Carvacrol has been shown to be effective on some inflammatory and neurodegenerative diseases of the brain, which makes it a good option for the treatment of MS. The characteristics of the EAE model were confirmed by the observed increase in neurological defects and clinical scoring, the presence of CNS-infiltrated immune cells and axonal degradation, which are consistent with results of previous studies(Mohajeri, Sadeghizadeh et al. 2015; Yang, Zheng et al. 2017).
Carvacrol has neuroprotective effects that are demonstrated in neurological models such as ischemia and Parkinson's disease(Baluchnejadmojarad, Hassanshahi et al. 2014; Dati, Ulrich et al. 2017). The carvacrol treatment results in the present study showed a decrease in clinical scores and histological changes. It also resulted in decreased expression of some proinflammatory genes and increased expression of genes involved in myelination.
CNS repair is the goal of treatment for CNS and autoimmune disorders. Different neuroprotective therapeutic strategies have been used in clinical trials but, as yet, no comprehensive and successful examination of remyelination has been proposed. In the case of MS, remyelination not only better protects from axonal damage, but also can halt progression of the disease.
Oligodendrocytes have been a focus of attention for the development of therapeutic approaches and treatments for neurodegenerative disease because of their myelination potential(Kotter, Stadelmann et al. 2011). One of the most important features of MS is demyelination, in which the axon-oligodendrocyte relationship in the CNS is disrupted(Shakhbazau, Schenk et al. 2016). Evaluation of the therapeutic effects of such research methods requires the identification of oligodendrocytes. For this purpose, the present study used MBP, PDGFR and OLIG2 markers.
The results of molecular analysis for the carvacrol treated group showed that the remyelination rate increased and demonstrated that carvacrol increased OLIG2 mRNA expression. It was found that clinical scoring in the carvacrol treated group was lower than in the EAE group, indicating that the clinical damage improved with carvacrol treatment. OLIG2 expression increased in the carvacrol treated group as the clinical scores decreased. OLIG2 encodes the transcription factor that is necessary for remyelination and is obtained through proliferation and differentiation of neuronal progenitors and oligodendrogenesis progenitor cells (OPCs). This factor is necessary for oligodendrogenesis in the spinal cord. OLIG2-expressing progenitor cells differentiate into oligodendrocytes in damaged lesions, leading to premature myelination of the CNS(Wegener, Deboux et al. 2015) .
In addition to OLIG2, PDGF is also involved in activating the oligodendrogenesis pathway and is involved in their proliferation. An increase in PDGF has been shown to increase the number of OPCs in demyelinated lesions. However, although PDGF infusion induces SVZ type B cell proliferation which is effective for remyelination, it can have side effects such as tumor formation(Rivera and Aigner 2012).
OPCs express various markers, the most important of which are NG2 and PDGFRα. Because NG2 can be present in other glia precursor cells, the PDGFRα marker is more specific for immature oligodendrocytes.
In this study PDGFRα was used to assess the amount of immature oligodendrocyte formation. Precursor oligodendrocytes were observed to attach to neurons and, after two to three days, myelinated them(Blakemore and Irvine 2008). However, contrary to the results of previous studies, PDGFR showed increased expression in the EAE group and decreased expression in the carvacrol treated group.
In agreement with the results of the present study, Qiang Zho et al. reported that transgenic mice showed that deletion of PDGFR caused precocious differentiation of OPCs. Their findings demonstrate that PDGFR is an important negative regulator of OPC maturation in the developing CNS. During OPC differentiation, PDGFR expression gradually decreases and is completely silent in adult oligodendrocytes. It is not clear how cessation of PDGFR expression or OPC signaling triggers oligodendrocyte differentiation. One possibility is that PDGFR signaling may inhibit the expression or function of oligodendrocyte maturation activators. These activators include myelin regulatory factor (MRF), sip1 and SOX10, each of which is sufficient to differentiate oligodendrocytes. If this hypothesis is correct, Nkx2.2 transcriptional inhibitor initiates internal differentiation by removing the inhibition of these regulatory factors and responding to axon signals or other environmental messages. It may initiate internal differentiation by removing the inhibition of these regulatory factors in response to axon signals.
