http://dx.doi.org/10.11594/jtls.13.03.03
How to cite:
Alnawajha A, Endharti AT, Santoso S, et al. (2023) A Comparison Study of the Influence of Caffeic Acid Phenethyl Ester and Research Article
A Comparison Study of the Influence of Caffeic Acid Phenethyl Ester and Mitoxantrone in Experimental Autoimmune Encephalomyelitis Balb/C Mice Model
Amin Alnawajha 1,2 *, Agustina Tri Endharti 3, Sanarto Santoso 3, Dewi Santosaningsih 3, Irawan Satriotomo 4
1 Medical Science, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
2 Department of Internal Medicine, Alnajar Hospital, Gaza, Palestine
3 Department of Parasitology, Faculty of Medicine, Universitas Brawijaya, Malang, 65145, Indonesia
4 Tissue ProTechnology, Institute Neuroscience Indonesia (INI), Malang, 65145, Indonesia
Article history:
Submission September 2022 Revised April 2023 Accepted April 2023
ABSTRACT
A common neurodegenerative condition that still presents clinical challenges is Mul- tiple Sclerosis (MS). Effective multiple sclerosis treatments are sorely needed in clin- ical settings. Experimental Autoimmune Encephalomyelitis (EAE) is an animal model of multiple sclerosis, a T-cell-mediated disease. Active T-cells differentiate into the Th9 and Th17 subsets, which are controlled by NF-kB and produce the proinflamma- tory cytokines IL9 and IL17. Because these cytokines are crucial to the pathophysiol- ogy of EAE, they have been used as targets for MS therapy. Caffeic acid phenethyl ester (CAPE) is an active ingredient of propolis that has been shown to have immuno- modulatory and anti-inflammatory activities. Mitoxantrone is a synthetic antineo- plastic agent and cytotoxic immunosuppressive effect used to treat MS. The study aimed to determine whether the two medications have superior efficacy and effect in the treatment of EAE mouse model MS compared to the other. After inducing EAE in mice, CAPE and mitoxantrone were administered to evaluate this therapeutic effec- tiveness. ELISA was used to measure IL9, IL17 levels and the activity of NF-kBp56.
H&E was used to evaluate cell infiltration T lymphocytes for histopathology of spinal cord tissue. Molecular docking was performed to predict the interaction between CAPE and a cytokine. We found that CAPE has a sufficient effect of reducing the level of IL9, IL17, active NF-kBp56, and inflammatory cell infiltration T-lympho- cytes in all groups of mice EAE treated with CAPE. In contrast, mitoxantrone reduced cytokines and cell infiltration, so EAE mice treated with both compounds were ob- served more improvement than other groups. Based on our findings, two medications demonstrated the same efficacy and effect in EAE mice model MS., whereas CAPE did not statistically reach a significant value. While the combination of two medica- tions has the optimal effect.
Keywords: Caffeic acid phenethyl ester, IL9, IL17, Mitoxantrone, NF-kB
*Corresponding author:
E-mail:
Introduction
Multiple sclerosis (MS) is a neurodegenera- tive, inflammatory autoimmune disorder that causes T-cell activation and attacks the central nervous system, resulting in inflammation and de- myelination of neurons [1] and axonal damage [2], leading to motor dysfunction, sensory perception, and mental disorder [3]. Experimental autoim- mune encephalomyelitis (EAE) is an animal
model of MS because there is a similar neuropa- thological change as in MS [4], and it is used for research in MS [5] and testing new therapeutic drugs.
In spite of more therapeutic drugs being avail- able for MS, there is still no therapeutic cure for MS [6, 7], and many of these drugs have been de- veloped based on studies using the EAE model [8].
Immune T cells become active when T cells are faced with antigens, then differentiate into subset Th such as Th9, Th17 [3]. It is suggested that Th9 and Th17 are important in the pathogenesis of MS [9, 10]. Th17 produces proinflammatory cytokine IL17 [9, 11], and also can secret IL9 [12]. Th9 re- leases strong proinflammatory IL9 [13].
Both IL9 and IL17 play an important role in MS; several studies reported that mice deficit IL9 or IL17 less development EAE [10, 11, 13, 14].
