Anti-Inflammatory Peptide Prevents Aβ25-35-Induced Inflammation in Rats via Lipoxygenase Inhibition

Abstract

Neuroinflammation, triggered by lipoxygenase (LOX), contributes to Alzheimer's disease (AD) progression. Overexpression of LOX-5 in patients with AD serum highlights its role. This study assessed the efficacy of the LOX-inhibitor-peptide YWCS in an AD rat model induced by Aβ_25-35 injection. Cognitive tests, magnetic resonance imaging (MRI) scans, and molecular analyses were conducted. YWCS treatment significantly improved cognitive function, as evidenced by improved performance in the open field, novel object recognition, elevated plus maze, and Morris water maze tests. MRI scans revealed hippocampal shrinkage in AD rats and no changes were observed from YWCS treatment. Molecular analysis revealed altered expression of LOX-5, LOX-12, Aβ, γ-secretase components, p-Tau¹⁸¹, Akt, p-Akt, and p53 in AD rats. Immunofluorescence staining confirmed increased expression of LOX, Aβ, and p-Tau¹⁸¹ in the hippocampus of AD rats, which was reduced by YWCS treatment. Serum LOX levels were elevated in AD rats and significantly decreased after YWCS treatment, aligning with previous findings in human AD patients and AD cell models. YWCS offered improvements in behavioral and inflammatory marker regulation and also prevented progression of the disease, as shown by MRI results. These results suggest that YWCS, by targeting LOX, has the potential to be a promising therapeutic agent for AD.

Keywords: AD rat model; MRI scan; cognitive behavioral assessment; immunoblot; immunofluorescence; lipoxygenase.

Figure 1

(A). Representative images of behavioral experiments with Bonferroni multiple comparison post hoc test. (A) Open field test: In the AD group, rats traveled less distance as compared to the control and sham groups, which upon giving the treatment of the peptide YWCS improved the activity of rats. (B) Elevated plus maze test: The ratio of time spent in the open vs. closed arm is less in AD rats compared to the control and sham groups due to fear and anxiety. The effect of the intranasal treatment of peptide YWCS was that it led to an improvement in the ratio of time spent in the open vs. closed arm. (C) Novel object recognition test: The recognition index declined in the AD group as compared to the control and sham groups and it increased in a dose-dependent manner after the intranasal treatment of peptide YWCS. (D) Morris water maze test: Latency to reach the platform, (E) distance traveled, and (F) time spent in AD rats were all higher compared to the control and sham groups. After the treatment of the peptide YWCS, the latency, distance traveled in the target zone, and time spent in the target zone declined significantly in both of the doses. (*—p < 0.05, **—p < 0.001, ***—p < 0.0001, non-significant (ns)—p > 0.05).

Figure 2

MRI images of AD rats exhibiting shrinkage of hippocampus as compared to baseline. After intranasal treatment of peptide YWCS at dose 10 mg/kg, no further shrinkage of the hippocampus was observed in AD rats. (A) Coronal Slice; (B) axial Slice; (C) bar diagram representing the changes in volume of hippocampus in 3 study groups, the red dotted shape represents the hippocampal region of the rat brain. (**—p < 0.001, non-significant (ns)—p > 0.05).

Figure 3

Immunoblots of the expression of Aβ-amyloid, γ-secretase components (Nicastrin, Presenilin-1, Presenilin-2, PEN-2), and p-Tau¹⁸¹ in (A) the hippocampus and (B) frontal cortex of a control, an AD, and an intranasal YWCS (5.0 mg/kg and 10 mg/kg) treated rat brain. These are normalized against β-actin. Comparative data are represented in bar diagram as mean ± SEM (n = 3). (*—p < 0.05, **—p < 0.001, ***—p < 0.0001, non-significant (ns)—p > 0.05).

Figure 4

Immunoblots of the expression level of LOX-5, LOX-12, Akt, p-Akt, p53 in (A) the hippocampus and (B) the frontal cortex of a control, AD, and intranasal YWCS (5.0 mg/kg and 10 mg/kg) treated rat brain. These are normalized against β-actin. Comparative data are represented in the bar diagrams as mean ± SEM, (n = 3). (*—p < 0.05, **—p < 0.001, non-significant (ns)—p > 0.05).

Figure 5

(A) The immunofluorescence of Aβ-amyloid in the DG, CA1, CA2, and CA3 regions of the hippocampus in an AD, control, and YWCS treated rat brain. Magnification 10X, scale bar 50 µm. (B–E) The comparative level of Aβ-amyloid in different regions is represented in the bar diagram (n = 3). Quantification of mean fluorescence intensity (MFI) with mean ± SEM. *—p < 0.05, **—p < 0.001, ***—p < 0.0001.

Figure 6

(A) Comparative difference in immunofluorescence staining of p-Tau¹⁸¹ in DG, CA1, CA2, and CA3 region of hippocampus in a control, AD, and YWCS nasal treated rat brain. (B–E) The quantification of mean fluorescence intensity (MFI) of p-Tau¹⁸¹ in different regions is represented in the bar diagrams. Magnification 20×, scale bar 50 µm. (F) The quantification of neuronal NeuN-positive p-Tau¹⁸¹ in different regions is represented in the scattered plot. (*—p < 0.05, **—p < 0.001, ***—p < 0.0001).

Figure 7

(A) Comparative difference in immunofluorescence staining of LOX-5 in the DG, CA1, CA2, and CA3 regions of the hippocampus in a control, AD, and YWCS nasal treated rat brain. (B–E) Quantification of the mean fluorescence intensity (MFI) of LOX-5 in different regions is represented in the bar diagrams. Magnification 20×, scale bar 50 µm. (F) Quantification of neuronal NeuN positive LOX-5 in different regions is represented in the scattered plot. *—p < 0.05, **—p < 0.001, ***—p < 0.0001.

Figure 8

(A) Quantification of mean fluorescence intensity (MFI) of LOX-12 in DG, CA1, CA2, and CA3 in the hippocampus in a control, AD, and YWCS treated rat brain by immunofluorescence. Magnification 20X, scale bar 50 µm. (B–E) The comparative level of LOX-12 in different regions is represented in the bar diagrams (n = 3). MFI with mean ± SEM. The expression level of serum (F) LOX-5 and (G) LOX-12 in control, AD, and YWCS treated rats by SPR. *—p < 0.05, **—p < 0.001, ***—p < 0.0001.