Vanillic Acid, a Bioactive Phenolic Compound, Counteracts LPS-Induced Neurotoxicity by Regulating c-Jun N-Terminal Kinase in Mouse Brain

Abstract

The receptor for advanced glycation end products (RAGE), a pattern recognition receptor signaling event, has been associated with several human illnesses, including neurodegenerative diseases, particularly in Alzheimer's disease (AD). Vanillic acid (V.A), a flavoring agent, is a benzoic acid derivative having a broad range of biological activities, including antioxidant, anti-inflammatory, and neuroprotective effects. However, the underlying molecular mechanisms of V.A in exerting neuroprotection are not well investigated. The present study aims to explore the neuroprotective effects of V.A against lipopolysaccharides (LPS)-induced neuroinflammation, amyloidogenesis, synaptic/memory dysfunction, and neurodegeneration in mice brain. Behavioral tests and biochemical and immunofluorescence assays were applied. Our results indicated increased expression of RAGE and its downstream phospho-c-Jun n-terminal kinase (p-JNK) in the LPS-alone treated group, which was significantly reduced in the V.A + LPS co-treated group. We also found that systemic administration of LPS-injection induced glial cells (microglia and astrocytes) activation and significantly increased expression level of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-KB) and secretion of proinflammatory cytokines including tumor necrosis factor alpha (TNF-α), interleukin-1 β (IL1-β), and cyclooxygenase (COX-2). However, V.A + LPS co-treatment significantly inhibited the LPS-induced activation of glial cells and neuroinflammatory mediators. Moreover, we also noted that V.A treatment significantly attenuated LPS-induced increases in the expression of AD markers, such as β-site amyloid precursor protein (APP)-cleaving enzyme 1 (BACE1) and amyloid-β (Aβ). Furthermore, V.A treatment significantly reversed LPS-induced synaptic loss via enhancing the expression level of pre- and post-synaptic markers (PSD-95 and SYP), and improved memory performance in LPS-alone treated group. Taken together; we suggest that neuroprotective effects of V.A against LPS-induced neurotoxicity might be via inhibition of LPS/RAGE mediated JNK signaling pathway; and encourage future studies that V.A would be a potential neuroprotective and neurotherapeutic candidate in various neurological disorders.

Keywords: amyloidogenesis; c-Jun N-terminal kinases; lipopolysaccharide; neurodegenerative diseases; neuroinflammation; synaptic and memory impairment; vanillic acid.

Figure 1

Vanillic acid treatment inhibited lipopolysaccharides (LPS)-activated receptor for advanced glycation endproducts (RAGE)/gliosis (microglia and astrocytes) in Mice Brain. (A–D) the western blot bands of the RAGE, glial fibrillary acidic protein (GFAP), and ionized calcium-binding adaptor molecule 1 (Iba-1) antibodies along with their relative histograms indicating their protein expression level in the cortex and hippocampus of mice brain. The western blot bands were cropped and quantified using ImageJ software, and the differences are shown in the histogram. The relative density values were expressed in comparison with control in arbitrary units (A.U). All the values were taken as the mean ± SEM of three repeated/independent experiments for the respective indicated protein. As a loading control, an anti-β-actin antibody was used. n = 8 mice/group, and the number (N) of experiments performed = 3. (E–G) the representative photomicrographs of immunofluorescence staining in different regions represent the immunoreactivity of RAGE (cortex, CA1, and dentate gyrus (DG) region) and GFAP (CA1 and DG) along with its relative histograms in various experimental brain groups (green, fluorescein isothiocyanate (FITC); blue, 4′, 6′-diamidino-2-phenylindole (DAPI)). White small arrows indicated the desired signals of the tested antibody. The relative integrated density values were expressed in comparison with control in A.U. All values were taken as the means (±S.E.M) for the respective indicated proteins. For nucleus staining, DAPI (blue) was used. n = 8 mice/group, and the number (N) of experiments = N = 3. Magnification = 10×. Scale bar; cortices/DG hippocampal regions = 50 μm. Asterisk sign (*) indicated significant difference from the normal saline-treated (Cont.) group; hash sign (#) indicated significant difference from LPS-alone treated group; while the phi sign (Φ) indicated no significance from normal saline-treated (Cont.) group. Significance: * # = p ≤ 0.05, ** ## = p ≤ 0.01, and *** = p ≤ 0.001.

