*3.6. Chronic Administration of Curcumin Reversed Synaptic Dysfunction and Memory Impairment in the Ethanol-Treated Mouse Brain*

Previous studies have shown that ethanol intoxication contributes to the loss of synaptic protein in animal models [39,43]. To explore the effects of curcumin on synaptic marker ethanol-treated mouse brains, we evaluated the expression of synaptic proteins via Western blot and confocal microscopy. According to our findings, the synaptic proteins, including PSD-95, synaptophysin, and SNAP-25, were significantly downregulated in the ethanol-treated mice brains compared with the control mice. Interestingly, curcumin significantly inhibited these effects. The confocal microscopic results of PSD-95 showed significant fluorescence in the curcumin- and ethanol-treated group compared with the ethanol alone group, showing that curcumin preserves the synaptic markers by regulating oxidative stress, neuroinflammation, and apoptotic cell death in mouse brains (Figure 6). Previously, it has been shown that chronic ethanol intoxication causes abnormalities in motor functions and impaired spatial learning and memory [39]. To show whether curcumin could rescue ethanol-induced memory impairment, we performed the MWM and Y-maze tests. First, we recorded the learning abilities of the mice (*n* = 12 mice/group) with the MWM test. We found that ethanol-treated mice had an enhanced latency to reach to the platform, and the mice that had received curcumin (50 mg/kg, i.p., six weeks) showed a decreased escape latency (Figure 6). Twenty-four hours after the fifth day of training, the platform was removed and the mice were allowed to swim freely. We found that the ethanol-treated mice spent less time in the target quadrant and showed less platform crossings, highlighting that ethanol induces memory impairments. Interestingly, curcumin improved the ethanol-induced memory impairments by increasing the total time spent in the target quadrant, and the number of crossings of the platform (Figure 6F,G). After that, we checked the spontaneous alternation behaviors (showing the spatial working memory or short-term memory) of mice (*n* = 12 mice/group) in the Y-maze test. In the ethanol-treated mice, there was a reduction in the spontaneous alternation behaviors compared with the saline-treated mice, showing a cognition decline in the ethanol treated mice. Interestingly, curcumin significantly increased the spontaneous alternation behaviors in the ethanol-treated mice compared

with the ethanol-treated group (Figure 6H). Collectively, our findings showed that the co-administration of curcumin with ethanol significantly prevents cognitive dysfunctions in mice.

**Figure 4.** Curcumin Abrogated Ethanol-Induced Apoptotic Cell Death in Mouse Brain: (**A** & **B**) Immunoblot results of B-cell lymphoma 2-associated X (Bax); , B-cell lymphoma 2 (Bcl-2), Caspase-3, and PARP-1 in the brains of the experimental groups, with their relative bra graphs; (**C** & **D**) Immunofluorescence results of activated Caspase-3 and poly (ADP-ribose)polymerase 1 (PARP-1) in the brains of the experimental mice, with their histograms (*n* = 12 mice/group), Magnification, 40×, scale bar 50 μm & 30 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.

**Figure 5.** Curcumin Ameliorates Neurodegeneration in Mouse Brains, as visualized by Nissl and FJB staining: (**A**) FJB stained sections from mice hippocampus, co-stained with DAPI and its histogram, the differences have been shown in the histogram; (**B**) Images of the Nissl stained sections (CA1, CA3 and DG) from different experimental groups with histograms, *n* = 12 mice/group, scale bar 50 μm & 100 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.

**Figure 6.** Curcumin Rescued Ethanol-Induced Synaptic Dysfunction and Memory Impairment in Mouse: (**A** & **B**) Immunoblot results of PSD95, SNAP23 and synaptophysin in mice brain (*n* = 10), with histograms; (**C**) Confocal images of PSD-95 in mouse brains, with its bar graph. Magnification, 40×, scale bar up to 50 μm; (**D**). Mean escape latency (shown in seconds) to the platform during a training session, *n* = 12 mice/group; (**E**). Latency on the final day of the probe test; (**F**). Histograms showing the number of platform crossings during the probe test; (**G**) Time spent in the target quadrant during the probe trial; (**H**) Spontaneous alternations (in percent) in the Y-maze test; (**I** & **J**). Trajectories of the MWM tests & Y-maze tests, respectively (*n* = 12 mice/group). Ω, significantly different from the vehicle-treated, Φ, significantly different from the ethanol-treated group. Significance = Φ *p* < 0.05, Ω, *p* < 0.05. eth: ethanol, curc: curcumin.

