Introduction
Multiple sclerosis (MS) is a chronic, inflammatory, demyelinating, autoimmune disease of the central nervous system (CNS) ( 1 , 2 ). It usually occurs in young adults between the ages of 20 and 40, and women are affected up to three times more often than men ( 3 , 4 ).
Multiple foci of inflammatory reactions, microgliosis, astrocytosis, oligodendrocyte depletion, demyelination, and axonal degeneration are part of the pathogenesis of MS ( 5 ). In addition, cytokines released by immune cells are among the other factors that play an important role in the pathogenesis of this pathological condition ( 6 ). IL-2 is a key factor in maintaining immunological tolerance, which together with IFN-γ, increases the inflammation of MS lesions ( 7 - 9 ).
In contrast to IL-2, IL-10, as a pivotal anti-inflammatory cytokine, plays a key role in suppressing the immune system ( 10 ). In addition, studies have shown that TGF-β, with its function of maintaining immune tolerance by regulating lymphocyte proliferation, differentiation, and survival, is one of the important factors that play a role in protecting against MS ( 6 ). Although the exact role of immunoglobulin-producing cells in the pathogenesis of MS is still unclear, studies suggest that some immunoglobulins act as beneficial factors in myelin regeneration ( 11 ). In recent years, stem cell therapy has become a very promising scientific research topic ( 12 ). Mesenchymal stem cells (MSCs), with their high proliferation and differentiation potential and immunomodulatory properties, are considered a promising source for replacing damaged oligodendrocytes in MS ( 13 ). Experimental studies have shown that human adipose-derived stem cells (hADSCs) can differentiate into myelin-producing cells and compensate for myelin loss in MS disease models ( 14 ). Compared with other cell types, the use of adipose-derived stem cells can be used for cell-based therapy in MS due to their ease of isolation using a minimally invasive method, high proliferation capacity, and ability to withstand a large number of passages ( 14 - 16 ). However, transplantation of these cells alone has not yielded significant results in treating the disease. Therefore, it is hypothesized that adding a complementary therapy to cell transplantation may enhance therapeutic efficacy.
Natural substances and supplements are commonly used for the treatment of various neurological disorders based on their medicinal properties ( 17 , 18 ). Vanillin (4-hydroxy-3-methoxybenzaldehyde), a phenolic compound and the most popular food flavoring, is extracted from the pods of the orchid Vanilla planifolia ( 19 - 21 ). This phenolic compound has been reported to possess anticancer, antidiabetic, antioxidant, antidepressant, and anti-inflammatory activities, along with neuroprotective and libido-enhancing effects ( 21 - 26 ). Furthermore, its ability to cross the blood-brain barrier has been demonstrated ( 22 ). In the present study, it was hypothesized that vanillin could be a promising neuroprotective agent in the management of demyelinating diseases such as MS. There are a wide range of models for inducing MS in laboratory animals. The cuprizone model is widely used in research to study the mechanisms of demyelination and remyelination in MS ( 27 ). Cuprizone, a copper chelator, induces demyelination in animal models by inducing metabolic stress and apoptosis in oligodendrocytes, leading to myelin degradation in specific regions of the white matter of the brain, including the corpus callosum (CC). The expression of myelin-related proteins, including myelin basic protein (MBP), myelin oligodendrocyte glycoprotein (MOG), and proteolipid protein, is reduced by cuprizone in the CC ( 28 ). Since, the in vivo effects of vanillin on stem cell differentiation in oligodendrocyte cells and myelination improvement have not been studied, the present study was designed and conducted to evaluate the role of vanillin on the differentiation of hADSCs into oligodendrocytes and remyelination in a cuprizone-induced mouse model ( Figure 1 ).
Materials and Methods
Experimental animals
This experimental study was conducted on 60 C57BL/6 mice, weighing 20–25 g in the animal house of the Medical Research Center, Isfahan University of Medical Sciences, according to the guidelines and regulations established by the Medical Research Ethics Committee (IR.MUI.AEC.1403.033) in 2024.