Expression of α4β1 integrin on T-cells contributes T-cells cross through the BBB to CNS [15]. Once these cells cross BBB, they attack neurons leading to neuroinflammation and demyelination [3]. Dur- ing disease development, NF-kB is necessary for immune cell activation and differentiation [16]. It has been demonstrated that the NF-kB controls a number of processes involved in T-cell formation, activation, differentiation, and survival [17].
Propolis contains the active ingredient caffeic acid phenethyl ester (CAPE) [18], and many stud- ies reported that CAPE has immunomodulatory, anti-inflammatory, antineoplastic, and antioxidant properties. DMT therapy is currently available for multiple sclerosis [19]. However, most trials of DMT have not succeeded for all forms of MS [20], and it has serious side effects, even fatal, and until now there are no specific therapeutic agents and no effective cure for MS [21]. Mitoxantrone is one DMT drug with a cytotoxic immunosuppressive effect [21]. It is a synthetic antineoplastic agent and has immunomodulatory effects [1]. Mitoxan- trone has a long half-life; tissues absorb it and ac- cumulate in the tissue for a long time (1 month) [22]. Because of the risks of cardiotoxicity and leukaemia, long-term drug use is limited to two or three years because the cumulative dose is 120mg/m2 [23]. Therefore, FDA recommended monitoring the cardiac function before each dose.
Another risk is amenorrhea, reduction of fertility and severe infections [22]. This study aimed to de- termine whether the two medications have supe- rior efficacy and effect in the treatment of EAE mouse model MS.
Material and Methods
This study used mice with twenty female mice at 6 to 8 weeks of age, healthy and active. Mice were divided into four groups (C+, T1, T2, T3). In each group were five mice. Food and water were provided. The light/dark cycle is maintained at 12- hour intervals. Figure 1 shows the overall research
flow chart in our study.
Induction of experimental autoimmune enceph- alomyelitis
Myelin oligodendrocyte glycoprotein MOG35-55 was used to induce EAE in mice. The mice were subcutaneously injected with 200 μg of MOG 35-55(cat. Number pro-371-a, Prospec- Tany TechnoGene Ltd, Ness Ziona 74140, IS- RAEL) in complete Freund’s adjuvant (cat. Num- ber 786-1682, G-Biosciences/ Geno Technology, St Louis, USA) 100 μL per site on the right and left flanks. The mice were injected i.p. with 400 ng pertussis toxin (GSB-1886, The Native Antigen Company Langford Locks, OxfordOX5 1LH UK) in Phosphate-Buffered Saline (PBS). The first dose was given on day 0, followed by the second dose on day 2 [24].
A veterinarian doctor conducted all proce- dures. The mice were monitored and scored every day for the onset of disease according to the fol- lowing scale: 0 = normal; 1 = tail tonicity loss; 2 = mild weakness in the hind limbs; 3 = partial paral- ysis of the hind limbs; 4 = paralysis of the entire hind limb; 5 = complete hind limbs paralyzed, with forelimb weakness or moribund [25]. CAPE was obtained from (code P2088 -Tokyo Chemical Industry-JAPAN). It was given to mice orally at a dose of 20 mg/kg/for two weeks for the T1 group [26]. Mitoxantrone was purchased from (code HY-13502-MedChem Express-Pricenton-USA) at doses of 1.6 mg/kg/day on day 1,5,9 IP for the T2 group, and a combination of Mitoxantrone at doses of 1.6 mg/kg/day on day 1,5,9 IP, and CAPE at a dose of10 mg/kg/for two weeks for the T3 group.
Measurement level of IL9, IL17, NF-kBp65 by ELISA
Quantitative enzyme-linked immunosorbent assay (ELISAs) for IL9 (cat. no. E0080Mo), IL-17 (cat. no. E0041Mo), NF-kBp65 (cat. no.
E0521Mo) were performed using Abs-specific for each of these cytokines according to manufac- turer’s recommendations (Bioassay Technology Laboratory- Shanghai, China).
