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BY 4.0 license Open Access Published by De Gruyter Open Access May 23, 2023

A phytoconstituent 6-aminoflavone ameliorates lipopolysaccharide-induced oxidative stress mediated synapse and memory dysfunction via p-Akt/NF-kB pathway in albino mice

  • Shakeel Ahmad EMAIL logo , Shahid Ali Shah , Naeem Khan EMAIL logo , Umar Nishan , Nargis Jamila and Amal Alotaibi
From the journal Open Chemistry

Abstract

The current work examined the therapeutic potential of 6-aminoflavone (6AF) against mouse model-based oxidative stress-driven synaptic and memory impairment caused by lipopolysaccharides (LPSs). In the brains of the experimental mice, LPS administration for 3 weeks significantly increased oxidative stress by inhibiting antioxidant enzymes, including superoxide dismutase, peroxidase, catalase, glutathione, and upregulating lipid peroxidase. Male albino mice were arbitrarily divided into four groups including (1) Control, (2) LPS treated (250 µg/kg, for 3 weeks), (3) LPS plus 6AF treated (30 mg/kg for 2 weeks), and (4) 6AF treated (30 mg/kg for 2 weeks). Different antioxidant enzyme assays, behavior tasks, and the western blotting technique were used to test the therapeutic potentials of this 6AF. Remarkably, the dosage of 6AF significantly reversed the activities of antioxidant enzymes and reduced neuroinflammation in adult albino mice. Additionally, 6HF also improved the synapse (both pre- and post-proteins) and restored the impaired memory against LPS. In short, these findings propose that 6AF is a natural, non-toxic, and potent therapeutic agent to treat neurodegenerative diseases.

1 Introduction

Alzheimer’s disease (AD), a prevalent ailment associated with aging, differs from other illnesses in a variety of respects, including cell death, beta-amyloid clumps, and hyperphosphorylated tau protein tangles [1]. One of the biggest dangers to mitochondrial dysfunction, which leads to the onset of AD, is assumed to be oxidative stress or the formation of reactive oxygen species (ROS). ROS can also trigger inflammation in the brain. ROS in excess impedes antioxidant defense systems, leading to protein, DNA, and lipid damage [2,3,4,5]. Neuroinflammation can also lead to synaptotoxicity and memory loss [6].

The polysaccharide portion of Gram-negative bacteria, lipopolysaccharides (LPS), triggers an innate immune response by attachment to Toll-like receptor 4 (TLR4) in microglia [7]. The interaction between TLR4 and LPS activates and stimulates nuclear factor kappa-B (NF-κB) and other kinases, which results in the release of pro-inflammatory cytokines like interleukin-1 (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor (TNF-α) [8]. As a result, LPS has been utilized in an animal model to study the molecular approaches to memory impairment for many years [9]. The efficacy of new and novel medications to treat AD in animals is best studied using LPS-induced AD neuropathology.

Plants are good sources of polyphenols and flavonoids, which are the most vital polyphenols and are an important part of the human diet [10,11,12]. 6-Aminoflavone (6AF) possesses potent and proliferative activity against selected human tumor cell lines both in vivo and in vitro [13]. The current study aims to evaluate the neurodegenerative potential of 6AF due to its reported anti-proliferative activity; the selected compound 6AF may act as a potential drug against neurodegenerative diseases.

2 Materials and methods

2.1 Chemicals

Phosphate buffer saline (PBS) tablets, LPS, ammonium persulfate (APS), acrylamide, sodium dodecyl sulfate (SDS), Trizma base, bis-acrylamide, potassium chloride (KCl), and sodium chloride (NaCl) were purchased from Dae-Jung Chemicals & Metals Co. Ltd (Gyeonggi-do, Shiheung, South Korea) and Sigma Aldrich Chemical Co. (St. Louis, MO, USA).

