Humic substances from composted fennel residues control the inflammation induced by Helicobacter pylori infection in AGS cells

Abstract

Helicobacter pylori (H. pylori) is a common human pathogen causing inflammation. Recent studies have suggested a sophisticated interplay between mitochondria, innate immunity and inflammatory response, thus proposing mitochondrial disfunction as the hallmark of severe inflammatory disorders. In this study, humic substances isolated from composted fennel residues (HS-FEN) were tested as potential therapeutical strategy to restore the mitochondrial physiology and control the inflammation associated with H. pylori infection. The molecular features of HS-FEN were characterized by infrared spectrometry, thermochemolysis-GC/MS, NMR spectroscopy, and high-performance size-exclusion chromatography (HPSEC), which revealed the presence of aromatic polyphenolic components arranged in a rather stable conformation. In vitro results showed antioxidant and anti-inflammatory properties of HS-FEN, that was found to increase the expression level of OPA-1 and SOD-2 genes and in AGS cells stimulated with H. pylori culture filtrate (Hpcf) and concomitantly decrease the expression level of Drp-1 gene and IL-12, IL-17 and G-CSF proteins. The hydrophobic features of HS, their conformational arrangement and large content of bioactive molecules may explain the beneficial effects of HS-FEN, that may potentially become an interesting source of anti-inflammatory agents capable to counteract or prevent the H. pylori-related inflammatory disorders.

Fig 1. FTIR-DRIFT spectrum of humic substances from fennel green compost.

Fig 2. ¹³C-CPMAS-NMR spectrum of HS-FEN.

Fig 3. HPSEC chromatograms of HS-FEN before and after addition of acetic acid (AcOH) to lower solution pH from 7 to 3.5.

Fig 4. Antioxidant activity of HS-FEN at different concentration (50, 30, 25 μg mL^-1).

Vertical bars represent the standard deviation (s.d.). Different capital letters indicate significant differences according to Tukey test (p ≤ 0.05).

Fig 5. Cell viability and CC₅₀.

MTT assay showing the AGS cells viability after incubation with increasing concentrations of HS-FEN (spanning from 6 μg mL^-1 to 500 μg mL^-1) for 24 hours. The concentration of 65.93 μg/mL was estimated as that inhibiting 50% of cell viability (CC₅₀).

Fig 6. Hpcf affects gastric epithelial cell viability.

MTT assay showing the AGS cells viability after incubation with different concentrations of Hpcf for 24 hours. The assay was performed testing Hpcf as it is (Hpcf) or diluted with uninoculated broth medium (Hpcf 1:2; Hpcf 1:4; Hpcf 1:8). One-way ANOVA followed by Bonferroni post hoc correction was used to determinate statistically significant differences (*** p <0.0001).

Fig 7. HS-FEN subverts detrimental effects elicited by Hpcf.

Cellular expression of OPA-1 (a), Drp-1 (b) and SOD2 (c) genes detected by quantitative PCR performed on RNA extracted from AGS cells cultured for 3 hours with HS-FEN 25 μg mL^-1 in presence or absence of Hpcf (1:2) stimulation. GAPDH was used as housekeeping gene to normalize all samples. Data were represented as means ± s.d. of three independent experiments, each performed in triplicate. One-way ANOVA followed by Bonferroni post hoc correction was used to determinate statistically significant differences (*** p <0.0001).

Fig 8. HS-FEN mitigates Hpcf-induced inflammation.

Expression level of cytokines IL-12, IL-17 and G-CSF detected in AGS culture medium. Graph reports pg of cytokine in mL of cell medium differently treated: 1) HS-FEN 25 μg mL^-1; 2) Hpcf 1:2 for 12 hours; 3) Hpcf 1:2 + HS-FEN 25 μg mL^-1 for 12 hours. Values were normalized to basal activity (control cells) and data were represented as means ± s.d. of three independent experiments, each performed in triplicate. One-way ANOVA followed by Bonferroni post hoc correction was used to determinate statistically significant differences (** p<0.001; *** p <0.0001).