The Effects of Lavender Oil on IL-10 mRNA, miR-378 in the Brain of Experimental Autoimmune Encephalomyelitis

The Effects of Lavender Oil on IL-10 mRNA, miR-378 in the Brain of Experimental Autoimmune Encephalomyelitis | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article The Effects of Lavender Oil on IL-10 mRNA, miR-378 in the Brain of Experimental Autoimmune Encephalomyelitis Adile Merve BAKI, Ilknur BINGUL, Sefika Nur GUMUS, Merva SOLUK TEKKESIN, and 2 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-9516476/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 4 You are reading this latest preprint version Abstract Objective To investigate the effect of lavender oil (LO) on expressions of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), microRNA-155 (miR-155) and miR-378 in brain tissue of experimental autoimmune encephalomyelitis (EAE). Materials and Methods EAE was provoked by s.c. injection containing encephalitogenic peptide MOG35–55 and Mycobacterium tuberculosis H37RA, followed by i.p. injection of pertussis toxin in C57BL/6 J mice. LO was administered i.p. daily for 28 days. mRNA expressions of pro-/anti-inflammatory cytokines (TNF-α, IL-10), and miR-155 and miR-378 expressions together with histopathology were investigated in the brain tissue. Results The clinical manifestation of symptoms commenced around day 10, with peak intensity observed during days 13 and 14 (clinical score 2). Although the clinical scores showed a remission on day 28, inflammatory infiltration was observed in the brain tissue histopathologically. TNF-α mRNA expression not changed, while IL-10 mRNA had an increasing pattern in EAE. miR-155 and miR-378 expressions were elevated in EAE. When LO administered to EAE animals IL-10 mRNA and mir-378 expressions increased. Conclusion Our results demonstrate that LO may have a neuroprotective effect in EAE, and exert this effect by increasing the expression of anti-inflammatory and immunomodulatory IL-10 mRNA and miR-378. Nevertheless, additional research is required to validate the results of this investigation. Experimental autoimmune encephalomyelitis IL-10 mRNA miR-155 miR-378 TNF-α mRNA Figures Figure 1 Figure 2 Figure 3 1. Introduction The pathological progression of multiple sclerosis (MS) and its preclinical counterpart, experimental autoimmune encephalomyelitis (EAE), is fundamentally driven by central nervous system inflammation, axonal decay, and the loss of myelin. Cytokines facilitate the entire progression of MS, mediating the early differentiation of peripheral pathogenic T-cells as well as the subsequent CNS inflammation and structural damage. Among these, Tumor necrosis factor α (TNF-α) serves as a versatile signaling molecule regulating diverse cellular processes, including viability and apoptosis. Given its pro-inflammatory properties, the secretion of TNF-α by immune cells is considered a key contributor to the inflammatory damage observed in both MS and EAE. Interleukin 10 (IL-10) acts as a key regulatory cytokine, playing a vital role in curbing inflammatory and autoimmune disorders. Comprising approximately 18–25 nucleotides, microRNAs (miRNAs) represent a class of small non-coding RNA molecules that function as master regulators of diverse cellular pathways. They achieve their regulatory role by targeting the 3' untranslated region (UTR) of messenger RNAs (mRNAs), where they initiate mRNA decay [ 1 , 2 ]. miRNAs emerge as key modulators of post-transcriptional gene expression during neurogenesis, neuronal differentiation, and synaptic plasticity, while also modulating gliogenesis and myelin repair. A disequilibrium in the expression of miRNAs can be involved in many neurological pathologies, neurodegenerative and autoimmune diseases [ 3 , 4 ]. The involvement of miR-155 is critical to the modulation of immune-mediated processes and the coordination of inflammatory reactions [ 5 ]in various tissues including CNS [ 5 , 6 , 7 , 8 ]. By driving the specification of pathogenic Th1 and Th17 cells, miR-155 acts as a critical regulator of the T-cell-driven immune responses characteristic of MS. Furthermore, a significant overexpression of miR-155 has been identified within MS lesions during the inflammatory activation of macrophages [ 9 ]. miR-378 facilitates cell endurance, tumor growth, and angiogenesis [ 10 , 11 ], and has neuroprotective and immunomodulatory effects by suppressing neuroinflammation [ 12 ]. On the other hand, pro-inflammatory cytokines was reported to upregulate the miR-378a