2.2 Mice and their grouping

Male adult Swiss albino mice were acquired from the veterinary division of the National Institute of Health (NIH) in Islamabad, Pakistan, and transported to the Neuro Molecular Medicines Research Centre (NMMRC, Peshawar), where the tests were conducted. The mice were housed in clearly labeled cages (Biobase, China) with free access to water and food and were given time to adjust to their new surroundings. Later, the mice were arbitrarily allocated into groups of four (each group consists of n = 10 mice) as given below. Group 1: normal mice (normal saline-treated); Group 2: LPS-injected mice (250 µg/kg); Group 3: LPS-injected mice (250 µg/kg) + 6-AF-injected mice (30 mg/kg); and Group 4: 6-AF-injected mice (30 mg/kg). The 30–32 g male adult albino mice were then housed in separate cages in the animal house under controlled conditions, such as a 12 h light/dark cycle, a constant temperature of 25°C, and ad libitum access to standardized pellet food and clean water. All of the study animals were handled with distinct care and empathy, in accordance with the recommendations of the NMMRC’s animal ethical committee. This study was permitted by the ethical committee of Kohat University of Science and Technology, Pakistan (dated 09/03/2020, No. 524).

2.3 Behavioral tests

Two well-known behavioral tests were used to illustrate the therapeutic benefits of 6-AF on LPS-induced memory impairment. LPS was given intraperitoneally (i.p.) to Groups 2 and 3 mice and 6-AF was given i.p. to Groups 3 and 4 mice. Then, all of the research mice were tagged, separated into four groups, and behavioral studies were performed as a single-blind experiment. The tags and treatment groups of mice were kept hidden from the researcher conducting the behavioral testing.

2.4 Morris water maze (MWM) test

The hippocampus learning skills of mice were tested using the MWM test. The design and size of the MWM test apparatus have recently been given in detail in the study of Shah et al. [14]. The animals were trained to swim twice a day for 3 days prior to the start of the testing to acclimate to the water tank and platform. Later, in order to find the hidden/submerged platform, the mean escape latency for each mouse was measured for 60 s, and this technique was repeated for 5 days. The mice were forcibly directed onto the platform and left there for a set amount of time if they failed to find it in the specified amount of time (60 s). The platform was hidden throughout the 2-day rest interval before the final probe testing and the amount of time each mouse spent in the target quadrant was recorded.

2.5 Y-maze

The Y-maze behavioral test was used, as previously reported [15]. The Y-maze device is made up of three arms that measure 50 × 10 × 20 cm3 (L × W × H) and are joined at a 1,200° angle. Mice were given 10 min to acclimate to their new surroundings each time. The mice were then placed in the maze’s center and given 8 min to freely travel in three arms. Each mouse’s total arm entries and following triplets were tallied using special software, and the proportion of alternations was determined using the formula:

Percentage alternations = Successive triple tests Total number of arm entries 2 × 100 .

The percentage of alternations was positively linked with the spatial memory function of mice.

2.6 Western blotting analysis

After the therapy, all of the animals were killed [14].

The brain tissue from the hippocampus was carefully taken from the beheaded mice and immediately transferred to the RNAlater solution and PBS (1:1) on ice. T-PER solution (Thermo Scientific) was used to homogenize the hippocampus brain tissue after which the supernatant was collected and stored at −20°C for future investigation. The protein concentration was determined using the Bio-Rad protein estimation assay, and the absorbance measurement was made at 595 nm. All protein samples were standardized to 30 g per group and separated using SDS-PAGE on gels with a 12–15% gel concentration. For the first 20–30 min of the run, running conditions were maintained at 50 mA, and then for the last hour to an hour and a half, running conditions were changed to 120 V. Proteins were moved from the gel to a PVDF membrane (Santa Cruz Biotechnology, USA) using the semi-dry trans blot technique (Bio Rad). Anti-SYP, anti-PSD95, anti-p-Akt, anti-actin, anti-NF-kB, anti-TNF-α, and IL-1 monoclonal antibodies (Santa Cruz, CA, USA) were used as primary antibodies, followed by anti-mice HRP conjugated (Santa Cruz, CA, USA) secondary antibodies. For the development of the results, X-ray films were employed [14].

