Abstract graphic file with name pt3c00226_0007.jpg Sleep deprivation (SD) has led to a rise in cognitive impairment (CI) cases. Kaempferol (KMP), known for its anti-inflammatory and antiapoptotic properties, holds promise in countering SD-induced CI. Experimental validation using a sleep-deprived CI model confirmed KMP’s efficacy in mitigating CI. Immunofluorescence investigations emphasized diminished activation of astrocytes and reduced the proliferation of microglia in the hippocampus of mice subjected to SD. Subsequently, network pharmacological analyses were conducted and found that KMP may be closely related to the mitogen-activated protein kinase (MAPK) pathway in SD-induced CI. The influence of KMP on the MAPK pathway was verified by the observed decrease in the expression of phosphorylated JNK (p-JNK) and p38 (p-p38). Analyzing hippocampal AMPARS and NMDARS expression indicated KMP’s ability to enhance GluA1 phosphorylation (Ser831 and Ser845) and GluN2A levels. Patch clamp assays demonstrated heightened excitatory transmitter transmission in the hippocampus, suggesting KMP’s positive influence. Overall, KMP combats neuroinflammation via MAPK inhibition, augments synaptic function, and addresses learning and memory dysfunction in sleep-deprived mice. Keywords: kaempferol, cognitive impairment, sleep deprivation, neuroinflammation, synaptic plasticity, MAPK pathway 1. Introduction In recent years, with the increase in pressure at work, the reduction in voluntary sleep has become a modern condition that leads to insufficient sleep. Sleep deprivation (SD) refers to a condition in which a person is unable to get enough sleep, leading to various physical, mental, and behavioral abnormalities, including cognitive impairment (CI). With the advancement of social life, increasing life pressures, and widespread use of electronic devices, SD has become increasingly prevalent in modern society. In recent years, there has been an increase in the incidence of learning and memory disorders caused by SD, and currently, there is no effective treatment available.^[35]1,[36]2 Neuroinflammation and synaptic damage play a critical role in CI.^[37]3 Chronic SD can induce a state of chronic inflammation in neurons, which can result in synaptic damage, neuronal dysfunction, neuronal death, and impaired neurogenesis.^[38]4 Neuroinflammation is closely associated with the activation of glial cells, which typically remain in a resting state but become activated and release pro-inflammatory molecules under stressful or pathological conditions.^[39]5 Various pro-inflammatory factors, including tumor necrosis factor-α (TNF-α), maintain a chronic state of neuroinflammation, which damages neurogenesis and spatial memory.^[40]6 Interleukin-1β (IL-1β) and TNF-α have been reported to inhibit memory formation and impair learning and memory function;^[41]7,[42]8 meanwhile, IL-1β and TNF-α influence downstream mitogen-activated protein kinase (MAPK) and NF-κB pathways involved in the regulation of learning and memory.^[43]9 Moreover, the release of inflammatory factors can adversely affect synaptic plasticity and hinder the expression or activity of the Glu system, resulting in impaired learning and memory function.^[44]10,[45]11 Additionally, these inflammatory factors significantly modulate micro and macro connections in brain circuits, leading to deficits in long-term potentiation (LTP) and long-term depression, both of which are crucial for learning and memory.^[46]12 During LTP, the rapid transport of AMPA receptors (AMPARs) to the postsynaptic density enhances synaptic transmission. AMPARs and NMDA receptors (NMDARs), which are widely expressed in the central nervous system,^[47]13,[48]14 are located in the presynaptic and postsynaptic regions, supporting the transmission of signals from one neuron to another. The downregulation of synaptic proteins leads to decreased intercellular signaling, ultimately resulting in CI. Plant flavonoids have garnered significant attention for their potential in improving human memory function and are considered one of the primary targets for the prevention and treatment of various neurodegenerative diseases due to their antioxidant, anti-inflammatory, and antiapoptotic properties, as well as their ability to cross the blood–brain barrier.^[49]15 Kaempferol (KMP), a type of flavonol mainly derived from the rhizome of Kaempferia galanga L. and widely found in various fruits and vegetables,^[50]16 has been shown to have promising prospects in the treatment of CI.^[51]17 A recent study has indicated that the intake of KMP and quercetin is associated with a slower decline in overall learning and memory function.^[52]18 Recent studies have shown that KMP can alleviate memory impairment in STZ-induced OVX rats, possibly by increasing the endogenous hippocampal antioxidants superoxide dismutase and glutathione and reducing neuroinflammation.