Original Paper
Nervonic acid regulates the oxidative imbalance in experimental allergic encephalomyelitis
2021 Volume 27 Issue 2 Pages 269-280
Details
2021 Volume 27 Issue 2 Pages 269-280
We aimed this study at investigating the effect of nervonic acid on inflammation, and the potential mechanisms underlying the action of nervonic acid in experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. During the study, we divided the mice into six groups: Model group, Control group, Drug group, and Nervonic acid groups(three different doses). This study observed the effects of nervonic acid (NA) on inflammatory infiltration and demyelination in the spinal cord of mice by Hematoxylin & Eosin (H&E) and Luxol Fast Blue (LFB). Combining biochemical evaluation and enzyme linked immunosorbent assay (ELISA) analysis was evaluated as the expression of antioxidant proteins and anti-inflammatory cytokines. Our results indicated that treatment with nervonic acid significantly inhibited the development and severity of experimental autoimmune encephalomyelitis in mice, accompanied by mitigating inflammatory infiltration and demyelination in the spinal cord of mice, and increased the expression of antioxidant proteins and anti-inflammatory cytokines. So, nervonic acid may be a promising candidate for intervention in multiple sclerosis and other autoimmune diseases as a potential new treatment option for treating multiple sclerosis.
Multiple sclerosis (MS) is the most common central nerve demyelinating disease and is characterized by loss of myelin, glial scar formation, subsequent axonal and progressive loss of nerve function (Dendrou et al., 2015; Lassmann and Bradl, 2017; Noseworthy et al., 2000). Experimental autoimmune encephalomyelitis (EAE) is one of the most mature animal models of MS (Baxter, 2007; Constantinescu et al., 2011). Cytokines are involved in regulating of inflammatory responses and are closely related to the acute phase of MS. Soluble products produced by infiltration of immune cells and glial cells are one of the causes of this tissue damage. Highly active molecules, such as reactive oxygen or reactive nitrogen, are thought to be involved in inflammatory processes, exacerbating the inflammatory responses and causing tissue damage (Atilla et al., 2004). Central nervous system tissue is susceptible to oxidative damage, suggesting that oxidation plays an important role in the pathogenesis of MS and its animal model EAE (Greter et al., 2005; Mahad et al., 2015). Cytokines associated with Th2 or Treg cells such as IL-4 and IL-10 can reduce inflammation and improve symptoms in MS patients (Brambilla et al., 2009; Bettelli et al., 2003; Hibbits et al., 2012).
Nervonic acid (NA, C24:1, Δ15, ω-9, cis-tetracos-15-enoic acid) is a kind of ω- 9 long-chain monounsaturated fatty acid, with a molecular formula of C24H46O2, Chemical formula of CH3-(CH3)7-CH=CH-(CH2)13-COOH, and a molecular weight of 366.6. Pure NA is a white solid under room temperature conditions (Yamazaki et al., 2014; Wang et al., 2006). NA is the core natural component of brain nerve tissue and nerve cells (Chivandi et al., 2008; Tanaka et al., 2007). It is a substance with the special effect of promoting the repair and regeneration of damaged nerve tissue; It improves the activity of the cranial nerves, preventing brain aging with a significant effect. NA is often implicated in many neurological disorders and in some mental illnesses, such as: MS, Adrenoleukodystrophy, Alzheimer's disease, Parkinson's syndrome, Schizophrenia and Attention Deficit Disorder (Penkowa et al., 2000; Steinman, 1996).
In our study, we immunized C57BL/6 mice with MOG35-55 peptide to induce EAE and tested the effect of treatment with NA on the development and progression of EAE and observed the effects of NA on inflammatory infiltration and demyelination in mice by Hematoxylin & Eosin (H&E) and Luxol Fast Blue (LFB). Combining biochemical evaluation and enzyme linked immunosorbent assay (ELISA) analysis was evaluated as the expression of antioxidant proteins and anti-inflammatory cytokines. Our test indicated that treatment with NA increased the expression of antioxidant proteins and anti-inflammatory cytokines. So, NA can regulate the oxidative imbalance in EAE.
Animals We purchased C57BL/6 mice (female, weighing 18–22 g, aged 6–8 weeks) from the Pengyue Laboratory Animal Co. Ltd. Jinan (Number: SCXK20190003). Mice were given food and drinking water ad libitum and maintained at a room temperature of 20–25 °C with 12 h dark/light cycles in the animal room of Shandong Academy of Medical Sciences. The Institutional Animal Care and Use Committee of Shandong Academy of Medical Sciences approved experimental protocols.