2.7 Antioxidant analysis of brain homogenates

2.7.1 Catalase (CAT) assay

A considerably modified technique was used to assess CAT [16] activity. A total of 2,500 mL of phosphate buffer (50 mm) at pH 5.0, 100 mL of the brain supernatant, and 400 mL of H2O2 were used in the 3 mL reaction mixture (5.9 mm). At 240 nm and 1-min intervals, the absorbance of the reaction mixture was measured as it changed. One unit of CAT activity was defined as a variation in the absorbance occurring at a rate of 0.01 units per minute.

2.7.2 Peroxidase (POD) assay

A significantly modified approach [16] was used to test POD activity. A total of 2,500 L of phosphate buffer (50 mM, pH 5.0), 300 µL of H2O2 (40 mM), 1,000 µL of the brain homogenate supernatant, and 100 µL of guaiacol were used to prepare the POD assay reaction mixture (20 mM). The absorbance of the reaction mixture at 470 nm was monitored each minute. One unit of POD activity was defined as a change in the absorbance of 0.01 units per minute.

2.7.3 Superoxide dismutase (SOD) assay

As previously stated [17], the SOD assay was carried out with a few modifications. A reaction mixture containing 300 L of the brain homogenate supernatant, 1,200 L of sodium pyrophosphate buffer (0.052 mM; pH 7.0), and 100 phenazine methosulfate was used to assess SOD activity. To initiate an enzyme reaction, 200 L of NADH (780 M) was mixed with the reaction mixture after a 1-min interval; 1,000 µL of glacial acetic acid was used as a stopping agent. By estimating the absorbance of the reaction mixture at 560 nm and translating the data into units/mg of protein, it was possible to calculate the amount of chromogen generated.

2.7.4 Reduced glutathione (GSH) assay

In a previous work [18], to determine the amounts of reduced GSH, the proteins in 1,000 L of brain homogenate were precipitated by adding an equivalent volume of sulfosalicylic acid solution (4%) to the mixture. The reaction mixture was then maintained at 4°C for a further hour, and then it was centrifuged at 1,200×g for 20 min at 4°C in a refrigerator. The reaction mixture contained 2,700 mL of phosphate buffer (pH 7.2) and 200 mL of 100 mM DTNB. The absorbance of the reaction mixture at 412 nm was determined as quickly as possible. GSH levels were determined in milligrams per gram of tissue.

2.7.5 Estimation of lipid peroxidase (TBARS)

A slightly modified approach was used for the lipid peroxidation thiobarbituric acid reactive substance (TBARS) test [19]. A 1,000 L reaction mixture containing 200 µL of 100 mM ascorbic acid, 200 µL of the brain homogenate supernatant, 580 L of 0.1 M phosphate buffer (pH 7.4), and 20 µL of 100 mM ferric chloride was used for the experiment. The reaction mixture was then heated to 37°C and agitated for an hour in a water bath. Then, 1,000 L of a 10% trichloroacetic acid solution was added to stop the reaction. Following that, 1,000 L of 0.67% thiobarbituric acid was added to the tubes, and they were centrifuged for 20 min at a speed of 2,500×g (95°C) in a hot water bath. The quantity of lipid peroxidase (TBARS) generated in each sample was calculated by measuring the absorbance of the supernatant at 535 nm and 37°C using a spectrophotometer.

2.7.6 Statistical analysis

After that, all of the X-ray findings were scanned. The scanned data were compiled and then statistical analysis was performed using computer-based software. Among the programs included are Adobe Photoshop, Prism 5 Graph Pad, and Image Jare. Normal saline-treated animals and LPS-injected mice had significantly different mean ± SEM in arbitrary units (AUs); P ≤ 0.05, P ≤ 0.01, and P ≤ 0.001, respectively.

2.7.7 All-atom molecular dynamics (MD) simulation

The therapeutic efficacy of 6-AF against oxidative stress-mediated neuronal synapse induced by LPS and the associated memory impairment in adult male albino mice was investigated in this work. By activating the phospho-Akt (p-Akt) signaling pathway, 6-AF can drastically reduce LPS-induced oxidative stress, synapse dysfunction, neuroinflammation, and memory impairment in an AD animal model. We employed extensive MD and molecular docking simulation with both the molecular operating environment (MOE®) and AMBER software package to investigate the stimulating mechanism of p-Akt by 6-AF.