Molecular docking
Molecular docking aims to know drug biolog- ical molecule interactions for rational drug design and discovery [27]. The molecular docking proto- col is divided into four major steps. The first is the
Preparation of the ligand and target. The second step is to create docking and scoring parameters.
Third, the docking process is run. The fourth step
is to analyze and evaluate the docking results [28].
In this study, CAPE was auto-docked to see how it interacted with the target ligand.
Ligand and protein acquiring
The caffeic acid phenethyl ester (CAPE) struc- ture was retrieved from PubChem NCBI Database with ID number 129688353. The three-dimen- sional structure of proteins was obtained from Pro- tein Data Bank. The protein involved interleukine- 9, interleukin-17 (PDB ID 4HR9) NFKB-P65 (PDB ID 1le9). Interleukin-9 was modelled using the Swiss Model with the protein sequences from NCBI (GenBank EDL41238.1). The interleukin-9 structure has assessed the quality by using MolProbidity.
Protein preparation and docking
All ligands and proteins were imported to the Molegro virtual Docker version 5 for docking analysis. A specific protein grid was used to re- dock the ligands. The binding cavities of proteins were predicted with the parameter van der Waals maximum of 5 cavities to identify the active sites of proteins. Then, the CAPE was redocked to the targeted protein. The specific grid of proteins in- cluding IL17 (X=-6.07; Y=28.29; Z=65.68; radius 9), and IL-9 (X=-18.73; y=7.27; Z=-28.95) radius 11; NFKB-P65 (X=68.38; Y=30.36; Z=22.69) ra- dius 16. The other docking parameters were Grid resolution 0,30A, RMSD was less than 2.0, the binding model maximum was five, and the num- ber of running was ten repetitions. Ligand-proteins interactions were analyzed by PyMol version 2.2 and Discovery Studio version 21.1.1.
Histopathology of spinal cord tissue
The spinal cords were removed and placed in 10% neutral buffered formalin at room tempera- ture. The sections were cut at 5µm thick sections from the level of the cervical spinal cord, mounted on glass slides and stained with hematoxylin and eosin (H&E) to identify infiltrating inflammatory cells. The inflammation scores were evaluated as follows: 0 indicates no inflammation; 1 indicates perivascular areas and meninges cellular infiltra- tion; 2 indicates inflammatory cells infiltration in less than one-third of the white matter; 3 indicates inflammatory cells infiltration in more than one- third of the white matter; and 4 indicates total white matter infiltration of inflammatory cells [11]. The tissue investigations were performed by
a pathologist who did not know about the mice group. The images were taken with an OLYMPUS microscope- Japan.
Results and Discussion Assessment of EAE symptoms
EAE symptoms were seen in the large number of the immunized mice. We monitored the appear- ance of clinical symptoms of the disease on daily observation of the tail, and paralysis of the hind limb. The disease appeared between day 14 and day 18 postinduction of EAE. Two mice died after the induction of EAE.
Analysis of proinflammatory cytokines produc- tion
The levels of cytokines after treatment explained the clinical condition. The CAPE treatment altered the levels of cytokines in all treated EAE mouse groups, as measured by ELISAs. Many studies have found evidence that IL9 and IL17 are crucial to EAE, indicating that IL17 as a targeted treatment is beneficial in EAE mice[9,29]. Our finding demonstrated a reduction in the level of IL9 and IL-17 in the treated EAE mice group with CAPE, which indicates that CAPE can interrupt the cascade pathogenesis.
This result is consistent with a previous study that reported mice lack IL9 or IL 17 less development EAE [10, 14].
The levels of IL9 and IL17 in CAPE-treated EAE mice were lower than the level in EAE mice control positive. The overall differences in the levels of IL9 and IL17 in each treatment group are depicted in Fig. 2A and B, respectively. Based on the results of ANOVA for the mean level of IL9, IL17 revealed a significance of 0.001, 0.000 (p<
0.05), respectively. Then, a post hoc test of multiple comparisons was conducted with Tukey’s test for the level of IL9 and IL17 in each treated EAE mice group. The result was that the levels of IL9 and IL17 in treated EAE mice T2 received mitoxantrone compared with EAE mice control positive showed significant (< 0.05), while the level of cytokines in T3 treated EAE mice received both medications compared with EAE mice control positive (< 0.05). However, the level of IL-9 and IL17 in T1 mice EAE treated with CAPE alone did not differ significantly from the
level of these cytokines in the mice EAE positive control group and treated mice T2,3 (p>0.05), although CAPE has the ability in a reduction in IL9 and IL17 level as revealed ELIZA result but did not reach significant value. However, the correlation result shows a strong relationship between the dose of CAPE and levels of IL9 and IL17, which means the higher dose of CAPE will be followed by decreased IL9 and IL17 levels.
