Preclinical Studies of Canagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, and Donepezil Combined Therapy in Alzheimer’s Disease
<p>Behavioral analysis for novel object recognition test (NORT). Panel: TA, denotes familiar object; TB, denotes the novel object. (<b>A</b>) Data are represented as exploration time (seconds) in the NORT, test session for the nootropic model compared to the scopolamine model after an interval of 60 min following the training period; (<b>B</b>) represents the graph plot for the exploration time (seconds) in the NORT, test session for the tested models (nootropic and scopolamine) after an interval of 24 h following the training period; (<b>C</b>) represents the graph plot for the novelty preference index for the nootropic model; (<b>D</b>) the novelty preference index for the scopolamine model. Data are expressed as mean ± SEM, and statistical analysis using a one-way ANOVA, ∗ <span class="html-italic">p</span> < 0.05 vs. ConG, ∗∗ <span class="html-italic">p</span> < 0.01 vs. ConG and ∗∗∗∗ <span class="html-italic">p</span> < 0.0001 vs. ConG; ∗∗∗∗∗ <span class="html-italic">p</span> < 0.00001 vs. ConG; # <span class="html-italic">p</span> < 0.05 vs. SG, ## <span class="html-italic">p</span> < 0.01 vs. SG and #### <span class="html-italic">p</span> < 0.0001 vs. SG. ConG: control group; SG: negative control group—scopolamine; DG: positive control group—donepezil; CanG: canagliflozin group; CanDG: canagliflozin–donepezil group.</p> "> Figure 2
<p>Representative images of the effect of treatments on the expression of muscarinic acetylcholine receptor M1 (M1mAChR), vascular endothelial growth factor A (VEGF-A), cyclooxygenase-2 (COX2) and the mammalian target of rapamycin (mTOR) in the studied groups. The M1mAChR marker showed positivity in the subcortical area and the hippocampus in the ConG. M1mAChR expression was lower to moderate in mice in the CanG and CanDG groups compared to the scopolamine group. VEGF-A labelled the endothelial cells in the capillaries in all areas of the nervous system, including the ciliary processes. The expression of the COX2 and mTOR markers registered close reactivity, more intense in the scopolamine mice and less in the CanG, DG and CanDG mice. The positive expression was recorded in the cortical, subcortical and hippocampal areas for the glial fibrillary acidic protein (GFAP). The denser cell population was observed in the control, scopolamine, donepezil groups and in the females exposed to canagliflozin. In the males treated with canagliflozin and in the CanDG mice, a reduction in the GFAP positive cell population was observed. ConG: control group; SG: negative control group—scopolamine; DG: positive control group—donepezil; CanG: canagliflozin group; CanDG: canagliflozin–donepezil group; SGLT2i: sodium-glucose cotransporter 2 inhibitor; M1mAChR: muscarinic acetylcholine receptor M1; VEGF-A: vascular endothelial growth factor A; COX2: cyclooxygenase-2; mTOR: mammalian target of rapamycin; GFAP: glial fibrillary acidic protein.</p> "> Figure 3
<p>The different expressions of the major histocompatibility complex class II (MHC II), cluster of differentiation 68 (CD68), nuclear factor erythroid 2-related factor 2 (Nrf2) and p65 expression in the control and experimental groups. MCH II labeled microglia were small with numerous short, branched extensions, the cells being grouped in the periventricular zone. CD68 positive microglia had an amoeboid appearance, present especially in the periventricular area. The frequency of the cell populations was lower in the CanG and CanDG groups. An inverse expression of the Nrf2 and p65 markers was observed in the areas studied. MHC II: major histocompatibility complex class II; CD68: cluster of differentiation 68; Nrf2: nuclear factor erythroid 2-related factor 2; ConG: control group; SG: negative control group—scopolamine; DG: positive control group—donepezil; CanG: canagliflozin group; CanDG: canagliflozin–donepezil group.</p> "> Figure 4
<p>Schematic representation of the overall experimental procedure. For 21 consecutive days mice were treated with either donepezil, canagliflozin, donepezil+ canagliflozin or the vehicle solution using gavage and with intraperitoneal injections with scopolamine for the last 9 days of the study. Behavioral tests were performed between days 10-12 and 19-21; as indicated in the inset. NORT: novel object recognition test; EPM: elevated plus maze.</p> ">
