The Combination of Neurotropic Vitamins B1, B6, and B12 Enhances Neural Cell Maturation and Connectivity Superior to Single B Vitamins

Abstract

Peripheral neuropathy (PN) is a prevalent condition characterized by damage to peripheral nerves, often linked to risk factors such as diabetes. This condition results from various forms of neural damage, including injury to the cell body, axons, or demyelination, frequently beginning with small and thinly or unmyelinated fibers. Such nerve damage disrupts normal signaling, leading to symptoms like numbness, tingling, and pain. Effective nerve repair and regeneration, particularly through remyelination, are essential therapeutic objectives. While vitamin B12's role in repair processes has been well established, emerging evidence suggests that other neurotropic vitamins, specifically B1 and B6, also contribute significantly to nerve health and symptom relief in PN. In this study, we demonstrate that a combination treatment of vitamins B1, B6, and B12 enhances repair and oxidative stress responses in co-cultures of neural and Schwann cells, leading to improved cell maturation and connectivity compared to vitamin B12 alone. Furthermore, proteomic analysis supports these observations at the molecular level, with enhanced cellular recycling processes like proteasome enhancement, as well as protein synthesis upregulation, needed to rebuild nerve connections and combatting oxidative stress. Our combined morphological and molecular results highlight the potential therapeutic advantage of the B1, B6, and B12 combination over vitamin B12 alone.

Keywords: nerve regeneration; neurotropic B vitamins; peripheral neuropathy; proteomics.

Figure A1

Differential protein expression in response to the combination vitamin treatment. The combination treatment markedly upregulated the proteins ALYREF, PSMC1, USP15, EIF3L, and THBS1 when compared to both the vitamin B12 treatment alone and the control group.

Figure 1

Two approaches for in vitro neuron cell cultures. (A) a three-dimensional assembly of neurons and Schwann cells embedded in a cellular matrix; (B) a single-cell two-dimensional culture.

Figure 2

Viability of neuron and Schwann cell 3D co-cultures under non-insulted (a) and hydrogen peroxide-insulted (b) conditions. Non-insulted and insulted cells were treated with control medium, individual vitamins B1, B6, or B12, or the B1/B6/B12 combination followed by alamarBlue^® staining. Absolute fluorescence intensity is shown. p-values above the brackets: * p < 0.05 vs. control; ** p < 0.01 vs. control. p-values above the brackets indicate statistical significance vs. B1/B6/B12 combination. Control = no vitamin treatment, control medium with low-level B vitamins. Treatment: B1 = 40 mM vitamin B1; B6 = 20 µM vitamin B6; B12 = 0.4 µM vitamin B12; B1/B6/B12 combination = combination of vitamins B1 (40 µM), B6 (20 µM), and B12 (0.4 µM).

Figure 3

3D co-cultures of neurons and Schwann cells were treated with control medium, individual vitamins B1, B6, or B12, or the B1/B6/B12 combination followed by staining with NeuroFluor to test for neuronal maturation. Neuron maturation under different treatment conditions was measured as integrated brightness of green fluorescence. p-values above the brackets: *** p < 0.001 vs. control. p-values above the brackets indicate statistical significance vs. B1/B6/B12 combination. Control = no vitamin treatment, control medium with low-level B vitamins. Treatment: B1 = 40 mM vitamin B1; B6 = 20 µM vitamin B6; B12 = 0.4 µM vitamin B12; B1/B6/B12 combination = combination of vitamins B1 (40 µM), B6 (20 µM), and B12 (0.4 µM).

Figure 4

Synapsing and networking of the neuron cells (non-insulted (a) vs. insulted (b)) treated with control medium, vitamin B12 alone, or the B1/B6/B12 combination, followed by morphological analysis. p-values above the brackets: * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control. p-values above the brackets indicate statistical significance vs. B1/B6/B12 combination. Control = no vitamin treatment, control medium with low-level B vitamins. Treatment: B12 = 0.4 µM vitamin B12; B1/B6/B12 combination = combination of vitamins B1 (40 µM), B6 (20 µM), and B12 (0.4 µM).

Figure 5

(a,b) Synapsing and networking of the neuron cells (non-insulted (a) vs. insulted (b)) treated with control medium, vitamin B12 alone, or the B1/B6/B12 combination, followed by morphological analysis. p-values above the brackets: * p < 0.05 vs. control; ** p < 0.01 vs. control; *** p < 0.001 vs. control. p-values above the brackets indicate statistical significance vs. B1/B6/B12 combination. (c,d) Live NG108 neuron images captured under phase contrast (non-insulted and treated with control medium (c) or insulted and treated with the B1/B6/B12 combination (d)). Neuron morphology in the micrographs was simplified, with dendrites represented as lines and cell bodies as circles. Control = no vitamin treatment, control medium with low-level B vitamins. Treatment: B12 = 0.4 µM vitamin B12; B1/B6/B12 combination = combination of vitamins B1 (40 µM), B6 (20 µM), and B12 (0.4 µM).

Figure 6

(a) PCA plot showing distinct clustering of the treatment groups, indicating significant differences in protein expression profiles; (b) Volcano plots highlighting upregulated proteins in the combination treatment versus B12.