3.2.1 Chronic EGCG treatment decreased hippocampal and cortical acetylcholinesterase activity in rats with chronic ethanol administration
A Significantly increased acetylcholinesterase activity was observed in hippocampal (2.28 fold) and cortical (3.66 fold) brain regions of rats with chronic ethanol administration as compared with respective samples from control group. A significant effect across the groups was observed on acetylcholinesterase activity in hippocampus and cerebral cortex after one-way ANOVA followed by Brown-Forsythe test [(F (5,24) = 27.57; DF = 5, p < 0.0001), (F (5,24) = 80.39; p < 0.0001) respectively. The EGCG (25, 50 and 100 mg/kg) treatment significantly (p < 0.001) decreased acetylcholinesterase activity dose-dependently in both brain areas of rats with chronic ethanol exposure. Whereas, EGCG per se group had a negligible effect on acetylcholinesterase activity of the brain as compared with control group. (Fig. 2A, 2B).
3.2.2 Chronic EGCG treatment enhances hippocampal and cortical antioxidant enzymatic activity in rats with chronic ethanol administration
Ethanol chronic exposure in rats significantly (p < 0.05) reduces the hippocampal and cortical enzymatic activity of catalase as compared to the rats in the control group A significant effect across the groups was observed on catalase activity in hippocampus and cerebral cortex after one-way ANOVA followed by Brown-Forsythe test [(F (5,24) = 53.47; DF = 5, p < 0.0001), (F (5,24) = 96.98; p < 0.0001) respectively (Fig. 2C, 2D). A similar trend was observed in levels of other major anti-oxidant enzymes GSH was shown in cerebral cortex and hippocampus was shown in the (Fig. 2E, 2F) A significant effect across the groups was observed on GSH activity in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 21; DF = 5, p < 0.0001), (F (8,24) = 115.8; DF = 5; p < 0.0001) respectively, however in the case of SOD in the cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 51; DF = 5, p < 0.0001), (F (5,24) = 167.5; DF = 5; p < 0.0001) respectively, was shown (Fig. 2G, 2H). Moreover, chronic treatment with EGCG (25, 50 and 100 mg/kg) significantly reverses these enzymatic activities in hippocampus and cortex brain areas of rats with ethanol administration (p < 0.0001).
3.2.3 Chronic EGCG treatment suppressed hippocampal and cortical nitrosative stress and LPO levels in rats with chronic ethanol administration
The ethanol administration for chronic-duration significantly increases the nitrite levels in hippocampus (1.78 fold) and cortex (1.89 fold) brain region of rats. A significant effect across the groups was observed on nitrile activity in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 113.28; DF = 5, p < 0.0001), (F (5,24) = 88.07; DF = 5; p < 0.0001) respectively, Moreover, EGCG (25, 50 and 100 mg/kg) treatments significantly (p < 0.0001) decreased the nitrite levels in the brain of chronic ethanol administered rats. This effect was dose-dependent. However, EGCG per se group rats were observed to have a negligible effect on brain nitrite levels. (Fig. 3A, 3B). Rats administered with chronic alcohol were found to have significant (p < 0.001 increase in lipid peroxidation in the hippocampus (3.57 fold) and cerebral cortex (3.51 fold) as compared to the control group rats. A significant effect across the groups was observed on LPO activity in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 75.44; DF = 5, p < 0.0001), (F (8,24) = 112.8; DF = 5; p < 0.0001) respectively. The EGCG treated (25, 50 and 100 mg/kg) rats were observed to have a significant (p < 0.001) reduction in lipid peroxidation in both brain regions of ethanol administered rats. Moreover, the EGCG per se group rats were observed to have a negligible effect on lipid peroxidation in the brain. (Fig. 3C, 3D).
3.2.4 Chronic EGCG treatment mitigates hippocampal and cortical tumor necrosis factor-alpha TNF-α in rats with chronic ethanol administration
Chronic ethanol administration in rats significantly (p < 0.05) increased the hippocampal (3.47 fold) and cortical (3.17 fold) TNF-α levels as compared to the control group rats. A significant effect across the groups was observed on TNF-α expression in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 320.8; DF = 5, p < 0.0001), (F (8,24) = 418.8; DF = 5; p < 0.0001) respectively, EGCG (25, 50 and 100 mg/kg) treatment decreases the TNF-α levels significantly (p < 0.05) in both the brain regions of ethanol administered rats dose dependently. Whereas, EGCG per se group rats were observed to have no change in the level of TNF-α in the rat brain. (Fig. 4A, 4B)
3.2.5 Chronic EGCG treatment suppressed hippocampal and cortical interleukin-1β (IL-1β) in rats with chronic ethanol administration
The IL-1β levels were significantly (p < 0.05) increased in the hippocampus (4.33 fold) and cerebral cortex (4.79 fold) of rats with chronic ethanol administration when compared to the rats in control. A significant effect across the groups was observed on IL-1β expression in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 1270; DF = 5, p < 0.0001), (F (8,24) = 326.8; DF = 5; p < 0.0001) respectively. Moreover, rats treated with EGCG (25, 50 and 100 mg/kg) showed significant (p < 0.05) reduction in hippocampal and cortical IL-1β levels. This effect was dose-dependent. (Fig. 4C, 4D).
3.2.6 Chronic EGCG treatment ameliorated hippocampal and cortical nuclear factor kappa beta (NF-κβ) p56 subunit levels in rats with chronic ethanol administration
Rats administered with chronic ethanol were observed to have significantly (p < 0.05) elevated NF-κβ p65 subunit levels in the hippocampus (7.62 fold) and cerebral cortex (2.38 fold) when compared to the control group. The significant effect across the groups was observed on NF-κβ expression in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 42; DF = 5, p < 0.0001), (F (8,24) = 98; DF = 5; p < 0.0001) respectively. Treatment with EGCG (25, 50 and 100 mg/kg) significantly (p < 0.001) reduced the hippocampal and cortical NF-κβ p56 subunit expression of chronic ethanol administered rats and this effect was dose-dependent. Whereas, no effect was observed in EGCG per se treatment group on the expression of NFκβ p56. (Fig. 4E, 4F)
The rats administered with chronic ethanol were found to have a significant (p<0.05) increase in caspase-3 level in the hippocampus (3.62 fold) and cortex (4.15 fold) brain regions as compared to the rats in control group. The significant effect across the groups was observed on caspase-3 expression in hippocampus and cerebral cortex after one-way ANOVA followed by Bartlett’s test [(F (5,24) = 14; DF= 5, p < 0.0001), (F (5,24) = 3342; DF= 5; p < 0.0001) respectively. A significant (p<0.05) reduction was observed with EGCG treatment (25, 50 and 100 mg/kg) in caspase-3 levels of hippocampus and cortex in a dose-dependent mode. However, EGCG per se treatment was not found to have any effect on levels of Caspase-3 in rat brain. (Fig 4.G, 4H).