*3.3. Chronic Administration of Curcumin Regulated Ethanol-Induced Inflammatory Markers in Mouse Brains*

Previous studies have shown that ethanol intoxication is responsible for the activation of stress-markers and inducing inflammatory cytokines [39]. To explore whether these markers may be inhibited with the administration of curcumin, we analyzed the expression of stress and inflammatory markers, such as p-JNK, p-NF-κB, cyclooxygenase-2 (COX-2), interleukin-1β (IL-1β), and tissue necrosis factor-α (TNF-α), in the mice brains. As shown here, treatment with ethanol elevated the expression of p-JNK, p-Nf-κB, IL-1β, COX-2, and TNF-α. However, the mice that were co-administered the curcumin and ethanol had a lower expression of these markers compared with the ethanol-treated group, supporting the hypothesis that curcumin inhibits the expression of inflammatory cytokines, thereby rendering protection to mice brains against ethanol-induced neurodegeneration. The Western blot results were further supported by the confocal microscopic analysis, which showed that curcumin inhibits the effects of ethanol against the activation of p-JNK in mice brains (Figure 3).

**Figure 1.** Chronic administration of curcumin inhibits ethanol-induced oxidative stress in mice brain: (**A** & **B**) Results of lipid peroxidation (LPO) and reactive oxygen species (ROS) assays, respectively; (**C** & **D**) Western blot results of nuclear factor erythroid 2-related factor 2 (Nrf2) & heme-oxygenase 1 (HO-1), in the mice brain; (**E** & **F**) Confocal microscopic results of HO-1, and Nrf2 in the experimental groups, with bar graphs, magnification 30×, scale bar 50 μm; (**G**) In vitro HT22 cells treated with ethanol. Curcumin or ethanol + curcumin, and Ω, significantly different from the vehicle-treated, Φ, significantly different from the ethanol-treated group, and Ψ, significantly different from the ethanol + Curc treated group. DG means dentate gyrus, Significance = Φ *p* < 0.05; Ω, *p* < 0.05; Ψ, *p* < 0.05. MDA: malondialdehyde, DCF: 2 7 dichlorofluorescein, eth: ethanol, curc: curcumin.

**Figure 2.** Curcumin Rescued activated Microglia and Astrocytes in the ethanol-treated Mouse Brains: (**A** & **B**) The Western blot results of toll-like receptor 4 (TLR4), Receptor for Advanced Glycations End Product (RAGE), glial fibrillary acidic protein (GFAP), and ionized calcium binding adaptor molecule 1 (Iba-1) in the brains of the experimental groups; (**C**) Immunofluorescence images of the expression of GFAP in the experimental groups; (**D**) Immunofluorescence images of Iba-1 in the experimental groups (*n* = 12 mice per group); (**E**) Immunoblot results of TLR4 and Iba-1 in BV-2 Cells, in different experimental groups. Magnification 30× objective field, scale bar = 50 μm & 30 μm. Ω, significantly different from the vehicle-treated, Φ, significantly different from the ethanol-treated group and Ψ, significantly different from the ethanol + Curc treated group. DG means dentate gyrus, Significance = Φ *p* < 0.05; Ω, *p* < 0.05; Ψ, *p* < 0.05. eth: ethanol, curc: curcumin, DAPI: 4 ,6-diamidino-2-phenylindole dihydrochloride. <sup>66</sup>

**Figure 3.** Curcumin Abrogates the Phosphorylation of c-Jun N-Terminal Kinase (JNK) and its Downstream Targets in Ethanol-Treated Mouse Brain: (**A** & **B**) Immunoblot results, showing the expression of active phospho c-Jun N-terminal kinase (p-JNK), nuclear factor kappa-light-chain enhancer of activated B cells (p-NF-kB), tumor necrosis factor alpha (TNF-α), cyclooxygenase-2 (COX-2), and IL- 1β and in mice that received saline, ethanol, and ethanol plus Curcumin for 6 weeks (*n* = 12 mice per group), normalized with its β-actin, as a loading control, with its Histograms; (**C** & **D**). Confocal images, showing active JNK and interleukin-1β (IL-1β) in the mouse brain. Magnification 30× objective field, scale bar = 50 μm, Ω, significantly different from the vehicle-treated, Φ, significantly different from the ethanol-treated group. DG means dentate gyrus, Significance = Φ *p* < 0.05, Ω, *p* < 0.05. eth: ethanol, curc: curcumin.
