Anti-Inflammatory and Antioxidant Effects of Carpesium cernuum L. Methanolic Extract in LPS-Stimulated RAW 264.7 Macrophages

Abstract

A hypernomic reaction or an abnormal inflammatory process could cause a series of diseases, such as cardiovascular disease, neurodegeneration, and cancer. Additionally, oxidative stress has been identified to induce severe tissue injury and inflammation. Carpesium cernuum L. (C. cernuum) is a Chinese folk medicine used for its anti-inflammatory, analgesic, and detoxifying properties. However, the underlying molecular mechanism of C. cernuum in inflammatory and oxidative stress conditions remains largely unknown. The aim of this study was to examine the effects of a methanolic extract of C. cernuum (CLME) on lipopolysaccharide- (LPS-) induced RAW 264.7 mouse macrophages and a sepsis mouse model. The data presented in this study indicated that CLME inhibited LPS-induced production of proinflammatory mediators such as nitric oxide (NO) and prostaglandin E₂ (PGE₂) in RAW 264.7 cells. CLME treatment also reduced reactive oxygen species (ROS) generation and enhanced the expression of heme oxygenase-1 (HO-1) protein in a dose-dependent manner in the LPS-stimulated RAW 264.7 cells. Moreover, CLME treatment abolished the nuclear translocation of nuclear factor-κB (NF-κB), enhanced the activation of nuclear factor-erythroid 2 p45-related factor 2 (Nrf2), and reduced the expression of extracellular signal-related kinase (ERK) and ERK kinase (MEK) phosphorylation in LPS-stimulated RAW 264.7 cells. These outcomes implied that CLME could be a potential antioxidant and anti-inflammatory agent.

Figure 1

CLME prevents septic death in an LPS-induced endotoxemia mouse model and inhibits the levels of NO, PGE₂, and iNOS in LPS-stimulated RAW 264.7 cells. (a) The graph represents survival rate after LPS injection (25 mg/kg) with or without CLME treatment (50 or 100 mg/kg). RAW 264.7 cells were pretreated with indicated concentrations of CLME for 1 h and followed by LPS stimulation (1 μg/mL) for indicated times. (b) NO production was analyzed indirectly by measuring the supernatants of cultured RAW 264.7 cells for nitrite using the Griess reagent. (c) The prostaglandin E₂ (PGE₂) production in the culture media was measured by ELISA kit. (d) The iNOS expression was analyzed by western blot analysis. Densitometric analysis was performed using ImageJ ver. 1.50i. The values are represented as the mean ± S.D. (n = 4). ^###P < 0.001 vs. the CON group; ^∗∗∗P < 0.001 vs. the LPS-treated group.

Figure 2

CLME inhibits the phosphorylation and degradation of IκB and nuclear translocation of NF-κB in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with indicated concentrations of CLME for 1 h and followed by LPS stimulation (1 μg/mL) for 30 min. (a) The IκB, p-IκB, and (b) p65 expressions were analyzed by western blot analysis. Densitometric analysis was performed using ImageJ ver. 1.50i. The values are represented as the mean ± S.D. (n = 3). ^###P < 0.001 vs. the CON group; ^∗∗∗P < 0.001 vs. the LPS-treated group.

Figure 3

CLME inhibits the generation of ROS in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with indicated concentrations of CLME for 1 h and followed by LPS stimulation (1 μg/mL) for 1 h. (a) The plot shows the histogram of gated cells with two markers providing data on two cell populations: ROS neg. (-) and ROS pos. (+) cells, indicating M1 and M2, respectively. ROS generations were analyzed by MUSE® oxidative stress assay kit. (b) The graph for percentage of ROS pos. (+) cells. The values are represented as the mean ± S.D. (n = 3). ^##P < 0.01 vs. the CON group; ^∗P < 0.05 vs. the LPS-treated group.

Figure 4

CLME regulates the expression of HO-1 and Keap1/Nrf2 in LPS-stimulated RAW 264.7 cells. (a–c) HO-1 expression in RAW 264.7 cells. (a) The cells were treated with CLME of 25 μg/mL for the indicated times. (b) The cells were treated with CLME of indicated concentrations for 12 h. The values are represented as the mean ± S.D. (n = 3). ^∗P < 0.05 and ^∗∗∗P < 0.001 vs. the CON group. (c) The cells were pretreated with indicated concentrations of CLME for 1 h and followed by LPS stimulation (1 μg/mL) for 12 h. (d, e) The cells were pretreated with indicated concentrations of CLME for 1 h and followed by LPS stimulation for 1 h. (d) Keap1 and (e) Nrf2 expressions were analyzed by western blot analysis. Densitometric analysis was performed using ImageJ ver. 1.50i. The values are represented as the mean ± S.D. (n = 3). ^###P < 0.001 vs. the CON group; ^∗∗P < 0.01 and ^∗∗∗P < 0.001 vs. the LPS-treated group.

Figure 5

CLME inhibits the phosphorylation of MEK/ERK in LPS-stimulated RAW 264.7 cells. RAW 264.7 cells were pretreated with indicated concentrations of CLME for 1 h and followed by LPS stimulation (1 μg/mL) for (a) 30 min and (b) 20 min. These MAPK and MEK protein expressions were analyzed by western blot. Densitometric analysis was performed using ImageJ ver. 1.50i. The values are represented as the mean ± S.D. (n = 3). ^###P < 0.001 vs. the CON group; ^∗∗∗P < 0.001 vs. the LPS-treated group.

Figure 6

Myrtenal inhibits the production of NO, TNF-α, and IL-6 in LPS-stimulated RAW 264.7 cells. HPLC chromatogram of CLME (a) and (1R)-(-)-Myrtenal (b) as a standard compound. (c) Chemical structures of Myrtenal. (d) The viability of Myrtenal in RAW 264.7 cells. The values are represented as the mean ± S.D. (n = 6). ^∗∗P < 0.01 vs. the CON group. (e) The nitrite production was determined using Griess reagent. (f) TNF-α and (g) IL-6 production in the culture media was measured by ELISA kit. The values are represented as the mean ± S.D. (n = 3). ^###P < 0.001 vs. the CON group; ^∗P < 0.05, ^∗∗P < 0.01, and ^∗∗∗P < 0.001 vs. the LPS-treated group.