The Inhibitory Effect Of Cucurbitacin E On The Inflammatory Response Of Lipopolysaccharides-induced Mouse Macrophage RAW264.7and Its Mechanism

Aim:Cucurbitacin E (CuE) is a member of natural compounds belonging to tetracyclictriterpenoid, which is derived from Trichosanthes Kirilowii Maximowicz (Cucurbitaceae family).It possesses a wide range of biological activities, including anti-inflammatory and anti-cancerproperties. It has been recently reported that cucurbitacins exhibt anti-cancer effect in varioustumor cell types by changing cellular morphology, disrupting the actin cytoskeleton andinducing cell cycle arrest. Yet, its anti-inflammatory mechanism is incompletely understood. Inthis study, we sought to investigate the potential effect and mechanism of anti-inflammatoryproperty of CuE on lipopolysaccharide (LPS)-stimulated RAW264.7macrophage cells. Weanalyzed the effect of CuE on the expression of pro-inflammatory cytokines such as tumornecrosis factor-(TNF-α) and interleukin-1（IL-1）in LPS-stimulated RAW264.7cells, thusto explore its anti-inflammatory mechanism on cellular and molecular levels.Methods:1. Murine RAW264.7macrophages were maintained in DMEM supplemented with10%FBS at37°C in a humidified incubator with5%CO2.2. RAW264.7cells were cultured with different concentrations of CuE (0.16mol/L、4mol/L、20mol/L、100mol/L). After48h, MTS was used to determine cell viability. Theabsorbance at490nm was measured by a microplate reader, and the50%inhibitionconcentration (IC50) was determined from the dose-response curve.3. RAW264.7cells were cultured with different concentrations of CuE for12h, andanalyzed using phase-contrast microscope. Western blotting and protein quantitative methodwere used to analyze G-actin and F-actin levels. Actin cytoskeleton was analyzed byimmunofluorescence microscopy.4. Propidium iodide (PI) staining was used to examine cell cycle distribution ofCuE-treated LPS-stimulated RAW264.7cells for24h. 5. LPS-activated RAW264.7cells were cultured with different concentrations of CuE for6h, and cytokine TNF-α and IL-1expression was analyzed with flow cytometry afterintracellular cytokine staining.6. Using Western blot analysis, we investigated the NF-κB nuclear translocation, theprotein expression of the phosphorylation levels of p38、JNK、ERK1/2of MAPKs signalingpathway, and NF-κB signaling pathway (p-IκB、p-p65、IκB、p65).Results:1. After treatment with LPS, RAW264.7cells were cultured under differentconcentrations of CuE for48h. The cell proliferation was reduced following treatment withCuE. When the concentration of CuE was100mol/L, the proliferation rate was reduced to16.540.70%. This result indicated that CuE could suppress the growth of RAW264.7cells ina dose-dependent manner. The IC50value was7.79mol/L.2. Cell morphology observation revealed that compared with morphological change ofcontrol group, LPS stimulation of RAW264.7cells extended with pseudopodia formation.When pretreated with CuE followed by LPS stimulation, however, the cells became roundupwith the disappearance of pseudopodia.3. Western blotting showed that the CuE treatment led to a decrease in G-actin but anincrease in F-actin levels, Immunofluorescence revealed that actin was difusely distributed inuntreated or LPS-stimulated cells, whereas CuE cotreatment induced actin aggregates.4. PI staining together with flow cytometry showed that CuE induced cell cycle arrest atG2/M phase in LPS-activated RAW264.7cells.5. Intracellular cytokine staining showed that CuE dose-dependently inhibited theexpression of TNF-α and IL-1in LPS-activated cells.6. Western blotting analysis of the nuclear translocation of NF-κB in LPS-inducedRAW264.7cells showed that CuE treatment for4h could significantly suppress the nucleartranslocation of NF-κB in a does-dependent manner.7. Western blotting analysis showed that CuE pretreatment did not inhibit LPS-inducedactivation of MAPK and NF-κB pathways. Conclusion1. CuE inhibited the proliferation of RAW264.7cells in a dose-dependent manner,induced cell morphology change and actin aggregates, and arrested cell cycle at G2/M phase.This may be one of the mechanisms for the anti-inflammatory activity of CuE.2. CuE treatment suppressed the expression of TNF-α and IL-1and nuclear translocationof NF-κB in LPS-activated RAW264.7cells, but had little effect on the activation of MAPKsand NF-κB. It is probable that CuE exhibits anti-inflammatory drug by suppressing nucleartranslocation of NF-κB, thereby leading to decreased expression of TNF-α and IL-1inLPS-stimulated RAW264.7cells.