Induction and Assessment of EAE and Treatment For use, 5 mg of MOG35-55 was dissolved in 1 mL of physiological saline, and Bacillus Calmette-Guerin(BCG) was dissolved in complete Freund's adjuvant (CFA) at a concentration of 10 g/L. Subsequently, the above two liquids were respectively mixed 1:1 to form an induced emulsifier. The EAE model was prepared by subcutaneous injection of 0.1 mL (0.05 mL each of the left and right dorsal sides) into the mouse's back with a syringe. The control group was injected with the same amount of normal saline. On the day of modeling and 2 days later, the mice were intraperitoneally injected with 0.5 mL of pertussis dilution to enhance immunity, and the control group was injected with the same amount of normal saline. Mice that successfully modeled were included in the experiment based on the incidence and methodology of our study.
Nervonic acids were separated from acer truncatum oil by urea complexation, molecular distillation and preparative high performance liquid chromatography. Finally, the purity of nervonic acid was 95.8%. Mice were given nervonic acid by intragastric administration.
1. Control group (n = 10): without immunization
2. EAE group (n = 10): immunized only.
3. Low dose of NA + EAE group (n = 10): Low dose of NA (197 mg/kg) was given on the third day of immunization (Day 3) and continued daily for the duration of the experiment.
4. Medium dose of NA + EAE group (n = 10): Medium dose of NA (394 mg/kg) was given on the third day of immunization (Day 3) and continued daily for the duration of the experiment.
5. High dose of NA + EAE group (n = 10): High dose of NA (788 mg/kg) was given on the third day of immunization (Day 3) and continued daily for the duration of the experiment.
6. Drug group (n = 10): Dexamethasone (6 mg/kg) was given on the third day of immunization (Day 3) and continued daily for the duration of the experiment.
Mice were given with above substances in accordance with the methodology of this study to achieve the peak period of disease, then the mice were sacrificed. The animals were weighed and examined daily for the severity of neurological dificts by two independent experienced observers who were not aware of the animal's treatment.The neurological deficit score was as follows :0 for no paralysis, 1 for loss of tail tension, 2 for weakness of hindlimb, 3 for paralysis of hindlimb, and 4 for paralysis of fore-limb and hindlimb. 5. for moribund and death.
The 9th day after immunization was recorded as the disease incubation period, the 17th day was recorded as the peak onset period, and the 28th day was recorded as the remission period. Each group was sacrificed at the peak of onset (day 17 was recorded as the peak of onset)
Histopathological Evaluation Animals from all experimental groups were sacrificed 17 days after EAE induction for further analysis. Blood was extracted from the heart for serum preparation. Their brains and spinal cords were removed carefully, and they were fixed in paraformaldehyde and embedded in paraffin. The 5 µm spinal cord sections of mice were stained with Hematoxylin & Eosin (H&E) for routine evaluation of histology and inflammation, and were stained with Luxol Fast Blue (LFB) for the evaluation of demyelination to assess the extent of cellular inflammatory lesions.
Electron Microscopy The spinal cord tissue washed with normal saline, and placed in 2% glutaraldehyde fixative solution. Then it was dehydrated and embedded in epoxy resin, and cut into 1 µm thick sections, and stained in 1% benzylamine blue. The tissue of the desired site was made into ultra-thin sections (80 nm) and stained with saturated uranyl acetate and Reynolds lead citrate, and examined with an electron microscope(Hitachi HT-7800 Japan).
Immunohistochemistry Analysis Immunohistochemistry analysis of Nrf2, NQO1 and HO-1. The main antibodies were rabbit anti-Nrf2 polyclonal antibody (Abcam), anti-HO-1 polyclonal antibody (Abcam) and anti-NQO1, (Abcam) at the dilution factor of 1:50. The secondary antibody was HRPlabeled goat anti-mouse IgG antibody with the dilution factor of 1: 100. Count positive cells under the light microscope.