We then analyzed the effect of CAPE on the
active NF-KB p65, and the analysis of data by ANOVA revealed a significance 0.000 (p < 0.05).
As shown in Figure 2C, the expression of NFkBp65 in the positive control group differs sig- nificantly from the expression of NFkBp65 in the treated groups T1,2, and 3, and showed signifi- cance 0.000 (p < 0.05), but expression of NF- KBp65 was lower in T3 than in the other treated groups (T1, T2).
Further, we analyzed NF-kBp56 expression Figure 2. Interaction of caffeic acid phenethyl ester with Interleukin 9(A), and Interaction of caffeic acid
phenethyl ester with Interleukine17(B), and NF-kB(C).
Figure 3. H&E-stained sections of the spinal cord at the cervical level of EAE mice. (A-B) image from non- treated groups C+ mice, shown extensively inflammatory cell infiltration in the spinal cord. (C-D) image from treated group T1 mice with CAPE. (E-F) image from second group T2 treated with mitoxantrone, and (G, H) image from third group T3 treated with both compounds, shown different degree a reduction of inflammatory cells infiltration in the spinal cord (black arrows indicate in- flammatory cell infiltration) a significance value of 0.000 (p < 0.05) (magnification 10×,40×).
Figure 1. The level of pro-inflammatory cytokines IL-9, IL17, NF-kBp65 in EAE mice. ELISA was used to measure the level of cytokines, the data was analyzed by ANOVA, comparison test with turkey test.
(A) as shown, the level of IL9 in treated EAE mice lower compared with control positive EAE, ANOVA for mean of IL9 level showed significance 0.001(p < 0.05), comparison test, *T3 compared with control positive show significant (p 0.001), **T2 mice compared with control positive (P 0.015),
***T1 compared control positive not significant (>0.05). (B) the data shown IL17 level lower in treated EAE mice compared with control positive. ANOVA result for mean of IL 17 show significant (0.000) (p < 0.05), comparison test shows, ♦p < 0.05 compared with control positive show significant (p 0.001), ♦♦p < 0.05 compared with C+ (p 0.036), ♦♦♦p compared C+ didn’t significant (> 0.05) despite the level lower than C+. (C) the result revealed NF-kBp65 expression lower in treated EAE mice compared with control positive. ANOVA result for mean NF-kBp65 expression show significant (p 0.000), a comparison test shows p compared with control positive significant (p 0.001), and NF-kBp65 expression in p compared with control positive significant (p 0.020),֍֍֍ pcompared control positive not significant (> 0.05) the data is presented as mean ± SD (n = 5).
50.56
29.25*** 24.09** 18.55*
0 10 20 30 40 50 60 70
C+ T1 T2 T3
The average of IL 9ng/l
EAE mice
A
149.29
108.75♦♦♦
79.21♦♦
52.34♦ 0
50 100 150 200
C+ T1 T2 T3
The average of IL 17pg/ml
EAE mice
B
1.84 1.5֍֍֍ 1.34֍֍ 1.16֍
0 0.5 1 1.5 2 2.5
C+ T1 T2 T3
Average of NF-kBp65
EAE mice
C
and showed that CAPE treatment suppressed the activation of NF-kB that suppresses T cell differentiation into subset cells, including Th9 and Th17—consequently, reduced production of proinflammatory cytokines IL9 and IL17.
Mitoxantrone exhibited a reduction in the level of IL9 and IL17 in T2, T3 treated mice EAE.