^[53]2 Previous studies have demonstrated the therapeutic potential of KMP in enhancing learning and memory. However, the specific mechanisms underlying KMP effects on learning and memory impairment induced by SD have not been investigated. Thus, this study aims to comprehensively investigate the therapeutic targets and biological signaling pathways of KMP in the treatment of SD-induced CI. The findings of this study offer both bioinformatics and experimental data, which are crucial for future translational and clinical research on KMP. 2. Materials and Methods 2.1. Animals The research encompassed male C57BL/6 mice aged within the range of 6–8 weeks. The animals were subjected to random grouping and were accommodated in cages housing six individuals per cage. A continuous and sufficient supply of food and water was ensured. Environmental parameters were diligently controlled, maintaining ambient conditions at 24 ± 2 °C, accompanied by a relative humidity spanning 50 to 60%. A well-regulated light-dark cycle of 12 h duration was instituted. Before commencing the experiment, the mice underwent a minimum 1 week acclimation period to the laboratory conditions, during which they were fed a commercially available diet. 2.2. Establishment of the SD Model and KMP Treatment The model of SD was established by a multiplatform water tank according to previous studies.^[54]3,[55]19 Briefly, a water-filled tank was utilized to study learning and memory, housing multiple platforms, each possessing a diameter of 2.5 cm. The water level was situated 3 cm below the platforms. The mice were situated on these platforms with unhindered access to food and water. Stringent regulation maintained the water temperature at 30 ± 1 °C throughout the entire experiment, which extended over a continuous duration of 5 consecutive days. Either KMP (0.4, 2, and 10 mg/kg, CAS No. 520-18-3, purity: 98%, Topscience, Shanghai, China) or 0.9% saline (vehicle, 0.2 mL) was administered intragastric (i.g.) during SD for a further 5 consecutive days. 2.3. New Object Recognition Test The main objective of this investigative study was to assess the memory recognition abilities of mice by using a novel object recognition test (NORT). The NORT protocol consisted of three distinct phases: habituation, training, and testing. During the habituation phase, which spanned 2 days, the mice were placed in a square enclosure (40 cm × 40 cm × 25 cm) without any objects for 5 min. On the training day, the mice were placed in the same enclosure and observed interacting with two identical objects for 5 min. The testing session took place the following day, where two different objects—one familiar and one novel—were introduced, and this lasted for 5 min. To determine exploratory behavior, the criterion was established that the mice must approach an object within a distance of 2 cm and orient their nose toward it. To ensure objectivity in the experimental conditions, two separate researchers who were unaware of the study details recorded the duration of object exploration. The recognition index (RI) was calculated by dividing the time spent investigating the novel object by the total exploration time for both objects. 2.4. Morris Water Maze Experiment In a previous study,^[56]20 researchers extensively described the Morris water maze (MWM) protocol used to assess the learning and memory performance of mice over a continuous 5-day period. The MWM apparatus consisted of a 120 cm diameter circular pool enclosed by blue drapes. The pool was divided into four quadrants (I, II, III, and IV) and filled with water maintained at 25 ± 1 °C and a depth of 45 cm. There were additional cues in each quadrant for the mice to recognize. Within one of these quadrants, a 6 cm diameter circular platform was positioned 2 cm below the water’s surface, making it invisible to the mice. The spatial learning test, conducted over 4 days, involved randomly placing the mice in any of the three quadrants without the platform. The mice had up to 60 s to navigate the pool and find the hidden platform. If they failed, an experimenter guided them to the platform. Each mouse underwent four training trials per day. On the fifth day, the submerged platform was removed, allowing the mice to swim freely for 60 s during a probe trial. The mice’s swimming trajectories were recorded using a digital camera and subsequently analyzed using the ANY maze behavioral tracking software (Stoelting, Co., Illinois, USA). 