Cell labeling with PKH26
hADSCs from our previous study was cultured with DMEM/F12(BIO-IDEA 25131) medium containing 10% fetal bovine serum (FBS) (Invitrogen 10270-106) and 1% penicillin/streptomycin at 37 °C in a humidified 5% CO2 incubator. After 80% cell confluency, hADSCs were detached and cell labeling was done with PKH26 (Sigma) before transplantation according to previous studies ( 29 , 30 ). To achieve this, after placing cells in a conical tube and washing once with PBS, we centrifuged the cells at 1500 rpm for 5 min. Then PKH26 was added to the cells and washed with PBS. After labeling cessation by 1% BSA (Sigma Aldrich A2153), the cells were washed by DMEM/F12. Fluorescent microscopy (Olympus BX51, Japan) was employed to assess the mean percentage of the labeled cells.
Induction of demyelination model by cuprizone and animal grouping
In this study, 60 female C57BL/6 mice weighing 20–25 g and aged approximately 8 weeks were used. The mice were provided free access to food and water and maintained under suitable health conditions and temperature control (23 °C [± 2] °C). The MS model lesion was induced using cuprizone (bis-cyclohexanone oxaldihydrazone, Sigma-Aldrich Inc. C9012) in all animals except control and sham groups (n = 8 in each group). To this end, 44 mice were gavaged with 400 mg/kg/day cuprizone solution in 200 µl corn oil for 5 weeks. After confirming of MS induction using the hanging wire test, demyelinated animals (n=32) were randomly divided into 4 groups (n = 8 in each group), including MS, MS/vanillin, MS/stem cells, and MS/ vanillin /stem cells. The control group animals received no intervention. Moreover, the mice in the sham group were gavaged daily with 200 µl corn for five weeks, followed by a single intravenous injection of 200 µl PBS into the tail vein. In vanillin receiving groups, vanillin (40 mg/kg dissolved in water) was injected daily for four weeks and in stem cell receiving groups, a single dose of PBS (200 µl) containing 1 × 10 6 stem cells were injected intravenously (IV) into the tail.
Motor function examination by behavioral tests
The hanging wire test was used to compare improvements in balance and motor function in the various experimental groups. Briefly, the animal was hung from a metal wire (90 cm long, 2 mm in diameter, and 50 cm high), and the latency to fall was recorded over a 300 sec period. The maximum hanging time was recorded. This test was performed weekly from one week before induction of MS model to the end of the study ( 31 ).
Histological studies
The mice were anesthetized by intraperitoneal injection of combination of ketamine (Sigma) and xylazine (Sigma) and mice brain fixation was performed through the cardiac perfusion method with ice-cold PBS (pH = 7.4) and 4% paraformaldehyde (PFA) (PFA, Sigma-Aldrich, P6148). After removal of the brain from the skull, they were fixed again in 10% formalin for 24 hr. Thereafter, paraffin sections were prepared from the CC in serial sections of 5–7 μm thickness.
Luxol fast blue staining
According to a previous study ( 32 ), after deparaffinization and clarification of the prepared tissue sections, the samples were placed in Luxol Fast Blue (LFB) solution (0.1% w/v, Sigma-Aldrich, USA) at 56 °C overnight. After that, the samples were washed in 70% ethanol, 0.05% lithium chloride solution for 30 sec, and then in 70% ethanol. After washing in distilled water, they were immersed in cresyl violet acetate solution for 60 sec. In order to assess the degree of demyelination, the stained sections were photographed under a microscope and myelin density was measured using ImageJ.
Immunohistochemical technique for identifying Mbp, Olig2, and Mog markers
Based on our previous studies ( 31 - 33 ), oligodendrocyte transcription factor 2 (Olig2), myelin oligodendrocyte glycoprotein (Mog), and MBP (myelin basic protein or myelin marker) were used to identify oligodendrocytes and myelin. After antigen retrieval using sodium citrate buffer, blocking of nonspecific antigens was performed using 10% goat serum albumin, diluted in PBS for 30 min at room temperature. Next, primary antibodies, including rabbit anti-Mog antibody (1:1000; Abcam, ab32760, RRID: AB -2142685); Rat anti-Mbp antibodies (1:1000; Abcam, ab7349 Cambridge, MA, USA, RRID: AB - 306726) and rabbit anti-Olig2 (1:1000; Abcam, ab109186, RRID: AB -10861310) were used overnight at 4 °C in a humidified environment. After washing with PBS, the slides were incubated with FITC-conjugated secondary antibodies (1:500; Abcam, Cambridge, MA, USA) for 1 hr at room temperature. Subsequently, the sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, D9542) for 2 min to reveal the cell nuclei, and the cells were examined using a fluorescence microscope (Olympus BX51, Japan). Finally, the average percentage of cells expressing Mog and Olig2 markers was determined from 200 cells per slide using ImageJ. In addition, the average of myelin density was calculated using ImageJ.