However, the T3 group mice EAE treated with both substances has the lowest amount of IL9 and IL17 and NF-kBp56 expression. A previous study
about the effect of mitoxantrone on cytokines profile reported that change was found for IL17, IL10, and TNF-α but increased Th2 cytokines secretion, which exerts an anti-inflammatory effect [30]. Our study reported that mitoxantrone can reduce levels of IL17 and IL9 in EAE mice model MS.
Molecular docking analysis
Interleukine9 Caffeic acid phenethyl ester Figure 2. Interaction of caffeic acid phenethyl ester with Interleukin 9(A), and Interaction of caffeic acid
phenethyl ester with Interleukine17(B), and NF-kB(C).
Figure 3. H&E-stained sections of the spinal cord at the cervical level of EAE mice. (A-B) image from non- treated groups C+ mice, shown extensively inflammatory cell infiltration in the spinal cord. (C-D) image from treated group T1 mice with CAPE. (E-F) image from second group T2 treated with mitoxantrone, and (G, H) image from third group T3 treated with both compounds, shown different degree a reduction of inflammatory cells infiltration in the spinal cord (black arrows indicate in- flammatory cell infiltration) a significance value of 0.000 (p < 0.05) (magnification 10×,40×).
bound to Interleukin 9 in several active sites.
Those were THR116, SER123, GLY119, ASN120, LYS111, PHE124, PRO67, and CYS68.
The interaction types were hydrogen bond, hydro- phobic interaction, and van der Waals. The bind- ing energy of those complexes was -331,2 kJ/mol (Figure 2A). Interleukin 17; Caffeic acid phenethyl ester interacted with Interleukin 17 in the active sites of interleukin –17, which were ASP84, CYS76, ILE77, ASN78, CYS76, MET87, and CYS123 (Figure 2B).
The ligand-protein complex generated binding energy -238 kJ/mol with hydrogen bonds, electro- static, and hydrophobic interactions Figure 2B.
NF-KBp65; Three-dimensional structure of CAPE-NFKBp65 complex performed nucleic acid binding region. Caffeic acid phenethyl ester inter- acted with NFKBp65 by five hydrogen bonds in residues ARG33, GLU193, PRO189, ARG33, and ARG305. ARG305 performed electrostatic inter- action with the benzene ring of CAPE. LYS218 posed Pi-Sigma and Pi-Lone Pair at the CAPE ar- omatic ring and also showed Pi-alkyl or hydropho- bic interaction with C-atom of CAPE Figure 2C.
Analysis of the spinal cord histopathology H&E was used to stain tissue sections of in- flamed spinal cords at the cervical level from EAE-induced mice to investigate the levels of im- mune cell infiltration in the spinal cords, revealed that cell infiltration was reduced in the spinal cords of EAE-treated mice, showed significant (p
< 0.05). Immune cell infiltration was significantly lower in the spinal cords of EAE mice T3(p ˂ 0.05) when compared to the EAE mice control positive group Figure 3. Immune cell infiltration in T1 EAE mice did not differ significantly from T2 EAE mice (p > 0.05).
Histopathology of the spinal cord, reflects the efficacy of therapy, as shown in Figure 3, less cell infiltrate in the spinal cord in all treated groups.
We observed that therapy in EAE mice T1, T2 in the form of CAPE and mitoxantrone, respectively, has the ability to decrease immune cell infiltration, whereas therapy in EAE mice T3 was given ther- apy in the form of a mix of both CAPE and mito- xantrone, and was observed to be more prominent in decreasing immune cell infiltration. This result suggests that the combination of two compounds has more therapeutic efficacy and gives optimal effects than a single compound.
Conclusion
This is the first study to compare CAPE and mitoxantrone efficacy in the EAE mouse model MS. Based on our findings. We concluded that the two medications have the same efficacy and effect in alleviating spinal cord lesions in the EAE mouse model of MS. Despite the demonstrated ef- fect, CAPE did not reach statistical significance.
The combination of two medications is having the optimal impact.
Acknowledgment
We would grateful to all DPN Indonesian Red Crescent, and branches of the Red Crescent for ad- vice, support, and for providing a research grant.