2.5. Y Maze The Y maze configuration encompasses three arms, all of uniform length, positioned at angles of 120° from one another. These arms measure 50, 18, and 35 cm, respectively. During 8 min, a camera system documented the behavioral changes exhibited by the animals in the maze, focusing on the following indicators: a mouse entering an arm with all four feet was considered to have entered the arm once, resulting in a count of total entries (Indicator 1). Moreover, the instances of individual arm entries made by the animals were meticulously documented (Indicator 2). Another option to assess behavior involved having the mice enter all three arms of the Y maze. The determination of the maximum number of alternations involved the division of the overall count of rotations by two (total rotations/2), resulting in a score that quantified the prevalence of self-initiated rotations. This score was computed as follows: Score = (total rotations/major rotations) × 100%. 2.6. Immunofluorescence Staining After sedation was induced with 3% pentobarbital sodium and transcardial perfusion was performed using sterile saline, the mice were subjected to administration of 4% paraformaldehyde (PFA). Then the brains were subjected to overnight postfixation in 4% PFA at a temperature of 4 °C. Following this, the brains were dehydrated by using a sucrose gradient solution until they sank to the bottom. Employing a freezing microtome (CM1950, Leica), a comprehensive series of coronal sections (20 μm) was obtained. Particular emphasis was placed on the selection of sections encompassing the hippocampal region. These sections were subsequently cleansed within a 0.1 mM phosphate-buffered saline (PBS) buffer and rendered permeable by treatment with 0.3% Triton in 5% normal goat serum, conducted at room temperature over a duration of 1 h. For immunostaining, we used polyclonal anti-goat IBa1 and polyclonal antimouse glial fibrillary acidic protein (GFAP) as the primary antibodies, which were diluted in phosphate buffered saline with Tween 20 (PBST) (0.1% Tween 20 in PBS) at a 1:200 ratio and incubated on the sections overnight at 4 °C. After conducting three successive rinses, the sections were subjected to an incubation period with secondary antibodies (goat anti-mouse antibody, donkey anti-goat antibody, Jackson ImmunoResearch) in darkness at room temperature, lasting for a span of 2 h. Following a 15 min wash utilizing PBS, the sections were affixed onto slides utilizing 50% glycerin, while cell nuclei were counterstained using DAPI, which was suitably diluted in a 0.1 mM PBS solution (1:1000). Subsequent to sample preparation, image acquisition was conducted by utilizing an Olympus FV1000 confocal laser microscope. The obtained images underwent analysis and manipulation through the utilization of ImageJ software, carried out by researchers who were unaware of the experimental conditions. 2.7. Network Pharmacology The Swiss database ([57]http://www.swisstargetprediction.ch/) and the TCMSP database ([58]https://old.tcmsp-e.com/) were used to obtain potential targets for KMP. Duplicate values were removed and the targets from both databases were merged. For CI targets, information was collected from DisGeNET ([59]https://www.disgenet.org/), GeneCards ([60]https://www.genecards.org/), and OMIM ([61]https://www.omim.org/). Disease targets specific to “cognitive impairment” were obtained separately from DisGeNET, GeneCards, and OMIM, and after eliminating duplicates, the disease targets were combined. The target lists for KMP and CI were determined using Venny ([62]https://bioinfogp.cnb.csic.es/tools/venny/). Protein–protein interaction (PPI) network analysis was performed using Online STRING 11.0 ([63]https://string-db.org/) to understand the biological functions and potential mechanisms of the identified targets. To further investigate pathway enrichment, a Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis was conducted through DAVID Bioinformatics Resources 6.8 ([64]https://david.ncifcrf.gov/). The results of the enrichment analysis were visualized using the bioinformatics online analysis platform ([65]https://www.bioinformatics.com.cn/). 2.8. Enzyme-Linked Immunosorbent Assay (ELISA) Blood samples were collected from each cohort and allowed to stand for 1 h. After that, the samples were centrifuged at 3000 rpm for 3 min to obtain serum. The serum was then stored at −80 °C. Levels of IL-1β, IL-6, and TNF-α in the serum were measured using ELISA kits obtained from Service, China, following the manufacturer’s instructions. Briefly, equal volumes of serum were added to designated wells, and the optical density at 450 nm was measured by using a plate reader. The concentration of the target proteins was determined by analyzing the standard curve. 