2.7.8 Molecular docking study

The binding mechanism of the 6-AF molecule in the active site of p-Akt was demonstrated using a molecular docking study using MOE-Dock® software. PDB code 3OCB was used to retrieve the crystal structure of p-Akt in complex with C20H23Cl1N6O1 (co-crystal) from the protein data bank (www.rcsb.org). Using the defaulting parameters of the MOE energy minimized algorithm (gradient: 0.05, force field: Amber99; for p-Akt and MMFF94s; for 6-AF), minimized energy of the crystal structure, the 3D protonation, and 6-AF were investigated. Finally, by applying the Triangular Matching docking method (default), 6-AF was docked into the site where the co-crystal bonded in the protein, yielding 100 distinct conformations. For further investigation, the top-ranked conformation with the highest docking score was chosen. In addition, to confirm the dynamic behaviors of 6-AF in the p-Akt active site, we conducted a MD simulation study.

2.7.9 Molecular dynamics simulation

Using the generalized Amber force field (GAFF), the small molecule 6-AF was first parameterized [20]. To calculate the partial atomic charges, single-point energy calculations were performed using Schrodinger’s quantum chemistry module, the Hartree–Fock level of theory, Jaguar, and the 6-311g** basis set. Assigning GAFF atom types was done using the Antechamber module [21], and creating the parameter files for overall rendering was done using the AMBER LeaP module [22]. All-atom MD simulations and research of the primary dynamics were conducted using AMBER version 2018 [23]. The crystal structure was amended with hydrogen atoms using the LeaP module. Counter ions were introduced to maintain the system’s equilibrium. The TIP3P water model’s truncated octahedral box was used to solve the system with a cut-off of 8.0 buffer. The particle mesh Ewald (PME) approach [24] was utilized to manage broad ranges of electrostatic interactions. The force field ff14SB was utilized for MD simulations [25]. With a tolerance of 10−5, the SHAKE method was utilized to safeguard all covalent bonds, including hydrogen atoms [26]. The PMEMD CUDA version was utilized to speed up each MD simulation. The NVT ensemble’s solvated systems were reduced in 20,000 steps using the steepest descent method, then heated for 400 ps, and then brought to equilibrium in 200 ps. With isotropic position scaling, a relaxation duration of 2.0 ps, and a constant time of 1.0 ps, Langevin’s approach [27] was employed to fix the pressure and temperature.

3 Results and discussion

3.1 6-AF restored LPS-induced synapse and memory dysfunction in adult mice brain

Inhibition of both pre- and post-neuronal synapse proteins is undoubtedly possible with LPS treatment [28]. For neuronal transmission, the proteins, synaptophysin and PSD95, which are located in the pre- and post-neuronal synapse, respectively, are necessary. Using the western blotting method, their expression was ascertained. LPS decreased the pre- and post-synapse protein in the male adult albino mouse brains, as per the findings of western blotting (Figure 1a–c). Treatment with 6-AF did, however, significantly preserve synaptic neuronal plasticity when LPS was present. After 6-AF was administered, it was seen that both pre- and post-synapse protein expression levels were upregulated (Figure 1a–c). The memory and behavior of experimental animals were impacted by LPS [29]. The Y-maze and the MWM, two well-known behavioral tests (MWM), were completed by the mice in the current investigation after they had been injected with LPS, LPS combined with 6-AF, control, and 6-AF alone. In the MWM test, mice were trained twice daily for 2 days, following which data were collected daily for 5 days. Their average daily escape times to the platform in 1 min were recorded. On day 1, control animals showed low mean escape latency, but mice treated with LPS had considerably increased mean escape latency (Figure 2a). While receiving LPS and 6-AF, the mean escape latencies of mice to reach the target platform were significantly lowered (Figure 2a). The control animals and the animals receiving 6-AF alone showed increasingly reduced mean escape latencies, and this trend was seen in all experimental animals. The mean escape latencies of the LPS-treated mice were generally longer than those of the control mice, even though they showed minimal improvement from day 1 to 5. Similarly, mice who were given 6-AF in addition to LPS had lower mean escape latencies than mice administered LPS alone, as shown in Figure 2a. After removing the submerged platform from the probe test, the control and mice administered 6-AFalone, both used up more time in the target quadrant, as shown in Figure 2b. The mice who were given LPS, on the other hand, seldom ever visited the target area. It is interesting to note that, as shown in Figure 2b, mice administered 6-AF and LPS spend more time in the target quadrant. As a measure of spatial memory performance, the Y-maze test yielded the spontaneous alternation percentage. According to Figure 2c, mice given 6-AF in addition to LPS showed a greater percentage of spontaneous alternation, but it was lower than in control mice. In contrast, mice given LPS alone showed a lower percentage of spontaneous alternation. The current study demonstrates that 6-AF has a protective effect against memory impairment and neural synapse dysfunction generated by LPS in adult albino mice. In albino mice, 6-AF alleviated LPS caused oxidative stress neuroinflammation, neuronal synaptotoxicity, and memory impairment.

