Mahanimbine Improved Aging-Related Memory Deficits in Mice Through Enhanced Cholinergic Transmission And Suppressed Oxidative Stress, Amyloid Levels, And Neuroinflammation Part 2

2.6. Evaluation of Malondialdehyde (MDA) and Glutathione (GSH) Levels

Using commercially available kits, the MDA and GSH levels were analyzed in brain samples (Cayman Chemical Company, Ann Arbor, MI, USA). 

As science and technology continue to advance, scientists are trying to uncover the mysteries of mysterious and important human psychological activities such as memory, thinking, and consciousness. Among them, the study of brain samples is considered to be a field of great concern because it can help us understand the relationship between memory and the brain more deeply.

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First of all, what is a brain sample? Simply put, it refers to a method of capturing, preserving, and analyzing neurons (brain cells) and their connections in the brain. In layman's terms, it is like a map of the brain that can show the connections and functions between different areas.

Next, let's talk about the relationship between brain samples and memory. Scientific research has shown that our memory is closely related to brain structure. When we learn new knowledge, new neuronal connections are formed in the brain, and these connections become stronger over time. Through the study of brain samples, scientists are working hard to study these brain connections and try to understand how to enhance learning and memory through them.

In addition, through the analysis of brain samples, scientists can also find some interesting things. For example, they found that the hippocampus in the human brain is an area closely related to memory. This is because the hippocampus has an important function in the human brain to convert information from short-term memory to long-term memory.

Therefore, the value of brain sample research is not only to help us better understand the relationship between the brain and memory but also to help us discover the root causes of memory disorders or cognitive diseases, thereby promoting the treatment and prevention of related diseases.

Finally, we should take a positive view of the help of brain sample research on memory. Although there are still many scientific problems to be solved, with the continuous improvement of science and technology, we believe that there will be more breakthroughs in the future. Let us look forward to the future together and look forward to making our brains smarter through scientific research! It can be seen that we need to improve memory. Cistanche can significantly improve memory because Cistanche has antioxidant, anti-inflammatory, and anti-aging effects, which can help reduce oxidative and inflammatory reactions in the brain, thereby protecting the health of the nervous system. In addition, Cistanche can also promote the growth and repair of nerve cells, thereby enhancing the connectivity and function of neural networks. These effects can help improve memory, learning ability, and thinking speed, and can also prevent the occurrence of cognitive dysfunction and neurodegenerative diseases.

MDA and GSH levels were measured in nanomoles/milligrams of protein and micromoles/milligrams of protein, correspondingly. Bradford technique was followed to estimate the protein content in the brain tissues.

2.7. Estimation of Acetylcholine (ACh) and Acetylcholinesterase (AChE) Activities

The ACh and AChE levels in the brain homogenate were analyzed using commercialized kits (BioAssay System, Hayward, CA, USA), which were EnzyChromTM Acetylcholine Assay kit and QuantiChromTM Acetylcholinesterase Assay kit, respectively. 

The intensity of color formation was detected in a microplate reader at 570 and 412 nm, respectively. The level of ACh obtained was expressed as µM, whereas AChE activity was expressed as U/L.

2.8. Measurement of β-Amyloid Levels

The ELISA kits from Cloud Clone Corp (Katy, TX, USA) were used to evaluate the levels of Aβ1-40 and Aβ1-42 in brain homogenates. A microplate reader set to 450 nm was used to measure the color formation. 

The concentration of Aβ1-40 and Aβ1-42 in the sample was inversely related to the color intensity. The results were represented in terms of pg/mL of total protein.

2.9. Assessment of β-Secretase (BACE-1) Activity in Mouse Brain

BACE-1 activity in the homogenate was quantified using a commercialized kit (SensoLyte® β-Secretase Assay Kit from AnaSpec, Fremont, CA, USA). The detection of the kit is based on a peptide, which is cleaved by BACE-1, and the signal is detected using a spectrophotometer with excitation/emission at 490/520 nm.