2.9. Western Blotting Analysis After completing the behavioral assessments, the mice were euthanized in a humane manner, and their brains were carefully removed. The hippocampal tissue was meticulously dissected on a chilled metal plate placed on ice and then stored at −80 °C until protein extraction. Each individual sample was mixed with a lysis buffer containing protease and phosphatase inhibitors. The mixture was then triturated and centrifuged. Protein concentration was determined using a BCA protein assay. Subsequently, 30 μg of the protein samples was separated on a 9% SDS-PAGE gel and transferred onto Millipore PVDF membranes. To prevent nonspecific binding, the membranes were blocked with a solution of 5% skim milk in PBST, and incubated at room temperature for 2 h. Afterward, the membranes were incubated overnight at 4 °C with primary antibodies diluted in PBST. The primary antibodies included p-GluA1R Ser831 rabbit monoclonal antibodies (1:1000), p-GluA1R Ser45 rabbit monoclonal antibodies (1:1000), GluA1 polyclonal antirabbit antibodies (1:1000), GluA2 rabbit monoclonal antibodies (1:1000), JNK polyclonal antirabbit antibodies (1:1000), p-JNK polyclonal antirabbit antibodies (1:1000), p38 polyclonal antirabbit antibodies (1:1000), p-p38 polyclonal antirabbit antibodies (1:1000), and anti-β-actin antibodies (1:10,000). These antibodies were obtained from Abcam, Cell Signaling, and Sigma-Aldrich. After rinsing with PBST, the membranes were incubated with appropriate horseradish peroxidase-conjugated secondary antibodies, either goat antirabbit or goat antimouse antibodies from Santa Cruz Biotechnology. The immunoreactive bands were visualized using enhanced chemiluminescence (ECL) obtained from PerkinElmer Life Sciences. Band intensities were quantified using a Tanon5200 imager and analyzed using ImageJ software provided by the National Institutes of Health. 2.10. Whole-Cell Patch-Clamp Recordings Cervical dislocation was used to euthanize the animals, and then coronal slices of the hippocampus (300 μm in thickness) were prepared in a chilled artificial cerebrospinal fluid (ACSF) solution. The ACSF solution contained 124 mM NaCl, 2.5 mM KCl, 2 mM CaCl[2], 2 mM MgSO[4], 25 mM NaHCO[3], 1 mM NaH[2]PO[4], and 10 mM glucose, with a pH of 7.2–7.4. The solution was saturated with 95% oxygen and 5% carbon dioxide. After incubating the slices in ACSF at room temperature for 1 h, they were transferred to a recording chamber on the stage of an Olympus microscope. Infrared digital interference contrast optics were used for better visualization during whole-cell patch-clamp recordings. The recording method involved using whole-cell voltage-clamp mode with a holding potential of −70 mV to monitor miniature excitatory postsynaptic currents (mEPSC) in hippocampal neurons. During the recordings, 100 μM picrotoxin and 1 μM TTX were added to the solution. Data inclusion criteria involved excluding cases where there were changes in the access resistance exceeding 15% during the experiment or when the resting membrane potential exceeded −60 mV. Afterward, offline analysis of the voltage clamp data was performed by using the specialized software Mini Analysis Program 6.0.7 for mEPSC analysis. 2.11. Statistical Analysis The data presented in this study are expressed as mean ± standard error of the mean, which were obtained from a compilation of three separate and independent experiments. The differences between the various groups were assessed using a one-way analysis of variance (ANOVA), followed by Tukey’s post hoc test. For the evaluation of the hidden-platform training in the MWM test, a two-way repeated-measures ANOVA was conducted, followed by Tukey’s post hoc analysis. Statistical significance was considered at a P value of less than 0.05. 3. Results 3.1. KMP Ameliorates Learning and Memory Impairments Induced by SD in Mice To evaluate cognitive deficits associated with SD, we conducted a series of conventional behavioral tests, including MWM, new object recognition tests, and Y maze tests. These tests were specifically designed to assess impairments in learning and memory. In the MWM experiment, we found no significant differences in escape latency between the sleep-deprived group and that treated with medication compared to the control group. This lack of significant divergence was observed throughout the 4 days of training. Nevertheless, the model mice displayed less time spent within the target quadrant and a decrease in the frequency of entry into the target region. Treatment, however, ameliorated these impairments ([66]Figure [67]1 A–E). Figure 1. [68]Figure 1 [69]Open in a new tab Impact of KMP on cognitive disorders caused by sleep deprivation. (A) Morris water maze experiment process is depicted. (B) Morris water maze trajectories of different groups of mice are shown. (C) Escape latency of mice in different groups is displayed for 1–4 days. (D) Time spent in the target quadrant by mice in different groups is presented. (E) Number of entries in the target zone by mice in different groups is reported. (F) Experimental process of novel object recognition and Y maze is