Figure 1 
                  Both pre-and post-synaptic protein expression enhanced by 6-AF against LPS in mice. (a) In the brain supernatant of homogenates treated with either LPS alone or in combination with 6-AF, the western blot approach revealed the expression of both pre- and post-synapse proteins, synaptophysin, and PSD95. (b and c) Histograms of SYP and PSD95, representing their respective relative densities. The densities were determined, and graphs were created using the Image J program. P values of 0.01 and 0.001 are considered significant for the results, which were derived using an arbitrary unit (AU) and a histogram that displays the mean in AU SEM.
Figure 1

Both pre-and post-synaptic protein expression enhanced by 6-AF against LPS in mice. (a) In the brain supernatant of homogenates treated with either LPS alone or in combination with 6-AF, the western blot approach revealed the expression of both pre- and post-synapse proteins, synaptophysin, and PSD95. (b and c) Histograms of SYP and PSD95, representing their respective relative densities. The densities were determined, and graphs were created using the Image J program. P values of 0.01 and 0.001 are considered significant for the results, which were derived using an arbitrary unit (AU) and a histogram that displays the mean in AU SEM.

Figure 2 
                  6-AF boosted memory in adult mice’s brains. The results of behavioral tests are offered as (a) mean escape latency from day 1 to day 5 in the Morris water maze test, (b) probe test, and (c) percentage of spontaneous modification in the Y-maze test. All verified data are shown as mean ± SEM. P values of 0.01 and 0.001 are significant.
Figure 2

6-AF boosted memory in adult mice’s brains. The results of behavioral tests are offered as (a) mean escape latency from day 1 to day 5 in the Morris water maze test, (b) probe test, and (c) percentage of spontaneous modification in the Y-maze test. All verified data are shown as mean ± SEM. P values of 0.01 and 0.001 are significant.

3.2 6-AF alleviated oxidative stress induced by LPS in adult mice brains

Various antioxidant assays, like SOD, POD, CAT, GSH, and lipid peroxidase assays, were performed on the brain homogenates of all experimental mice. The findings demonstrated that LPS significantly increased oxidative stress in adult mice brains by reducing SOD (P < 0.001), POD (P < 0.001), CAT (P < 0.01), and GSH (P < 0.01), as well as activating lipid peroxidase activity (P < 0.001). Surprisingly, 6-AF dramatically enhanced the activity of anti-oxidant enzymes SOD (P < 0.001), POD (P < 0.001), CAT (P < 0.01), and GSH (P < 0.01), while decreasing the lipid peroxidase activity (P < 0.01), as shown in Figure 3a–e. In this study, we used a variety of anti-oxidant enzyme assays to see whether LPS can cause oxidative stress. The findings show that LPS inhibits anti-oxidant enzymes such as SOD, POD, CAT, and GSH while upregulating lipid peroxidation, implying that oxidative stress generated by LPS inhibits these enzymes while upregulating LPO. Similarly, our neuroprotective agent, 6-AF, greatly reduces oxidative stress by restoring the activities of various enzymes like POD, SOD, GSH, and CAT, as well as significantly lowering LPO activities, indicating that 6-AF is an effective anti-oxidant. In this line, previous studies [2,30,31] also proved that melatonin is an effective neuroprotective agent by lowering LPS-induced oxidative stress.