To Prof. Dr. dr. Basuki Supartono, Sp.OT, FICS, MARS for support during the study. To dr. Fajar Shodiq of the Faculty of Veterinary Medicine, Universitas Brawijaya for the support during this research, also dr. Aina Department of Pathology Anatomy of Faculty of Medicine, Universitas Brawijaya, and Heni Endrawati (Clinical Parasit- ology Laboratory). The authors would like to acknowledge Universitas Brawijaya for providing a research grant.
References
1. Garg N, Smith TW (2015) An update on immunopatho- genesis, diagnosis, and treatment of multiple sclerosis.
Brain and Behavior 5(9): e00362. doi: 10.1002/brb3.362.
2. Kawamoto E, Nakahashi S, Okamoto T et al. (2012) Anti- Integrin Therapy for Multiple Sclerosis. Autoimmune diseases 2012: 357101. doi: 10.1155/ 2012 /357101.
3. Palmer AM (2013) Multiple Sclerosis and the Blood- Central Nervous System Barrier.Cardiovascpsychiatry Neurol 2013: 530356. doi: 10.1155/ 2013 /530356.
4. Peiris M, Monteith GR, Thomson SJR, Cabot PJ (2007) A model of experimental autoimmune Encephalo myelitis (EAE) in C57BL/6 mice for the characterization of inter- vention therapy. Journal of neuroscience methods 163 (2): 245-54. doi: 10.1016/j.jneumeth.2007.03. 013.
5. Berenice AS, Carina CF (2016) Gr upSM Animal exper- imental models for understanding and treating Multiple Sclerosis.
6. Scruggs BA, Semon JA, Zhang X et al. (2013) Age of the donor reduces the ability of human adipose-derived stem cells to alleviate symptoms in the experimental autoimmune encephalomyelitis mouse model. Stem cells translational medicine 2(10): 797-807. doi: 10.5966 / sctm.2013-0026.
7. Belloli S, Zanotti L, Murtaj V et al. (2018) 18F-VC701- PET and MRI in the in vivo neuroinflammation assessment of a mouse model of multiple sclerosis.
Journal of neuroinflammation 15(1): 33. doi:
10.1186/s12974-017-1044-x.
8. Constantinescu CS, Farooqi N, O'Brien K, Gran B (2011) Experimental autoimmune encephalomyelitis (EAE) as a model for multiple sclerosis (MS). British journal of
pharmacology 146(4): 1079-1106). doi: 10.1111/j.1476- 5381.2011. 01302.x.
9. McGinley AM, Sutton CE, Edwards SC et al. (2020) In- terleukin-17A Serves a Priming Role in Autoimmunity by Recruiting IL-1β-Producing Myeloid Cells that Promote Pathogenic T Cells.j. Immunity 52(2): 342-356.e6. doi:
10.1016/j.immuni.2020.01.002.
10. Li H, Nourbakhsh B, Cullimore M et al. (2011) IL-9 is important for T-cell activation and differentiation in autoimmune inflammation of the central nervous system.
European journal of immunology 41(8): 2197-2206. doi:
10.1002/eji.201041125.
11. Seno A, Maruhashi T, Kaifu T et al. (2015) Exacerbation of experimental autoimmune encephalo myelitis in mice deficient for DCIR, an inhibitory C-type lectin receptor.
Experimental animals 64(2): 109-119. doi:
10.1538/expanim.14-0079.
12. Elyaman W, Khoury SJ (2017) Th9 cells in the pathogenesis of EAE and multiple sclerosis. Seminars in immunopathology 39(1): 79-87. doi: 10.1007/s00281- 016-0604-y.
13. Pan HF, Leng RX, Li XP et al. (2013) Targeting T-helper 9 cells and interleukin-9 in autoimmune diseases.
Cytokine & growth factor reviews 24(6): 515-522.
doi: 10.1016/j.cytogfr.2013.09.001.
14. Hofstetter H, Ralf G, Hans H (2009) Th17 Cells in MS and Experimental Autoimmune Encephalomyelitis. The International MS Journal 16(1): 12-18. doi:
10.2332/allergolint. R-07-159.