Biochemical Evaluation The thiobarbituric acid reactive substances (TBARS), reactive oxygen species (ROS), superoxide dismutase (SOD), and catalase (CAT) activities in the brain were measured to evaluate the degree of oxidative stress in the brain. Mice were decapitated at the peak of EAE, periventricular brain tissue of 0.5 g was obtained, and immediately the tissue were crushed into a powder with a grinding rod (note: do not freeze-thaw the tissue). The sample was placed in a 15 mL conical centrifuge tube and add 500 µL of lysate placed in an ice bath, and vortex vigorously for 5 s. Then the homogenate was centrifuged at 3 000 × g for 10 min to collect supernatant. The supernatant was stored at −80 °C for further analysis. The concentrations of SOD, CAT, TBAR and ROS were measured using the assay kit (Bioss, China). All analyses were performed according to the kit instructions.
Enzyme Linked Immunosorbent Assay (ELISA) Analysis The whole blood sample collected in the serum separation tube were left at room temperature for 2 hours, and the sample was centrifuged at 1 000 × g for 20 min. The supernatant was stored at −80 °C(avoid repeated freeze-thaw cycles). The mouse ELISA kit (Bioss, China) was used to detect the levels of inflammatory factors IFN-γ / TNF-α and IL-4 / IL-10 in serum.
Statistical Analysis The measurement data were expressed as the mean plus or minus the standard deviation (x ± s). We used the ANOVA single factor analysis of variance to analyse the comparison of multiple groups of measurement data, and the LSD method to test the pairwise comparison between groups; Correlations between variables were analysed using Pearson linear correlation analysis. To perform the statistical processing we used the SPSS17.0 software. Test level α = 0.05, p <0.05 was considered statistically significant.
Nervonic Acid Reduces the Severity of EAE The mice in the control group had no abnormal performance, and the EAE group and the drug intervention group had different degrees of disease severity. In the early stage of the onset, the main manifestations were reduced activity, reduced food intake and weight loss. Some mice had sclerosis, swelling, hair loss, or even ulceration at the site of antigen injection. The symptoms of neurological dysfunction were mostly manifested by the decline in rat tail tension. Subsequently, the mice gradually developed paralysis of the hind-limbs or fore-limbs to varying degrees, and even quadriplegic and incontinence in individual mice. There were no dead mice in both groups and we didn't observe any effects associated with the NA treatment.
In fig. 1A, the weight of mice in the control group increased normally. After the induction of EAE model, mice in each group did not show obvious symptoms of weight loss during the observation period. The body weight of the EAE group, drug group and NA groups began to drop on the 11th day after modeling, and the body weight of the EAE group was the most obvious.
A Weight changes of mice in each group. B Mean clinical score; data were given as mean clinical disease scores,where 0 signified no paralysis, 1 signified loss of tail tone, 2 signified hindlimb weakness, 3 signified hindlimb paralysis, 4 signified hindlimb and forelimb paralysis; and 5 signified moribund and death. (n = 10. *p < 0.05 vs. Control group; #p < 0.05 vs. EAE group. Different colors represent different groups.
The development and severity of clinical signs in the different groups of mice were monitored in Fig. 1B. Clearly, treatment with NA reduced the clinical scores at post-treatment and the mean clinical scores in the NA-treated mice were lower than that in the EAE group mice throughout the observation period. Collectively, these data indicated that treatment with NA inhibited the development and severity of EAE in mice.
Effect on Central Nervous System Inflammatory Response We used histological methods to detect the pathological changes of spinal cord in different groups of mice on the 17th day after immunization. There was no abnormality in the spinal cord tissue of control group mice. There was no detectable inflammatory infiltrate in the central nervous system, and myelin in the spinal cord of the control group mice was arranged regularly. In the EAE group, could see the vasculature, inflammatory cells, and congestion in the white matter under the spinal cord. There were infiltrations of inflammatory cells around the blood vessels. In the drug group and the high dose of NA group, the amount inflammatory cells were obviously reduced and the myelin sheath was arranged as a stratiform in the spinal cords. There were still many inflammatory infiltrates, vasodilatation and hyperemia in the spinal cords of the medium dose of NA group and the low dose of NA group. The results revealed that the pathological of inflammation and demyelination in the NA-treated mice were lower than that in the EAE mice. So, treatment with NA mitigated autoimmunity-related inflammation in the spinal cords of mice (Croxford et al., 2015; Penkowa and Hidalgo, 2000) (Fig. 2).
H&E analysis of the spinal cord sections (× 400).