Figure 3 
                  6-AF restored the activities of LPS-suppressed antioxidant enzymes in adult male mice brains. The activities of antioxidant enzymes (a) CAT, (b) SOD, (c) POD, (d) GSH, (e) and lipid peroxidase (TBARS) were measured in mice brain supernatant homogenates administered with either LPS alone or in combination with 6-AF. The technique is detailed in Section 2. Each group’s findings are expressed as mean ± SEM of (n = 5) mice. P values of 0.01 and 0.001 are significant.
Figure 3

6-AF restored the activities of LPS-suppressed antioxidant enzymes in adult male mice brains. The activities of antioxidant enzymes (a) CAT, (b) SOD, (c) POD, (d) GSH, (e) and lipid peroxidase (TBARS) were measured in mice brain supernatant homogenates administered with either LPS alone or in combination with 6-AF. The technique is detailed in Section 2. Each group’s findings are expressed as mean ± SEM of (n = 5) mice. P values of 0.01 and 0.001 are significant.

3.3 6-AF inhibited NF-kB activation to abrogate LPS-induced neuroinflammation in mice

As LPS is responsible for producing neuroinflammation in a mouse model [29], we found that LPS administration via oxidative stress promoted neuroinflammation in adult mice brains by activating NF-kB, which was followed by the stimulation of TNF-α (Figure 4a–d). LPS produced neuro-inflammatory indicators like NF-kB motivation and translocation into the nucleus, as well as TNF-α and IL-1 protein activation. 6-AF treatment, on the other hand, totally blocked the activation of neuroinflammatory indicators such as NF-kB, TNF-α, and IL-1-proteins (Figure 4a–d). LPS has been reported to induce neuroinflammation in male adult albino mice brains [22]. Similarly, we also observed that the expression of neuroinflammatory markers such as NF-kB, TNF-α, and IL-1β was upregulated by LPS, which is assessed through western blotting, while the administration of 6-AF significantly reversed these changes induced by LPS. In this regard, Lee et al. [29] also reported that LPS can cause inflammation, and the methanolic extract of Carpesium cernuum L. is a protective agent and reduces the inflammation induced by LPS. So, 6-AF is a good anti-neuroinflammatory agent, which can reduce the inflammation induced by LPS in mice brains.

Figure 4 
                  6-AF inhibited NF-kB and revoked LPS-induced neuroinflammation in experimental mice. The western blot analysis of the neuro-inflammatory markers NF-kB, TNF-α, and IL-1 (a) is displayed, as well as histograms of the corresponding relative densities, in (b–d). The loading control used was β-actin. The data were computed using the Image J program, and a histogram that provided the mean in arbitrary units (AU) and the standard deviation (SEM) in AU. P values of 0.01 and 0.001 indicate statistical significance.
Figure 4

6-AF inhibited NF-kB and revoked LPS-induced neuroinflammation in experimental mice. The western blot analysis of the neuro-inflammatory markers NF-kB, TNF-α, and IL-1 (a) is displayed, as well as histograms of the corresponding relative densities, in (b–d). The loading control used was β-actin. The data were computed using the Image J program, and a histogram that provided the mean in arbitrary units (AU) and the standard deviation (SEM) in AU. P values of 0.01 and 0.001 indicate statistical significance.

