Ambroxol hydrochloride (CAS 23828-92-4) was purchased from Santa Cruz Biotechnology. LPSs from Escherichia coli (E. coli O111:B4/L2630.), and all the remaining chemicals used in current study design were acquired from Sigma-Aldrich (St. Louis, MO, USA).

The primary antibodies used in western blot and confocal microscopy in our present study are listed in Table 1.

Wild-type C57BL/6N male 8-week-old mice (n = 32, with eight mice per group) weighing between 26 and 28 g were acquired from Samtako Bio, Osan, South Korea. First, the mice were acclimatized for one week, keeping control of temperature and humidity (23°C ± 2°C and 60 ± 15%, respectively) with a regulated 12-h light/12-h dark cycle. All the mice were allowed to freely eat standard pellet food and drink water ad libitum. All experimental procedures were performed keeping the standardization of the Institutional Animal Care and Use Committee (IACUC) of the Division of Applied Life Sciences, Department of Biology, at Gyeongsang National University, South Korea.

All the mice were randomly divided into four distinct group: (a) a control group treated with 0.9% normal saline; (b) LPS-injected group (LPS 250 µg/kg IP every alternative day for 2 weeks); (c) LPS + Ambroxol co-treated group (LPS 250 µg/kg and Ambroxol 30 mg/kg, IP); for 14 days (Ambroxol was administered simultaneously with LPS); (d) Only Ambroxol treated group (30 mg/kg for 2 weeks consecutively). Both the LPS and Ambroxol was dissolved in sufficient amount of normal saline and administered IP route ( Figure 1 ).

The Morris water maze (MWM) test was performed as discussed previously with little modification (24). The MWM was used to measure declarative learning and memory in laboratory rodents. This setup consists of a circular water tank (width 100 cm, height 40 cm) having four different quadrants and filled with water dissolved in non-toxic white ink. A hidden platform (4.5 cm in diameter and 14.5 cm in height) was placed slightly below the surface of water in specific quadrants. The mice were freely allowed to move in order to locate the hidden platform. All the mice were individually receive four training trials for five consecutive days. On the fifth day, the hidden platform was removed, allowing the mice to swim freely for 1 min. The time spent by each mouse in the targeted quadrant and the number of crossings of over the location of the hidden platform were measured. All the procedures were recorded on the video graphic camera attached above. The data were analyzed by motion tracking software (SMART 3.0, Panlab Harvard Apparatus, Bioscience Company, and Holliston, MA, USA).

To explore spontaneous alteration behavioral changes and exploratory activity, the Y-Maze test was performed as previously reported (25). The Y-maze assembly consisted of three equal arms (each arm is 50 cm in length, 10 cm in width, and 20 cm in height). All the mice were placed in the center of the Y-maze assembly one by one, allowing movement with an interval of 8 min. All arm entries and alterations were recorded with the motion sensor attached above. The spontaneous alteration percentage (%) was calculated as (entries into three different arms consecutively/total number of arm entries-2) × 100. The increase in percentage of spontaneous behavior reflects the cognitive and memory dysfunction and vice versa.

Post-behavioral analysis, all the mice were sacrificed, and both the cortexes and hippocampal tissue were isolated as described previously (8). The cortical and hippocampal tissues were then homogenized using PRO-PREP (iNtRON Biotechnology, Dallas, Texas, MA, USA) solution. After that, the samples were centrifuged for a duration of 25 min at 4°C with a speed of 13,000 revolutions per minute (r.p.m.). The supernatants were collected and preserved at −80°C.

The protein concentrations were measured by using the BioRad Kit as reposted previously (26). Equal amounts of protein were loaded in 12%/10% (depending upon the molecular weight of protein) sodium dodecyl sulfate–poly-acrylamide gels and then transferred to polyvinylidene fluoride (PVDF) membranes (Immobilon-PSQ, Transfer membrane, Merck Millipore, Burlington, MA, USA). The PVDF membranes were blocked with 5% skim milk w/v dissolved in 1X TBST solution and incubated with primary antibodies overnight at 4°C. On the next day, the membranes were washed with a 1X TBST solution and again incubated with the secondary antibodies for a duration of 1 to 2h (either anti-mouse/anti-rabbit depending on the primary source of the antibody). Furthermore, it was washed with 1X TBST, and to detect the protein level, the ECL fluorescence reagent (EzWestLumiOne, ATTO, Tokyo, Japan) was used. The x-ray films were quantified using ImageJ software (v. 1.50, NIH, Bethesda, MD, USA). Whereas β-actin normalizes the expression levels of the protein of interest, guarantees uniform protein loading and transfer across lanes, and acts as an internal reference to take experimental condition heterogeneity into account.

