Clematichinenoside AR improves isoflurane-stimulated cognitive dysfunction in aged mice by ameliorating neurotoxicity through ERK-CREB pathway

2.1 Establishment of isoflurane animal model

Male C57BL/6 mice, aged seven to eight months, were acquired from BIORAY LABORATORIES. The experiments were carried out in accordance with the Animal Care and Use Guidelines [13].

Thirty mice were divided into 5 groups: sham, isoflurane, isoflurane + AR (8 mg/kg, GC46698, GlpBio, Montclair, CA, USA), isoflurane + AR (16 mg/kg) and isoflurane + AR (32 mg/kg). Mice in the isoflurane-stimulated groups were subjected to a specified protocol [14]. Specifically, the mice were placed in an anesthesia tank and injected with isoflurane and oxygen, and the isoflurane concentration was maintained at 1.5% for 4 hours.

2.2 Drug administration

On the first day after isoflurane treatment, isoflurane + AR (8 mg/kg), isoflurane + AR (16 mg/kg) and isoflurane + AR (32 mg/kg) groups received AR administration.

2.3 Fear conditioning test

14 days after isoflurane anesthesia and drug administration, the animals underwent a fear conditioning test (FCT) based on established protocol [15]. Each mouse was placed in a dark room and subjected to tone-foot shock at 1-minute intervals. After two hours, the mice were transferred to a different, well-lit room, where they experienced three cycles of tone and shock accompanied by a smell cue.

2.4 Morris water maze test

The Morris water maze (MWM) test was conducted 2 days after the FCT was completed according to established protocol [16]. During each trial, each mouse was placed in the water facing the maze wall in a quadrant without a platform. A camera connected to a computer video system recorded the mouse’s swimming behavior. On the 6th day, the mice were subjected to a probe trial (120 s) without the platform. Memory performance was quantified by the percentage of time spent in the target quadrant, the number of target zone transitions, and freezing time in the fear conditioning context test.

2.5 TUNEL staining

The hippocampus sections were treated with TUNEL (ab66108, Abcam, Cambridge, UK) to elucidate the effects on cell apoptosis.

2.6 Western blot

The proteins extracted from brain tissues were separated using Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), transferred to Polyvinylidene Fluoride (PVDF) membranes (Millipore), and then blocked with 5% non-fat milk for 1 h. The proteins was identified by antibodies, including Caspase-3 (ab4051, 1:1200; Abcam, Cambridge, UK), Bax (ab53154, 1:3000; Abcam, Cambridge, UK), Bcl-2 (ab182858, 1:1200; Abcam, Cambridge, UK), Tumor Necrosis Factor-Alpha (TNF-α) (ab205587, 1:3000; Abcam, Cambridge, UK), Interleukin-6 (IL-6) (AF506, 1:2500; R&D Systems, Minneapolis, MN, USA), IL-1β (ab205924, 1:1200; Abcam, Cambridge, UK), p-NF-κB p65 (ab86299, 1:1500; Abcam, Cambridge, UK), NF-κB p65 (ab16502, 1:1500; Abcam, Cambridge, UK), Ionized Calcium Binding Adaptor Molecule 1 (Iba1) (ab153696, 1:2500; Abcam, Cambridge, UK), p-ERK (ab201015, 1:3000; Abcam, Cambridge, UK), ERK (ab17942, 1:1200; Abcam, Cambridge, UK), p-p90RSK (ab32413, 1:3000; Abcam, Cambridge, UK), p90RSK (ab32114, 1:1200; Abcam, Cambridge, UK), p-CREB (ab10564, 1:1200; Abcam, Cambridge, UK), CREB (ab178322, 1:1200; Abcam, Cambridge, UK) and β-actin (ab8227, 1:1200; Abcam, Cambridge, UK) overnight at 4 °C. Then, the membranes were incubated with Horseradish Peroxidase (HRP)-conjugated goat anti-rabbit Immunoglobulin G (IgG) secondary antibody (ab205718, 1:2000; Abcam, Cambridge, UK), and the bands on the membranes were visualized by the Enhanced Chemiluminescence (ECL) reagent (P0018, Beyotime, Beijing, China).

2.7 qPCR

Total RNA was extracted using Trizol reagent (15596026, Invitrogen, Carlsbad, CA, USA). The mRNA levels of TNF-α, IL-6, IL-1β, CD86 and CD206 was detected through Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR) by the SYBR Premix EX Taq (RR420A, Takara, Kusatsu, Japan) and analyzed by the 2^−ΔΔCT method. Primer sequences are shown in Table 1.