Figure 2

Vanillic acid treatment inhibited LPS-induced elevated expression level of stress kinase (c-Jun N terminal kinase (p-JNK)) in mice brain. (A,B) representative western blot band of p-JNK antibody along with its relative histogram indicating its protein expression level in both cortex and hippocampus regions of mice brain. The western blot bands were cropped and quantified using ImageJ software, and the differences are shown in the histogram. The relative density values were expressed in comparison with control in A.U. All values were taken as the mean ± SEM of three repeated/independent experiments for the respective indicated protein. As a loading control, an anti-β-actin antibody was used. n = 8 mice/group, and the number (N) of experiments performed (N) = 3. (C,D) the representative photomicrographs of immunofluorescence staining represent the immunoreactivity of p-JNK protein along with its relative histogram in different regions (cortex and hippocampus) in various experimental brain groups (green, FITC; blue, DAPI). White small arrows indicated the desired signals of the tested antibody. The relative integrated density values were expressed in comparison with control in A.U. All values were taken as the means (±S.E.M) for the respective indicated proteins. For nucleus staining, DAPI (blue) was used. n = 8 mice/group, and the number (N) of experiments = N = 3. Magnification = 10×. Scale bar; cortices/DG hippocampal regions = 50 μm. Asterisk sign (*) indicated significant difference from the normal saline-treated (Cont.) group; hash sign (#) indicated significant difference from LPS-alone treated group; while the phi sign (Φ) indicated no significance from normal saline-treated (Cont.) group. Significance: * # = p ≤ 0.05, ** ## = p ≤ 0.01, and *** = p ≤ 0.001.

Figure 3

Vanillic acid treatment inhibited LPS-induced activation of nuclear factor nuclear factor-kappa B (p-NF-κB) and neuroinflammatory mediators/cytokines in mice brain. (A–E) the western blot bands of p-NF-κB, tumor necrosis factor alpha (TNF-α), interleukin-1 βeta (IL-1βeta), and cyclooxygenase-2 (COX-2) antibodies along with their relative histograms indicating their respective proteins expression level in both cortex and hippocampus regions of mice brain. The western blot bands were cropped and quantified using ImageJ software, and the differences are shown in the histogram. The relative density values were expressed in comparison with control in A.U. All values were taken as the mean ± SEM of three repeated/independent experiments for the respective indicated protein. As a loading control, an anti-β-actin antibody was used. n = 8 mice/group, and the number (N) of experiments performed (N) = 3. (F,G) the representative photomicrographs of immunofluorescence staining represent the immunoreactivity of p-NF-κB protein along with its relative histogram in different regions (cortex, CA1, and DG region) in various experimental brain groups (green, FITC; blue, DAPI). White small arrows indicated the desired signals of the tested antibody. The relative integrated density values were expressed in comparison with control in A.U. All values were taken as the means (±S.E.M) for the respective indicated proteins. For nucleus staining, DAPI (blue) was used. n = 8 mice/group, and the number (N) of experiments = N = 3. Magnification = 10×. Scale bar; cortices/DG hippocampal regions = 50 μm. Asterisk sign (*) indicated significant difference from the normal saline-treated (Cont.) group; hash sign (#) indicated significant difference from LPS-alone treated group. Significance: * # = p ≤ 0.05, ** ## = p ≤ 0.01, and *** = p ≤ 0.001.

Figure 4

Vanillic acid treatment inhibits LPS-induced elevated protein expression of BACE-1 and β-amyloid (Aβ) in mice brain. (A–C) the western blot bands of BACE-1 and Aβ antibodies along with their relative histograms indicating their protein expression level in the cortex and hippocampus of mice brain. The western blot bands were cropped and quantified using ImageJ software, and the differences are shown in the histogram. The relative density values were expressed in comparison with control A.U. All values were taken as the mean ± SEM of three repeated/independent experiments for the respective indicated protein. As a loading control, an anti-β-actin antibody was used. n = 8 mice/group, and the number (N) of experiments performed = 3. (D,E) the representative photomicrographs of immunofluorescence staining represent the immunoreactivity of Aβ protein along with its relative histogram in different regions (cortex, CA1, and DG region) in various experimental brain groups (green, FITC; blue, DAPI). White small arrows indicated the desired signals of the tested antibody. The relative integrated density values were expressed in comparison with control in A.U. All values were taken as the means (±S.E.M) for the respective indicated proteins. For nucleus staining, DAPI (blue) was used. n = 8 mice/group, and the number (N) of experiments = N = 3. Magnification = 10×. Scale bar; cortices/DG hippocampal regions = 50 μm. Asterisk sign (*) indicated significant difference from the normal saline-treated (Cont.) group; hash sign (#) indicated significant difference from LPS-alone treated group. Significance: * # = p ≤ 0.05.