(The black arrows represent inflammatory infiltrates. The red arrows represent vasodilatation and hyperemia. Scale bar = 50µm)
Treatment with NA Mitigates Inflammatory Infiltrates and Demyelination in the Central Nervous System The control group showed no abnormalities in spinal cord tissue. There was normal myelin structure, light and dark concentric circular myelin lamellar layers arranged closely. In the EAE group was a partial loss of the myelin sheath; We observed the myelin lamellar, the myelin lamellar rupture, and the vacuolation of the myelin sheath. In the drug group and the high dose of NA group, there was minor loss of the myelin sheath. The myelin sheath was slightly loose, and it was not broken. The gap between the sheets was slightly increased, and the number of layer turns were not obviously reduced. Compared with the EAE group, the degree of lesions was reduced. The gap between the sheets was increased, the myelin lamellar arrangement was loose in the medium dose of NA group, and we didn't observe any obvious myelin lamellar rupture and myelin cavitation. In the low dose of NA group, the myelin sheath was partially detached and edema. The gap between the sheets was increased. The myelin sheath was loose and broken, and the vacuolation of the myelin sheath was seen. Overall, the drug group and the high dose of NA group were obviously improved, and the medium dose of NA group and the low dose of NA group were slightly improved than the EAE group. Treatment with NA mitigated demyelination in the spinal cords of mice (Bettelli et al., 2003). (Fig. 3)
Electron microscopic analysis of the spinal cord sections (× 25 000).
The red arrows represent the loss of myelin sheath. Scale bar = 2.0µm.
The Luxol Fast Blue staining showed that the myelin sheath was stained blue, and the myelin loss was manifested as white vacuoles in the blue area. There were no abnormalities in the spinal cord tissue and normal myelin structure in control mice. In the EAE group, the myelin sheath was obviously lost during the peak period. There were minor part of the myelin sheath lost in the drug group and the high dose of NA group, and compared with the EAE group, the degree of lesions was reduced. Part of the myelin sheath was lost in the medium dose of NA group and the low dose of NA group. The results of the drug group and the high dose of NA group were obviously improved than those of the EAE group, and the results of the medium dose of NA group and the low dose of NA group were slightly improved than those of the EAE group (Hibbits et al., 2012; Sargent et al., 1994). (Fig. 4)
LFB analysis of the spinal cord tissue sections (× 400). Scale bar = 50µm.
Expression of Nrf2, NQO1 and HO-1 in the spinal cord We determined the number of spinal cord NQO1, Nrf2, and HO-1 positive cells in each group of mice at the peak of disease (Fig. 5). Compared with the control group, the number of NQO1, Nrf2, and HO-1 positive cells in the spinal cord of EAE mice increased significantly (p < 0.05). The number of NQO1, Nrf2, and HO-1 positive cells in the NA groups were higher than EAE group, and the highest dose of NA group was the most significant (p < 0.05). In the control group, Nrf2 expression were mainly detected in the cytoplasm of cells, in the EAE group, Nrf2 expression were detected in both the nucleus and cytoplasm.
The number of spinal cord NQO1, Nrf2, and HO-1 positive cells in different groups at the peak stage of EAE. (n = 10, *p < 0.05 compared with control group. #p < 0.05 compared with EAE group. The blue arrows represent the NQO1, Nrf2, and HO-1 positive cells in the spinal cord. Scale bar = 50µm.
The number of spinal cord NQO1, Nrf2, and HO-1 positive cells in different groups at the peak stage of EAE. (n = 10, *p < 0.05 compared with control group. #p < 0.05 compared with EAE group. The blue arrows represent the NQO1, Nrf2, and HO-1 positive cells in the spinal cord. Scale bar = 50µm.
Nervonic Acid Treatment Suppresses Oxidative Stress of EAE The TBARS, ROS, SOD, and CAT expression in the brain were measured to evaluate the degree of oxidative stress in the brain. At the peak of the EAE, from Fig. 6, the average TBARS and ROS concentrations of the mice in the NA groups were significantly lower than EAE group (p < 0.05, Fig. 6 A, B), and the SOD and CAT levels of the mice in the NA groups were significantly higher than EAE group (p < 0.05, Fig. 6 C, D). So, NA decreased the concentration of TBARS and ROS, and increased the levels of SOD and CAT. And the most significant treatment effect was high dose of NA group.
NA treatment inhibited the oxidative stress status of EAE. The TBARS, ROS, SOD, and CAT expression in different groups at the peak stage of EAE (n = 10, each group). *p < 0.05 compared with control group. #p < 0.05 compared with EAE group.
