α-Mangostin Alleviated Lipopolysaccharide Induced Acute Lung Injury in Rats by Suppressing NAMPT/NAD Controlled Inflammatory Reactions

Abstract

α-Mangostin (MAN) is a bioactive xanthone isolated from mangosteen. This study was designed to investigate its therapeutic effects on acute lung injury (ALI) and explore the underlying mechanisms of action. Rats from treatment groups were subject to oral administration of MAN for 3 consecutive days beforehand, and then ALI was induced in all the rats except for normal controls via an intraperitoneal injection with lipopolysaccharide. The severity of disease was evaluated by histological examination and hematological analysis. Protein expressions in tissues and cells were examined with immunohistochemical and immunoblotting methods, respectively. The levels of cytokines and nicotinamide adenine dinucleotide (NAD) were determined using ELISA and colorimetric kits, respectively. It was found that MAN treatment significantly improved histological conditions, reduced leucocytes counts, relieved oxidative stress, and declined TNF-α levels in ALI rats. Meanwhile, MAN treatment decreased expressions of nicotinamide phosphoribosyltransferase (NAMPT) and Sirt1 both in vivo and in vitro, which was accompanied with a synchronized decline of NAD and TNF-α. Immunoblotting assay further showed that MAN downregulated HMGB1, TLR4, and p-p65 in RAW 264.7 cells. MAN induced declines of both HMGB1/TLR4/p-p65 and TNF-α were substantially reversed by cotreatment with nicotinamide mononucleotide or NAD. These results suggest that downregulation of NAMPT/NAD by MAN treatments contributes to the alleviation of TLR4/NF-κB-mediated inflammations in macrophage, which is essential for amelioration of ALI in rats.

Figure 1

Therapeutic effects of MAN on ALI in rats. (a) Histological examination of lung in rats: A-D (100 × magnification), E-H (400 × magnification), representative images selected from normal control, ALI model, MAN treated (low), and MAN treated (high) groups, respectively; (b) quantitative evaluation of pathological changes based on histological examination; (c) main cell types with significant population differences among groups; (d) serological differences among groups. Statistics significance: ^∗∗p < 0.01 compared with ALI models.

Figure 2

Regulation of MAN on NAMPT and Sirt1 in vivo. (a) Expression of iNAMPT in lung; (b) expression of Sirt1 in lung; (c) quantitative results of immunohistochemical assays; (d) levels of eNAMPT in serum. Photographs A-D represent normal control, ALI model, MAN treated (low), and MAN treated (high), respectively. Statistical significance: ^∗∗p < 0.01 compared with ALI models.

Figure 3

Regulation of MAN on NAMPT and Sirt1 in RAW 264.7 cells in vitro. (a) Expression of iNAMPT in cells; (b) levels of eNAMPT in culture medium; (c) expression of Sirt1 in cells; (d) deacetylation capability of Sirt1 indicated by expression of ace-p65 in cells. Statistical significance: ^∗p < 0.05 and ^∗∗p < 0.01 compared with LPS treated cells.

Figure 4

Direct interaction between MAN and NAMPT revealed by molecular docking simulation analysis.

Figure 5

Downregulation of MAN on NAD production contributed to reduce TLR4/NF-κB activation in RAW 264.7 cells in vitro. (a) Production of NAD in cells; (b) MAN inhibited activation of TLR4/NF-κB in a concentration-dependent manner; (c) cotreatments with NAD/NMN reversed MAN induced inhibition on TLR4/NF-κB: A, secretion of TNF-α by cells; B, expression of p-p65 in cells; C, expression of HMGB1 and TLR4 in cells. Statistical significance: ^∗∗p < 0.01 compared with LPS treated cells; ^##p < 0.01 compared with LPS+MAN treated cells.