15. Bowles AC, Strong AL, Wise RM et al. (2017) Adipose Stromal Vascular Fraction-Mediated Improvements at Late-Stage Disease in a Murine Model of Multiple Sclerosis. Stem cells 35(2): 532544. doi:
10.1002/stem.2516.
16. Mc Guire C, Prinz M, Beyaert R Van, Loo G (2013) Nuclear factor kappa B (NF-κB) in multiple sclerosis pathology. Trends in molecular medicine 19(10): 604- 613. doi: 10.1016/j.molmed.2013.08.001.
17. Oh H, Ghosh S (2013) NF-κB: roles and regulation in different CD4(+) T-cell subsets. Immunological reviews 252(1): 41-51. doi: 10.1111/imr.12033.
18. Armutcu F, Akyol S, Ustunsoy S, Turan FF (2015) Therapeutic potential of caffeic acid phenethyl ester and its anti-inflammatory and immunomodulatory effects (Review). Experimental and therapeutic medicine 9(5):
1582-1588. doi: 10.3892 /etm.2015.2346.
19. Hassan SG, Douglas MR (2011) Management and prognosis of multiple sclerosis. British journal of hospital medicine 72(11): M174-6.
20. Clare BA, Belinda JK, Howard LW (2018) Multiple Sclerosis: Mechanisms and Immunotherapy. J.Neuron 97(4): 742-768. doi: 10.1016/j.neuron.2018.01.021.
21. Edan G (2016) Chapter 18 - Currently Approved Injectable Disease-Modifying Drugs:Cytotoxic Immuno suppressive Drugs. Translational Neuroimmunology in Multiple Sclerosis. San Diego, Academic Press 245-257.
22. Esposito F, Radaelli M, Martinelli V et al. (2010) Comparative study of mitoxantrone efficacy profile in patients with relapsing-remitting and secondary progressive multiple sclerosis. Journal Multiplesclerosis 16(12): 1490-1499. doi: 10.1177/1352458510379613.
23. Jain KK (2000) Evaluation of mitoxantrone for the treatment of multiple sclerosis. Journal Expert opinion on investigational drugs 9(5): 1139-1149. doi:
10.1517/13543 784.9.5.1139.
24. Bittner S, Afzali AM, Wiendl H, Meuth SG (2014) Myelin oligodendro cyteglycoprotein (MOG35-55) induced experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice. Journal ofvisualized experiments (86). doi: 10.3791/51275.
25. Gibson C, Katherine N, Boyden AW et al. (2016) A method for histopathological study of the multifocal nature of spinal cord lesions in murine experimental autoimmune encephalomyelitis. PeerJ.4 e1600. doi:
10.7717/peerj.1600.
26. Jae HP, Jong KL, Hyung SK et al. (2004) Immunomodulatory effect of caffeic acid phenethyl ester in Balb/c mice. International Immunopharmacology 4(3):
429-436. doi: 10.1016/j.intimp.2004.01.013.
27. Ayaz MD, Shafia M (2017) Molecular Docking:
Approaches, Types, Applications and Basic Challenges.
Journal of analytical and bioanalytical techniques 8(2): 1- 3. doi: 10.4172/2155-9872.1000356.
28. El-Hachem N, Haibe-Kains B, Khalil A et al. (2017) AutoDock and AutoDockTools for Protein-Ligand Docking: Beta-Site Amyloid Precursor Protein Cleaving Enzyme 1 (BACE1) as a Case Study. Methods in molecular biology 1598: 391-403. doi: 10.1007/978-1- 4939-6952-4_20.
29. Liu X, Lee YS, Yu CR, Egwuagu CE (2008) Loss of STAT3 in CD4+ T cells prevents development of experimental autoimmune diseases. Journal of immunology 180(9): 6070-6076. doi:
10.4049/jimmunol.180.9.6070.
30. Vogelgesang A, Rosenberg S, Skrzipek S et al. (2010) Mitoxantrone treatment in multiple sclerosis induces TH2-type cytokines. Acta neurologica Scandinavica 122(4): 237-243. doi: 10.1111/j.1600-0404.2009.
01295.x.
This page is intentionally left blank.
