Neuroprotection effect of bovine umbilical mesenchymal stem cell-conditioned medium on the rat model of Alzheimer's disease mediated by upregulation of BDNF and NGF and downregulation of TNF-α and IL-1β

Abstract

Background: Neurodegenerative diseases (NDDs) are distinguished by impairment and depletion of nerve cells; one of the most common NDDs is Alzheimer's disease (AD), which can appear in early onset or late onset. In recent years, the secretome or conditioned medium of mesenchymal stem cells has provided new hope for improving conditions and preventing AD. One of the secretomes is bovine umbilical mesenchymal stem cells-conditioned medium (BUMSC-CM), where BUMSC is predicted to promote neuronal proliferation potentially.

Aim: This study analyzes the therapeutic efficiency of conditioned medium or secretome produced from BUMSC-CM in treating neurodegeneration in animal models of AD.

Methods: Five groups consisting of 12 male rats were assigned: untreated (Group A, n = 5), positive control group given normal saline 1 ml/100 g BW (Group B, n = 5), AD rats model followed by Donepezil treatment (Group C, n = 5), AD rats model with BUMSC-CM 0.2 ml/kg BW post-trimethyltin (TMT) induction (Group D, n = 5), and AD rats model with BUMSC-CM 0.5 ml/kg BW post-TMT induction (Group E, n = 5). The brain samples were analyzed for neuronal density using cresyl violet staining. The expression and activity of brain-derived neurotrophic factor (BDNF) were analyzed by ELISA; in addition, interleukin 1beta (IL-1β), tumor necrotic factor-alpha (TNF-α), and neural growth factor (NGF) were analyzed by quantitative polymerase chain reaction. Interactions between the main substances of BUMSC-CM and beta-amyloid protein were visualized using in silico molecular docking.

Results: Our result demonstrated that BUMSC-CM with the dosage of 0.5 ml/kg BW significantly increased BDNF concentration. We also found that BUMSC-CM with dosage 0.2 ml/kg BW and 0.5 ml/kg BW down-regulated IL-1β and TNF-α and upregulated NGF expression. Additionally, the number of neurons in AD rats post-treated with BUMSC-CM was significantly increasing. Furthermore, the amino acids in BUMSC-CM, including isoleucine, leucine, and valine, bind to the amyloid beta protein via interactions that are hydrophobic and hydrogen-bonded.

Conclusion: In this study, the neuroprotective potential of BUMSC-CM was demonstrated by its ability to upregulate BDNF and NGF while downregulating IL-1β and TNF-α. Additionally, BUMSC-CM showed potential to promote neuron proliferation in the hippocampus regions of a rat AD model. The main constituents in BUMSC-CM adhere to amyloid beta protein, hence diminishing the likelihood of ND disorders, specifically AD.

Keywords: Alzheimer’s disease; BUMSC-CM; IL-1β; Rat model; TNF-α.

Fig. 1.. The neuronal density in the CA1, CA3, and DG regions of the hippocampus in the rat model of AD. Cresyl violet staining shows the (A, F, K) control group; (B, G, L) positive control (TMT); (C, H, M) treatment A (TMT + BUMSC-CM 0.2 mg/kg BW); (D, I, N) ) treatment B (TMT + BUMSC-CM 0.5 mg/kg BW); (E, J,O) commercial drug comparator (TMT + Donepezil); SO = stratum oriens; SR = stratum radiatum.

Fig. 2.. The graphic represents the quantification of neuron cell density in the CA1, CA3, and DG areas. There is an increase of nerve cells in the non-treated group, the comparison drug group (Donepezil), and the BUMSC-CM treatment group with doses of 0.2 and 0.5 mg/kg BW compared to the positive control (TMT) (*: p < 0.0001), ns = no significant.

Fig. 3.. Interaction between the essential amino acids isoleucine, valine, leucine, and donepezil on beta-amyloid protein. Tosca represents beta-amyloid protein, red indicates target ligand. A. isoleucine—B-amyloid, B. valine—B-amyloid. C. leucine—B-amyloid, D. donepezil—B-amyloid. Both the 3D and 2D structures of the ligand-protein complex are symbolized as 1 and 2, respectively.

Fig. 4.. BUMSC-CM graph increases the BDNF concentration in the brain of rats modeled for AD induced with TMT. Mice were induced with TMT single dose intraperitoneally as an Alzheimer’s model animal, and donepezil was given orally 14 days post-TMT induction as a commercial drug comparison group, BUMSC-CM with 0.2 ml/kg, and 0.5 ml/kg dosage was given 14 days intraperitoneally post-TMT induction as a treatment group. Rat brain lysate was analyzed for BDNF by sandwich ELISA. The concentration data were computed using one-way ANOVA and then analyzed further using Tukey’s post hoc test (NT = non-treated; **, and ***, are the statistically significant values of the TMT group as positive control group with treatment and negative control groups, p values < 0.0038; 0.0008; n.s. = not significant, respectively).

Fig. 5.. BUMSC-CM reduced the expression of IL-1ß, TNF-α, and increased NGF expression in rats induced by neurotoxic agent TMT. Gene expression results were taken based on the fold change, and then calculated with one-way ANOVA followed by Tukey’s post hoc test (*, **, ***, and ****, are the statistically significant values of the TMT group as positive control vs. BUMSC-CM 0.2 ml/kg, BUMSC-CM 0.5 ml/kg, and Donepezil groups, with an optimal BUMSC-CM dosage of 0.5 ml/kg).