expression [ 13 ]. Aromatic plants, and particularly their volatile essential oils (EOs), have demonstrated remarkable pharmacological potential, including notable antioxidant, antimicrobial, antineoplastic, and anti-inflammatory properties [ 14 , 15 ]. Today, many EOs have been found to be associated with inflammation, autophagy, and apoptosis by causing changes in the expression of miRNAs [ 14 , 15 , 16 ]. Extracted from Lavandula angustifolia, lavender oil (LO) stands as a premier essential oil with widespread applications across both the pharmaceutical sector and therapeutic aromatherapy [ 17 ]. In traditional herbal medicine, LO exhibits a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, sedative, antidepressive, antimicrobial, antifungal and analgesic properties [ 18 ]. The LO contains over 100 chemical constituents [ 19 ]. Linalool and linalyl acetate are primary components of LO related to its anti-inflammatory activity [ 20 ]. However, there is no knowledge in the literature regarding the effect of LO on epigenetic regulation of inflammatory processes, namely miR-155 and miR-378. Consequently, The objective of the present study was to examine the therapeutic potential of LO on inflammation by measuring of mRNA expressions of pro- and anti-inflammatory cytokines (TNF-α, IL-10) as well as miR-155 and miR-378 expressions in the brain tissue of EAE induced mice. 2. Materials and Methods 2.1. Chemicals and kits Chemicals and reagents were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Kits and primers were obtained from Qiagen Technologies (USA). 2.2 Animals C57BL/6J mice were housed at 22 ± 1°C with a 12-h light/dark cycle and provided with ad libitum food and water. All protocols were approved by the Institutional Experimental Animal Ethics Committee (BVUHDEK-2022/105) and performed following veterinary and ethical guidelines 2.3. Induction of Experimental autoimmune encephalomyelitis To establish the EAE model, 6–8 week-old female C57BL/6J mice were immunized with 100 µg of an myelin oligodendrocyte glycoprotein (MOG) 35–55 (MOG 35–55 ) (#SCP0195) peptide per animal emulsified in complete Freund's adjuvant (CFA) containing 300 µg of attenuated Mycobacterium tuberculosis H37RA (#F5881). Each mouse received 0.1 mL of emulsion (s.c.) at two sites. After immunization, 400 ng of pertussis toxin (PTx) (#516551) in 100 µL of PBS was injected intraperitoneally (i.p.) into mice on day 0 and 2 [ 21 , 22 ]. Consistent with previously validated methodologies, the clinical progression of each subject was evaluated individually [ 22 , 23 , 24 ]. The clinical progression of the disease was evaluated individually each day for up to 28 days, employing the following 0-to-5 point scoring system based on symptomatic presentation: 0, no disease; 1, weak tail or unsteady gait; 2, hind-limb paresis; 3, hind-limb paralysis; 4, hind- and fore-limb paralysis; and 5, severe paralysis or death [ 25 ]. 2.4. Lavender oil treatment LO was diluted in 5% dimethyl sulfoxide (DMSO) and administered i.p. 100 mg/kg to each mouse (#W262218) [ 17 ]. 2.5. Groups Mice were randomly allocated into experimental groups and maintained in standard cages. Animals were divided into five groups: 1) Control (n = 7): Mice were provided with standard mice chow and drinking water ad libitum were injected to animals for 28 days; 2) DMSO (as a vehicle, n = 7): 0.1 mL of 5% DMSO was injected into mice daily for 28 days; 3) LO (n = 7): The i.p. administration of LO was performed daily for 28 days; 4) EAE group (n = 7): The EAE model in mice was induced as referenced above; 5) EAE + LO (n = 7): LO was administered daily for 28 days to EAE-induced mice two hours after the last PTx injection. Following the experimental period (at 28th day), blood was taken from the hearts of the mice under xylazine HCl (5mg/kg) and ketamine (100mg/kg) anesthesia and brain tissues were rapidly removed. For RNA isolation, brain tissue pieces were stored in “RNA protect tissue reagent” (#172034595, Qiagen, USA) at -35°C until the measurements. 2.6. Histological examination Brain samples were fixed in a 10% formalin buffer solution for 24 hours prior to embedding in paraffin blocks. Subsequently, 5 µm sections were obtained from each paraffin block and stained for histological examination. These sections were then evaluated using hematoxylin and eosin (H&E) staining to assess the presence of inflammation. 