Mahanimbine Improved Aging-Related Memory Deficits in Mice Through Enhanced Cholinergic Transmission And Suppressed Oxidative Stress, Amyloid Levels, And Neuroinflammation Part 3

3.4. Mahanimbine Inhibited Aβ1-40 and Aβ1-42 in the Aged Mouse Brain

Figure 5A shows the effect of mahanimbine against the Aβ1-40 levels in brain homogenate of aged mice. There was no considerable difference in the Aβ1-40 level in aged control mice (0.58 ± 0.06 pg/mL) compared with young control mice (0.43 ± 0.04 pg/mL). 

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Nevertheless, treatment with mahanimbine 1 mg/kg and 2 mg/kg significantly reduced (0.38 ± 0.03 pg/mL, p < 0.05; 0.35 ± 0.03 pg/mL, p < 0.01; respectively) the level of Aβ1-40 when linked with the aged control. 

Figure 5B represents the potential of mahanimbine in suppressing the Aβ1-42 level in the brain of aged mice. The level of Aβ1-42 was considerably higher in the aged control (524.9 ± 11.11 pg/mL, p < 0.001) when related to the young control (209.2 ± 10.81 pg/mL). 

The treatment with mahanimbine 1 and 2 mg/kg, however, significantly reduced (313.2 ± 8.72 pg/mL, p < 0.001; 187.3 ± 7.97 pg/mL, p < 0.001; respectively) the level of Aβ1-42 when compared with the aged control group.

3.5. Mahanimbine Inhibited BACE-1 Activity and Expression in the Aged Mouse Brain

Figure 6A highlights the effect of mahanimbine on BACE-1 activity. The activity of BACE-1 was significantly elevated in the aged control group (63,996 ± 6608 RFU/Unit; p < 0.05) compared with the young control (41,632 ± 4532 RFU/Unit). 

However, administration of 1 and 2 mg/kg of mahanimbine comparably inhibited (p < 0.05 and p < 0.01; respectively) the activity of BACE-1 when compared with the aged control group. 

The value of BACE-1 activity was 43,210 ± 3629 RFU/Unit in the 1 mg/kg mahanimbine group and 40,362 ± 2988 RFU/Unit in the 2 mg/kg mahanimbine-treated group.

To validate BACE-1 activity in the brain homogenate, expression of the BACE-1 gene was quantified using the RT-PCR method. Figure 6B displays the effect of mahanimbine on BACE-1 gene expression. 

Statistical analysis of BACE-1 expression in brain homogenate revealed that the aged control group displayed significantly higher BACE-1 expression (1.44 ± 0.03; p < 0.001) when related to the young control. 

Treatment with 1 and 2 mg/kg mahanimbine significantly reduced the expression of BACE-1 (1.19 ± 0.04 (p < 0.01), 1.03 ± 0.07 (p < 0.001); respectively) when matched with the aged control. From the findings, administration of a high dosage of 2 mg/kg mahanimbine showed greater prevention against BACE-1 activity and expression.

3.6. Effects of Mahanimbine on Total Cyclooxygenase (COX) Activity and COX-2 Expression in the Aged Mouse Brain

The ability of mahanimbine to suppress neuroinflammation is indicated by the reduced activity of total COX activity in the brain homogenate. Figure 7A shows the total COX activity in the aged control group significantly increased (27.96 ± 0.89 nmol/min/mL; p < 0.001) compared with the young control (12.34 ± 0.07 nmol/min/mL). Treatment of mahanimbine at 1 mg/kg significantly reduced the total COX activity (18.87 ± 0.53 nmol/min/mL; p < 0.001) compared with the aged control, but there was no considerable difference with 2 mg/kg of mahanimbine (26.84 ± 0.37 nmol/min/mL) compared with the aged control.

4. Discussion

These study results have demonstrated that mahanimbine improves cognitive functions in aged mice. These conclusions specified that the administration of mahanimbine influenced the learning and memory functions of aged mice via reduced oxidative stress, increased antioxidant level, improved cholinergic activity, attenuated deposition of Aβ, declined BACE-1 activity, and reduced total COX activity. It is widely accepted that aged rodents, like elderly humans, show aging-associated declines in cognitive functions with memory impairment [3,21,22]. 