Abstract
:1. Introduction
2. Results
2.1. Nootropic and Anti-Amnesic Effects of Canagliflozin and Donepezil, under Separate or Combined Therapy
2.2. Paraclinical Evaluation: Biochemistry Assay and Immunohistochemistry Analysis
2.2.1. Biochemistry Profile
2.2.2. Immunohistochemistry Analysis
3. Discussion
4. Materials and Methods
4.1. Animal Care
4.2. Drugs
4.3. Experimental Design
4.4. Paraclinical Evaluation: Biochemistry Assay and Immunohistochemistry Analysis
4.5. Data Analysis and Statistics
5. Concluding Remarks
Author Contributions
Funding
Institutional Review Board Statement
Informed Consent Statement
Data Availability Statement
Acknowledgments
Conflicts of Interest
References
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Dose/Parameter | ConG | SG | DG | CanG | CanDG |
---|---|---|---|---|---|
CRE (mg/dL) | 0.29 ± 0.04 | 0.23 ± 0.03 | 0.21 ± 0.03 | 0.17 ± 0.04 | 0.19 ± 0.05 |
AST (U/L) | 64.76 ± 25.33 | 78.81 ± 44.82 | 69.38 ± 52.38 | 71.03 ± 0.3 | 61.85 ± 85 |
ALT (U/L) | 53.23 ± 166.98 | 56.45 ± 106.47 | 51.01 ± 134.61 | 52.35 ± 152.80 | 48.58 ± 89.14 |
TC (mg/dL) | 229 ± 50.96 | 151.22 ± 15.07 | 155.5 ± 16.79 | 125 ± 13.04 | 121.40 ± 10.30 |
GLU (mg/dL) | 245.3 ± 21.46 | 183.22 ± 13.42 | 209.44 ± 12.50 | 176 ± 17.88 * | 170.70 ± 15.71 ** |
ALB (g/L) | 42.71 ± 5.49 | 45.56 ± 2.36 | 39.59 ± 4.69 | 34.62 ± 5.19 | 32.37 ± 5.49 |
TP (g/L) | 84.87 ± 9.51 | 74.76 ± 2.58 | 76.95 ± 2.13 | 64.98 ± 4.51 @ | 69.61 ± 2.66 @ |
UREA (mg/dL) | 64.09 ± 11.53 | 65.78 ± 3.55 | 53.78 ± 7.06 # | 49.13 ± 7.40 ## | 42.43 ± 4.48 ### |
Biomarkers | Experimental Animal Groups | ||||
---|---|---|---|---|---|
ConG | SG | DG | CanG | CanDG | |
M1 AChR H | +++ | ++++ | + | ++ | ++ |
M1 AChR SC | ++ | ++++ | ++ | + | + |
VEGF-A H | ++ | ++ | ++ | ++ | ++ |
VEGF-A SC | ++ | ++ | ++ | ++ | ++ |
COX-2 H | +++ | ++++ | + | + | + |
COX-2 SC | ++ | +++ | + | + | + |
mTOR H | +++ | ++++ | ++ | + | + |
mTOR SC | ++ | ++++ | +++ | + | + |
GFAP H (females) | +++ | +++ | +++ | +++ | + |
GFAP H (males) | +++ | +++ | +++ | - | - |
GFAP SC (females) | ++++ | +++ | ++ | +++ | + |
GFAP SC (males) | +++ | ++++ | +++ | - | ++ |
MHC II H | +++ | +++ | ++ | + | + |
MHC II SC | ++ | ++ | + | + | + |
CD68 H | +++ | +++ | ++ | + | ++ |
CD68 SC | +++ | +++ | + | + | + |
Nrf2 H | +++ | ++ | + | ++ | ++ |
Nrf2 SC | ++ | - | ++ | ++++ | +++ |
P65 H | +++ | ++ | + | + | + |
P65 SC | +++ | +++ | + | + | + |
Primary Antibody | Dilution | Secondary Antibody | Dilution | |
---|---|---|---|---|
1. | mTOR (ab109268) | 1:70 | Goat anti Rabbit | 1:100 |
2. | P65 (AA 143-158) | 1:100 | Goat anti Rabbit | 1:100 |
3. | Anti VEGFA (ABIN2788641) | 1:250 | Goat anti Rabbit | 1:1000 |
4. | GFAP (Cat.nr.173002) | 1:500 | Goat anti Rabbit | 1:500 |
5. | MHC II (Dako M0746) | 1:100 | Goat anti Rabbit | 1:100 |
6. | Nrf-2 (WJ3412022B) | 1:100 | Goat anti Rabbit | 1:100 |
7. | COX2 (ab16701 SP-21) | 1:100 | Goat anti Rabbit | 1:100 |
8. | M1mAChR (SC365966) | 1:250 | Goat anti Mouse | 1:250 |
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Stanciu, G.D.; Ababei, D.C.; Solcan, C.; Bild, V.; Ciobica, A.; Beschea Chiriac, S.-I.; Ciobanu, L.M.; Tamba, B.-I. Preclinical Studies of Canagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, and Donepezil Combined Therapy in Alzheimer’s Disease. Pharmaceuticals 2023, 16, 1620. https://doi.org/10.3390/ph16111620
Stanciu GD, Ababei DC, Solcan C, Bild V, Ciobica A, Beschea Chiriac S-I, Ciobanu LM, Tamba B-I. Preclinical Studies of Canagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, and Donepezil Combined Therapy in Alzheimer’s Disease. Pharmaceuticals. 2023; 16(11):1620. https://doi.org/10.3390/ph16111620
Chicago/Turabian StyleStanciu, Gabriela Dumitrita, Daniela Carmen Ababei, Carmen Solcan, Veronica Bild, Andrei Ciobica, Sorin-Ioan Beschea Chiriac, Loredana Maria Ciobanu, and Bogdan-Ionel Tamba. 2023. "Preclinical Studies of Canagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, and Donepezil Combined Therapy in Alzheimer’s Disease" Pharmaceuticals 16, no. 11: 1620. https://doi.org/10.3390/ph16111620
APA StyleStanciu, G. D., Ababei, D. C., Solcan, C., Bild, V., Ciobica, A., Beschea Chiriac, S.-I., Ciobanu, L. M., & Tamba, B.-I. (2023). Preclinical Studies of Canagliflozin, a Sodium-Glucose Co-Transporter 2 Inhibitor, and Donepezil Combined Therapy in Alzheimer’s Disease. Pharmaceuticals, 16(11), 1620. https://doi.org/10.3390/ph16111620