Activation of cholinergic anti-inflammatory pathway involved in therapeutic actions of α-mangostin on lipopolysaccharide-induced acute lung injury in rats

Abstract

Introduction: Alpha-mangostin (MAN) possesses a wide variety of pharmacological effects. In this study, we investigated its effect on cholinergic anti-inflammatory pathway (CAP), and tested if CAP regulation was involved in the therapeutic action on acute lung injury (ALI).

Methods: Male Sprague Dawley rats were pre-treated with MAN (40 mg/kg) for 3 days and ALI was induced with an intraperitoneal injection of lipopolysaccharide (LPS). Certain rats received monolateral vagotomy or sham surgery. The effects on inflammatory reactions and relevant pathways in ALI rats or LPS pre-treated RAW 264.7 cells were investigated by histological, immunohistochemical, immunoblotting, RT-qPCR, and immunofluorescence assays, while levels of proinflammatory cytokines, acetylcholine (Ach) and the enzymatic activity of acetylcholinesterase (AchE) were determined by corresponding quantitative kits.

Results: Oral administration of MAN reduced the severity of ALI, while vagotomy surgery antagonized this effect. MAN restored the decline in α7 nicotinic acetylcholine receptor (α7nAchR) in the lungs of ALI rats, and promoted the expression of α7nAchR and choline acetyltransferase (CHAT) in RAW 264.7 cells. Although AchE expression was barely affected by MAN at 5 μg/ml, its catalytic activity was reduced by almost 95%. Extracellular rather than intracellular Ach was notably raised shortly after MAN treatment. Furthermore, MAN at 5 μg/ml effectively inhibited LPS-induced increase in phosphorylation and nucleus translocation of p65 subunit, and secretion of TNF-α and IL-1β, which was then offset by methyllycaconitine citrate hydrate.

Conclusion: MAN activated CAP by increasing peripheral Ach and up-regulating α7nAchR expression, which eventually led to NF-κB inhibition and remission of acute inflammations.

Keywords: alpha7 nicotinic acetylcholine receptor; cholinergic anti-inflammatory pathway; immunity; inflammation; α-mangostin.

Figure 1.

Therapeutic effects of MAN on ALI rats and its regulatory effects on CAP in vitro. (a) Histological examination of rat lungs (H&E staining); (b) quantitative analyses of serological biomarkers in rats, assessed by the ELISA or colorimetric methods (n = 6); (c) α7nAchR expression in rat lungs, investigated by the immunohistochemical method; (d) quantification of experiment C (n = 3). For a and c: a-f represented healthy controls, healthy rats receiving vagotomy, healthy rats receiving sham surgery, ALI models, MAN treated ALI rats, and ALI rats receiving MAN treatment together with vagotomy surgery, respectively. Statistical significance: ^*P < 0.05 and ^**P < 0.01 compared with ALI models in b; ^*P < 0.05 and ^**P < 0.01 compared with normal healthy controls in d.

Figure 2.

MAN up-regulated α7nAchR/CHRNA7 expression in RAW 267.4 cells in vitro. (a) Levels of gene CHRNA7 expression, investigated by RT-qPCR (n = 3); (b) levels of protein α7nAchR expression, investigated by immunoblotting assay (n = 3). Statistical significance: ^**P < 0.01 compared with untreated cells; ^#P < 0.05 and ^##P < 0.01 compared with cells treated by MAN at 5 μg/ml.

Figure 3.

Possible mechanism involved in the regulation of MAN on Ach levels. (a) Quantitative results of intracellular and extracellular Ach levels in RAW 264.7 cells under MAN treatments at 5 μ/ml for varied time, determined by the colorimetric method (n = 3); (b) mRNA expression of gene CHAT and AchE, investigated by RT-qPCR (n = 3); (c) protein expression of CHAT and AchE, investigated by immunoblotting assay (n = 3); (d) enzymatic activity of AchE in RAW 264.7 cells under MAN treatments in vitro (n = 3). Statistical significance: ^*P < 0.05 and ^**P < 0.01 compared with untreated cells; ^#P < 0.05 compared with cells treated by MAN at 2.5 μg/ml.

Figure 4.

CAP activation contributed to the inhibition of MAN on NF-κB signaling in vitro. (a) Levels of pro-inflammatory cytokines secretion in LPS pre-treated RAW 264.7 cells receiving either LPS or in combination with test chemicals, determined by the ELISA method (n = 3); (b) levels of p-p65 expression in RAW 264.7 cells receiving treatments with either LPS or in combination with test chemicals, assessed by immunoblotting assay (n = 3); (c) effects of MAN and α7nAchR agonist/antagonist on p65 intracellular distribution in LPS pre-treated RAW 264.7 cells, investigated by immunofluorescence assay; D, nuclear distribution of p65 subunit in RAW 264.7 cells under different treatments, investigated by immunoblotting assay (n = 3). The concentration of MAN, Nic and MLA were 5 μg/ml, 5 μM, and 5 μM, respectively. Statistical significance: ^**P < 0.01 compared with LPS treated cells, ^##P < 0.01 compared with LPS+MAN treated cells.