Ferulic Acid Metabolites Attenuate LPS-Induced Inflammatory Response in Enterocyte-like Cells

Abstract

Ferulic acid (FA) is a polyphenol pertaining to the class of hydroxycinnamic acids present in numerous foods of a plant origin. Its dietary consumption leads to the formation of several phase I and II metabolites in vivo, which represent the largest amount of ferulates in the circulation and in the intestine in comparison with FA itself. In this work, we evaluated their efficacy against the proinflammatory effects induced by lipopolysaccharide (LPS) in intestinal Caco-2 cell monolayers, as well as the mechanisms underlying their protective action. LPS-induced overexpression of proinflammatory enzymes such as inducible nitric oxide synthase (iNOS) and the consequent hyperproduction of nitric oxide (NO) and cyclic guanosine monophosphate (cGMP) were limited by physiological relevant concentrations (1 µM) of FA, its derivatives isoferulic acid (IFA) and dihydroferulic acid (DHFA), and their glucuronidated and sulfated metabolites, which acted upstream by limiting the activation of MAPK p38 and ERK and of Akt kinase, thus decreasing the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-ĸB) translocation into the nucleus. Furthermore, the compounds were found to promote the expression of Nrf2, which may have contributed to the downregulation of NF-ĸB activity. The overall data show that phase I/II metabolites retain the efficacy of their dietary free form in contrasting inflammatory response.

Keywords: MAP kinases; inflammation; intestinal cells; lipopolysaccharide; metabolites; nitric oxide; nitric oxide synthase; polyphenols.

Figure 1

Percentage of cell viability compared with the control (0 μM, 100% viable cells) of Caco-2 cells incubated for 48 h with different concentrations of ferulic acid (FA), dihydroferulic acid (DHFA), isoferulic acid (IFA), ferulic acid glucuronide (FA glu), dihydroferulic acid glucuronide (DHFA glu), and isoferulic acid sulfate (IFA sulf; 0.5–5 μM). Each column represents the mean ± SD of the independent experiments (n = 12).

Figure 2

Level of iNOS dosed by Western blot and reported as percentage of the control, in Caco-2 cells treated with lipopolysaccharide (LPS) (1 μg/mL) for 48 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of the independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; * = significant vs. LPS (p < 0.001); # = significant vs. control (p < 0.001); ° = significant vs. LPS, DHFA glu + LPS and IFA sulf + LPS (p < 0.01); § = significant vs. LPS (p < 0.05), DHFA glu + LPS and IFA sulf (p < 0.001); a = significant vs. FA glu + LPS and IFA + LPS (p < 0.05). A representative WB picture of the experiment is shown. β-actin detection was used as a loading control for each sample.

Figure 3

Expression of iNOS dosed by qRT-PCR and reported as a percentage of the control, in Caco-2 cells treated with LPS (1 μg/mL) for 48 h and pretreated for 30 min with with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) prior to LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; # = significant vs. LPS (p < 0.001); ° = significant vs. control (p < 0.001); * = significant vs. FA glu + LPS (p < 0.05); § = significant vs. DHFA + LPS (p < 0.001); a = significant vs. FA (p < 0.01).

Figure 4

NO release (expressed as μM of nitrites) in Caco-2 cells treated with LPS (1 μg/mL) for 48 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 16). Significant differences among groups are described using different superscript symbols; * = significant vs. control (p < 0.01); ° = significant vs. control (p < 0.05); # = significant vs. LPS (p < 0.001); § = significant vs. IFA sulf + LPS (p < 0.05); a = significant vs. IFA sulf + LPS (p < 0.001).

Figure 5

cGMP release (pmol/mg proteins) in Caco-2 cells incubated for 48 h with LPS (1 µg/mL) after pretreatment with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) (n = 9). Control and LPS groups were pretreated with an equivalent volume of methanol. Significant differences among groups are described using different superscript symbols; * = significant vs. control (p < 0.001); # = significant vs. LPS (p < 0.001); ° = significant vs. IFA + LPS (p < 0.05); § = significant vs. LPS and DHFA + LPS (p < 0.01); a = significant vs. DHFA + LPS (p < 0.001); b = significant vs. control (p < 0.01).

Figure 6

Degradation of IĸBα reported as percentage of the control phospho IĸBα/total IĸBα ratio in Caco-2 cells treated with LPS (1 μg/mL) for 48 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; * = significant vs. LPS (p < 0.001); # = significant vs. control (p < 0.001); ° = significant vs. FA glu + LPS (p < 0.05); § = significant vs. LPS and FA glu + LPS (p < 0.01). Representative WB picture of the experiment is shown.

Figure 7

Activation of Akt reported as percentage of the control phospho Akt/total Akt ratio in Caco-2 cells treated with LPS (1 μg/mL) for 2 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; # = significant vs. control (p < 0.001); * = significant vs. LPS (p < 0.001); § = significant vs. LPS (p < 0.05) and vs. FA glu + LPS (p < 0.001); ° = significant vs. FA + LPS, IFA + LPS, DHFA glu + LPS and IFA sulf + LPS (p < 0.01). Representative WB picture of the experiment is shown.

Figure 8

Modulation of MAPK p38 reported as a percentage of the control phospho p38/total p38 ratio in Caco-2 cells treated with LPS (1 μg/mL) for 2 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of the independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; * = significant vs. LPS (p < 0.001); # = significant vs. control (p < 0.05); ## = significant vs. control (p < 0.01). Representative WB picture of the experiment is shown.

Figure 9

Modulation of MAPK ERK1/2 reported as a percentage of the control phospho ERK1/2/total ERK1/2 ratio in Caco-2 cells treated with LPS (1 μg/mL) for 2 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; * = significant vs. LPS (p < 0.001); # = significant vs. control, FA + LPS and IFA sulf + LPS (p < 0.001); § = significant vs. FA + LPS (p < 0.05). Representative WB picture of the experiment is shown.

Figure 10

Level of Nrf2 dosed by Western blot and reported as a percentage of the control in Caco-2 cells treated with LPS (1 μg/mL) for 48 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; * = significant vs. control (p < 0.001) and LPS (p < 0.001). Representative WB picture of the experiment is shown. β-actin detection was used as a loading control for each sample.

Figure 11

Expression of Nrf2 dosed by qRT-PCR and reported as a percentage of the control in Caco-2 cells treated with LPS (1 μg/mL) for 6 h and pretreated for 30 min with FA, DHFA, IFA, FA glu, DHFA glu, and IFA sulf (1 μM) preceding LPS co-exposure. Control and LPS groups were pretreated with an equivalent volume of methanol. Each column represents the mean ± SD of independent experiments (n = 6). Significant differences among groups are described using different superscript symbols; # = significant vs. control (p < 0.001); * = significant vs. LPS (p< 0.001); ° = significant vs. LPS (p < 0.01); § = significant vs. FA + LPS (p < 0.001) and vs. DHFA + LPS (p < 0.01); a = significant vs. IFA + LPS and DHFA glu + LPS (p < 0.01).