2.7. mRNA and miRNA expressions Total RNA was purified from samples using miRNeasy Tissue/cell Advanced Kit (#217604) following manifacturer’s instruction. Quantitative real time polymerase chain reaction (qPCR) were performed on a Qiagen Rotor-Gene Q device (Qiagen Hilden, Germany). For mRNA synthesis, cDNA was sythesized using QuantiTect Reverse Transcription kit (#205310). TNF-α and IL-10 expressions were quantified with QuantiNova LNA PCR assays TNF-α, (#SBM0788439-200) and IL-10 (#SBM1004151-200). The 2 −ΔΔCt method was employed to quantify mRNA expression levels, which were normalized against GAPDH as an internal control. For miRNA synthesis, cDNA was sythesized using miRCURY LNA RT Kit; (#YP02119464). For miR-155-3p and miR-378a-3p expressions, miR-155-3p and miR-378a-3p miRCURY LNA™ miRNA PCR Assays were used (#YP02104818 and #YP00204179, respectively). The expression levels of the miRNAs were calculated using the 2 −ΔΔCt method. The analytical data were calibrated against U6 snRNA expression levels, which served as an internal control. 3. Results 3.1. EAE clinical score Clinical symptoms manifested around day 10, peaking between days 13 and 14 with a maximum clinical score of 2, followed by a period of moderate remission (Fig. 1 ). The clinical scores of the EAE and EAE + LO groups were of a similar degree. 3.2. Histological assessment The control and DMSO groups exhibited similar morphology to normal histology while the LO group displayed characteristics that were relatively close to normal histology. While inflammatory infiltration was observed in the EAE group, the number of inflammatory cells was declined in the EAE + LO group in contrasted to the EAE group (Fig. 2 , original magnification H&E x200). 3.3. mRNA expressions of TNF-α and IL-10 No discernible alterations were observed in TNF-α mRNA levels among the experimental groups. There was an increasing pattern in IL-10 expression in the EAE group compared to the control group, but it did not reach significance level. IL-10 expression in EAE + LO group was higher than EAE group. In addition, when LO was used alone, it increased the IL-10 mRNA expression compared to the control (Fig. 3 ). 3.4. miR-155 and miR-378 expression miR-155 and miR-378 expressions were similar between the control and DMSO groups. They were found to be elevated in the EAE group in comparison with control (Fig. 3 ). miR-378 expression was higher in the EAE + LO group than in EAE group. In addition, when LO was applied alone, the miR-378 expression increased compared to the control (Fig. 3 ). Significant correlation between IL-10 mRNA and miR-378 was found (r = 0.651, p < 0.05). 4. Discussion MS represents a widespread neurodegenerative disorder, with a global prevalence exceeding one million individuals [ 26 ]. The etiology of MS is hypothesized to stem from a multifaceted interaction between genetic susceptibility and environmental triggers that precipitate immune system activation. For several decades, the EAE model has served as the primary paradigm for investigating the underlying mechanisms and developmental stages of the disease [ 27 ]. In one of the most prevalent experimental models, EAE is elicited through the subcutaneous administration of an encephalitogenic peptide, commonly MOG 35–55 or (PLP) 139−151 . This peptide is formulated as an emulsion in CFA (comprising mineral oil and Mycobacterium tuberculosis H37RA), followed by supplementary i.p. injections of pertussis toxin [ 28 ]. In our study, EAE was induced by this model as depicted by Dias et al. [ 21 ] and Tan et al. [ 22 ]. Dias et al. [ 21 ] have reported that clinical symptoms initially emerged around day 10, reaching peak severity by day 19 (mean clinical score 4.8) and continued with fluctuations until day 58. Tan et al. [ 22 ] found that clinical symptoms appeared on day 10, reached maximum values on day 16 (clinical score 2–3) and lasted until day 28. Similar to their results, in our study the emergence of clinical symptoms occured around day 10, and peak clinical score attained on day 13–14 (clinical score 2). Although the clinical scores showed a remission on day 28, inflammatory infiltration was observed in the brain tissue, histopathologically. The precise mechanism for the initiation and progression of MS is still not clear, although intertwined mechanisms of innate and adaptive immunity are thought to be involved. The immunopathology of MS is characterized by the involvement of T helper (CD4+) lymphocytes and essential antigen-presenting cells (APCs), such as macrophages, B cells, dendritic cells, and CNS-resident microglia. As a consequence of the interaction