Ambroxol attenuates detrimental effect of LPS-induced glia-mediated neuroinflammation, oxidative stress, and cognitive dysfunction in mice brain

Abstract

Neurodegenerative diseases, such as Alzheimer's disease (AD) and Parkinson's disease (PD), are multifactorial. Among various factors, lipopolysaccharides (LPSs) from Gram-negative bacteria, such as E. coli, are considered potential causative agents. Despite significant advancements in the field, there is still no cure. In this study, we investigated the neuroprotective effects of ambroxol against LPS-induced neuroinflammation, oxidative stress, neurodegeneration, and the associated cognitive dysfunction. Intraperitoneal injection of LPS (250 µg/kg every alternative day for a total of seven doses over 14 days) triggered glial cell activation, neuroinflammation, oxidative stress, and neurodegeneration in the mouse brain. Ambroxol treatment (30 mg/kg/day for 14 days) significantly reduced neuroinflammation and oxidative stress compared to LPS-treated mice. Immunoblotting and immunofluorescence results showed that ambroxol reduced levels of Toll-like receptor 4 (TLR4) and oxidative stress kinase phospho-c-Jun N-terminal Kinase 1 (p-JNK). It also decreased astrocyte and microglia activation in the cortex and hippocampus of LPS+ Amb-treated mice, as indicated by the downregulation of GFAP and Iba-1. Furthermore, ambroxol-reversed LPS-induced neuroinflammation by inhibiting inflammatory mediators, such as IL-1β and TNF-α, through regulation of the transcription factor p-NFkB. Persistent neuroinflammation disrupted the natural antioxidant mechanisms, leading to oxidative stress. Ambroxol treatment upregulated antioxidant markers, including Nrf-2, HO-1, and SOD, which were downregulated in the LPS-treated group. Additionally, ambroxol-inhibited lipid peroxidation, maintaining malondialdehyde levels in the mouse brain. Ambroxol also improves synaptic integrity by upregulating synaptic biomarkers, including PSD-95 and SNAP-23. Overall, ambroxol demonstrated anti-inflammatory, antioxidant, and neuroprotective effects in LPS-treated mice, highlighting its potential benefits in neurological disorders.

Keywords: ambroxol; cognitive; cognitive impairment; glial cells; lipopolysaccharide; neurodegeneration; neuroinflammation; oxidative stress.

Figure 1

A schematic representation of the experimental designed to investigate the effectiveness of ambroxol against LPS-induced neuroinflammation, oxidative stress, and memory impairments.

Figure 2

Ambroxol regulates neuroinflammation in the LPS-induced mice. (A) Showing Immunoblot results with respective histograms of TLR4, GFAP, Iba-1, p-NFkB, and p-JNK mice brains (n = 4). (B) Immunofluorescence images of GFAP with respective graphs describe relative integrated density for the cortex and hippocampus (DG) in the LPS-treated mice brain (n=4). Beta-actin was used as a loading control. All the data were measured in mean ± S.E.M. ^#significantly different from the saline-injected group, *significantly different from the LPS-injected group. Significance: ^# p ≤ 0.05, ^## p ≤ 0.01 and ^### p ≤ 0.001; *p ≤ 0.05, **p ≤ 0.01 and ***p ≤ 0.001.

Figure 3

Ambroxol regulates the expression of inflammatory cytokines. (A) Immunoblot analysis of TNF-α and IL-1β in the experimental groups, accompanied by corresponding bar graphs (n = 4). (B) Immunoreactivity of TNF-α in the cortex and hippocampus (n = 4), as illustrated by immunofluorescence photographs with corresponding bar graphs scale bar 50 µm. All data were expressed as mean ± S.E.M. ^#significantly different from the saline-injected group, *significantly different from the LPS-injected group. Significance: ^# p ≤ 0.05, ^## p ≤ 0.01, and ^### p ≤ 0.001; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 4

Ambroxol mitigates oxidative stress induced by LPS treatment. (A) Representing SOD and LPO assay respectively. (B) Immunoblot analysis and bar graphs of the expression of the proteins Nrf2 and HO-1 in the mouse cortex and hippocampus (n = 4). (C) Immunofluorescence images showing of Nrf2 in the experimental group, with a scale bar of 50 µm. All data were presented as mean ± S.E.M. (n = 4). Significant differences were seen between different experimental groups. ^#significantly different from the saline-injected group, *significantly different from the LPS-injected group. Significance: ^# p ≤ 0.05, ^## p ≤ 0.01 and ^### p ≤ 0.001; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 5

Ambroxol regulated synaptic integrity. (A) Western blots analysis of PSD-95 and SNAP-23 in the cortex and hippocampus of mice brain (n = 4). (B) Immunofluorescence images and bar graphs of PSD-95 in the cortical and hippocampal regions of the mouse brain (n = 4), with a scale bar of 50 µm. All data were expressed as mean ± S.E.M. ^#significantly different from saline-injected group, *significantly different from LPS-injected group. Significance: ^# p ≤ 0.05, ^## p ≤ 0.01, and ^### p ≤ 0.001; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 6

Ambroxol improves LPS-induced memory and cognitive dysfunction. (A) Visuals of the trajectory paths in the Y-maze tasks. (B) Percentage of spontaneous alternation behavior and Y-maze analysis. (C) Time spent in the targeted quadrant (D) Number of crossings over the hidden platform in MWM. (E) Line graph describing the average time taken to reach the platform in targeted quadrant. Mean ± S.E.M. was used to measure all the results (n = 8 per group). ^#significantly different from saline-injected group, *significantly different from LPS-injected group. Significance: ^# p ≤ 0.05, ^## p ≤ 0.01, and ^### p ≤ 0.001; *p ≤ 0.05, **p ≤ 0.01, and ***p ≤ 0.001.

Figure 7

The possible neuroprotective mechanism of Ambroxol’s against LPS-induced neuroinflammation, oxidative stress, and cognitive impairment in the mouse brain.