illustrated. (G) Sketch representing novel object recognition is provided. (H) Discrimination index of novel object recognition is depicted. (I) Sketch of the Y maze is presented. (J) Spontaneous alternation rate of the Y maze is shown. The values are presented as means ± standard deviation (SD). *P < 0.05, **P < 0.01 compared to the control group. ^#P < 0.05, ^##P < 0.01 compared to the group exposed to sleep deprivation (SD). In the context of the new object recognition experiment, a notably reduced RI was noted in sleep-deprived mice compared to that in the normative mice during the 24 h post-training assessment. However, the RI was reversed following the administration of KMP ([70]Figure [71]1 F–H). Finally, in the Y maze experiment, the sleep-deprived mice displayed a significantly reduced spontaneous alternation rate compared to that of the normative group. However, this rate was significantly increased after the administration of KMP ([72]Figure [73]1F,I,J). These observations imply that KMP enhances learning and memory capabilities in sleep-deprived mice. 3.2. KMP Mitigates Neuroinflammation Induced by SD in the Hippocampus Neuroinflammation assumes a crucial role in the etiology of learning and memory impairments.^[74]21 Prior investigations have substantiated that SD has the potential to incite neuroinflammation.^[75]22 Neuroinflammation is an immune response activated by microglia and astrocytes in the central nervous system. Therefore, to detect the regulatory effect of KMP on neuroinflammation, we measured the expression of Iba1 and GFAP, which are used as specific markers to activate microglia and astrocytes in the hippocampus ([76]Figure [77]2 A,B). Immunofluorescence staining ([78]Figure [79]2C–F) showed that compared to those in the control group, GFAP- and IBa1-labeled astrocytes and microglia in the hippocampus of the model group were increased. However, his trend of increasing was decreased after KMP treatment. Our results indicated that KMP reduced the inflammatory response caused by SD in the hippocampal CA1 region. Figure 2. [80]Figure 2 [81]Open in a new tab Effect of KMP on astrocyte and microglia activation in sleep-deprived mice. (A) Immunofluorescence staining for activated astrocyte GFAP in the hippocampal CA1 area. GFAP is represented in green, and DAPI in blue. (B) Immunofluorescence staining for activated microglia IBa1 in the hippocampal CA1 area. IBa1 is represented in green, and DAPI in blue. Scale bar = 50 μm. (C) GFAP positive cell number (per 0.1 mm^2). (D) GFAP relative fluorescence intensity (fold of vector). (E) Number of microglia proliferating (per 0.1 mm^2). (F) IBa1 relative fluorescence intensity (fold of vector). 3.3. KMP Suppresses the Expression of Pro-Inflammatory Cytokines Induced by SD in Serum and Hippocampal Tissue The presence of pro-inflammatory cytokines is a crucial aspect of the inflammatory process. To determine the levels of these pro-inflammatory factors, we conducted ELISA tests on serum samples and western blotting on hippocampal tissue. The results of the ELISA tests revealed a significant increase in the levels of pro-inflammatory cytokines, such as IL-1β, TNF-α, and IL-6, in the SD model group compared to those in the control group [determined using one-way ANOVA, F (4, 10) = 12.74, P = 0.0002, P = 0.0095, P = 0.0007, [82]Figure [83]3 A]. Similarly, the western blotting analysis produced consistent results with the ELISA findings [one-way ANOVA, F (4,10) = 10.85, P = 0.0060, P = 0.0395, P = 0.5476, [84]Figure [85]3B,C]. Importantly, the introduction of KMP resulted in a significant reduction in the expression of pro-inflammatory factors ([86]Figure [87]3A,B), suggesting its potential to effectively inhibit the release of inflammatory cytokines in the hippocampus induced by SD. Figure 3. [88]Figure 3 [89]Open in a new tab Reduction of pro-inflammatory factors following the KMP intervention. (A) Expression of pro-inflammatory factors in the serum of each group was detected using ELISA. (B) Utilized western blotting to determine the expression of pro-inflammatory factors in the hippocampal tissue. (C) Quantified the pro-inflammatory factors detected by western blotting. The values are presented as means ± SD (n = 3). Statistical significance was denoted by *P < 0.05 or **P < 0.01 compared to the control group and ^#P < 0.05 or ^##P < 0.01 compared to the SD group. 