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. 2016 Jul;12(1):499-505.
doi: 10.3892/etm.2016.3305. Epub 2016 Apr 28.

Tiliroside, the major component of Agrimonia pilosa Ledeb ethanol extract, inhibits MAPK/JNK/p38-mediated inflammation in lipopolysaccharide-activated RAW 264.7 macrophages

Xin Jin  1 , Shiqing Song  2 , Jing Wang  1 , Qingzhen Zhang  1 , Feng Qiu  3 , Feng Zhao  1
Affiliations

Tiliroside, the major component of Agrimonia pilosa Ledeb ethanol extract, inhibits MAPK/JNK/p38-mediated inflammation in lipopolysaccharide-activated RAW 264.7 macrophages

Xin Jin et al. Exp Ther Med. 2016 Jul.

Abstract

In the present study, the in vivo anti-inflammatory activity of Agrimonia pilosa Ledeb (AP) ethanol extract was confirmed in experimental animal models, including xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. Tiliroside, the major component of AP extract, was isolated and purified by high-performance liquid chromatography. The anti-inflammatory mechanism of tiliroside was then examined using lipopolysaccharide (LPS)-activated RAW 264.7 macrophage cells. An MTT assay was used to determine cytotoxicity and a Griess assay was used to determine nitric oxide (NO) production. Concentration levels of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) were determined by enzyme-linked immunosorbent assay. Protein expression levels of inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX-2), phosphorylated (p)-extracellular signal-regulated kinase (ERK) 1/2, p-c-Jun N-terminal kinases (JNK), p-p38 and inhibitor of κB-α were detected by western blot analysis. AP ethanol extract was revealed to inhibit xylene-induced ear edema in mice and carrageenan-induced paw edema in rats. Tiliroside significantly suppressed the overproduction of NO (P<0.01), but revealed no notable inhibition of the release of TNF-α and IL-6. In addition, tiliroside significantly downregulated the elevated expression levels of iNOS and COX-2 induced by LPS (P<0.01). The phosphorylation of JNK and p38 proteins were also significantly inhibited (P<0.01), however, tiliroside exhibited no obvious inhibition on the phosphorylation of ERK 1/2 and the degradation of IκB-α protein. In conclusion, the anti-inflammatory molecular mechanism of tiliroside may involve the downregulation of iNOS and COX-2 protein expression levels, and the inactivation of mitogen-activated protein kinase (MAPK)/JNK, in addition to the MAPK/p38 signaling pathway.

Keywords: Agrimonia pilosa Ledeb; cyclooxygenase-2; inducible nitric oxide synthase; mitogen-activated protein kinase; tiliroside.

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Figures

Figure 1.
Figure 1.
Chemical structure of tiliroside.
Figure 2.
Figure 2.
Effect of AP ethanol extract on xylene-induced ear edema in mice. Mice (n=8/group) were treated with 250, 500, 1,000 and 2,000 mg/kg AP ethanol extract or 5 mg/kg DEX-5. Both ears were harvested after 10 µl xylene was applied to the right ear. Data are presented as the mean ± standard deviation. **P<0.01 vs. vehicle control group. AP, Agrimonia pilosa; DEX-5, dexamethasine-5.
Figure 3.
Figure 3.
Effect of AP ethanol extract on carrageenan-induced paw edema in rats. Rats (n=8/group) were treated with 250, 500, 1,000 and 2,000 mg/kg AP ethanol extract or 5 mg/kg DEX-5. The paw volume was measured at selected intervals after 0.1 ml carrageenan was injected into the right-hind paw. Data are presented as the mean ± standard deviation. **P<0.01 vs. vehicle control group. AP, Agrimonia pilosa; DEX-5, dexamethasone-5.
Figure 4.
Figure 4.
Inhibitory effect of tiliroside on NO2− production induced by LPS. RAW 264.7 cells were treated with 1 µg/ml LPS with or without tiliroside (12.5, 25, 50 and 100 µM) or hydrocortisone (100 µM) for 24 h. NO2− concentrations were measured in triplicate. Data are presented as the mean ± standard deviation of three separate experiments. **P<0.01 vs. LPS treatment group; ##P<0.01 vs. untreated group. LPS, lipopolysaccharide; Hydro, hydrocortisone.
Figure 5.
Figure 5.
Effect of tiliroside on the release of (A) TNF-α and (B) IL-6. RAW 264.7 cells were treated with 1 µg/ml LPS with or without tiliroside (12.5, 25, 50 and 100 µM) or hydrocortisone (100 µM) for 6 h. The supernatant was analyzed to determine the expression levels of TNF-α and IL-6. Data are presented as the mean ± standard deviation of three separate experiments. **P<0.01 vs. LPS treatment group; ##P<0.01 vs. untreated group. LPS, lipopolysaccharide; Hydro, hydrocortisone; IL-6, interleukin-6. TNF-α, tumor necrosis factor-α.
Figure 6.
Figure 6.
Effect of tiliroside on the expression of iNOS and COX-2 proteins. (A) RAW 264.7 cells were treated with LPS 1 µg/ml with or without tiliroside (12.5, 25, 50 and 100 µM) for 24 h and the expression levels of iNOS and COX-2 were assessed by western blot analysis. Detection of β-actin was performed to confirm the equal loading of proteins. Densitometric analysis of (B) iNOS and (C) COX-2 protein expression levels. Blots are representative of the± standard deviation of three separate experiments. Data were normalized with respect to β-actin levels. **P<0.01 vs. LPS treatment group; ##P<0.01 vs. untreated group. LPS, lipopolysaccharide; iNOS, inducible nitric oxide synthase; COX-2, cyclooxygenase-2.
Figure 7.
Figure 7.
Effect of tiliroside on the phosphorylation of mitogen-activated protein kinase-ERK/JNK/p38 proteins. (A) RAW 264.7 cells were treated with LPS 1 µg/ml with or without tiliroside (12.5, 25, 50, and 100 µM) for 45 min and the expression of p-ERK1/2, p-JNK and p-p38 was assessed by western blot analysis. Detection of β-actin was conducted to confirm the equal loading of proteins. Densitometric analysis of p-ERK1/2 (B) p-JNK (C) and p-p38 (D) expression represent the mean±standard deviation of three separate experiments. Data were normalized with respect to β-actin levels. **P<0.01 vs. LPS treatment group; ##P<0.01 vs. untreated control group. LPS, lipopolysaccharide; p-, phosphorylated-; ERK 1/2, extracellular signal-regulated kinase 1/2; JNK, c-Jun N-terminal kinase.
Figure 8.
Figure 8.
Effect of tiliroside on the degradation of IκB-α protein. (A) RAW 264.7 cells were treated with LPS 1 µg/ml with or without tiliroside (12.5, 25, 50 and 100 µM) for 15 min and the expression of IκB-α protein was assessed by western blot analysis. Detection of β-actin was conducted to confirm the equal loading of proteins. (B) Densitometric analysis of IκB-α protein. Expression represent the mean ± standard deviation of three separate experiments. Data were normalized with respect to β-actin levels. ##P<0.01 vs. untreated control group. LPS, lipopolysaccharide; IκB-α, inhibitor of κB-α.
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