Abstract
Background/Aim
Acute lung injury (ALI) is associated with a high mortality rate and cancer patients who receive chemotherapy are at high risk of ALI during neutropenia recovery. Galantamine is a cholinesterase inhibitor used for Alzheimer’s disease treatment. Previous studies have shown that galantamine reduced inflammatory response in lipopolysaccharide (LPS)-induced ALI in rats. Mer protein was negatively associated with inflammatory response. The aim of the study was to investigate whether galantamine is effective in LPS-induced ALI during neutropenia recovery and its effect on Mer tyrosine kinase (MerTK) expression in mice.
Materials and Methods
Intraperitoneal cyclophosphamide was given to mice to induce neutropenia. After 7 days, LPS was administered by intratracheal instillation. Intraperitoneal galantamine was given once before LPS administration and in another group, galantamine was given twice before LPS administration.
Results
Galantamine attenuated LPS-induced ALI in histopathological analysis. The neutrophil percentage was lower in the group where galantamine was injected once, compared to the LPS group (p=0.007). MerTK expression was also higher in the group where galantamine was injected once but did not reach statistical significance (p=0.101).
Conclusion
Galantamine attenuated inflammation in LPS-induced ALI during neutropenia recovery.
Keywords: Cholinesterase inhibitor, acute lung injury, neutropenia recovery, mer tyrosine kinase
Neutropenia is common in cancer patients who receive chemotherapy. Respiratory deterioration occurs during resolution of neutropenia which might progress to acute lung injury (ALI) and acute respiratory distress syndrome (ARDS) (1). ALI was associated with significant mortality and the incidence of ALI has increased annually, over the past few years (2,3).
The process of ALI is mediated by the increase of proinflammatory cytokines. Cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1,6 are classified as early proinflammatory cytokines and high mobility group box (HMGB)1 is classified as a late pro-inflammatory cytokine (4,5). Therapeutics targeting early proinflammatory cytokines have not resulted in clinical improvement since these early cytokines resolved first and anti-cytokine agents were given after the early cytokines peaked (6). HMGB1 is one of the late mediators of lipopolysaccharide (LPS) lethality and sepsis (7,8). Inhibition of HMGB1 activity has been reported to increase survival in a murine sepsis model (9).
Galantamine is an acetylcholinesterase (AChE) inhibitor which ameliorates cognitive impairment in Alzheimer’s disease (AD) (10). A previous study has shown that the vagus nerve downregulates inflammation by a mechanism termed “cholinergic anti-inflammatory pathway” (11). Acetylcholine is a neurotransmitter in the brain and acts through muscarinic and nicotinic receptors. The α7 subunit of the nicotinic acetylcholine receptor mediates the anti-inflammatory activity of the vagus nerve (11). Galantamine is a nicotinic acetylcholine receptor agonist which enhances cholinergic anti-inflammatory pathway and downregulated HMGB1 expression in LPS-induced acute lung injury (2). Galantamine has also had an inhibitory effect on TNF- α in LPS-induced peritonitis in rats (12).
Mer is a tyrosine kinase receptor (MerTK) and Mer signaling plays an important role in the intrinsic inhibition of the inflammatory response to Toll-like receptor activation. Lung Mer protein expression decreases after LPS injection and upregulation of Mer signaling suppresses lung inflammatory response (13). Phosphatidylserine (PS) activates Mer kinase and neutrophil elastase (NE) promotes inflammation by cleaving the PS receptor (14). NE inhibition upregulates the Mer signaling pathway and attenuates LPS-induced ALI during neutropenia recovery in mice (15).
A previous study has shown that galantamine has a protective effect on LPS-induced ALI in rats (2). However, no previous studies have investigated whether galantamine attenuates LPS-induced ALI during neutropenia recovery in mice and the effect of galantamine on Mer signaling. In this regard, the aim of this study was to investigate whether galantamine is effective in LPS-induced ALI during neutropenia recovery and whether it enhances Mer expression and activates the Mer signaling pathway.