Based on the MWM data in the present study, the thirty-day treatment of mahanimbine improved memory parameters in aged mice since it decreased the EL and SD of mice as well as improved the time spent in the targeted quadrant compared with the aged control. Based on the average swimming speed of mice, there was no difference among groups. 

This indicated that the performance reduction in old mice was not caused by any perceptual deficiencies, such as vision or motor debits, and the performance gain produced by mahanimbine was not caused by swimming acceleration. From MWM findings, mahanimbine represents the first evidence of ameliorating cognitive deficits in aged mice. 

An increase in oxidative stress is reported during aging. Oxidative stress is caused by an imbalance between ROS generation and elimination, which involves a significant role in age-related diseases [23]. Preventing oxidative stress and maintaining oxidative state is essential for optimal physiological function and preventing age-related diseases [24]. 

Antioxidants are considered a protective system in a rat's brain while aging [25], and therefore, intake of antioxidant supplementation might protect against the effects of ROS that inspire the progression of numerous chronic diseases [26]. Our present results verified the antioxidant effect of mahanimbine in aged mice. The decreased level of malondialdehyde (MDA) in the brain homogenate of aged mice treated with mahanimbine as matched to aged controls indicates the reversal of ROS activity in treated groups. 

MDA is one of the primary intermediates in free radical damage, which results in oxidative stress [27]. It also obstructs cerebral function by disrupting the balance of inhibitory and excitatory neurons in the brain [28]. Additionally, our results showed an elevation in reduced glutathione (GSH) levels in mahanimbine-treated aged mice compared with aged control. 

These results specified that mahanimbine enhanced free radical scavenging functions in aged mice. This may be due to mahanimbine scavenging the activity of free radicals and restoring GSH levels. Based on a previous report, the downregulation of GSH was apparent in the organs of aged rats matched to those of younger rats [29]. GSH is an endogenous antioxidant that protects against damage produced by oxygen-free radicals. Mahanimbine seems to have the ability to increase GSH levels to counter oxidative stress in the aged brain. 

Our present findings were consistent with previous reports that observed the antioxidative property of carbazole alkaloids from M. koenigii leaves [30]. These results strongly suggested that mahanimbine's antioxidant potential was due to declining MDA levels in aged mice brains. Mahanimbine probably lowered MDA levels due to its antioxidant activity, which improved animal performance in the MWM task. 

The enhanced memory in aged mice with mahanimbine treatment may also be due to improvement in central cholinergic transmission by increasing the acetylcholine (ACh) level and inhibiting the activity of acetylcholinesterase (AChE) in the aged mouse brain. Both ACh and AChE in the synapse were associated with the learning and memory functions [31]. Memory dysfunction is directly correlated with a decrease in the release of ACh at the neurons [32]. From a previous report, a 25% to 30% decrease in levels of ACh leads to severe memory loss in the AD animal model [33]. 

The present results highlighted a 50% decrease in the concentration of ACh in aged mice brains compared to young mice, which could have resulted in a more serious loss of cognitive function. However, treatment with mahanimbine recovered the ACh level in aged mice and led to an improvement in cognitive functions. One of the important strategies to elevate the cholinergic function is by inhibiting AChE. 

This involves the breakdown of ACh into choline in the neural synapse, which causes cholinergic deficit and contributes to cognitive impairment [34]. Based on the AChE data, treatment with mahanimbine significantly decreased AChE activity in the aged mouse brain and concurrently increased the concentration of ACh in the brain. This finding is parallel with the study by Kumar et al. (2010) [35] in that inhibition of AChE helps to enhance ACh activity, which is one of the main approaches in the management of AD. 

On the other hand, it is well known that β-amyloid (Aβ) is continuously synthesized from its precursor and catabolized under normal settings, whereas the aging process leads to the pathological deposition of Aβ, which results from a defective metabolism [36]. Accumulation of Aβ induced abnormalities of neuronal function, thus resulting in cognitive dysfunction [37]. 

Consumption of dietary supplements can help reduce aging-related Aβ accumulation, which is a key method for preventing cognitive impairment. Therefore, the amount of Aβ1-40 and Aβ1-42 has been measured in brain homogenate of aged mice supplemented with mahanimbine. The Aβ1-40 denotes the most abundant isoform in brain tissue, whereas Aβ1-42 shows a considerable increase in the brain of AD patients [38]. 