3.4. Identification of Key Molecular Targets and Pathways for KMP’s Potential Anticognitive Impairment Effects: KMP Modulates the JNK/p38 Signaling Pathway to Mitigate the Inflammatory Response in Sleep-Deprived Mice After deduplication, 192 potential targets of the KMP compound were retrieved from the Swiss and TCMSP databases ([90]Table S1). A total of 577 distinct disease targets were acquired through data extraction from the DisGeNet, GeneCards, and OMIM databases ([91]Table S2). The outcome from the intersection of these sources is visually depicted within the Venn diagram ([92]Figure [93]4 A), highlighting a convergence of 61 potential targets that share associations with both KMP and CI. Subsequently, these overlapping targets were harnessed to construct a PPI network, a task accomplished through the utilization of the STRING database ([94]Figure [95]4B). Through a degree-based approach, we discerned the 10 foremost hub genes ([96]Figure [97]4C), including MMP9, STAT1, PPARG, MAPK1, AKT1, ESR1, TNF, SRC, JUN, and PTGS2. These hub genes are anticipated to exert pivotal functions in the context of KMP’s potential anticognitive impairment effects. Additionally, the outcomes of the KEGG pathway enrichment analysis pertaining to the 61 common targets are graphically illustrated in [98]Figure [99]4 D, underscoring a substantial linkage to the MAPK signaling pathway. Figure 4. [100]Figure 4 [101]Open in a new tab Suppression of the MAPK pathway in the hippocampus of mice subjected to sleep deprivation. (A) Venn diagram depicting the targets associated with KMP and cognitive impairment. (B) Protein–protein interaction (PPI) network consisting of 61 shared targets. (C) Identification of 10 prominent hub genes. (D) Top 20 pathways resulting from the KEGG enrichment analysis of the shared targets. (E) Western blotting for JNK and p38. (F, G) Analyses for JNK and p38. The values, expressed as means ± SD (n = 3), reveal that *P < 0.05 and **P < 0.01 indicate significance when compared to the control group, whereas ^#P < 0.05 and ^##P < 0.01 indicate significance when compared to the sleep deprivation group. The MAPK pathway consists of four main branches, known as ERK, JNK, p38/MAPK, and ERK5. JNK and p38 have similar functions related to inflammation, apoptosis, and growth.^[102]23 Previous studies^[103]24−[104]26 have shown that inhibiting MAPK activation can regulate the production of pro-inflammatory agents in microglial cells. In our study, we conducted western blot analysis ([105]Figure [106]4 E–G) and found that the SD group had levels of phosphorylated JNK (p-JNK) and phosphorylated p38 (p-p38) that were higher than those of the control group. However, treatment with KMP was able to counteract this increase. Additionally, we did not observe significant changes in the overall concentrations of p38 or JNK across the groups, suggesting that KMP may alleviate the inflammatory response in sleep-deprived mice by modulating the JNK/p38 signaling pathway. 3.5. KMP Modulates AMPA Receptor Phosphorylation and Expression in the Hippocampus to Enhance Synaptic Plasticity To investigate the potential influence of KMP on the functionality of AMPA within hippocampal neurons and its implications for enhancing learning and memory, we conducted an evaluation of the levels of total and phosphorylated GluA1R and GluA2R expressions ([107]Figure [108]5 A). The administration of KMP did not show any significant impact on the levels of total GluA1R or GluA2R expression compared to those in both the control and model groups ([109]Figure [110]5A,D,E). However, phosphorylation, which is known to play a pivotal role in synaptic plasticity, contributes significantly to modifications in AMPAR functionality. In comparison to that in the control group, we observed a noticeable decrease in the model group’s GluA1R phosphorylation levels at Ser 831 and Ser 845 (confirmed by one-way ANOVA, F (4,10) = 4.904, P = 0.0288; F (4,5) = 101.6, P = 0.0001, [111]Figure [112]5A–C). Figure 5. [113]Figure 5 [114]Open in a new tab Applying KMP treatment can enhance the expression of AMPAR in the hippocampus of sleep-deprived mice. (A, F) Western blotting reveals variations in the expression levels of GluA1R, GluA2R, p-GluA1R (Ser831), p-GluA1R (Ser845), GluN2A, and GluN2B. (B, C, D, E, G, and H) Western blotting analysis results for GluA1R, GluA2R, p-GluA1R (Ser831), p-GluA1R (Ser845), GluN2A, and GluN2B. The values are represented as means ± standard deviation (n = 3). The results indicate statistical significance with *P < 0.05 or **P < 0.01 compared to the control group and with ^#P < 0.05 or ^##P < 0.01 compared to the sleep deprivation (SD) group. Interestingly, the administration of KMP resulted in a discernible increase in these phosphorylation levels, with the most prominent effects observed at doses of 0.4 and 10 mg/kg for Ser831 (P = 0.0281, P = 0.0423), and 2 and 10 mg/kg for Ser845 (P = 0.0001 and P = 0.0020, respectively). Furthermore, our analysis revealed no significant variations in the expression of GluN2A [as determined using one-way ANOVA, F (4,5) = 4.935, P = 0.055]. In contrast, the model group exhibited a notable decrease in the expression levels of GluN2B compared to the control group [determined through one-way ANOVA, F (4,10) = 10.94, P = 0.0491]. However, treatment with KMP significantly increased GluN2B expression, particularly at a dose of 2 mg/kg (P = 0.0008), as shown in [115]Figure [116]5 F–H. Based on these findings, it can be concluded that treatment with KMP effectively modulates the expression of AMPA in the hippocampus. 