Flavonoids of Polygonum hydropiper L. attenuates lipopolysaccharide-induced inflammatory injury via suppressing phosphorylation in MAPKs pathways

Abstract

Background: Polygonum hydropiper L. is widely used as a traditional remedy for the treatment of dysentery, gastroenteritis. It has been used to relieve swelling and pain, dispel wind and remove dampness, eliminate abundant phlegm and inflammatory for a long time. Previous study showed that antioxidants especially flavonoids pretreatment alleviated sepsis-induced injury in vitro and in vivo. In the present study, the possible anti-inflammatory effect of flavonoids from normal butanol fraction of Polygonum hydropiper L. extract (FNP) against inflammation induced by lipopolysaccharide (LPS) was evaluated in vivo and in vitro.

Methods: The content of total flavonoid of FNP was determined by the aluminum colorimetric method. The content of rutin, quercetin and quercitrin was determined by HPLC method. Mice received FNP orally 3 days before an intra-peritoneal (i.p.) injection of lipopolysaccharide (LPS). Total superoxidase dismutase (T-SOD), total antioxidant capacity (T-AOC), glutathione peroxidase (GSH-PX), glutathione (GSH), myeloperoxidase (MPO) and malondialdehyde (MDA) levels were measured. Tumor necrosis factor-α levels in serum and tissue was measured. mRNA expressions of pro-inflammatory cytokines in lung were assessed by Real-Time PCR. Histopathological changes were evaluated in lung, ileum and colon. We also investigated FNP on reactive oxygen species (ROS), nitric oxide (NO) and pro-inflammatory cytokines (TNF-α, IL-1β, IL-6 and IL-8) production, inducible nitric oxide synthase (iNOS), Cyclooxygenase-2 (COX-2) protein expression, phosphorylation of MAPKs and AMPK in LPS-stimulated RAW264.7 cells.

Results: FNP increased the levels of T-SOD, T-AOC, GSH-PX and GSH, decreased the levels of TNF-α, MPO and MDA, attenuate the histopathological lesion in LPS-stimulated mice. FNP inhibited production of inflammatory cytokines, ROS and NO, protein expressions of iNOS and COX-2, phosphorylation of ERK, JNK and c-JUN in MAPKs, promoted phosphorylation of AMPKα suppressed by LPS.

Conclusion: These results suggested in vivo anti-inflammatory activities of FNP might contributed to its enhancement in antioxidant capacity, its inhibitory effects may be mediated by inhibiting the phosphorylation of JNK, ERK and c-JUN in MAPKs signaling pathways.

Fig. 1

HPLC analysis of FNP. Notes: The isolated compounds were identified in the extract by comparing their retention times. The chromatograms were obtained at a wavelength of 254nm. (1) rutin (2) quercitrin (3) quercetin

Fig. 2

Protective effect of FNP on lung, ileum and colon in LPS-stimulated mice. Notes: The mice in the LPS group were injected intraperitoneally with 17 mg/kg (body weight) of LPS, while control group were injected with PBS (10 mL/kg). FNP50, FNP100 and FNP200 group were orally administrated with diverse dose FNP (50 mg/kg, 100 mg/kg and 200 mg/kg) for 3 days before instillation of LPS. Histopathological studies by light microscope showing morphologically normal lung, ileum and colon tissues from mice in the control group (a, d and g). Incrassation of alveolar wall, serous exudation, hemorrhage and infiltration of PMN in lung from LPS-treated group (b) and no serous exudation or hemorrhage and less incrassation of alveolar wall and infiltration by PMN in lung from LPS-treated mice treated with FNP (c). Necrosis of epithelial cell, disorder of epithelial cell arrangement and fall of epithelium of intestinal villus in ileum and colon from LPS-treated group (e and h) and less necrosis of epithelial cell in ileum and colon from LPS-treated mice treated with FNP (f and i). Tissue sections were stained with hematoxylin and eosin and view by light microscopy (200 and 400×)

