Prim-O-glucosylcimifugin Attenuates Lipopolysaccharideinduced Inflammatory Response in RAW 264.7 Macrophages

Abstract

Background: Radix Saposhnikoviae (RS) exerts anti-inflammatory, analgesic, antipyretic, antioxidation effects and has been used in traditional Chinese medicine to treat common colds, headache, and rheumatoid arthritis. Prim-O-glucosylcimifugin (POG) is the highest content chromone and one of the major active constituents in RS.

Objective: The study was aimed to explore the anti-inflammation effects of POG in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages.

Materials and methods: Cell viability was detected by Cell Counting Kit-8 assay. Production of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and IL-6 was assessed by enzyme-linked immunosorbent assay. Real-time polymerase chain reaction and Western blot were performed to analyze mRNA and protein levels, respectively.

Results: During the whole experiment, 15, 50, and 100 μg/mL of POG had no cytotoxicity on RAW 264.7 cells. POG dose-dependently inhibited the production of NO, TNF-α, IL-1β, and IL-6 that were induced by LPS. POG treatment downregulated the mRNA and protein expression inducible NO synthase (iNOS) and cyclooxygenase 2 (COX-2) in LPS-activated RAW 264.7 macrophages in a concentration-dependent manner. Furthermore, LPS-induced JAK2/STAT3 activation was prevented in RAW 264.7 macrophages by POG treatment. STAT3 overexpression significantly reversed the effects of POG on LPS-activated RAW 264.7 macrophages.

Conclusion: These results demonstrate that POG exerts anti-inflammatory effects through the inhibition of iNOS and COX-2 expression by inhibiting the phosphorylation of JAK2/STAT3.

Summary: POG exerts anti-inflammatory effects in RAW 264.7 macrophages through the inhibition of iNOS and COX-2 expression by inhibiting JAK2/STAT3 signaling. Abbreviations used: LPS: Lipopolyssacharide; NO: Nitric oxide; TNF-α: Tumor necrosis factor-α; IL: Interleukin; RS: Radix Saposhnikoviae; POG: Prim-O-glucosylcimifugin; iNOS: Inducible NO synthase; COX2: Cyclooxygenase; FBS: Fetal bovine serum; DMSO: Dimethylsulfoxide; CCK-8: Cell Counting Kit; RIPA: Radio immunoprecipitation assay buffer; ECL: Enhanced chemiluminescence; SD: Standard deviation; ELISA: Enzyme-Linked immunosorbent assay.

Keywords: Anti-inflammation; JAK2/STAT3; cytokines; nitric oxide; prim-O-glucosylcimifugin.

Figure 1

Structure of prim-O-glucosylcimifugin

Figure 2

Effects of prim-O-glucosylcimifugin on cell viability with a Cell Counting Kit. RAW 264.7 cells were exposed with lipopolyssacharide (1 μg/mL) and various concentrations of prim-O-glucosylcimifugin (15, 50 and 100 μg/mL) or dimethyl sulfoxide alone. Cell viability was assessed 24 h after treatment and expressed as percentage of the dimethyl sulfoxide control. All values are means ± standard deviation (n = 3)

Figure 3

Effects of prim-O-glucosylcimifugin on lipopolyssacharide-induced NO and cytokine production. Raw 264.7 cells were incubated in a medium containing lipopolysaccharide (1 μg/mL) and various concentrations of prim-O-glucosylcimifugin (15, 50, and 100 μg/mL). Cells treated with dimethyl sulfoxide were set as control. The amount of nitrite (a), tumor necrosis factor-α (b), interleukin-6 (c), and interleukin-1β (d) in the medium was monitored at 24 h after exposure as described in Materials and Methods. All values are means ± standard deviation (n = 3). ^&&&P < 0.001 versus control; *P < 0.05, **P < 0.01, and ***P < 0.001 versus lipopolysaccharide-treated cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus lipopolysaccharide and 15 μg/mL prim-O-glucosylcimifugin-treated cells; ^$$P < 0.01 and ^$$$P < 0.001 versus lipopolysaccharide and 50 μg/mL prim-O-glucosylcimifugin-treated cells

Figure 4

Effects of prim-O-glucosylcimifugin on the inducible nitric oxide synthase and cyclooxygenase 2 expressions of lipopolysaccharide-stimulated RAW 264.7 cells. RAW 264.7 cells were treated with lipopolysaccharide (1 μg/mL) and various concentrations of prim-O-glucosylcimifugin (15, 50, and 100 μg/mL) and harvest at 24 h posttreatment. Cells treated with dimethyl sulfoxide only were set as control. (a) mRNA expression of inducible nitric oxide synthase and cyclooxygenase 2 was detected by real-time polymerase chain reaction with GAPDH as internal control; All values are means ± standard deviation (n = 3). (b) Protein expression of inducible nitric oxide synthase and cyclooxygenase 2 was detected by Western blotting. Representative Western blots (upper panel) and quantitative results (lower panel) were shown. All values are means ± standard deviation (n = 3). ^&&&P < 0.001 versus control; *P < 0.05, **P < 0.01, and ***P < 0.001 versus lipopolysaccharide-treated cells; ^#P < 0.05, ^##P < 0.01, and ^###P < 0.001 versus lipopolysaccharide and 15 μg/mL prim-O-glucosylcimifugin-treated cells; ^$P < 0.05, ^$$P < 0.01, and ^$$$P < 0.001 versus lipopolysaccharide and 50 μg/mL prim-O-glucosylcimifugin-treated cells

Figure 5

Involvement of JAK2/STAT3 signaling. (a) RAW 264.7 cells were treated with lipopolysaccharide (1 μg/mL) and various concentrations of prim-O-glucosylcimifugin (15, 50 and 100 μg/mL) and harvest at 4 h posttreatment. Cells treated with dimethyl sulfoxide were set as control. p-JAK2, JAK2, p-STAT3, and STAT3 were detected by Western blotting. Representative Western blots (left panel) and quantitative results (right panel) were shown. All values are means ± standard deviation (n = 3). ^&&&P < 0.001 versus control; *P < 0.05, **P < 0.01, and ***P < 0.001 versus lipopolysaccharide-treated cells; ^#P < 0.05 and ^##P < 0.01 versus lipopolysaccharide and 15 μg/mL prim-O-glucosylcimifugin-treated cells;^$P < 0.05 versus lipopolysaccharide and 50 μg/mL prim-O-glucosylcimifugin-treated cells. (b-d) RAW 264.7 cells were infected with STAT3 expression lentivirus or control vector lentivirus (Vector). After 24 h, cells were treated with lipopolysaccharide (1 μg/mL), and dimethyl sulfoxide or 100 μg/mL prim-O-glucosylcimifugin. The amount of nitrite (b) and cytokines (c) in the medium was monitored at 24 h after exposure. Protein expression of inducible nitric oxide synthase and cyclooxygenase 2 (d) in RAW 264.7 cells was detected by Western blotting. **P < 0.01 and ***P < 0.001 versus Vector + lipopolysaccharide; ^#P < 0.05 and ^###P < 0.001 versus STAT3 + lipopolysaccharide;^$P < 0.05,^$$P < 0.01, and ^$$$P < 0.001 versus POS + lipopolysaccharide