All the mice for the confocal study were perfused transcardially with normal saline (0.9%) following 4% paraformaldehyde for a duration of 4 min. The brain was isolated from each mouse fixed with 4% paraformaldehyde for 72h. Similarly, all the brains were kept in 20% sucrose for a duration of 72h. Now, the brain was blocked with optimal cutting temperature compound (O.C.T. compound) (Sakura Finetek USA, Inc., Torrance, CA, USA) and stored at −80°C. The brain was sliced (14 µm) using a CM3050C cryostat (Leica, Germany). All the brain sections were thaw-mounted on gelatin-coated slides (Fisher, Rockford, IL, USA) and stored again at −80°C.

Immunofluorescence analysis was performed as described previously (27). The slides were washed twice with filtered phosphate buffer saline (PBS, 1%) for 10 min at room temperature. Apply the proteinase K solution for 5 min and wash again with 1% PBS. The slides were then incubated for 1h with a specific solution consisting of 2% normal goat serum and 0.3% Triton X-100 dissolved in 1% PBS. The slides were then incubated with required antibodies (diluted in PBS, 1:100) overnight and kept at 4°C. On the next day, the slides were washed and incubated with secondary antibodies for 2h. The nuclear staining dye 4′, 6-diamidino-2-phenylindole was applied for 10 min. Now, the slides were dried, a few drops of mounting media (DAKO) were put on, and they were covered with coverslips. The slides were ready to observe under confocal laser microscopes, and the images were quantified by using ImageJ software. All the images were graphically and statistically analyzed by GraphPad Prism software (ver. 8.0, San Diego, CA, USA) (28).

Investigations were conducted on the impact of ambroxol treatment on SOD activity and malondialdehyde (MDA), which is the oxidative stress marker. The SOD assay was carried out according to previous studies (29). In summary, the 96 well plates (200 μl reaction mixture) were filled with a mixture of tris-EDTA (50 mM, pH 8.5), pyragallol (24 mM), and sample. The absorbance was recorded at 420 nm. Each activity was conducted three times independently. Additionally, the LPO rate was calculated using modified Utley et al. protocols by calculating the MDA concentration. The assay mixture is made up of 200 μl supernatant, 20 μl mM ferric chloride, 200 μl 100 mM ascorbic acid, and 580 μl of 0.1 mM phosphate buffer (pH 7.4). It is then incubated for 60 min at 37°C in a water bath. Following 1h of incubation, samples were treated with 1000 μl of 10% trichloroacetic acid and 1000 μl of 0.66% thiobarbituric acid (TBA) to stop the reaction. The tubes were kept in the water bath for 20 min, chilled in an ice bath, and then centrifuged for a 10-min period at 3000 × g. The concentration of thiobarbituric acid reactive substances (TBARS) was determined at 535 nm utilizing supernatant absorbance and a blank solution containing all reagents except the test sample. The result was expressed as nM TBARS/min/mg protein.

The data were analyzed through ImageJ software in order to measure the density, which is expressed as an arbitrary unit (AU) for both morphological and Western blot analyses. All the data were shown in mean ± standard error mean (SEM) of eight mice per group. All the groups are compared with each other by using one-way analysis of variance with Tukey’s post hoc test. P-values less than 0.05 were considered to be statistically significant. The symbol # indicates a significant difference from the saline-injected group, while the symbol * indicates a significant difference 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.

Previous literature has shown that there is enhanced expression of TLR4 with LPS, due to which activation of astrocytes and microglia occurs (30). To analyze the effects of LPS on TLR4 and its downstream inflammatory markers in the mouse cortex and hippocampus, we performed Western blotting for TLR4, Iba-1, GFAP, p-NFkB, and p-JNK. According to our results, there was increased expression of the above-mentioned inflammatory biomarkers with LPS administration, which was significantly downregulated in the LPS+Amb co-treated group ( Figure 2A ). These beneficial effects were further confirmed by fluorescent microscopy, using GFAP antibody, showing elevated immunoreactivity in the cortical and hippocampal regions in the LPS–injected groups that were significantly attenuated with the LPS+Amb group. Both WB and IF results suggest that Ambroxol may inhibit the upregulation of TLR4 and p-NFkB, which may aid in the neuroprotective mechanisms of Ambroxol ( Figure 2B ).