Serum Concentration of Th1/Th2 Cytokines IFN-γ/TNF-α and IL-4/IL-10
To test the effect of NA on inflammatory cytokines in serum of EAE mice. We determined the levels of Th1/Th2 cytokines IFN-γ/TNF-α and IL-4/IL-10 by ELISA. At the peak of the EAE, from Fig. 7, the levels of IFN-γ/TNF-α in the NA groups were significantly lower than EAE group (p < 0.05, Fig. 7 A,B), and the levels of IL-4/IL-10 in the NA groups were significantly higher than EAE group (p < 0.05, Fig. 7 C, D). We can conclude that treatment with NA decreased the levels of IFN-γ/TNF-α and increased the levels of IL-4/IL-10. And the most significant treatment effect was high dose of NA group.
NA treatment inhibited the inflammatory cytokines of EAE. The levels of IFN-γ, TNF-α, IL-4, and IL-10 in different groups at the peak stage of EAE (n = 10, each group). *p < 0.05 compared with control group. #p < 0.05 compared with EAE group.
Multiple sclerosis is the most common central nerve demyelinating disease and is characterized by loss of myelin, glial scar formation, subsequent axonal and progressive loss of nerve function. Inflammation causes neuronal myelin loss, inflammation and myelin destruction damage axons, eventually leading to neurodegenerative lesions and injuries (Ontaneda et al., 2017). Oxidative stress plays an important role in the pathogenesis of inflammatory infiltrates and demyelination of EAE. Inhibition of oxidative stress pathway can inhibit the symptoms of EAE. The Nrf2/ARE signaling pathway is an important endogenous antioxidant stress pathway. The nuclear factor NF-E2 related factor (Nrf2) plays an important role in defending against various external damages through the antioxidant response element (ARE) signaling pathway. In the Nrf2/ARE signaling pathway, Nrf2 can control the peripheral lymphatic system and maintain the stability of the internal environment. NQO1, HO-1, SOD, and CAT are important antioxidant proteases in the Nrf2/ARE pathway, they have anti-stress response and anti-oxidative damage function.
From the clinical manifestations of the mice, treatment with NA significantly reduced the clinical scores. The results showed that NA had a certain preventive and therapeutic effect on the relief of clinical symptoms in EAE group mice. Treatment with NA decreased the expression of ROS and inflammatory cytokines, and increased the expression of Nrf2, NQO1, HO-1, SOD, and CAT. It is possible that NA inhibits oxidative stress through the Nrf2/ARE signaling pathway, which significantly decrease the concentration of TBARS and ROS and increase the levels of SOD and CAT in the brain of EAE mice. NA can regulate the expression of inflammatory factors, which can reduce the inflammatory response of the nervous system, thereby reducing the loss of myelin in the white matter of the nervous system (Lin et al., 2007). And the most significant treatment effect was high dose of NA group.
Inflammatory mediators can produce a large amount of ROS during the pathogenesis of MS. High levels of ROS can cause demyelination, subsequent axonal and progressive loss of nerve function, and enhance inflammation. Studies have found that there was a large amount of lipid peroxidation in the cerebrospinal fluid and plasma of MS patients (Haider et al., 2011), and the serum ROS levels were significantly increased (Langemann et al., 1992). So, anti-oxidant stress is an effective measure for MS (Long et al., 2018). In our studies, treatment with NA can reduce the level of ROS and inhibited the progression of EAE in mice. It is possible that NA reduced ROS production by inhibiting oxidative stress. The TBARS, ROS, SOD, and CAT expression in the brain were measured to evaluate the degree of oxidative stress in the brain. SOD and CAT are anti-oxidant enzymes that can inhibit oxidative stress induced by inflammation. Compared with the EAE group, We found that treatment with NA decreased the concentration of TBARS and ROS and increased the levels of SOD and CAT. NA can increase the antioxidant enzyme activity and inhibit oxidative stress. So, NA can balance oxidation and antioxidant activity in MS (Long et al., 2018).