The LPS animal model is a good model for AD-like memory impairment [32,33,34]. Similarly, gypenosides have been shown to reduce LPS-induced neuroinflammation and memory impairment in mice [29]. In this study, 6-AF was found to restore neural synapse proteins and improve memory and behavior in male adult albino mice exposed to LPS. This finding also parallels a previous study on Ibrutinib [28] that also reduces LPS-induced synaptotoxicity by boosting the countenance of both the pre- and post-synapse proteins, as well as improving memory. It means that 6-AF is an effective therapeutic drug for reducing synaptic loss and restoring memory and behavior in animals suffering from LPS-induced neurotoxicity. LPS dramatically decreased p-Akt protein in adult albino mice brains when administered to normal adult male albino mice. Supplementation of 6-AF, on the other hand, dramatically increased the p-Akt protein, reducing LPS-induced oxidative stress-mediated neural synapse and memory impairment. Similarly, the full signaling route of 6-AF against LPS-induced neurotoxicity was discovered. LPS injection promotes oxidative stress by blocking enzymes involved in antioxidant activities, as indicated in the figure. In adult albino mice, inhibition of this enzyme increased the expression of neuroinflammatory markers such as NF-kB, IL-1β, and TNF-α, which was accompanied by synapse and memory impairment. As a result, this entire route shows that 6-AF prevents LPS-induced synapse and memory impairment via p- Akt activation. According to the findings in this study; 6-AF is a strong drug that lowers LPS-induced oxidative stress by up-regulating anti-oxidant enzyme expression/activation, which leads to a decrease in neuroinflammation. Furthermore, 6-AF improved memory deficits in adult albino mice by restoring the neural synapse. For the first time, this work shows that 6- AF is a promising therapeutic candidate for treating LPS-induced neuronal synapse and memory impairment. This study also found that 6-AF decreases neuroinflammation by activating the p-Akt pathway, which protected male albino mice from LPS. Last, but not least, 6-AF promotes the p-Akt signaling pathway to prevent LPS-induced neurotoxicity, which results in memory impairment. More research on the anti-inflammatory and anti-oxidative properties of 6-AF is needed.

3.4 6-AF stimulated phospho-Akt activation to abrogate LPS-induced neurotoxicity in mice

The p-Akt proteins, which are assumed to be involved in the cell’s survival process, were drastically decreased by LPS treatment. In contrast, as shown in Figure 5a and b, 6-AF treatment greatly boosted the production of p-Akt proteins, thereby stimulating the signaling pathway and rescuing adult albino mice against LPS. In the same way, the proposed mechanism of 6-AF against LPS-induced neurotoxicity is depicted in Figure 6.

Figure 5 
                  In the adult mouse brain, 6-AF increased phospho-Akt signaling pathways against LPS-induced memory impairment. (a and b) The immunoblots for p-Akt are provided, along with the corresponding histogram. β-Actin served as the loading control. Using Image J software, the data were calculated, and a histogram displaying the mean in arbitrary units (AU) and expressed in SEM was created. The P-values of 0.01 and 0.001 indicate statistical significance.
Figure 5

In the adult mouse brain, 6-AF increased phospho-Akt signaling pathways against LPS-induced memory impairment. (a and b) The immunoblots for p-Akt are provided, along with the corresponding histogram. β-Actin served as the loading control. Using Image J software, the data were calculated, and a histogram displaying the mean in arbitrary units (AU) and expressed in SEM was created. The P-values of 0.01 and 0.001 indicate statistical significance.

Figure 6 
                  The signaling route illustrates the suggested mechanism of 6-AF against LPS-induced memory impairment in the brain of adult male Albino mice. It demonstrates how LPS-induced multiple AD neuropathology in mouse models was reversed by 6-AF through a p-Akt-dependent mechanism.
Figure 6

The signaling route illustrates the suggested mechanism of 6-AF against LPS-induced memory impairment in the brain of adult male Albino mice. It demonstrates how LPS-induced multiple AD neuropathology in mouse models was reversed by 6-AF through a p-Akt-dependent mechanism.

3.5 Inspecting the stability of p-Akt in the presence of 6-AF

According to the findings in this study, 6-AF is a strong drug that lowers LPS-induced oxidative stress by upregulating anti-oxidant enzyme expression/activation, which leads to a decrease in neuroinflammation. Additionally, 6-AF improved memory deficits in adult albino mice by restoring the neural synapse. For the first time, this work shows that 6-AF is a promising therapeutic candidate for treating LPS-induced neuronal synapse and memory impairment.