between APCs with T lymphocytes Th cells differentiate into either Th1, Th2 or Th17 phenotypes. While Th1 and Th17 are phenotypes promoting inflammation, Th2 is anti-inflammatory one. Pro-inflammatory cytokines such as interferon-gamma and TNF-α are secreted by Th1 cells, whereas Th2 cells provide an anti-inflammatory response via IL-4, IL-10, and IL-13. Furthermore, the IL-17, IL-21, IL-22, and IL-26 cytokines are produced by the Th17 lineage of CD4 + T cells. Th1 cytokines promote the CNS recruitment of Th17 subsets, thereby driving the neuro-inflammatory cascade that characterizes the EAE model [ 29 ]. Destroying of the blood-brain barrier in EAE leads to the cerebral infiltration of autoreactive T cells and increases the pro-inflammatory cytokine levels. As consequence, initiated molecular mechanism produces the CNS immune self-attack, which is most prominent at the first phases of MS related with the microglia activation and macrophages infiltration promoting demyelination [ 30 ]. Many studies reported elevated TNF-α level in the CSF of MS patients, correlating with both disease severity and progression [ 31 , 32 ]. Since blocking TNF-α signaling has been established to attenuate EAE development [ 33 ], TNF-α inhibitors were suggested to be useful in MS treatment. Unfortunately, clinical trials demonstrated that MS patients treated with recombinant TNF receptor antibody experienced augmented disease pathology [ 34 ], suggesting that TNF-α exerts pleiotropic, cell-specific effects within the neuro-inflammatory landscape, including potential neuroprotective functions [ 35 ]. Conversely, IL-10 plays a fundamental role in EAE by modulating autopathogenic Th1 responses. Consistently, IL-10-deficient mice display exacerbated disease severity. [ 36 ]. Moreover, virus-induced encephalitis is reported to respond to IL-10 treatment [ 37 ] and significantly reduce EAE severity [ 38 ]. In our study, TNF-α mRNA expression did not change in the EAE group relative to the control, despite the presence of inflammatory infiltration in the brain tissue. In addition, IL-10 mRNA ​​showed an increasing pattern (28%, but not significant) in EAE animals at 28 day of immunization. Regarding to our clinical scores, we suggest that TNF-α mRNA and IL-10 mRNA expressions probably occurred on days 13–14, when clinical symptoms were more prominent. During the remission period, probably a compensation appeared in the progression of the disease and the cytokine pattern reversed on day 28. De Waal Malefyt et al. [ 39 ] reported that IL-10 suppresses the expression of multiple inflammatory cytokines, including IFN-γ, TNF-α, IL-1, and IL-6, in monocytes and T cells ad the earlier stage of EAE. In our study, we suggest that elevated IL-10 inhibited the TNF-α synthesis in the course of the disease. Many physiological processes such as neurogenesis, neuronal differentiation, synaps formation and myelinisation are regulated by miRNAs. Therefore disequilibrium between miRNAs expressions may result to various neurological pathologies, neurodegenerative and autoimmune processes [ 3 , 4 ]. miR-155 and miR-378 are important in brain inflammation and immune responses [ 5 , 6 , 7 , 8 , 12 , 25 ]. Many studies demonstrated a link between miR-155 expression and T-cell differentiation [ 25 , 40 ]. miR-155 was among the first miRNAs implicated in CD4 + T cells activation [ 25 , 41 ] and unveiled to be upregulated in Th1 and Th17 cells concurrent with EAE [ 25 ]. Moreover, in mice deficient in miR-155, increased numbers of Th2 cells and resistance to EAE was reported [ 40 , 42 ]. Also, miR-155 deficiency has been shown to modulate the differentiation of Th1 cells [ 42 ]. With regard to miR-378, it has been shown that this small molecule supports cell survival and angiogenesis [ 10 , 11 ] and suppress neuro-inflammation [ 12 ]. On the other hand, pro-inflammatory mediators, specifically TNF-α and IL-6 lead to upregulation of miR-378 expression [ 13 ]. In our study, miR-155 and miR-378 expressions were found to be boosted in the brain tissue of EAE mice. Based on our current understanding, miR-378 expression was analysed for the first time in an EAE model. In addition, a correlation between miR-378 and IL-10 mRNA expressions was found. The elevation of both miR-378 and IL-10 mRNA probably is a compensatory mechanism against inflammation seen in EAE. It can be suggest that along with anti-inflammatory properties miR-378 and IL-10 mRNA may have also neuroprotective effects on CNS. Aromatic plants and their