3.6. KMP Restores SD-Induced Decrease in Excitatory Synaptic Currents in the Hippocampus The primary goal of this study was to examine the impact of KMP treatment on neuronal excitability in the hippocampal regions of sleep-deprived individuals. We recorded mEPSC using whole-cell patch-clamp techniques ([117]Figure [118]6 A,B). Our observations revealed a significant decrease in mEPSC frequency in the SD group compared to that in the control group (established through one-way ANOVA, F (2,12) = 8.843, P = 0.0076, as shown in [119]Figure [120]6C,E). However, the introduction of KMP at a concentration of 10 μM effectively reversed the diminished mEPSC frequency induced by SD (P = 0.0102). Importantly, neither the model group nor the administration of KMP had any impact on the amplitude of mEPSC [evaluated using one-way ANOVA, F (2,12) = 3.588, P =0.0601, as shown in [121]Figure [122]6D,F]. These findings strongly suggest that KMP treatment effectively restores the decrease in excitatory synaptic currents caused by SD. Figure 6. [123]Figure 6 [124]Open in a new tab Effect of KMP on basal glutamate synaptic transmission. (A) Preparation and electrophysiological recording of the hippocampal brain section. (B) Miniature excitatory postsynaptic currents (mEPSC) were measured in mouse hippocampal neurons at a holding potential of −70 mV. (C, D) Cumulative frequency and amplitude histograms of mEPSC in mouse neurons, respectively. (E, F) Summary of mEPSC frequency and amplitude in mouse neurons, with six neurons from three mice included in the analysis. The values are presented as means ± SD, with significance indicated by *P < 0.05 and **P < 0.01 compared to the control group and ^#P < 0.05 and ^##P < 0.01 compared to the SD group. 4. Discussion Chinese herbal medicine and its effective natural compounds have the characteristics of easy accessibility, multi-target, and multi-mechanism treatment for human diseases. They offer significant advantages in the prevention and treatment of complex diseases, including CI.^[125]27 KMP has attracted attention because of its high anti-inflammatory activity. In an Alzheimer’s disease (AD) model, KMP has been shown to reduce neuroinflammation and oxidative stress^[126]2 and improve mitochondrial dysfunction and cognitive decline.^[127]28 However, the detailed molecular mechanism underlying the beneficial effects of KMP on SD-induced CI remains unclear. To assess alterations in cognitive functionality induced by a 5-day period of SD, the MWM behavioral test was utilized. Of notable significance, the administration of varied doses of KMP (0.4, 2.0, and 10 mg/kg) prominently mitigated the learning and memory deficits induced by SD. These findings align with those of previous studies^[128]2 that have demonstrated the efficacy of KMP in restoring spatial memory capacity in the animal model of AD. It is noteworthy that neuroinflammation has been discerned as a pivotal component in the pathogenesis of SD. Existing studies have emphasized the involvement of microglial activation in SD-mediated alterations of inflammatory molecules, neurogenesis, and spatial memory.^[129]6 In particular, activated microglia are responsible for the secretion of pro-inflammatory mediators, including IL-1β, interleukin-6 (IL-6), and TNF-α, which subsequently exacerbate neuronal deterioration.^[130]29 Our results demonstrated increased microglia and astrocytes labeled with Iba1 and GFAP, respectively, in the SD model, whereas KMP treatment successfully mitigated microglial and astrocytic activation. Furthermore, an examination of pro-inflammatory factor expression levels was undertaken, revealing that SD induced an elevation in IL-1β, IL-6, and TNF-α, all of which were notably mitigated by the administration of KMP. Network pharmacology has emerged as an invaluable tool for unraveling complex mechanisms that underlie therapeutic interventions.^[131]30 In this study, we successfully identified 61 potential targets of KMP that were closely associated with CI. Analysis focused on the enrichment of the constructed KMP-target-pathway network demonstrated that KMP’s potential to CI might operate via modulation of the MAPK-regulated signaling cascades. Therefore, we focused on understanding the role of MAPKs, which are important signaling molecules that mediate various cellular responses. In mammals, there are over a dozen MAPK enzymes that regulate processes, such as cell growth, differentiation, motility, and survival.