2.10. Measurement of Total Cyclooxygenase (COX) Activity

The activity of total COX in the brain homogenate was estimated using an ELISA kit (Cayman Chemical Company, Ann Arbor, MI, USA). The color formation was directly proportional to the total COX activity. A microplate reader was utilized to read the absorbance at 590 nm.

2.11. Gene Expression of BACE-1 and COX-2

The expression of BACE-1 and COX-2 in isolated brain RNA was analyzed using RT-PCR. The three major steps involved were: RNA extraction, conversion of RNA to cDNA, and real-time polymerase chain reaction (RT-PCR). 

All the steps involved kits provided by Qiagen Valencia, CA, USA. Firstly, RNA was extracted from the brain sample preserved in the RNA. In this step, approximately 100 mg of brain tissue sample was homogenized using a TissueRuptor in 1 mL of QIAzol lysis reagent and incubated for 5 min at room temperature. 

The mixture of brain lysate and QIAzol lysis reagent was added with chloroform and shaken vigorously for 15 s, followed by incubation at room temperature for 2–3 min. 

The mixture was centrifuged at 12,000× g for 15 min at 4 ◦C, and the three phases formed a colorless phase (upper phase), which consisted of RNA and was collected with 1 volume of 70% ethanol added to it. 

Next, 700 µL of the sample was transferred into a spin column and centrifuged at room temperature at 8000× g for 15 s; the flow-through was discarded. Following the addition of 700 µL of buffer RW1 (provided in the kit) and centrifugation for 15 s at 8000× g, the flow-through was discarded. The step was continued with the addition of 500 µL buffer RPE (provided in the kit) and centrifugation for 15 s at 8000× g. 

This step was repeated twice to wash any organic contamination in the RNA. Lastly, RNA was eluted by adding 50 µL of RNAse-free water and centrifuged for 1 min at 8000× g. 

The concentration of RNA was measured using a nanodrop (nanodrop 2000c, Thermofisher Scientific, Waltham, MA, USA). The extracted RNA was then converted into cDNA, which was utilized to perform RT-PCR. 

Firstly, 14 µL of template RNA was added with the 6 µL reverse-transcription master mix (1 µL of Quantiscript reverse transcriptase, 4 µL of Quantiscript RT buffer, 1 µL of RT primer mix). After mixing, it was incubated for 15 min at 42 ◦C, followed by incubation at 95 ◦C for 3 min to inactivate the Quantiscript reverse transcriptase. 

The product formed was cDNA, and finally, RT-PCR was conducted. The RT-PCR conditions were fixed as 95 ◦C for 5 min followed by 40 cycles at 95 ◦C for 10 min and 60 ◦C for 30 min. 

The expression of BACE-1 and COX-2 genes was quantified and normalized against the two housekeeping genes (β-actin and GAPDH). The primers of nucleotide sequences were based on mouse BACE-1 (front 5 0 -GCATGATCATTGGTGGTATC-30: reverse 50 -CCATCTTGAGATCTTGAC-CA-30 ) and COX-2 (forward 50 -GTGTGCGACATACTCAAGCAGGA) cDNA sequences. -30 : reverse 50 - TGAAGTGGTAACCGCTCAGGTG-30 ), GAPDH (forward 50 -TGACAGGATGCAGAAGGAGA-30 : reverse 50 -GCTGGAAGGTGGACAGTGAG-30 ), and -actin (forward 50 -TGACAGGATGCAGAAGGAGA-30 : reverse 50 -GCTGGAAGGTGGACAGTGAG-30 ). 

Fluorescence measurements were obtained, and Rotor-Gene 6000 software was used to evaluate them (Qiagen, Hilden, Germany). 

Results were presented as the levels of expression following normalization to the housekeeping gene, which were B-actin and GAPDH using the comparative CT (threshold cycle) approach. Equation (1) was used to calculate the gene expression of the sample. 