Nkx2.2 is a transcriptional inhibitor in cell-fate control during CNS development and determines the time of OPC differentiation. Overexpression of Nkx2.2 in transgenics will extinguish PDGFR expression. It is possible that, when Nkx2.2 binds to the binding site on the PDGFR promoter, it inhibits PDGFR transcription. By repressing the expression of the membrane receptor PDGFR, the OPCs will no longer respond to mitogen PDGFR, which will cause the proliferation and activation of cell differentiation to stop.
On the other hand, an increase in Nkx2.2 expression and inhibition of PDGFR expression has been observed to increase MBP expression in primary OPCs. Therefore, the possibility that Nkx2.2 may influence other factors simultaneously to affect differentiation and maturation of OPCs cannot be ignored (Zhu, Zhao et al. 2014). After demyelination, OPCs switch from neutral to active and mitotic cells by upregulating OLIG2 and Nkx2.2 (Göttle and Küry 2015). In general, the reduction in PDGFR expression by carvacrol in this study could provide a pathway for the differentiation of OPCs and help to myelinate and repair damage caused by the model induction.
MBP plays the most important role in the formation of the three-dimensional (3D) structure of the axial membrane of myelin. This protein binds to membrane lipids as it enters the oligodendrocyte membrane and strengthens the 3D structure of the myelin membrane(Czepiel, Boddeke et al. 2015). MBP was used in the present study as an adult oligodendrocyte marker to evaluate the final remyelination rate. The goal was to determine which changes occur during the process of remyelination from immature oligodendrocytes to adults. MBP mRNAs are located at one point in the cytoplasm of oligodendrocytes before myelination and then disperse into the cytoplasm at the beginning of myelination. This spatial separation differentiates oligodendrocytes into complete myelinating cells (Musse and Harauz 2007) .
It has been shown in MS patients and animal models that MBP decreased about three-fold compared to normal levels. In glial cells and macrophages, the group of enzymes called protein arginine deiminase (PAD) becomes uncontrolled for unknown reasons in inflammatory diseases such as MS and produces immunogenic citrullinated epitopes. PAD2 and PAD4 levels increase in MS. It is possible that these two enzymes alter the structure of the third MBP, and predispose them to proteolysis so that MBP cannot properly form the multilayer structure of a myelin sheath. Eventually, the accumulation of nerve cells will decrease(Jones, Causey et al. 2009).
In our study induction of the EAE model reduced MBP expression in the EAE group and further study showed that carvacrol administration increased MBP expression in the carvacrol treated group. These results were confirmed by both molecular methods and immunohistofluorescence analysis. The increase in OLIG2 expression increased the production of differentiated oligodendrocytes as well as MBP expression, which indicates the activity of oligodendrocytes in the myelination pathway. The increase of these two factors demonstrates the process of myelination. However, molecular processes are so complex that these observations cannot easily be attributed to an increase or decrease in the expression of a gene. In such cases, many factors must be considered.
Studies have shown that carvacrol has an effect on inflammatory factors and oxidative stress. Gholijani et al. (2016) caused inflammation in mice and cultured their spleen cells to show that carvacrol reduced the formation of IL-17 as well as the expression of T-bet, GATA-3 and RORƔc transcription factors that necessary for the maturation and function of Th1, Th2 and Th17 cells, respectively)(Gholijani and Amirghofran 2016). The Th17 lymphocyte has multiple pathogenic roles, most of which are attributed to IL-17 secretion. These include neutrophil recall, activation of innate immune cells, increased B lymphocyte function and stimulation of the release of inflammatory cytokines, including IL-1β and TNF-α, which are the cause of many autoimmune diseases and the induction of EAE and are present in many MS lesions. IL-1 acts directly on T lymphocytes and causes the secretion of IL-17; therefore, it plays a pivotal role in the progress of autoimmunity in EAE and initiates the differentiation of pathogenic Th17 cells(Sutton, Brereton et al. 2006). Th17 causes BBB disruption and promotes CNS immune cell traffic and tissue inflammation through IL-17 and IL-22. Il-17 exerts its pathological effects through chemokine stimulation and the adhesin molecules involved in the neutrophils penetration from the surrounding environment to the CNS(Raphael, Nalawade et al. 2015) .