^[132]31 Among these, MAPKs such as ERK, JNK, and p38 have been found to play a key role in controlling inflammation in microglia, the immune cells of the brain, when triggered by external signals.^[133]32 Our in vitro experiments revealed that KMP treatment resulted in a significant reduction in the level of excessive activation of p38 and JNK signaling pathways. This suggests that KMP protects the brain in the SD model by modulating the overactivated MAPK pathway, leading to a decrease in neuronal damage and an improvement in learning and memory. Therefore, targeting the MAPK pathway could be a potential therapeutic strategy for KMP, with promising possibilities for intervention. Research has established the indispensable contribution of neuronal and synaptic plasticity to brain function and cognitive abilities. Insufficient sleep has been shown to elevate neuronal excitability through oxidative stress, thereby potentially causing neurodegeneration, altering neuronal physiology, impairing synaptic plasticity, and inducing cell death, consequently affecting synaptic efficiency.^[134]22 The interplay between the brain and immune system encompasses bidirectional communication mechanisms that are orchestrated by glial cells, the intrinsic immune components of the brain, signifying their pivotal role in this interaction.^[135]33,[136]34 Neurons and glia interact to maintain brain homeostasis and collaborate in promoting neurogenesis and memory formation.^[137]21 The hippocampus, a vital cerebral region intricately linked with cognitive function, is susceptible to synaptic impairment, thereby being intricately associated with the onset of learning and memory disorders. Studies have indicated that restoration of AMPAR and NMDAR functions holds promise as a therapeutic strategy for these conditions, as it has been demonstrated to alleviate core symptoms in various models of cognitive dysfunction.^[138]35 AMPAR and NMDAR, pivotal components for rapid excitatory synaptic transmission within the central nervous system, hold the responsibility of fostering synaptic plasticity as well as facilitating the intricate processes of learning and memory formation. It represents a fundamental aspect of the brain’s experience-based regulation of information processing and storage.^[139]36 Our investigation unveiled a noteworthy reduction in the expression levels of synaptic-associated proteins in the hippocampus of the model group compared to those in the control group. Remarkably, upon the administration of 2.0 mg/kg of KMP, a substantial augmentation in these protein levels was observed. Additionally, we observed reduced excitatory synaptic transmission in the hippocampus due to SD, which improved after administration of 10 μM KMP. These findings suggest that KMP treatment can enhance impaired hippocampal synaptic plasticity caused by SD. In summary, the findings of this study are significant, because they are the first to illustrate the efficacy of KMP in alleviating memory deficits and synaptic plasticity impairment in sleep-deprived mice. The underlying mechanism is attributed to the downregulation of inflammatory factors by inhibition of the JNK/p38-mediated inflammatory pathway. However, these investigations did not address specific molecular mechanisms through which KMP operates within the context of the MAPK pathway. Therefore, further research is necessary to understand the therapeutic properties of KMP comprehensively and facilitate the development of novel derivatives. 5. Conclusions The administration of KMP demonstrated significant efficacy in ameliorating CI in sleep-deprived mice. This effect was achieved through inhibition of the JNK/p38 signaling pathway, resulting in the attenuation of the inflammatory response within the hippocampus and the promotion of synaptic plasticity preservation. The results suggest that KMP has potential therapeutic benefits in alleviating the CI associated with SD. Data Availability Statement Upon request, the data will be made available to interested parties. Supporting Information Available The Supporting Information is available free of charge at [140]https://pubs.acs.org/doi/10.1021/acsptsci.3c00226. * Potential targets of Kaempferol, and potential targets of cognitive impairment ([141]PDF) Author Contributions L.-X.L. and L.Y. designed the study. Q.Y., Q.-Q.L., and J.-M.L. assisted in carrying out the experiments. Y.-Y.D. and T.S. wrote the manuscript. L.-X.L. and L.Y. helped to proofread the article. All authors contributed to the analysis and interpretation of the data and approved the final manuscript. Y.-Y.D. and T.S. contributed equally to this study. This study was supported by the National Natural Science Foundation Grant No. 31800887 (to L.Y.). The authors declare no competing financial interest. Supplementary Material [142]pt3c00226_si_001.pdf^ (91KB, pdf) References