The levels of Aβ1-40 and Aβ1-42 were significantly decreased in the mahanimbine-treated group of aged mice compared to the aged control. Additionally, the present study also evaluated the activity of BACE-1, which is an enzyme that contributes to the formation of Aβ [39]. 

Aβ was produced from APP by proteolytic cleavage by β-secretase (BACE-1) and followed by γ-secretase [40]. The generation of Aβ was initiated by BACE-1; it metabolizes APP to APPβ, Aβ N terminus, and a C-terminal fragment, C99. 

Then, γ-secretase produces Aβ [41]. The current study showed that oral administration of mahanimbine in aged mice declined the activity of BACE-1 compared with an aged control. The gene expression of BACE-1 was also lower with the administration of mahanimbine, further confirming the results obtained. Thus, decreased activity of BACE-1 and expression of the BACE-1 gene consequently reduced the formation of Aβ and significantly attenuated spatial learning and memory deterioration. 

The present study also studied the effect of mahanimbine on inflammatory markers, which were total COX activity and COX-2 expression. From our findings, aged mice showed a high level of total COX activity, but treatment with mahanimbine could attenuate total COX activity at only the 1 mg/kg level. 

However, expression of the COX-2 gene in aged mice brains did not exhibit any notable differences as matched to young controls. Several epidemiological findings link inflammation and aging to predict a variety of aging phenotypes, including changes in neuronal health, metabolic homeostasis, body composition, and immune senescence [5]. 

The present study has some limitations; recently, a few reports have highlighted that anti-inflammatory agents do not support AD management. Moreover, this initial evaluation resulted in using a mouse model, where some discrepancy between mice and humans might be due to the different functions of microglial cells, particularly microglial cells triggering the inflammatory process and aggravating AD, but they neither express iNOS nor produce NO, but protect neurons via growth factors (GF) and neurotrophic factors (NF) in humans [42]. However, the current results support the further evaluation of mahanimbine on more specific targets, including aging-related memory functions.

5. Conclusions

Overall, the present study indicates that mahanimbine could improve spatial learning and memory since it showed shorter EL and SD and enhanced time spent in the targeted quadrant during the MWM test in aged mice. In biochemical analysis, mahanimbine decreased the levels of MDA, AChE, Aβ1-40, Aβ1-42, BACE-1, and total COX but raised the level of GSH and ACh in aged mice's brains compared to aged controls. In conclusion, mahanimbine could protect against learning and memory impairment in aged mice through attenuation of oxidative stress (MDA), deposition of Aβ1-42, AChE level, and BACE-1 activity while increasing antioxidant (GSH) and ACh levels. Therefore, mahanimbine could be a potential substitute for treating aging-related conditions. However, the mechanistic aspect of the neuroprotective effect of mahanimbine for improving cognitive function needs to be further evaluated.

Author Contributions: Conceptualization, V.M.; Formal analysis, V.M., N.S.M.A., and S.M.L.; Funding acquisition, V.M., K.R., and A.B.A.M.; Investigation, V.M., N.S.M.A., and K.R.; Methodology, V.M., N.S.M.A., K.R., and S.M.L.; Project administration, V.M., K.R., and A.B.A.M.; Supervision, V.M. and A.B.A.M.; Validation, V.M., N.S.M.A., and K.R.; Writing-original draft, V.M., and K.R.; Writing-review and editing, V.M., K.R., S.M.L., and A.B.A.M. All authors have read and agreed to the published version of the manuscript.

Funding: The researcher (Vasudevan Mani) would like to thank the Deanship of Scientific Research, Qassim University, for funding the publication of this project.

Institutional Review Board Statement: This study was conducted according to the guidelines of the Declaration of Helsinki and approved by the Research Committee on Ethical Use in Research (approval number: UiTM Care-372014), UiTM, Malaysia.

Data Availability Statement: The data presented in this study are available from the corresponding author upon reasonable request.

Acknowledgments: The researcher (Vasudevan Mani) would like to thank the Deanship of Scientific Research, Qassim University, for funding the publication of this project.

Conflicts of Interest: All authors declare that they have no conflict of interest.

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