essential oils (EOs) have long been utilized for their antioxidant, antimicrobial, anticancer, and anti-inflammatory properties [ 15 ]. Today, there are various studies suggesting that some EOs may also be effective on epigenetic mechanisms. For instance, it has been shown that miRNAs (such as miR-155, miR-146a, miR-16, miR-21) may be implicated in inflammation, autophagy and apoptosis by causing changes in their expressions [ 14 , 15 , 16 ]. LO is believed to exert antibacterial, antifungal properties and effective for burns and insect bites [ 18 , 43 ]. The anti-inflammatory effect of LO has been shown in former reports in experimental renal ischemia [ 44 ], thrombosis [ 45 ], and stroke [ 46 ] studies. Therefore, the aim of this study was to delineate the influence of LO on EAE. Although LO had no noticeable efficacy on the clinical score of EAE, in histopathological examination, brain leukocyte infiltration was observed to be less severe in the EAE + LO group compared to the EAE group. While there was no change in TNF-α mRNA expression in the EAE + LO group relative to EAE group, a notable increase in IL-10 mRNA expression was found. Although there was no statistically significant deviation in miR-155 expression between EAE and EAE + LO, an increase in miR-378 expression was observed in EAE + LO. When LO was used alone, it increased in IL-10 mRNA and miR-378 expressions compared to the control. Furthermore, a correlation between IL-10 and miR-378 mRNA expressions was found. This finding suggested that increased IL-10 and miR-378 may have neuroprotective impacts on the CNS along with anti-inflammatory functions. Indeed, Sun et al. [ 12 ] reported that overexpression of miR-378a-5p attenuated neuronal damage, whereas knockdown of miR378a-5p reversed this neuroprotective effect. Our study supports their findings. In the current study, the observations and pathogenetic mechanisms of the LO administration were assessed in EAE model. It was observed that LO per se increase the anti-inflammatory and immunomodulatory IL-10 mRNA and miR-378 levels, therefore we suggest that these RNAs contribute to anti-inflamatory effects of LO. In addition, the rise of IL-10 mRNA and miR-378 leves in EAE + LO and the presence of the correlation between them suggest that these RNAs, together with anti-inflammarory function, may have also neuroprotective efects on CNS. Additional research is warranted to substantiate our current observations. Declarations Data availability statement The authors confirm that data supporting the findings of this study are available upon request. Funding This work was financially supported by the Research Fund of Istanbul University (Project No. TSA-2022-39004). Ethical approval This study was approved by the Animal Experimentaion Local Ethics Board of Istanbul Bezmialem Foundation University (27.05.2022-E.63382). Conflicts of Interest No potential conflict of interest relevant to this article was reported. Author contributions Conception/Design of study: AMB, PV Data Acquisition: AMB, IB, MST, SNG Data Analysis/Interpretation: AMB, PV, IB, SDA Drafting Manuscript: AMB, PV Critical Revision of Manuscript: IB, SDA References Lin HE, Gregory JH (2004) MicroRNAs: small RNAs with a big role in gene regulation. Nat Rev Genet 5(7):522–531. https://doi.org/10.1038/nrg1379 Kenneth SK (2006) The neuronal microRNA system. Nat Rev Neurosci. ; 7 (12): 911 – 720. https://doi.org/10.1038/nrn2037 Barbato C, Giorgi C, Catalanotto C, Cogoni C (2008) Thinking about RNA? MicroRNAs in the brain. Mamm Genome 19(7–8):541–551. https://doi.org/10.1007/s00335-008-9129-6 Varma-Doyle AV, Lukiw WJ, Zhao Y, Lovera J, Devier D (2021) A hypothesis-generating scoping review of miRs identified in both multiple sclerosis and dementia, their protein targets, and miR signaling pathways. 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Science 316(5824):608–611. https://www.science.org/doi/ 10.1126/science.1139253 Xu P, Wang K, Lu C, Dong L, Gao L et al (2016) Protective effect of lavender oil on scopolamine induced cognitive deficits in mice and H2O2 induced cytotoxicity in PC12 cells. J Ethnopharmacol 193:408–415. https://doi.org/10.1016/j.jep.2016.08.030 Aboutaleba N, Jamali H, Abolhasani M, Toroudi HP (2019) Lavender oil (Lavandula angustifolia) attenuates renal ischemia/reperfusion injury in rats through suppression of inflammation, oxidative stress and apoptosis. Biomed Pharmacother 110:9–19. https://doi.org/10.1016/j.biopha.2018.11.045 But VM, Bulboacă AE, Rus V, Ilyés T, Gherman