The CT value of each sample was obtained based on the standard curve that was generated for each gene (target gene and reference gene).

∆∆CT = ∆CT sample − ∆CT control (1)

where

∆CT sample: CT value of target gene − CT value of reference gene

∆CT control: CT value of target gene − CT value of reference gene

2.12. Statistical Analysis

Experimental data were presented as mean ± SEM. One-way ANOVA procedure (Graph Pad version 9, GraphPad Software Inc., La Jolla, CA, USA) and Tukey–Kramer post hoc test were utilized to categorize statistical variations. Significant was defined as a probability value of 0.05.

3. Results

3.1. Mahanimbine-Enhanced Memory in Aged Mice

The spatial learning and memory ability of mahanimbine in the mouse model was examined using the MWM test. Escape latency (EL), escape distance (ED), and time spent in the target quadrant were measured. 

Moreover, locomotor activity was also considered based on the average swimming speed of mice. Figure 2A shows that the aged control group had markedly longer EL from day 1 to day 3 (22.05 ± 1.70 s, 21.35 ± 1.85 s, 19.96 ± 2.34 s; p < 0.001; respectively) when compared to the young control (12.75 ± 1.30, 10.12 ± 0.98, 7.60 ± 1.31; respectively). 

It revealed that aged mice established a substantial deficit in learning and memory function in the MWM test. However, oral treatment with different dosages (1 and 2 mg/kg) of mahanimbine showed a reversal of the aforementioned changes in EL of aged mice. 

The EL values for the 1 mg/kg mahanimbine group were 22.01 ± 1.60 s, 15.88 ± 1.54 s (p < 0.01), and 8.26 ± 0.93 s (p < 0.001) for days 1, 2, and 3, correspondingly compared with the aged control, whereas the EL values for the 2 mg/kg mahanimbine group were 16.09 ± 0.33 s (p < 0.05), 17.14 ± 0.57 s (p < 0.05), 10.57 ± 0.86 s (p < 0.01), respectively, when paralleled to the aged control. On day 3, the EL of both groups (1 and 2 mg/kg) of mahanimbine was not significantly different from the young control.

Figure 2. Effect of mahanimbine on (A) escape latency, time taken to find the hidden platform. (B) Escape distance, distance traveled from starting point until finding the hidden platform; (C) average speed, swimming speed of animal to find the hidden platform; (D) probe test, aged mice's percentage time spent in the target quadrant using Morris water maze test. There were significant improvements in escape latency and escape distance with mahanimbine in aged mice. 

Additionally, treatment increased the time spent in the target quadrant of aged mice. All data were expressed as mean ± SEM (n = 6). One-way ANOVA (day 1: F(3,20) = 11.74, p < 0.001, day 2: F(3,20) = 17.38, p < 0.001 and day 3: F(3,20) = 14.88, p < 0.001 for escape latency; day 1: F(3,20) = 12.22, p < 0.001, day 2: F(3,20) = 28.62, p < 0.001, and day 3: F(3,20) = 23.15, p < 0.001 for escape distance; day 1: F(3,20) = 1.939, p > 0.05, day 2: F(3,20) = 3.467, p > 0.05, and day 3: F(3,20) = 2.497, p > 0.05 for average speed; F(3,20) = 7.185, p < 0.01 for probe test followed by Tukey–Kramer multiple comparisons test. ** p < 0.01 and *** p < 0.001 in comparison with young control, # p < 0.05, ## p < 0.01, and ### p < 0.001 in comparison with aged control.

Figure 2B represents the effect of mahanimbine on the distance traveled from the starting point until finding the hidden platform. The aged control group traveled the longest distance before finding the hidden platform on days 1, 2, and 3 (4.79 ± 0.73 m, 4.34 ± 0.11 m, and 4.38 ± 0.59 m; p < 0.001; respectively) when related to the young control (1.36 ± 0.31 m, 1.35 ± 0.24 m, and 0.78 ± 0.14 m; respectively). 