Figure 5

Vanillic acid treatment inhibited LPS-induced synaptic and memory dysfunction in mice brain. (A–C) the western blot bands of postsynaptic density proteins (PSD95), synaptophysin (SYP), antibodies along with their relative histograms indicating respective protein expression level in the cortex and hippocampus of mice brain. The western blot bands were cropped and quantified using ImageJ software, and the differences are shown in the histogram. The relative density values are expressed in comparison with control in A.U. All the values were taken as the mean ± SEM of three repeated/independent experiments for the respective indicated protein. As a loading control, an anti-β-actin antibody was used. n = 8 mice/group, and the number (N) of experiments performed = 3. (D,E) the representative photomicrographs of immunofluorescence staining represent the immunoreactivity of PSD95 in different regions (cortex and DG region) in various experimental brain groups (green, FITC; blue, DAPI). White small arrows indicated the desired signals of the tested antibody. The relative integrated density values were expressed in comparison with control arbitrary units (A.U). All values were taken as the means (±S.E.M) for the respective indicated proteins. For nucleus staining, DAPI (blue) was used. n = 8 mice/group, and the number (N) of experiments = N = 3. Magnification = 10×. Scale bar; cortices/DG hippocampal regions = 50 μm. Morris water maze (MWM) parameters are indicating (F,G) average escape latency (in a sec) and (H) probe test indicating time spent in target quadrant, (I) number of target crossings, and (J) Swim speed (cm/s) among the experimental groups. The Y-maze analysis represented (K) total distance covered (cm) and (L) spontaneous alteration behaviors. (M,N) indicating MWM and y-maze trajectories. For the behavioral study, the number of mice (n = 16/experimental group) was used. Asterisk sign (*) indicated significant difference from the normal saline-treated (Cont.) group; hash sign (#) indicated significant difference from LPS-alone treated group. Significance: * # = p ≤ 0.05 and **= p ≤ 0.01. n.s = non significance.

Figure 6

Suggested graphical representation of neuroprotective mechanism of vanillic acid against LPS-induced neurotoxicity in mice brain. ① LPS (intraperitoneal injections (I.P.) 250 μg/kg/day) via RAGE result in neurodegeneration. ② in this process, LPS-activated p-JNK has a central role in activating various downstream signaling cascades lea ding to ③ neuroinflammation: Resulting from increased in expression level of p-NF-κB, TNFα, IL-1β, and COX-2. ④ amyloidogenesis: Resulting from increased in expression level BACE-1 and Aβ. ⑤ Synaptic dysfunction: Resulting from reduction in the expression level of synaptic makers (PSD-95, SYP). ⑥ vanillic acid (I.P. 30 mg/kg) reduced LPS-induced neurotoxicity might via inhibition of LPS/RAGE-mediated elevated c-Jun N-terminal kinase in mice brain.

Figure 7

Schematic design of the current research study, classification of animals into the experimental grouping, drug dosage treatment, behavioral analyses, biochemical and morphological experimental approach for the entire research study. The experimental mice were randomly divided into four groups. (i) mice treated with normal-saline as a vehicle (two weeks; I.P.); normal saline-treated (Cont.) group. (ii) mice treated with vehicle (one week) and LPS (I.P.: 0.25 mg/kg/day; one week); LPS-alone treated group. (iii) mice treated with LPS (0.25 mg/kg/day; one week) and V.A (I.P.: 30 mg/kg/day; two weeks); i.e., one week before the LPS treatment and one week co-treated with LPS); V.A + LPS co-treatment group. (iv) mice treated with V.A (I.P.: 30 mg/kg/day; two weeks); V.A-alone treatment group. After completion of behavioral analyses, experimental mice were euthanized and further subjected to immunoblotting and immunofluorescence analyses.