ML et al (2023) Anti-inflammatory and antioxidant efficacy of lavender oil in experimentally induced thrombosis. Thromb J 21(1):85. https://doi.org/10.1186/s12959-023-00516-0 Vakili A, Sharifat S, Akhavan MM, Bandegi AR (2014) Effect of lavender oil (Lavandula angustifolia) on cerebral edema and its possible mechanisms in an experimental model of stroke. Brain Res 1548:56–62. https://doi.org/10.1016/j.brainres.2013.12.019 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Under Review Version 1 posted Reviewers invited by journal 29 Apr, 2026 Editor assigned by journal 28 Apr, 2026 Submission checks completed at journal 28 Apr, 2026 First submitted to journal 24 Apr, 2026 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-9516476","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":633177029,"identity":"1e625d6b-d2f0-4d14-96b9-cae7d98ace31","order_by":0,"name":"Adile Merve BAKI","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAABD0lEQVRIie3RvUoDQRSG4W8Z2DQn2q4IyS1MKhF/ciszLJjORgiChSOCafYCVgK5hnRiN8sp0oTYWqTYIKROug0omGFTyWa1tJi3OwMPc4YBfL5/mX6wgEUkhLVulhAGCGuJ2ZFQWShHgpLQXqJQEpDEn8jhIDZ2hXn7aEDrbFNcXp9E2mDVZ3SPbSWJpguTpVh2hqI5ZlLxzWmqTZDOGHSgqq9514YJHIwcgRJ6vD0Rzact2bNZ25FPcHckKM8KdV+SrxoiHQFYDwXBkuKSBDWk496SyGX8/BhKpquJfk3cyaxHNK0mrUlvkRe384v0jT/WxfmdfmnEWV70z1qNpJrs1vsxW9T9pM/n8/l+7RvIb2n6XWLLpQAAAABJRU5ErkJggg==","orcid":"","institution":"Istanbul University","correspondingAuthor":true,"prefix":"","firstName":"Adile","middleName":"Merve","lastName":"BAKI","suffix":""},{"id":633177031,"identity":"2bd5e825-a599-4c64-85ba-39ca8fcb3bda","order_by":1,"name":"Ilknur BINGUL","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Ilknur","middleName":"","lastName":"BINGUL","suffix":""},{"id":633177032,"identity":"e2bf3d2a-3abc-4998-93b5-327bb719c097","order_by":2,"name":"Sefika Nur GUMUS","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Sefika","middleName":"Nur","lastName":"GUMUS","suffix":""},{"id":633177033,"identity":"78b665be-c31c-4674-bf6c-3ee018c4be76","order_by":3,"name":"Merva SOLUK TEKKESIN","email":"","orcid":"","institution":"Ankara University","correspondingAuthor":false,"prefix":"","firstName":"Merva","middleName":"SOLUK","lastName":"TEKKESIN","suffix":""},{"id":633177034,"identity":"22e45a68-95f9-46f8-be02-5bcbfa47f4af","order_by":4,"name":"Semra DOGRU ABBASOGLU","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Semra","middleName":"DOGRU","lastName":"ABBASOGLU","suffix":""},{"id":633177035,"identity":"e80b5c51-6b79-4353-a20e-ec4d33c48861","order_by":5,"name":"Pervin VURAL","email":"","orcid":"","institution":"Istanbul University","correspondingAuthor":false,"prefix":"","firstName":"Pervin","middleName":"","lastName":"VURAL","suffix":""}],"badges":[],"createdAt":"2026-04-24 11:11:25","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-9516476/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-9516476/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":108726012,"identity":"57badcde-e272-41f4-a455-1cd992895746","added_by":"auto","created_at":"2026-05-07 17:08:03","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":49463,"visible":true,"origin":"","legend":"\u003cp\u003eThe clinical scores of the experimental autoimmune encephalomyelitis (EAE) group\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9516476/v1/8c12ceac448b6487fd5a7ca9.jpg"},{"id":108806673,"identity":"a67f286f-a40c-4f2c-863f-465bb98383c5","added_by":"auto","created_at":"2026-05-08 15:29:14","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":398928,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of lavender oil (LO) administration on brain tissue histopathology of experimental autoimmune encephalomyelitis (EAE) induced mice(H\u0026amp;E x200).\u003c/p\u003e\n\u003cp\u003eA: Control, B: Dimethyl sulfoxide, C: LO, D: EAE, E: EAE+LO\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9516476/v1/44d868da957320e5880e786a.jpg"},{"id":108726013,"identity":"c0182fe7-016b-4f42-a67f-c40acb2c4c5a","added_by":"auto","created_at":"2026-05-07 17:08:03","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":177353,"visible":true,"origin":"","legend":"\u003cp\u003eThe effect of lavender oil (LO) administration on relative expressions of TNF-α mRNA,\u0026nbsp; IL-10 mRNA, miR-155 and miR-378 of experimental autoimmune encephalomyelitis (EAE) induced mice (DMSO, dimethyl sulfoxide).