2.8 Histopathological analysis

Hippocampus tissues were fixed in 4% (v/v) Paraformaldehyde (PFA) and embedded in paraffin. Next, the tissues were cut into 3 μm sections and stained with hematoxylin and eosin solution (HHS32, Sigma-Aldrich, St. Louis, MO, USA). Lastly, they were observed under a microscope (BX53, Olympus, Tokyo, Japan).

2.9 Statistical analysis

The data were analyzed with GraphPad 5.0 software (GraphPad Software, San Diego, CA, USA). Error bars represent the means ± Standard Errors of the Mean (SEMs). Statistical significance between two groups was determined via an unpaired Student’s t-test. One-way Analysis of Variance (ANOVA) with Tukey’s post hoc test was applied for multiple comparisons. A p < 0.05 was considered statistically significant.

3.1 Clematichinenoside AR improves isoflurane-stimulated cognitive dysfunction in mice

The animal POCD model was established, and behavioral tests were conducted to evaluate the behavioral performance following isoflurane administration. The MWM test was first performed to evaluate POCD (Fig. 1A). The results indicated that mice treated with isoflurane required more time to locate the hidden platform compared to mice in the control group, suggesting that isoflurane impairs spatial learning in the aged mice (Fig. 1B). Furthermore, the mice treated with isoflurane spent less time in the target quadrant. They demonstrated a reduced frequency of transitions to the target zone (Fig. 1C,D). In contrast, treatment with AR improved the time taken to find the hidden platform and increased the time spent in the target quadrant, indicating that AR can mitigate the cognitive deficits caused by isoflurane in aged mice.

Subsequently, FCT was conducted to assess POCD. The FCT assay revealed that mice treated with isoflurane exhibited decreased freezing times compared to mice in the sham group, indicating that isoflurane impaired associative learning and memory in aged mice. However, AR treatment improved isoflurane-stimulated short freezing times in the mice, suggesting that AR effectively rescued the isoflurane-stimulated declines in learning and memory in aged mice (Fig. 1E). AR, therefore, can improve isoflurane-stimulated cognitive dysfunction in mice.

Fig. 1.Clematichinenoside AR can improve isoflurane-stimulated cognitive dysfunction in mice. (A) Representative swim paths in the MWM. (B) Escape latency in the MWM. (C) The percentage of time spent in the target quadrant in the probe trial of the MWM. (D) The number of target zone transitions in the probe trial of the MWM. (E) Freezing time in the fear conditioning context test. Data were presented as the mean ± SD with three independent experiments. *p < 0.05, ***p < 0.001, versus sham group, ^#p < 0.05, ^###p < 0.001, and versus isoflurane group. AR: Clematichinenoside AR; MWM: Morris Water Maze.

3.2 Clematichinenoside AR can inhibit isoflurane-stimulated apoptosis in mice hippocampus

Isoflurane treatment substantially increased the number of TUNEL-positive neurons, whereas AR treatment resulted in a notable decrease in TUNEL-positive neurons, demonstrating significant inhibitory effects (Fig. 2A). Additionally, isoflurane treatment markedly upregulated the expression levels of Bax and cleaved caspase-3, while downregulating Bcl-2. However, the expression of Bax and cleaved caspase-3 were dramatically reduced while Bcl-2 was enhanced by AR treatment in the isoflurane administration group (Fig. 2B). Therefore, AR can inhibit isoflurane stimulated apoptosis in mice hippocampus.

Fig. 2.Clematichinenoside AR can inhibit isoflurane-stimulated apoptosis in mice hippocampus. (A) TUNEL assay was conducted to determine cell apoptosis for each group. (B) Western blot was conducted to detect cell apoptosis-related proteins for each group. Relatively quantitative results were determined by Image J and shown as a histogram. Data were presented as the mean ± SD with three independent experiments. ***p < 0.001 versus sham group, ^###p < 0.001 versus isoflurane group. AR: Clematichinenoside AR; Bcl-2: B-cell lymphoma 2.