ELISA test for interleukin evaluation
This method was used to assess the serum levels of inflammatory factors (IL-2, interferon gamma (IFN-γ)), anti-inflammatory factors (IL-10 and transforming growth factor beta (TGF-β)) and immunoglobulin G factor. First of all, washing buffer (KPG-WB, karmaniaparsgen, Iran) and avidin-HRP solution (KPG-HA, karmaniaparsgen, Iran) were prepared according to the company's protocol. In the first step, 50 μl of standard solutions 1 to 4 (KPG- MIL2 NS1-4, karmaniaparsgen, Iran) were added to the first to fourth wells, and the fifth well was considered a control. In the next step, 50 μl of the sample was added to the remaining wells and incubated for 50 min on a shaker (speed 200 rpm). After that, the plates were washed 3 times using washing solution (step 3). Next, 50 μl of conjugated antibody (Detection ab, KPG- MIL2D, karmaniaparsgen, Iran) was added to all wells except the control and incubated again for 50 min under the previous conditions (step 4). After incubation, the plates were washed 3 times using washing solution (step 5). Next, 50 μl of HRP-avidin solution (HAA, karmaniaparsgen, Iran) was added to all wells except the control well and placed on a shaker for 50 min (step 6). After incubation, the plates were washed 5 times (step 7) and then 50 μl of substrate was added to all wells and incubated for 15 min (step 8). Finally, 2 μl of stopping solution (KPG-ST, karmaniaparsgen, Iran) was added to all wells (step 9) and the absorbance of the samples was measured in an ELISA reader at a wavelength of 4.0 nm. It should be noted that all steps except steps 4 and 6 were performed for the control well.
Statistical analysis
Statistical differences between groups were determined using one-way analysis of variance (ANOVA) using SPSS version 25 (IBM, Armonk, NY, USA). Results were presented as mean ± standard error, and P -values less than 0.05 were considered significant.
Results
Effects of stem cells and vanillin on mouse motor function improvement
Data evaluation showed that the motor function of the mice significantly decreased following the administration of cuprizone; however, it improved with cell transplantation and vanillin consumption. As shown in Figure 2 , In the fourth week a significant increase of hanging time in mice was seen in MS/Vanillin group (133±4.4) ( P ≤0.05), Ms/Stem group (130±4.2) ( P ≤0.05) and in Ms/stem/Vanillin group (153±3) ( P ≤0.01) in compared to the Ms group (105±3), Additionally Ms/Stem/Vanillin group had a statistically significant increase compared to MS/Vanillin and Ms/Stem groups ( P ≤0.05).
Vanillin and hADSCs reduced demyelination and increased oligodenderogenesis
Analysis of the results showed the average percentage of Olig2 and Mog positive cells increased significantly in treated groups, especially in the Ms/stem/Vanillin group (27.5 ±.6 for Olig 2 and 24.5± .7 for Mog) compared to the Ms group (12±.8 for Olig2 and 7.5±.8 for Mog) ( P ≤0.01) ( Figures 3A and 4A ). Furthermore, quantification of the data showed that PKH26/Olig 2 and PKH26/Mog positive cells were significantly higher in Ms/stem/Vanillin groups (12.5±.4 for PKH26/Olig2 and 10.5±.7 for PKH26/Mog) ( P ≤0.05) than in the Ms/stem group (7.5±.3 for PKH26/Olig2 and 6.3±.1 for PKH26/Mog) ( P ≤0.05) ( Figures 3B and 4B ).
In order to assess myelin density and the extent of demyelination/remyelination in mice, the average myelin density was measured in sections stained with LFB and in sections stained using the myelin-specific marker ( Figure 5 ). The results showed that the density of myelin in CC sections significantly increased in treated groups, especially in the Ms/stem/Vanillin group (153000±98) compared to the Ms group (52000±120) ( P ≤0.01) ( Figure 5A, B ).