\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ea\u003c/sup\u003ep\u0026lt;0.05\u0026nbsp; LO group vs Control,\u003c/p\u003e\n\u003cp\u003e\u003csup\u003eb\u003c/sup\u003ep\u0026lt;0.05\u0026nbsp; EAE group vs Control,\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ec\u003c/sup\u003ep\u0026lt;0.05\u0026nbsp; EAE+LO vs Control,\u003c/p\u003e\n\u003cp\u003e\u003csup\u003ed\u003c/sup\u003ep\u0026lt;0.05 EAE+LO vs EAE group\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-9516476/v1/189a45d33e73e6d92eff8887.jpg"},{"id":108809754,"identity":"ec094745-ef6a-4d33-b66e-f201d2aeaf60","added_by":"auto","created_at":"2026-05-08 15:55:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":850110,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-9516476/v1/748ad501-a3e1-4e32-9d1e-9179f87ebffb.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"The Effects of Lavender Oil on IL-10 mRNA, miR-378 in the Brain of Experimental Autoimmune Encephalomyelitis","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe pathological progression of multiple sclerosis (MS) and its preclinical counterpart, experimental autoimmune encephalomyelitis (EAE), is fundamentally driven by central nervous system inflammation, axonal decay, and the loss of myelin. Cytokines facilitate the entire progression of MS, mediating the early differentiation of peripheral pathogenic T-cells as well as the subsequent CNS inflammation and structural damage. Among these, Tumor necrosis factor α (TNF-α) serves as a versatile signaling molecule regulating diverse cellular processes, including viability and apoptosis. Given its pro-inflammatory properties, the secretion of TNF-α by immune cells is considered a key contributor to the inflammatory damage observed in both MS and EAE. Interleukin 10 (IL-10) acts as a key regulatory cytokine, playing a vital role in curbing inflammatory and autoimmune disorders.\u003c/p\u003e \u003cp\u003eComprising approximately 18\u0026ndash;25 nucleotides, microRNAs (miRNAs) represent a class of small non-coding RNA molecules that function as master regulators of diverse cellular pathways. They achieve their regulatory role by targeting the 3' untranslated region (UTR) of messenger RNAs (mRNAs), where they initiate mRNA decay [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e]. miRNAs emerge as key modulators of post-transcriptional gene expression during neurogenesis, neuronal differentiation, and synaptic plasticity, while also modulating gliogenesis and myelin repair. A disequilibrium in the expression of miRNAs can be involved in many neurological pathologies, neurodegenerative and autoimmune diseases [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. The involvement of miR-155 is critical to the modulation of immune-mediated processes and the coordination of inflammatory reactions [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e]in various tissues including CNS [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e]. By driving the specification of pathogenic Th1 and Th17 cells, miR-155 acts as a critical regulator of the T-cell-driven immune responses characteristic of MS. Furthermore, a significant overexpression of miR-155 has been identified within MS lesions during the inflammatory activation of macrophages [\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e]. miR-378 facilitates cell endurance, tumor growth, and angiogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e], and has neuroprotective and immunomodulatory effects by suppressing neuroinflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. On the other hand, pro-inflammatory cytokines was reported to upregulate the miR-378a expression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eAromatic plants, and particularly their volatile essential oils (EOs), have demonstrated remarkable pharmacological potential, including notable antioxidant, antimicrobial, antineoplastic, and anti-inflammatory properties [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Today, many EOs have been found to be associated with inflammation, autophagy, and apoptosis by causing changes in the expression of miRNAs [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. Extracted from Lavandula angustifolia, lavender oil (LO) stands as a premier essential oil with widespread applications across both the pharmaceutical sector and therapeutic aromatherapy [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e]. In traditional herbal medicine, LO exhibits a broad spectrum of pharmacological activities, including anti-inflammatory, antioxidant, sedative, antidepressive, antimicrobial, antifungal and analgesic properties [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e]. The