Materials and Methods
Animals. Six-week-old female mice (18g-20g) were obtained from The Orient Bio Experimental Animal Center (Gyonggi, Republic of Korea). The mice were housed in pathogen-free cages and were fed with food and water ad libitum. Mice were randomly assigned into four groups: (A) control (n=7) (B) cyclophosmide+LPS (n=7), (C) cyclophosimide+LPS+galantamine (5 mg/kg) and (D) cyclophomide+ LPS+galantamine (5 mg/kg) injected twice. To induce neutropenia, all mice except control mice were given intraperitoneal cyclo-phosphamide (150 mg/kg on day 5 before neutropenia, followed by 100 mg/kg on day 2 before neutropenia). In groups (B), (C) and (D), mice were administered 2 μg LPS by intratracheal instillation 2 days after neutropenia. In group (C), 5mg/kg galantamine was given by intraperitoneal injection 30 minutes before LPS administration. In group (D), 5mg/kg galantamine was given twice: 5hours and 30 minutes before LPS administration. All experimental mice were treated according to the guidelines approved by the Animal Subjects Committee of the Catholic University of Korea (UJA2023-03A).
Bronchoalveolar lavage (BAL). The left lung was subjected to BAL. After washing the lung with 0.8 ml of sterile saline, BAL fluid was collected. The number of total cells in BAL was counted by a hemocytometer. BAL fluid was processed by cytospin (5 minutes at 750 rpm) onto microscope slides and stained with Diff-Quick (Sysmax, Tokyo, Japan).
Histopathological analysis. Mice were sacrificed 24 h after LPS administration. The obtained lung tissue specimens were fixed with 4% formaldehyde for 24 h, embedded in paraffin and cut into 4 μm thick sections using a microtome. Deparaffinized tissue sections were then stained with hematoxylin and eosin (H&E).
Statistical analysis. All statistical analyses were performed using R (version 4.02; The R foundation, Vienna, Austria). All data are expressed as mean±standard deviation. p-Values <0.05 indicate statistical significance. Differences between groups were evaluated by the Kruskal-Wallis test with Dunn’s test used for post hoc analysis.
Results
Histopathological analysis. The cyclophosmide+LPS group showed destruction of pulmonary cell walls, edema, and infiltration of inflammatory cells. Mice pre-treated with galantamine 30 min before the LPS injection (group C) showed significant attenuation of inflammation. Mice pre-treated with galantamine 5 h and 30 min before the LPS injection (group D) also showed an attenuation of inflammation (Figure 1).
Figure 1. Effect of galantamine on histopathological changes in 4 groups. Hematoxylin and eosin staining: (A). Control group; normal lung structure. (B). Lipopolysaccharide injected group; infiltration of inflammatory cells. (C) Galantamine 5 mg/kg injected-once group. (D) Galantamine 5 mg/kg injected-twice group; inflammation was attenuated by galantamine.
Effects of galantamine on inflammatory cells. As shown in Table I, intratracheal LPS administration resulted in a significant increase in the total number of cells, number of neutrophils and proportion of neutrophils in BAL fluid. Differences between the total cell count and neutrophil count were not statistically significant between groups. The proportion of neutrophils was significantly lower in group C (p=0.017, Figure 2).
Table I. Results of BAL fluid analysis (105/ml).
Results are presented as mean±standard deviation. LPS: Lipopolysaccharide.
Figure 2. Effects of galantamine on the neutrophil percentage in bronchial alveolar lavage fluid. LPS: Lipopolysaccharide.
Effects of galantamine on MerTK mRNA expression. Real time RT-PCR was conducted to evaluate the effects of galantamine on the mRNA expression of MerTK. MerTK expression was decreased after LPS administration and increased in group C. MerTK mRNA expression in group D was similar to the LPS injected group. However, these results were not significant (p-value=0.101) (Figure 3).
Figure 3. Effects of galantamine on MerTK mRNA expression. Error bars represent standard error of the mean (SEM). LPS: Lipopolysaccharide.
