Exploring the Anti-Inflammatory Activity of the Heat-Processed Gynostemma pentaphyllum Extract (Actiponin®) in RAW264.7 Cells and Carrageenan-Induced Rat Models

Abstract

Gynostemma pentaphyllum (GP) is a medicinal plant that has long been used as drug for the treatment of rheumatism, liver disease, and diabetes. In this study, GP was extracted with 50% ethanol extract, and then the extract was heat-processed under high pressure to analyze the anti-inflammatory potential of these extract (named actiponin (AP)) and its derived components, damulin A and damulin B, in RAW264.7 cells and carrageenan-induced rat models. Ap had no effect on RAW264.7 cells up to 180 μg/mL, but DA and DB showed cytotoxicity from 18 μM. Pretreatment with AP significantly suppressed the LPS-induced increase in nitric oxide (NO) and inducible nitric oxide synthase (iNOS) protein expression via griess reagent and Western blot analysis, and these effects were similar to those of DA and DB. AP, DA, and DB also significantly suppressed the expression of prostaglandin E2 (PGE2) and cyclooxygenase-2 (COX-2) protein, which were increased by LPS, in a concentration-dependent manner. In addition, AP, DA, and DB inhibited the LPS-induced increase in pro-inflammatory cytokines, tumor necrosis factor (TNF)-α, and interleukin (IL)-6 in RAW264.7 cells. The anti-inflammatory activities of AP, DA, and DB are mediated by the suppression of the nuclear factor (NF)-κB and phosphorylation of mitogen-activated protein kinases (MAPKs) signaling pathways. Oral administration of 30, 50, 100, or 200 mg/kg (AP) suppressed carrageenan-induced edema in a concentration-dependent manner. Collectively, these results suggest that AP exerts potential anti-inflammatory activity by suppressing the inflammatory-mediators and pro-inflammatory cytokines via the NF-κB and MAPK pathways in vitro and by reducing the thickness of carrageenan-induced paw edema in vivo.

Keywords: Gynostemma pentaphyllum; MAPKs; NF-κB; anti-inflammatory; carrageenan-induced paw edema.

Figure 1

Effects of AP, DA, and DB on viability of RAW264.7 cells. (A) Chemical formular of DA. (B) Chemical formular of DB. The cells (2.5 × 10⁵ cells/well) were cultured in 48-well plates and treated with AP, DA, and DB at the indicated concentrations for 24 h. Thereafter, cell viability of (C) AP, (D) DA, and (E) DB was analyzed using the CCK-8 as-say and graphed as a percentage. Data are presented as the mean ± SEM of three independent experiments. a–b Mean values designated by different superscript letters were significantly different between groups at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 2

Effects of AP, DA, and DB on NO production and iNOS expression in LPS-stimulated RAW264.7 cells. The cells (2.5 × 10⁵ cells/well in 48-well plate for NO assay, 2 × 10⁶ cells/100 mm dishes for Western blot) are cultured and treated with AP, DA, and DB for 4 h, then incubated with LPS (500 ng/mL) for another 20 h. Nitrite accumulated in the culture medium is analyzed using Griess reagent, and calibration curve is based on NaNO₃; (A) AP, (B) DA, and (C) DB. Protein expression of iNOS is determined using Western blot analysis; (D) AP, (E) DA, (F) DB. (G–I) Quantitative data of (D–F) are analyzed using ImageJ bundle with Java 1.8.0_172 software. GAPDH served as an internal control. The data are presented as the mean ± SEM of three independent experiments. a–e Mean values designated by different superscript letters are significantly different between groups at p < 0.05 as determined by Duncan’s multiple-range test.

Figure 3

Effects of AP, DA, and DB on PGE2 production and COX-2 expression in LPS-stimulated RAW264.7 cells. The cells (2 × 10⁶ cells) are cultured in 100 mm dishes and treated with AP, DA, and DB for 4 h, then incubated with LPS (500 ng/mL) for another 20 h. PGE2 accumulated in the culture medium is analyzed using PGE2 ELISA; (A) AP, (B) DA, (C) DB. Protein expression of COX-2 is determined using Western blot analysis; (D) AP, (E) DA, (F) DB. (G–I) Quantitative data of (D–F) were analyzed using ImageJ software. GAPDH served an internal control. The data are presented as the mean ± SEM of three independent experiments. a–e Mean values designated by different superscript letters are significantly different between groups at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 4

Effects of AP, DA, and DB on pro-inflammatory cytokines in LPS-stimulated RAW264.7 cells. The cells (2 × 10⁶ cells) are cultured in 100 mm dishes and treated with AP, DA, and DB for 4 h, then incubated with LPS (500 ng/mL) for another 20 h. Protein expression of TNF-α and IL-6 is determined using Western blot analysis; (A) AP, (B) DA, (C) DB. (D–F) Quantitative data of (A–C) are analyzed using ImageJ software. GAPDH served as an internal control. The data are presented as the mean ± SEM of three independent experiments. a–d Mean values designated by different superscript letters are significantly different between groups at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 5

Effects of AP, DA, and DB on MAPKs and NF-κB pathways in LPS-stimulated RAW264.7 cells. The cells (2 × 10⁶ cells) are cultured in 100 mmm dishes and treated with AN, DA, and DB for 1 h, then incubated with LPS (500 ng/mL) for another 30 min. Protein expression of MAPKs (t/p-ERK, t/p-JNK, and t/p-p38) and NF-κB (t/p-IκB-α and t/p-p65) is determined using Western blot analysis. MAPKs: (A) AP, (B) DA, (C) DB, NF-κB (t/p-IκB-α and t/p-p65); (G) AP, (H) DA, (I) DB. (D–F,J–L) Quantitative data of (A–C,G–I) are analyzed using ImageJ software. GAPDH served as an internal control. The data are presented as the mean ± SEM of three independent experiments. a–e Mean values designated by different superscript letters are significantly different between groups at p < 0.05, as determined by Duncan’s multiple-range test.

Figure 6

Effects of AP on carrageenan-induced paw edema. Paw edema is induced by subcutaneous injection of a 1% carrageenan solution (100 uL/animal) into the plantar area of rats, followed by oral administration of AP and diclofenac (10 mg/kg) 1 h later. (A) Morphological characteristics of the paw of rats 4 h after carrageenan injection. (B) Paw thickness is measured using digital calipers each hour after carrageenan injection for a total of 4 h. The data are presented as the mean ± SEM of four animals. a–d Mean values designated by different super-script letters are significantly different between groups at p < 0.05, as determined by Duncan’s multiple-range test.