In the pathological process of EAE, the relationship between Th1 type and Th2 type cells was unbalanced. Pro-inflammatory cytokines such as TNF-α and IFN-γ produced by Th1 type cells can promote the activation of T cells and macrophages, and they can directly destroy the blood-brain barrier, allowing inflammatory cells to enter the nervous system and causing loss of myelin sheath. Anti-inflammatory cytokines such as IL-4/IL-10 produced by Th2 cells played an irreplaceable role in delaying the pathology of MS and promoted the recovery of EAE. In our study, we have found that treatment with NA decreased the levels of IFN-γ/TNF-α and increased the levels of IL-4/IL-10 (Li et al., 2013; Liu et al., 2014). It can be shown that NA can induce the expression of anti-inflammatory cytokines and inhibit oxidative stress.
Multiple sclerosis is a demyelinating disease that occurs in the central nervous system (Moriguchi et al., 2013; Ma et al., 2017). As the sphingomyelin of nerve fibers is destroyed, the myelin sheaths of the nerve fibers fall off in blocks, which interrupts nerve transmission (Miyamoto et al., 2009). The central nervous system is composed of white matter and gray matter, while NA is a component of white matter and a structural component of white matter in the brain. It can synthesize gangliosides, cerebrosides, and sphingomyelin in the body, which can improve medulla dystrophy and inhibit damage and loss of myelin sheath, improve the symptoms of multiple sclerosis. Sargent had found that patients with multiple sclerosis and adrenal white matter dystrophy (ALD) have low levels of NA in the brain phospholipids, which to a certain extent also indicates that NA can improve the condition of patients with MS and ALD (Sargent et al., 1994). Intake of NA can promote the synthesis of glycosphingolipids and sphingomyelin in the body, and then promote the myelinization of nerve fibers and regenerate the lost myelin sheath. Erlwanger KH et al. (2008) thought that NA can improve demyelination.
Nervonic acid is an essential molecule for the growth and maintenance of the brain and peripheral nervous tissue enriched in sphingomyelin and related to multiple sclerosis (Yuki et al., 2018). NA can completely pass through the blood-brain barrier, directly act on nerve fibers for repair and unblocking, and regenerate damaged and shed protective sheaths. NA dissolves the necrotic tissue that blocks the channel, induces the self-growth and division of nerve fibers, so that information generated by nerve cells and external information can be smoothly transmitted through the nerve fibers. This activated damaged and diseased nerve cells, and it can reshape the neural network and restore some or all of the patient's functions in language, memory and sensations (Emmanouil et al., 2009). NA is the world's first recognized by scientists, but also the only one can repair and unblock damaged nerve pathways in the brain and promote nerve cell regeneration.
The pathogenesis of EAE/MS, most scholars believed that susceptible factors cause the body's autoimmune response, then, lymphocytes and monocytes in peripheral blood are activated (Trapp et al., 2008). Activated T cells can express a variety of adhesion molecules and bind to receptors on the vessel wall (Becher et al., 2006; Way et al., 2015). Vascular endothelial cells can also express selective binding to T cells, and chemokiness can also promote T cells to enter the CNS (Croxford et al., 2015; Furlan et al., 2001). The T cells secrete inflammatory cytokines and chemokiness in the CNS, which further chemotactic and activate other inflammatory cells to produce a series of complex cascade immune responses, resulting in damage to the myelin sheath and even axons. NA can effectively inhibit T cell proliferation and promote the secretion of anti-inflammatory factors, thereby reducing the inflammatory response of the central nervous system (DeLeo et al., 2001; Raghavendra et al., 2004). NA is difficult to produce in the body itself, and can only be supplemented by ingestion in vitro. Sargent have found that NA can not only accelerate the metabolism of essential fatty acids in the body, reduce the concentration of total cholesterol in the serum, but also improve the microcirculation of the blood, thereby preventing diabetes, hypertension, and hyperlipidemia (Sargent et al., 1994).
In conclusion, our results indicated that treatment with NA obviously inhibited the development and severity of EAE in mice, accompanied by mitigating inflammatory infiltration and demyelination in the spinal cord of mice. Apparently, NA may inhibit the development and severity of EAE by its potent anti-oxidant and anti-inflammation activity. Therefore, NA, especially high dose of NA, may be a promising candidate for intervention of multiple sclerosis and other autoimmune diseases, which has potential as a novel therapeutic option for multiple sclerosis.
Acknowledgements This study was supported by the Shandong province key research and development plan (Number: 2018YYSP023) and Breakthrough breeding of new woody oilseed species (Number: 2020LZGC009).
Conflict of interest The authors have no potential conflicts of interest.