Discussion
In this study, we evaluated the effect of galantamine in LPS-induced ALI during neutropenia recovery in a murine model. Galantamine has been approved as a treatment for AD and has a half-life of approximately 7 hours. AD patients have cognitive deficits which are related to decreased numbers of cholinergic neurons. By inhibiting the AchE enzyme, which catalyzes the breakdown of acetylcholine, the availability of acetylcholine increases. This inhibits the degeneration of cholinergic neurons (16). The increase of acetylcholine increases the cholinergic system and vagus nerve activity which leads to the inhibition of the inflammatory response. Galantamine significantly lowered the levels of serum pro-inflammatory cytokines and alleviated the severity of ARDS in mice (17). Galantamine also ameliorated inflammation caused by hyperoxia in mice (18).
AD and cancer are one of the leading causes of death worldwide. Previous studies have shown that there is an inverse association between AD and cancer. Cancer survivors had a lower risk of AD and AD patients had a lower risk of cancer (19,20). However, since cancer and AD are common in ageing populations, there is a portion of patients who have both AD and cancer. AD patients with cancer can be treated with chemotherapy; development of ALI during neutropenia recovery might be critical to those patients. In this study, we have shown that galantamine, which is a commonly prescribed drug in AD patients, decreased inflammation and contributed to the attenuation of LPS-induced ALI during neutropenia recovery in mice.
Mice were randomly assigned to 4 groups: (A) control, (B) cyclophosmide+LPS, (C) cyclophosimide+LPS+galantamine (5mg/kg) injected 30 minutes before LPS administration and, (D), cyclophosmide+LPS+galantamine (5mg/kg) injected 5 hours and 30 minutes before LPS. To evaluate the dose effect of galantamine, galantamine was given twice in group (D). Considering the half-life and peak time of galantamine, it was administered 5 hours and 30 minutes before LPS injection. As shown in Figure 2, inflammation was reduced in both group C and D.
Neutrophils produce elastase, reactive oxygen species and antimicrobial peptides which can activate cell surface receptors or modulate signal transduction pathways. However, under pathological situations, unregulated release of these compounds can paradoxically damage the host tissues (21). Neutrophil elastase induces the production of inflammatory cytokines and enhances neutrophil migration (22). Lee et al. have shown that inhibition of neutrophil elastase reduced neutrophil activity and alleviated lung inflammation in a murine model (15). As shown in Table I and Figure 2, the proportion of neutrophils was significantly lower in the galantamine pretreatment group. Galantamine is a cholinesterase inhibitor and inhibits TNF-α release through the cholinergic anti-inflammatory pathway (12,23). Decrease of TNF-α is related to the decrease of neutrophil recruitment (24). Inhibition of neutrophil recruitment alleviates paradoxical damage and acute lung injury.
Mer protein expression suppressed the lung inflammatory response, and the expression was decreased after LPS administration (13). Neutrophil elastase attenuated LPS-induced ALI and this effect was associated with the increased expression of Mer (15). MerTK mRNA expression was decreased after LPS injection and galantamine increased its expression in our study. Therefore, the effect of galantamine on the attenuation of inflammation was associated with MerTK induction.
There are several limitations in this study. Firstly, the administration dose of galantamine was higher than clinical dosage. Galantamine could ameliorate ALI during neutropenia by enhancing the cholinergic anti-inflammatory pathway at the dose we used in this study. Secondly, there could be differences between mice and humans. Thirdly, we have shown that galantamine has increased MerTK mRNA expression but there were not enough samples to reach statistical significance. Further study is needed to investigate the pathway of galantamine and MerTK. Lastly, a previous study has shown that suppressing NF-ĸB/TLR4 pathway has protective effects against ALI (25). The effect of galantamine on the NF-ĸB/TLR4 pathway should also be investigated in future studies.
Conclusion
Galantamine had a protective effect against LPS-induced ALI during neutropenia recovery in mice. Histopathological analysis revealed that galantamine significantly attenuated inflammation in lung and the proportion of neutrophils was significantly lower when galantamine was administered. This effect may be explained by the increase of MerTK expression induced by galantamine.
Conflicts of Interest
The Authors have no potential conflicts of interest to report.
Authors’ Contributions
Conceptualization: OBK, JWK. Formal analysis: OBK, HJ. Investigation: KYK, WS, CDY. Software: OBK. Writing-original draft: OBK. Writing-review and editing: CDY, JWK. Supervision: JWK. All Authors read and agreed to the submitted version of the manuscript.
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