Magnolol protects against acute gastrointestinal injury in sepsis by down-regulating regulated on activation, normal T-cell expressed and secreted

Abstract

Background: Sepsis is a major medical challenge. Magnolol is an active constituent of Houpu that improves tissue function and exerts strong anti-endotoxin and anti-inflammatory effects, but the mechanism by which it reduces intestinal inflammation in sepsis is yet unclear.

Aim: To assess the protective effect of magnolol on intestinal mucosal epithelial cells in sepsis and elucidate the underlying mechanisms.

Methods: Enzyme-linked immunosorbent assay was used to measure tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), IL-6, and regulated on activation, normal T-cell expressed and secreted (RANTES) levels in serum and ileal tissue in animal studies. The histopathological changes of the ileal mucosa in different groups were observed under a microscope. Cell Counting Kit-8 and cell permeability assays were used to determine the concentration of drug-containing serum that did not affect the activity of Caco2 cells but inhibited lipopolysaccharide (LPS)-induced decrease in permeability. Immunofluorescence and Western blot assays were used to detect the levels of RANTES, inhibitor of nuclear factor kappa-B kinase β (IKKβ), phosphorylated IKKβ (p-IKKβ), inhibitor of nuclear factor kappa-B kinase α (IκBα), p65, and p-p65 proteins in different groups in vitro.

Results: In rats treated with LPS by intravenous tail injection in the presence or absence of magnolol, magnolol inhibited the expression of proinflammatory cytokines, IL-1β, IL-6, and TNF-α in a dose-dependent manner. In addition, magnolol suppressed the production of RANTES in LPS-stimulated sepsis rats. Moreover, in vitro studies suggested that magnolol inhibited the increase of p65 nucleation, thereby markedly downregulating the production of the phosphorylated form of IKKβ in LPS-treated Caco2 cells. Specifically, magnolol inhibited the translocation of the transcription factor nuclear factor-kappa B (NF-κB) from the cytosol into the nucleus and down-regulated the expression level of the chemokine RANTES in LPS-stimulated Caco2 cells.

Conclusion: Magnolol down-regulates RANTES levels by inhibiting the LPS/NF-κB signaling pathways, thereby suppressing IL-1β, IL-6, and TNF-α expression to alleviate the mucosal barrier dysfunction in sepsis.

Keywords: Anti-inflammation; Lipopolysaccharide; Magnolol; Nuclear factor-kappa B; Regulated on activation, normal T-cell expressed and secreted; Sepsis.

Figure 1

Hematoxylin and eosin staining of ileal tissues. A: Control group shows orderly arranged ileal mucosal villi; B: Model group shows damaged ileal mucosal villi. The epithelial cells at the villus tips were wiped off, the subepithelial capillaries exhibited congestion, the central lacteals were expanded, the lamina propria was exposed and disintegrated, and blood capillaries were bleeding, with ulcer formation and widened intracellular tight junctions; C-E: Ileal mucosal structure in rats of the LM (C), MM (D), and HM (E) groups, respectively. The morphology of ileal mucosal villi and intestinal glands was improved to varying degrees compared to panel B. The ileal mucosal morphology was improved in panel E compared to D and in D compared to C. Magnification, × 100. Control: The control group; Model: Severe sepsis group; LM: Low-dose magnolol group; MM: Middle-dose magnolol group; HM: High-dose magnolol group.

Figure 2

Effects of different concentrations of magnolol on Caco2 cell permeability induced by lipopolysaccharide. Cell permeability was evaluated to determine the concentration of magnolol that inhibited the lipopolysaccharide (LPS)-induced increased permeability of Caco2 cells. After adding LPS, the permeability of the cells was significantly increased (^bP < 0.01). After adding magnolol to LPS-treated Caco2 cells, the permeability of the cells was decreased in a dose-dependent manner (^bP < 0.01). Control: Treated with solvent only; 2 μmol/L, 5 μmol/L, and 10 μmol/L magnolol: Treated with different concentrations of magnolol; LPS: Treated with solvent and LPS (100 μg/mL); LPS + magnolol (2 μmol/L, 5 μmol/L, and 10 μmol/L): Treated with magnolol (2 μmol/L, 5 μmol/L, and 10 μmol/L) and LPS (100 μg/mL); OD520nm: Optical density at 520 nm; FITC: Fluorescein isothiocyanate isomer. LPS: Lipopolysaccharide.

Figure 3

Expression of regulated on activation, normal T-cell expressed and secreted protein in Caco2 cells in each group. A: The cells were pretreated with magnolol or solvent for 8 h, followed by lipopolysaccharide (LPS) (100 μg/mL) for 24 h. The content of RANTES in the supernatant of the four groups of cells was determined by ELISA. Data are presented as the mean ± SD; the comparison between different groups was performed using the LSD method in one-way ANOVA (^bP < 0.01); B: The cells were pretreated with magnolol or solvent for 8 h and then with LPS (100 μg/mL) for 24 h. The expression of RANTES protein in Caco2 cells in each group was detected by Western blot analysis. 0 or Solvent: Treated with solvent only; 10 μmol/L magnolol: Treated with 10 μmol/L magnolol; LPS: Treated with solvent and LPS (100 μg/mL); LPS + magnolol 10 μmol/L: Treated with magnolol 10 μmol/L and with LPS (100 μg/mL); GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; RANTES: Regulated on activation, normal T-cell expressed and secreted.

Figure 4

Nuclear factor-kappa B pathway-related protein expression and p65 nucleation in Caco2 cells in each group. A: The cells were treated with magnolol or solvent for 8 h and then with lipopolysaccharide (LPS) (100 μg/mL) for 24 h. The protein levels of phosphorylated inhibitor of nuclear factor kappa-B kinase β and p65 were assessed by Western blot; B: Fluorescence distribution of p65 in the nucleus of Caco2cells treated with magnolol or solvent for 8 h and then with LPS (100 μg/mL) for 24 h. Control or Solvent: Treated with solvent only; 10 μmol/L magnolol: Treated with 10 μmol/L concentration of magnolol; LPS: Treated with solvent and LPS (100 μg/mL); LPS + magnolol 10 μmol/L: Treated with magnolol 10 μmol/L and with LPS (100 μg/mL); GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; DAPI: 4,6-guanidine-2-phenylindole; IKKβ: Inhibitor of nuclear factor kappa-B kinase β; p-IKKβ: Phosphorylated inhibitor of nuclear factor kappa-B kinase β; IκBα: Inhibitor of nuclear factor kappa-B kinase α; LPS: Lipopolysaccharide.