Fig. 3

Effects of FNP pretreatment on TNF-α levels in blood and tissues, mRNA expression in lung. Notes: The mice in the LPS group were injected intraperitoneally with 17 mg/kg (body weight) of LPS, while control group were injected with PBS (10 mL/kg). FNP50, FNP100 and FNP200 group were orally administrated with diverse dose FNP (50 mg/kg, 100 mg/kg and 200 mg/kg) for 3 days before instillation of LPS. Serum and lung were collected, liver and intestine homogenates were prepared. TNF-α levels of serum and homogenates were shown in (a). The level of mRNA expression in lung is expressed as fold-change relative to the control group (b). The mRNA expression of TNF-α, IFN-α, IFN-γ and IL-2 was analyzed by qRT-PCR. Data are presented as the means ± SD from three independent experiments (n = 10). _## P <0.01 vs control group; _* P <0.05 vs LPS group; _** P <0.01 vs LPS group

Fig. 4

Effects of FNP on antioxidant capacity in LPS-stimulated mice. Notes: The mice in the LPS group were injected intraperitoneally with 17 mg/kg (body weight) of LPS, while control group were injected with PBS (10 mL/kg). FNP50, FNP100 and FNP200 group were orally administrated with diverse dose FNP (50 mg/kg, 100 mg/kg and 200 mg/kg) for 3 days before instillation of LPS. Serum, homogenates of small intestine and liver were prepared. (a) MDA level in small intestine. (b) MPO level in small intestine. (c) T-AOC level in liver. (d) T-SOD level in liver. (e) GSH-PX level in liver. (f) GSH level in serum. (g) ACP level in serum. (h) LZM level in serum. Each column represented as the means ± SD from three independent experiments (n = 10). _## P <0.01 vs control group; _* P <0.05 vs LPS group; _** P <0.01 vs LPS group

Fig. 5

Cytotoxic effects of FNP in RAW264.7 cells (a) and the effects of FNP on LPS-induced NO (b), and ROS (c) productions in RAW264.7 macrophages. Notes: The cells were incubated for 24 h with 1 μg/mL of LPS in the absence or presence of FNP (20, 40, and 80 μg/mL). FNP was added 1 h before incubation with LPS. Cell viability assay was performed by using MTT assay. Nitrite concentration in the medium was determined by using Griess reagent. ROS production was determined by fluorescent probe DCFH-DA. Each column represented as the means ± SD from three independent experiments. _## P <0.01 vs control group; _* P <0.05 vs LPS group; _** P <0.01 vs LPS group

Fig. 6

Effects of FNP on LPS-induced TNF-α (a) and IL-6, IL-8, IL-10 and IL-1β (b) productions in RAW264.7 macrophages. Notes: The cells were incubated for 24 h with 1 μg/mL of LPS in the absence or presence of FNP (20, 40, and 80 μg/mL). FNP was added 1 h before incubation with LPS. Inflammatory cytokine level were determined by cytokine-specific ELISA kit. Each column represented as the means ± SD from three independent experiments. _## P <0.01 vs control group; _* P <0.05 vs LPS group; _** P <0.01 vs LPS group

Fig. 7

Effects of FNP on iNOS and COX-2 protein expression in LPS-stimulated RAW264.7 cells. Notes: The cells were pretreated with different concentrations (40 μg/mL, 80 μg/mL) of FNP for 2 h or 4 h and stimulated with 1 μg/mL of LPS for 24 h. Total cellular proteins (50 μg) were separated by SDS-PAGE, then transferred to PVDF membrane and detected by Western blot analysis. Quantification of iNOS and COX-2 protein expression was normalized to β-actin using a densitometer. Each column represented as the means ± SD from three independent experiments. _## P <0.01 vs control group; _* P <0.05 vs LPS group; _** P <0.01 vs LPS group

Fig. 8

Effects of FNP on phosphorylation of MAPKs and AMPK in LPS-stimulated RAW264.7 cells. Notes: The cells were pretreated with different concentrations (40 μg/mL and 80 μg/mL) of FNP for 2 h or 4 h and stimulated with 1 μg/ml of LPS for 1 h. Total cellular proteins (50 μg) were separated by SDS-PAGE, then transferred to PVDF membrane and detected by Western blot analysis. Western blot results were shown in (a). Quantification of p-AMPKα, p-ERK1/2, p-P38, p-JNK and p-c-JUN protein expression was normalized to AMPKα, ERK1/2, P38, JNK and c-JUN using a densitometer. Each column represented as the means ± SD from three independent experiments. Results of 4h pretreatment were shown in (b) and results of 2 h pretreatment were shown in (c). _# P <0.05 vs control group; _##P <0.01 vs control group; _* P <0.05 vs LPS group; _** P <0.01 vs LPS group