ELISA test results
The findings showed that the serum level of proinflammatory cytokine (IL-2 and IFN- γ) in the MS group were considerably higher (76.98±.9 for IL-2 and 77.89±1.1 for IFN- γ) compared to other groups ( P ≤0.01) ( Figure 6 A, B ). Moreover, the serum levels of the anti-inflammatory factors (IL-10 and TGF-β) in the treated groups showed a significant increase especially in the Ms/stem/Vanillin group (78.52±.88 for IL-10 and 73.82±.9 for TGF-β) compared to the MS group (30.91±.58 for IL-10 and 30.77±.79 for TGF-β) ( P ≤0.01) ( Figure 6 C, D ). Also, a significant difference was seen in the serum levels of IgG in the MS group (70.91±.67) compared to the other groups (57.34±.9 in Ms/Vanillin group, 32.81±.77 in Ms/stem group, and 34.83±.55 in Ms/stem/Vanillin group) ( Figure 7 ).
Discussion
Neurological diseases are among the major threats to human health. During neurodegenerative diseases, lymphocyte cell infiltration into the nervous tissue and specific interleukins secretion lead to the initiation of oligodendrocyte cell death ( 34 ). This process leads to myelin tissue destruction and the development of functional disorders in the CNS. Among these abnormal conditions, MS can be mentioned. MS is an autoimmune disease in which the immune system reacts against oligodendrocyte cells and the myelin sheath ( 35 ). The role of pro-inflammatory cytokines is very important in the development of this abnormal condition. Pro-inflammatory cytokines such as TNF-α, IL-2, IL-6, IL-17, and IFN-γ play an important role in myelin destruction by activating immune cells and increasing inflammation, followed by disrupting the transmission of nerve signals, leading to the development of sensory-motor problems. The role of anti-inflammatory cytokines such as IL-10 in reducing inflammation and myelin damage has also received special attention ( 35 ). Although the role of immunoglobulin-producing cells is still unclear, some studies suggest that some of the immunoglobulins produced may be beneficial in myelin regeneration ( 11 ). Therefore, there may be a relationship between the onset or prevention of MS and immunoglobulins. Current published data suggest that cell-based and drug-based therapies could be a new paradigm for the treatment of MS.
For example, transplantation of human adipose stem cells (hADSCs) into a cuprizone model of MS has been shown to improve motor function and reduce pathological symptoms such as demyelination ( 32 ). This finding is consistent with a previous study which demonstrated that intravenous injection of hADSCs can reverse the clinical course of EAE ( 36 ). Taken together, ADSCs, with their wide range of valuable properties, can replace degenerated neurons, provide a suitable environment for the maintenance of remaining neurons, and promote tissue regeneration. Therefore, ADSCs are a promising cell source for cell-based therapies in MS, although further studies are needed to confirm their efficacy and safety in humans. In line with the results of the present study, Razavi et al . in 2018 showed a significant increase in remyelination in the groups receiving adipose-derived stem cells ( 37 ). Also, another study showed that intravenous administration of adipose tissue-derived MSCs increased remyelination and improved motor and cognitive function ( 38 ).
In addition, the use of specific drugs or agents can enhance the differentiation of stem cells into target cells. Therefore, the present study aimed to evaluate the role of vanillin as a neuroprotective, anti-inflammatory, and immunomodulatory agent in the differentiation of hADSCs into oligodendrocytes and remyelination in a cuprizone-induced mouse model.
Vanillin is an aromatic organic compound with various therapeutic effects, including anticancer ( 24 ), antidiabetic ( 26 ), antioxidant ( 23 ), antidepressant ( 39 ), antibacterial ( 40 ), and neuroprotective properties ( 41 ). Several studies suggest that vanillin has promising potential in the prevention and treatment of various metabolic and nonmetabolic diseases ( 42 ). It has been reported that this natural compound significantly protects the brain by reducing the level of lipid peroxidation and increasing the activity of antioxidant enzymes. Furthermore, it improves motor and non-motor deficits by reducing apoptosis, reducing stress, and reducing mitochondrial dysfunction in the brain ( 19 ).