3.3 Clematichinenoside AR can reduce isoflurane-stimulated inflammatory response in mice

The qPCR and western blot analyses showed that the isoflurane group’s mRNA and protein expression levels of TNF-α, IL-6 and IL-1β were significantly increased. However, AR markedly reduced the levels of these inflammatory factors in the isoflurane administration group. Furthermore, the inflammatory factors stimulated by isoflurane administration showed a dose-dependent change (Fig. 3A,B). The phosphorylation levels of NF-κB p65 were also detected by western blot. The phosphorylation of NF-κB p65 was notably increased in the isoflurane administration group, but it was dramatically reduced after AR administration (Fig. 3C). Therefore, AR has a significant anti-inflammatory effect on the isoflurane-treated mice.

Fig. 3.Clematichinenoside AR can reduce isoflurane-stimulated inflammatory response in mice. (A) The mRNA expression levels of TNF-α, IL-6 and IL-1β in mice in each group. (B) The protein expression levels of TNF-α, IL-6 and IL-1β in mice in each group. (C) The protein expression levels of NF-κB p65 in mice in each group. Data were presented as the mean ± SD with three independent experiments. ***p < 0.001 versus sham group, ^#p < 0.05, and ^##p < 0.01, ^###p < 0.001 versus isoflurane group. AR: Clematichinenoside AR; TNF: Tumor Necrosis Factor; IL: Interleukin; NF-κB: Nuclear Factor-Kappa B.

3.4 Clematichinenoside AR can inhibit isoflurane-stimulated activation of microglia cells in mice hippocampus

H&E staining and western blot analysis were conducted on the hippocampus of mice in each group to investigate the neuroprotective effect of AR in POCD mice. The H&E staining results indicated that the nuclei of hippocampal neurons of mice in the isoflurane group were shrunken and damaged. However, mice in the isoflurane administration group treated with AR showed preserved nuclear structures within their hippocampal neuron (Fig. 4A). Furthermore, mice in the isoflurane administration group showed elevated expression levels of Iba1, indicating an increased number of activated microglia cells (Fig. 4B). Following AR treatment in isoflurane administration group, the expression levels of Iba1 markedly reduced. The mRNA expression levels of CD86 were significantly increased, while the levels of CD206 profoundly decreased after isoflurane treatment, which revealed that an increasing number of microglia cells became activated in response to the isoflurane administration. After AR treatment, the mRNA expression levels of CD86 and CD206 were reversed, which indicated that microglia cells in the hippocampus of mice were gradually shifted from M1 to M2 phenotype (Fig. 4C). Therefore, AR can induce neuroprotective effects in isoflurane-treated mice.

Fig. 4.Clematichinenoside AR can inhibit isoflurane-stimulated activation of microglia cells in mice hippocampus. (A) H&E staining of the hippocampus of mice in each group. (B) The protein expression levels of Iba1 in the hippocampus of mice in each group. Relatively quantitative results were determined by Image J and shown as a histogram. (C) The mRNA expression levels of CD86 and CD206 in mice in each group. Data were presented as the mean ± SD with three independent experiments. *p < 0.05, ***p < 0.001 versus sham group, ^#p < 0.05, and ^##p < 0.01, ^###p < 0.001 versus isoflurane group. AR: Clematichinenoside AR; Iba1: Ionized Calcium Binding Adaptor Molecule 1.

3.5 Clematichinenoside AR can activate the ERK-CREB pathway

Western blot analysis was conducted to investigate the mechanism by which AR attenuates isoflurane-stimulated cognitive dysfunction in aged mice by ameliorating neurotoxicity through the ERK-CREB pathway. The phosphorylation levels of ERK1/2, p90RSK and CREB were dramatically downregulated in the hippocampus after isoflurane administration. However, the phosphorylation levels of ERK1/2, p90RSK and CREB were reversed in the isoflurane administration group after AR treatment, indicating that AR could activate ERK1/2, p90RSK and CREB axis (Fig. 5).

Fig. 5.Clematichinenoside AR can activate the ERK-CREB pathway. The protein levels of p-ERK1/2, ERK1/2, p-p90RSK, p90RSK, p-CREB and CREB in each group. Relatively quantitative results were determined by Image J and shown as a histogram. Data were presented as the mean ± SD with three independent experiments. ***p < 0.001 versus sham group, ^###p < 0.001, and ^##p < 0.01 versus isoflurane group. AR: Clematichinenoside AR; ERK: Extracellular Signal-Regulated Kinase; p90RSK: p90 Ribosomal S6 Kinase; CREB: cAMP Response Element-Binding Protein.