#### **4. Discussion**

The current study has the following main findings. (i) Curcumin has strong anti-oxidant potentials against ethanol-induced oxidative stress, in vivo mouse brains, and in vitro mouse hippocampal HT22 cells, and the antioxidant effects of curcumin are solely dependent on Nrf2. (ii) Curcumin has rescuing effects against ethanol induced activated astrocytes and microglia, in vivo and in vitro, as revealed by the reduced expression of TLR4/RAGE, GFAP, and Iba-1. (iii) Curcumin may rescue the mice brain from ethanol-induced apoptotic cell death, synaptic dysfunction, and memory impairment in mice brains. Several lines of studies have also reported the neuroprotective effects of curcumin against neuroinflammation and apoptotic cell death [44–46]. One study completely focused on oxidative stress mediated neuroinflammation [47]. However, they have not further extended their study to the main antioxidant enzymes, such as Nrf2/HO-1. Curcumin was administered intraperitoneally, and although it is considered a parenteral route of administration, the pharmacokinetics of substances administered intraperitoneally are more similar to those seen after oral administration. In both cases, the primary route of absorption is into the mesenteric vessels, which drain into the portal vein and pass through the liver [48].

Curcumin has long since been reported to be a potent antioxidant, but no studies have yet reported its effects on the endogenous antioxidant mechanisms (Nrf2/HO-1) and TLR4/RAGE mediated neurodegeneration. A wide range of effects are associated with ethanol intoxication, including oxidative stress, apoptotic neurodegeneration, excitotoxicity, and the disruption of cell to cell interactions [40,49]. The production of ROS may cause other serious consequences, such as an altered metabolism, deleterious structural modifications of proteins, DNA mishandlings, and altered mitochondrial homeostasis. Here, we report that ethanol-induced oxidative stress mediated neuroinflammation via Nrf-2/TLR4 signaling. The induction of oxidative stress with the administration of ethanol is in accordance with previous studies [50]. Interestingly, the in vitro findings also support the results, showing antioxidant effects against ethanol induced oxidative stress. For more confirmatory purposes, we used specific Nrf2 siRNA, and the findings showed that curcumin may relieve the elevated ROS level through Nrf2.

The other outcome of our study is that curcumin may inhibit the TLR4/RAGE triggered neuroinflammation in mice brains and in in vitro microglial cells. TLR4, which is mainly expressed in the microglial cells, induces microglial activation and the expression of proinflammatory cytokines, such as TNF-α and p-NF-kB, in response to a variety of stimuli [51]. Besides TLR4, other receptors are also involved in neurodegenerative conditions, such as RAGE. Here, we also evaluated the expression of RAGE in the experimental groups. According to our findings, there was an enhanced expression of TLR4/RAGE and its downstream inflammatory mediators in the ethanol-treated mice brain. Interestingly, these markers were significantly reduced with the administration of curcumin. For more confirmatory purposes, we used a specific TLR4 inhibitor (TAK242). Interestingly, the rescuing effects of curcumin against TLR4/iba-1 in the microglial cells were comparable to TAK 242. GFAP and Iba-1, which are the assigned markers for the activated astrocytes and microglia, were significantly upregulated in the ethanol-treated group, however, there was a significantly lower expression in the curcumin-treated group. The upregulation of GFAP and Iba1 in the ethanol-treated group is in accordance with the previous reports [4]. The inhibition of TLR4 further inhibited the expression of its downstream effectors (Iba-1) in the microglial cells. The upregulation of TLR4, RAGE, and the iba-1 in the ethanol-treated mice brains is in accordance with the previous reported effects of ethanol [52].

The other inducer of the neurodegenerative conditions are as follows: the phosphorylation of MAP kinases, which phosphorylates and translocates the NF-kB to the nucleus, playing a pivotal role in the release of inflammatory mediators [53]. According to our findings, there were enhanced expressions of p-JNK and p-NF-kB, as well as other inflammatory mediators (TNF-α, Cox-2, and IL-1β) in the ethanol treated mice, which is in accordance with previous studies [39]. Another main contributor to the neurodegeneration is apoptotic cell death in neurodegenerative conditions [54], as apoptotic cell death has closely been linked to the oxidative damage [55]. For the evaluation of the effects of curcumin against apoptotic cell death, the expression of proapoptotic (Bax, Caspase-3, and PARP-1) and antiapoptotic (Bcl-2) markers were evaluated; according to our findings, ethanol promoted proapoptotic cell death, which was significantly inhibited with the administration of curcumin. For more confirmatory purposes, we performed Nissl and FJB staining, which showed that curcumin significantly inhibited the neurotoxic effects of ethanol on mice brains. The ultimate consequence of neurodegeneration is synaptic dysfunction and loss of memory, which have been extensively reported with ethanol intoxication [56].

For the evaluation of synaptic dysfunction and memory impairment, we performed behavioral studies in Morris water maze and Y-maze tests, as well as the protein markers related to synaptotoxicity. According to our results, curcumin significantly improved the behavioral alterations and synaptic markers (postsynaptic density protein-95, SNAP-23, and synaptophysin) in the mice brain. The protective effects of curcumin on the behavioral changes and synaptic dysfunction are in accordance with previous studies [43,57].