The results of the present study showed that co-treatment of stem cells with vanillin increased oligodendrocyte differentiation and enhanced remyelination. As shown in immunohistochemical images ( Figure 2 ), the mean percentage of cells which expressing Olig2 and Mog markers was significantly reduced in the cuprizone-treated groups. In justification of this, cuprizone may cause mitochondrial enzyme dysfunction associated with oxidative stress, leading to metabolic stress in oligodendrocytes and myelin loss due to selective oligodendrocyte death in several brain regions ( 43 ).
Our findings further revealed that cuprizone has an important function in increasing serum IgG levels which play an important role in demyelination by forming immune complexes and causing cytotoxicity. As seen in figure 7 , serum levels of IgG significantly decreased in the groups receiving vanillin. Furthermore, the duration of hanging in the groups that received vanillin significantly increased compared to the cuprizone group. In justification of these results, it can be said that vanillin has significant immunomodulatory and antioxidative effects, thus, with inhibitory effects on immune cell activity ( 44 ) can lead to decreased immunoglobulins secretion. On the other hand, vanillin, due to its neuroprotective effects, is able to prevent cell death by inhibiting ERK1/2, P38 and the NF-κB Signaling Pathway ( 45 ). Therefore, by preserving neuroglia cells and the myelin sheath, muscle contractile strength will increase and the mice's motor balance will be maintained.
Inflammation plays a main role in the MS pathogenesis ( 46 ). As seen in Figure 6 , proinflammatory factors, including IL-2 and IFN-gamma were significantly increased following the use of cuprizone. Another relevant result of our study was that, serum levels of anti-inflammatory factors were significantly increased in the groups that used vanillin and stem cells. In justification of these findings, it can be concluded that vanillin reduced neuroinflammation with reducing proinflammatory factors and neutralizing the effects of cuprizone. Also, vanillin improves mitochondrial function in neuroglia cells by neutralizing free radicals and increasing endogenous antioxidant enzymes. So, vanillin probably suppresses apoptosis of oligodendrocytes and myelin tissue through its antioxidant and anti-inflammatory activities. The results of the present study showed that hADSCs are able to reduce proinflammatory cytokines and IgG factor. In addition, they increase serum levels of anti-inflammatory factors. In justification of these results, it can be said that hADSCs probably modulate the function of monocytes/macrophages with paracrine functions and therefore play an important role in the immune response and are able to suppress inflammation ( 47 ).
Conclusion
The results of this study showed that vanillin, with its anti-inflammatory and neuronal modulatory properties, can induce differentiation of hADSCs into oligodendrocytes and improve remyelination in the brain tissue. Also, hADSCs are able to facilitate the reduction of inflammation by reducing inflammatory cytokines and immunoglobulin G. Therefore, the use of vanillin alone and simultaneously with hADSCs transplantation will be an effective step towards improving the health of MS patients.
Acknowledgment
The results presented in this paper were part of a student thesis, and we acknowledge the facility and funding provided by Isfahan University of Medical Sciences, Iran.
Ethical Approval and Consent to Participate
This research project, titled "The Synergistic myelin regenerative, immunomodulatory and Anti-inflammatory Effects of vanillin and adipose derived stem cells in Cuprizone Animal Model of Multiple Sclerosis" was carried out in strict accordance with the "Guideline for the Care and Use of Laboratory Animals in Iran". This experimental study was conducted on the female C57BL/6 mice model of MS at the Central Laboratory of Isfahan University of Medical Sciences, Iran. All procedures of research were conducted according to the guidelines of the Iranian Committee of Animal Care approved by the Ethics Committee (Ethics# IR.MUI.AEC.1403.033).
Funding
Isfahan university of Medical Sciences, Iran, supported this work (grant number: 3403223).
Authors' Contributions
All authors contributed to the study conception, design, statistical analysis, and drafting of the manuscript. S G and N G contributed to data collection and manuscript drafting. M M revised the manuscript. All authors read and approved the final manuscript.
Conflicts of Interest
All authors confirm that they have reviewed and understood the journal's conflicts of interest and authorship policies. They declare no competing interests related to this work. Each author has made significant contributions to the research and the manuscript, and has reviewed and approved the final version for publication.
Declaration
We have not used any AI tools or technologies to prepare this manuscript.
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