Molecular docking results discovered a strong binding pattern of 6-AF with the highest docking score (Figure 7a–c) by setting basic interactions with active site residues, including Gly157, Val164, and Glu234 through phi-stacking interactions, while Met227 and Glu228 through hydrogen bonding (Figure 7d). Additionally, a MD simulation was run to demonstrate the 6-AF complex’s stability with the p-Akt protein in a clear water environment. In general, it was practically observed that the aniline moiety of 6-AF remains attached to the active site, which is considered to be the moiety of entrance first to the active site of the protein. For ease of support and clarification of dynamics results, we also analyze the dynamics trajectory. When the system’s root-mean-square deviation (RMSd) was compared with actual structures, it was discovered that a simulation duration of 50 ns is adequate to attain equilibrium at 310 K (Figure 7e). The deviation of the backbone atoms was then examined using the RMSD technique. The RMSd value decreases when conformation stability increases and vice versa [35,36,37]. Since the RMSd curve continually rises and oscillates at 1.2 Å at the start, we recovered that conformation and compared it to the other conformations produced during succeeding simulation periods. We discovered variations in the 6-AF conformation, where the non-substituted benzene ring rotates by 20°, but not in the other conformations. The RMSd curve depicts the complex’s overall conformation being stabilized, with the 6-AF interacting closely with the bound residues at the active site over the majority of the simulation. The 6-AF molecule’s strong attachment to the active site demonstrates its importance in protein stability, as it rescues the protein from suppression. We used this molecule to explore the carbon-alpha (CA) distance research of active site residues in order to better understand the selectivity of the 6-AF molecule’s dynamic bonding capabilities in the active site. The results show extremely robust and consistent behavior at the active site with the bound residues, and the simulation time oscillated (Figure 7f). These findings show that the binding of the 6-AF molecule rescues the protein from LPS-induced suppression by stabilizing the overall structure of the pAkt protein.

Figure 7 
                  (a) Docking score of selected top 20 conformers. (b and c) Cartonic view of p-Akt in complex with 6-AF and the top 20 6-AF conformers were superposed in the active site of p-Akt. (d) Protein and 6-AF interaction profile. (e) Graph of the system of RMSd. (f) The dynamic CA distance. (d) Graph for 6-AF and interactive residues of the p-Akt protein.
Figure 7

(a) Docking score of selected top 20 conformers. (b and c) Cartonic view of p-Akt in complex with 6-AF and the top 20 6-AF conformers were superposed in the active site of p-Akt. (d) Protein and 6-AF interaction profile. (e) Graph of the system of RMSd. (f) The dynamic CA distance. (d) Graph for 6-AF and interactive residues of the p-Akt protein.

4 Conclusions

This study’s findings suggest that 6-AF is a potent drug that reduces LPS-induced oxidative stress by upregulating anti-oxidant enzyme expression/activation, which reduces neuroinflammation. Also, by repairing the neuronal synapse, 6-AF helped adult albino mice with memory problems. This study determines for the first time the therapeutic potential of 6-AF in the treatment of memory loss and impaired neuronal synapses caused by LPS. Also, it was shown that 6-AF reduces neuroinflammation via activating the p-Akt pathway, protecting male albino mice against LPS. Last, but not the least, to stop LPS-induced neurotoxicity, which impairs memory, 6-AF stimulates the p-Akt signaling pathway. The anti-inflammatory and anti-oxidative effects of 6-AF require more study.

Acknowledgment

The authors extend their appreciation to the Researchers Supporting Project Number (PNURSP2023R33) at Princess Nourah bint Abdulrahman University Saudi Arabia for financial support.

  1. Funding information: Researchers supporting project number (PNURSP2023R33) Princess Nourah bint Abdulrahman University Saudi Arabia.

  2. Author contributions: Data curation: S.A.S. and N.J.; formal analysis: U.N., A.A.; funding acquisition: A.A.; investigation: S.A.; methodology: N.K.; resources: A.A.; software: U.N.; supervision, S.A.S. and N.K.; validation: N.K. and A.A.; writing – original draft, N.J. and S.A.

  3. Conflict of interest: The authors declared no conflict of interest.

  4. Ethical approval: This study was permitted by the ethical committee of Kohat University of Science and Technology Pakistan (dated 09/03/2020, No. 524).

  5. Data availability statement: All the available data incorporated in the manuscript.

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Received: 2023-01-23
Revised: 2023-04-27
Accepted: 2023-04-30
Published Online: 2023-05-23

© 2023 the author(s), published by De Gruyter

This work is licensed under the Creative Commons Attribution 4.0 International License.

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