Aged mice that were fed 1 mg/kg of mahanimbine significantly reduced the ED (2.17 ± 0.11 m (p < 0.01), 2.10 ± 0.24 m (p < 0.001), 0.89 ± 0.18 (p < 0.001); respectively) for day 1 until day 3 as matched to the aged control. 

A significant parallel decline in ED was observed in mice administered 2 mg/kg of mahanimbine for days 1, 2, and 3 (2.43 ± 0.28 (p < 0.01), 2.78 ± 0.32 (p < 0.001), 1.47 ± 0.30 (p < 0.001); respectively).

Concerning the average swimming speeds during the task, no differences were observed among the groups (Figure 2C). This indicates that mahanimbine did not alter any locomotor activity related to motor function in aged mice. 

After 24 hours of the actual task, a probe test session (day 30) was conducted to evaluate the memory retention of animals. The aged control group (Figure 2D) spent less time in the target quadrant (10.10 ± 1.14% (p < 0.001)) and showed a significantly reduced time spent in the targeted quadrant compared to the young control (19.53 ± 2.56%), whereas aged mice treated with 1 mg/kg (20.27 ± 4.84%, p < 0.01) and 2 mg/kg (17.85 ± 1.56%, p < 0.05) of mahanimbine by oral gavage showed a significantly longer time spent in the targeted quadrant compared with the aged control.

3.2. Effect of Mahanimbine on MDA and GSH Levels in the Aged Mouse Brain

The MDA levels in the aged control (Figure 3A) were considerably higher (11.54 ± 0.88 µM; p < 0.001) related to the young control (4.37 ± 0.09 µM). The increase in the MDA level in the brain homogenate of aged mice reflected a higher LPO activity, which indicated an elevation in oxidative stress in the brain tissue. 

Conversely, the administration of mahanimbine (1 mg/kg, p.o) significantly declined brain MDA levels in aged mice (7.56 ± 0.35 µM; p < 0.001) compared to the aged control. No considerable changes were noted with 2 mg/kg of mahanimbine (9.65 ± 0.51 µM).

The quantity of glutathione (GSH) in the brain homogenate suggested mahanimbine's antioxidant potential. Statistical analysis of GSH (Figure 3B) showed a significant attenuation between the aged control group (0.0028 ± 0.0004 µmoles/mg; p < 0.001) when associated with the young control (0.0447 ± 0.0029 µmoles/mg). 

The present study also found that treatment with mahanimbine (1 and 2 mg/kg, p.o) reversed the GSH level, which was significantly higher (0.0372 ± 0.0038 µmoles/mg, p < 0.001 and 0.0282 ± 0.0041 µmoles/mg, p < 0.001; respectively) as matched to the aged control.

3.3. Mahanimbine Improved the Cholinergic Activity in the Aged Mouse Brain

Figure 4A shows the ACh levels in the mouse brain. ACh was comparably reduced (p < 0.001) in the aged control (17.79 ± 1.54 µM) as matched to the young control (34.82 ± 0.31 µM). Administration of mahanimbine (1 and 2 mg/kg), however, significantly increased (35.61 ± 0.85 µM, (p < 0.001); 39.42 ± 0.88 µM (p < 0.001); respectively) the level of ACh in the brain as associated with the aged control. 

Figure 4B represents the effect of mahanimbine against ACHE activity in the brain. The activity of AChE in the aged control group (204.80 ±1.55 U/L) was considerably elevated (p < 0.001) as paralleled with the young control (51.06 ± 1.36 U/L). 

Nonetheless, the AChE level was suggestively inhibited with the treatment of 1 mg/kg (62.03 ± 4.64 U/L; p < 0.001) and 2 mg/kg (43.73 ± 4.76 U/L; p < 0.001) mahanimbine when associated with the aged control. Overall, administration of a high dose of mahanimbine (2 mg/kg, p.o) produced better results for cholinergic activity since it enhanced the ACh level and suppressed AChE activity.

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