Due to the decrease in IL-17 expression caused by carvacrol both in RT and immunohistochemical histological analysis in the present study, it appears that the effectiveness of carvacrol on reducing IL-17 production may relate, at least partially, to the modulation of auto-reactive Th17 and Th1. Carvacrol also diminish the flow of leukocytes to the CNS by reducing the inflammatory cytokines that are participated in the production of chemokines and adhesion molecules(Yang, Zheng et al. 2017). Studies have shown that carvacrol reduces IL-1 and TNF-α and inhibits dendritic cell maturation and function as well as the selection of T cell responses.
The results of this research on the NF-ҚB and IL-1 gene expression are consistent with the results of Gholijani et al. and indicated that carvacrol reduced the expression of these genes(Raphael, Nalawade et al. 2015). NF-ҚB plays an important role in inflammatory processes. Aristatile et al. Induced inflammation in rat liver cells and showed that treatment of these cells with carvacrol reduced NF-Қβ and TNF-α expression(Aristatile, Al-Assaf et al. 2013). The TNF-α gene promoter contains the NF-Қβ response element. Under normal condition, NF-β is present in the cytoplasm of cells and binds to IҚBα and IҚBβ, which prevents it from entering the nucleus. Separation of NF-ҚB from IҚB leads to NF-ҚB entry into the nucleus and its binding to specific sequences in the promoters of specific genes and triggers gene expression. Aristatile et al. showed that carvacrol inhibits NF-ҚB expression by inhibiting IҚB degradation, which inhibits TNF-α gene expression(Gimenez, Sim et al. 2006).
When EAE mice are immunized with myelin peptides, increased TNF-α expression exacerbates demyelination . However, the results of analysis of TNF-α gene in the present study was not consistent with the results of previous studies. In the carvacrol treated group, the carvacrol did not change the expression of this gene compared to the EAE group.
Despite the proinflammatory effect of TNF-α, which has been the subject of numerous articles, Arnet et al.,(Arnett, Mason et al. 2001) got unexpected results.They induced a demyelinated model in mice using cuprizone and deliberately inhibited TNF-α using XPro1595. Contrary to expectations, they found that the lack of TNFα caused significant delays in remyelination. These results were confirmed by histological and immunohistochemical analysis for myelin proteins. The reason for this failure in repair was shown to be the reduction of the pool of proliferating oligodendrocyte precursors, which resulted in a decrease in the number of adult oligodendrocytes.
TNF-α actually initiates remyelination through the TNFR2 signaling pathway and TNFR2 is necessary for the maturation of oligodendrocytes(Kircik and Del Rosso 2009). MCP inflammatory factor also is involved in the development of MS. It is broadly expressed in brain of MS patients, but not in the white matter of CNS tissue of healthy individuals. MCP is a β-chemokine that is a potent monocyte and T-cell chemoattractant and is secreted by active hypertrophic astrocytes(Van Der Voorn, Tekstra et al. 1999). Altered astrocyte functions, such as loss of normal activity or gaining abnormal functions could be involved in the onset and progression of MS(Mostafavi, Khaksarian et al. 2014). MCP activates and invokes myelin-destroying macrophages and is directly involved in the progression of MS.
MCP causes the secretion of lysosomal enzymes in monocytes and provides the signal needed to activate T lymphocytes, thereby facilitating antigen presentation. On the other hand, it also increases the secretion of MMP-9 by T-cells. MMP-9 has the ability to destroy the basal membrane and other matrix compounds, which will cause inflammatory cells to migrate to such tissue. MCP-1 can activate microglia cells in the tissue and in the newly arrived lymphocytes in MS wounds, causing tissue damage and demyelination. MCP-1 also increases inflammation, stimulates the secretion of enzymes, which is likely involved in BBB degradation and activates monocytes(Mostafavi, Khaksarian et al. 2014).
The effect of carvacrol on MCP as related to inflammatory factors in patients exposed to mustard gas has been shown to decrease. The antioxidant effect of carvacrol on MCP-1 also has been demonstrated in mice infected with Campylobacter jejuni. However, the results of this study did not agree with those results. On the contrary, carvacrol not only failed to reduce this factor in the carvacrol treated group, but actually increased it compared to the EAE group. Little study has been done thus far on the efficacy of carvacrol on MCP-1 in an EAE model and this finding requires further investigation. The favorable effects of carvacrol observed on some inflammatory factors and myelin repair factors show promise for the therapeutic effects of carvacrol. In this regard, additional clinical and molecular evidence must be gathered.