Figure 4.

Effects of liposomal and free curcumin on experimental models of neuroinflammation (A, B) and reactive gliosis (C, D). (A) Upon stimulation with 100 ng/mL LPS for 24 h, HMC3 cells showed the typical amoeboid morphology of reactive microglia, which could be alleviated by pre-incubation with 0.01 µM free (Cur) or liposomal curcumin (LipoCur). Representative images of n = 2 independent stimulations, scale bar indicate 20 µm. (B) Stimulation with 100 ng/mL LPS for 24 h also induced expression of pro-inflammatory cytokines IL6, IL1β, TNFα and TGFβ, as monitored by qPCR. These effects could be reduced by pre-incubation with 0.01 µM curcumin and, in trend even more effective, by corresponding amounts of LipoCur. Please note that due to high variations of LPS-induced cytokine expression curcumin and LipoCur mediated reduction is not always significant, but trends can be seen for all investigated cytokines. Graphs show ΔΔCT values representing the n-fold expression in comparison to unstimulated controls. In non-inflammatory conditions (without LPS stimulation), neither curcumin, nor LipoCur, nor empty liposomes induced any changes in expression of these genes. (C) Stimulation with a combination of IL1β, TNFα and TGFβ (10 ng/mL each) for 24 h served to induce activation of human astrocyte cell line SVGA. Cells show nuclear deformations as a sign of cellular stress. Effects were almost completely abolished upon pre-incubation with 0.01 µM free curcumin or LipoCur. Representative images from n = 2 experiments, scale bar indicates 20 µm. (D) As representatives for reactive astrogliosis associated genes, expression of nestin, tenascin C and fibronectin was analyzed by qPCR and found to be induced upon stimulation with IL1β, TNFα and TGFβ (10ng/mL each) for 24 h. These effects were significantly alleviated by pre-incubation with 0.01 µM curcumin or LipoCur. LipoCur showed higher significance levels compared to soluble curcumin and was in case of tenascin C even significantly more effective. In “normal” conditions (without cytokine stimulation), neither curcumin, nor LipoCur, nor empty liposomes induced any changes in expression of these genes. Shown are mean values ± SD from n = 5–15 independent experiments, asterisks indicate significant changes of stimulations compared to LPS control (B) and cytokines (D) or between the groups under the brackets. Data have been analyzed by Two-Way-ANOVA followed by Bonferroni’s multiple comparison test (*p<0.05, **p<0.001, ***p<0.001, ****p<0.0001).