Glial cell activation leads to the synthesis and secretion of TNF-α and interleukin-1β (IL-1β) and takes part in inflammatory diseases. To investigate the level of TNF- α and IL-1β we performed western blotting and immunofluorescences. Our western blot result revealed that both the levels of TNF- α and IL-1β were increased in the LPS-only subjected group. Similarly, this elevated level of TNF- α and IL-1β was remarkably decreased in the LPS+Amb co-treated group ( Figure 3A ). Additionally, we performed the immunofluorescences of brain tissues of different groups of experimental animals. The LPS-treated group showed high reactivity of TNF- α as compared to the controlled group. Whereas the LPS+Amb treated group showed less immunoreactivity as compared to the LPS-only treated group, as shown in Figure 3B .

Neuroinflammation leads to elevated oxidative stress and downregulates the level of antioxidant protein (NRF2 and HO-1) (31). The antioxidant activity of SOD in cortical and hippocampal tissues was significantly reduced following LPS injection. Ambroxol significantly alleviated this reduction. Furthermore, cells experience severe stress when phospholipid peroxidation leads to the production of MDA. The MDA levels were significantly elevated in the LPS group compared to the normal control group (p < 0.001). The attenuative potential of Ambroxol was further supported by the significant reduction (p < 0.001) in MDA levels, highlighting its effective role in mitigating oxidative stress ( Figure 4A ). Similarly, we assessed the concentrations of nuclear factor erythroid 2-related factor 2 (NRF2) and its subsequent antioxidant enzyme, oxygenase-1 (HO-1), in the cortical and hippocampal tissues of mice. Our Western blot result revealed decreased expression of NRF2 and HO-1 in LPS-injected mice as compared to the normal control, whereas Ambroxol attenuated the NRF2 and HO-1 expression caused by LPS in the brain tissue of the LPS-Amb co-treated mice. These results show that Ambroxol mitigated the level of oxidative stress by regulating the NRF2 and HO-1 expression level in the cortical and hippocampal tissues induced by LPS administration ( Figure 4B ). Additionally, in order to reinforce the western blot results, we performed the immunofluorescence analysis, which confirmed that the LPS-injected mice showed decreased immunoreactivity of NRF2, while the LPS+Amb co-treated group significantly reversed these effects and increased immunofluorescence reactivity of NRF2 in the cortex and hippocampus, suggesting its antioxidant potential ( Figure 4C ).

LPS-induced neuroinflammation and elevated oxidative stress collectively leads to neuronal toxicity and synaptic dysfunction. To measure integrity of synapsis we measured level of both pre and post synaptic biomarkers such as SNAP-23 and PSD-95 respectively. The Western blot results showed a reduced in expression of SNAP-23 and PSD-95 in the LPS-treated group as compared to the normal saline-injected group. Notably, the expressions of these synaptic biomarkers were upregulated in the LPS+Amb-cotreated group ( Figure 5A ). These effects of ambroxol were further confirmed by performing immunofluorescence of PSD-95 in cortex and hippocampus of different mice group. Our findings, suggest that PSD-95 expression was reduced in LPS-treated mouse brains compared to the saline-injected control group. Notably, the immunofluorescence of the PSD-95 was markedly increased in the LPS + Amb-cotreated group as shown in Figure 5B .

To examine the effects of ambroxol on learning and memory dysfunctions in LPS-injected mice, we performed the Y-maze tests and the MWM. First, we performed the Y-maze test, which showed that LPS leads to short-term special memory impairment. Interestingly, ambroxol treatment increased the percentage of spontaneous alternation behavior, which enhanced spatial working memory function ( Figures 6A, B ). In MWM, after the initial training, the animals were allowed to find the hidden platform, and the latency time was recorded in the MWM task. Our result showed that the LPS-injected mice spent more time in the target quadrant, while LPS+Amb-treated mice took less time to reach the target quadrant where the hidden platform was placed ( Figures 6C–E ). All of these results showed that ambroxol treatment improved learning and memory impairments in the LPS-injected animal model.