$25-35-induced PC12 cells. However, these changes would disappear due to the inhibition of Akt. Collectively, these findings provide further evidence that ECR controls Nrf2 by activating the Akt/GSK3$\beta$ pathway to protect PC12 cells from damage induced by A$\beta$25-35.

Fig. 7.

ECR regulates Nrf2 by activating the Akt/GSK3$\beta$ pathway to protect cell damage of A$\beta$25-35-induced PC12 cells. (A) Determination of the effect of high concentration of ECR on cell viability of PC12 cells induced by A$\beta$25-35 before and after Akt inhibitor treatment with MTT assay (n = 6). (B) Effect of high concentration of ECR on ROS levels in PC12 cells induced by A$\beta$25-35 before and after Akt inhibitor treatment was analyzed by flow cytometry (n = 3). (C) Western blotting analysis of the effects of high ECR concentration on levels of Cytosolic-Nrf2, Nuclear-Nrf2, P-GSK3$\beta$ and GSK3$\beta$ expressions in PC12 cells before and after Akt inhibitor treatment. Normalization of the level of nuclear-Nrf2 protein to LaminB (n = 3). Normalization of the level of P-GSK3$\beta$ protein to GSK3$\beta$ (n = 3). Normalize Cytosolic-Nrf2 protein level to GAPDH (n = 3). The data were presented as the mean $\pm$ SD after being subjected to one-way ANOVA and Tukey’s post hoc test analysis. ${}^{**}$p 0.01, means significant difference.