LO contains over 100 chemical constituents [\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e]. Linalool and linalyl acetate are primary components of LO related to its anti-inflammatory activity [\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e]. However, there is no knowledge in the literature regarding the effect of LO on epigenetic regulation of inflammatory processes, namely miR-155 and miR-378. Consequently, The objective of the present study was to examine the therapeutic potential of LO on inflammation by measuring of mRNA expressions of pro- and anti-inflammatory cytokines (TNF-α, IL-10) as well as miR-155 and miR-378 expressions in the brain tissue of EAE induced mice.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Chemicals and kits\u003c/h2\u003e \u003cp\u003eChemicals and reagents were obtained from Sigma-Aldrich (St Louis, Missouri, USA). Kits and primers were obtained from Qiagen Technologies (USA).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Animals\u003c/h2\u003e \u003cp\u003eC57BL/6J mice were housed at 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C with a 12-h light/dark cycle and provided with ad libitum food and water. All protocols were approved by the Institutional Experimental Animal Ethics Committee (BVUHDEK-2022/105) and performed following veterinary and ethical guidelines\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Induction of Experimental autoimmune encephalomyelitis\u003c/h2\u003e \u003cp\u003eTo establish the EAE model, 6\u0026ndash;8 week-old female C57BL/6J mice were immunized with 100 \u0026micro;g of an myelin oligodendrocyte glycoprotein (MOG) 35\u0026ndash;55 (MOG\u003csub\u003e35\u0026ndash;55\u003c/sub\u003e) (#SCP0195) peptide per animal emulsified in complete Freund's adjuvant (CFA) containing 300 \u0026micro;g of attenuated Mycobacterium tuberculosis H37RA (#F5881). Each mouse received 0.1 mL of emulsion (s.c.) at two sites. After immunization, 400 ng of pertussis toxin (PTx) (#516551) in 100 \u0026micro;L of PBS was injected intraperitoneally (i.p.) into mice on day 0 and 2 [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Consistent with previously validated methodologies, the clinical progression of each subject was evaluated individually [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e]. The clinical progression of the disease was evaluated individually each day for up to 28 days, employing the following 0-to-5 point scoring system based on symptomatic presentation: 0, no disease; 1, weak tail or unsteady gait; 2, hind-limb paresis; 3, hind-limb paralysis; 4, hind- and fore-limb paralysis; and 5, severe paralysis or death [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Lavender oil treatment\u003c/h2\u003e \u003cp\u003eLO was diluted in 5% dimethyl sulfoxide (DMSO) and administered i.p. 100 mg/kg to each mouse (#W262218) [\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Groups\u003c/h2\u003e \u003cp\u003eMice were randomly allocated into experimental groups and maintained in standard cages. Animals were divided into five groups: 1) Control (n\u0026thinsp;=\u0026thinsp;7): Mice were provided with standard mice chow and drinking water ad libitum were injected to animals for 28 days; 2) DMSO (as a vehicle, n\u0026thinsp;=\u0026thinsp;7): 0.1 mL of 5% DMSO was injected into mice daily for 28 days; 3) LO (n\u0026thinsp;=\u0026thinsp;7): The i.p. administration of LO was performed daily for 28 days; 4) EAE group (n\u0026thinsp;=\u0026thinsp;7): The EAE model in mice was induced as referenced above; 5) EAE\u0026thinsp;+\u0026thinsp;LO (n\u0026thinsp;=\u0026thinsp;7): LO was administered daily for 28 days to EAE-induced mice two hours after the last PTx injection.\u003c/p\u003e \u003cp\u003eFollowing the experimental period (at 28th day), blood was taken from the hearts of the mice under xylazine HCl (5mg/kg) and ketamine (100mg/kg) anesthesia and brain tissues were rapidly removed. For RNA isolation, brain tissue pieces were stored in \u0026ldquo;RNA protect tissue reagent\u0026rdquo; (#172034595, Qiagen, USA) at -35\u0026deg;C until the measurements.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Histological examination\u003c/h2\u003e \u003cp\u003eBrain samples were fixed in a 10% formalin buffer solution for 24 hours prior to embedding in paraffin blocks. Subsequently, 5 \u0026micro;m sections were obtained from each paraffin block and stained for histological examination. These sections were then evaluated using hematoxylin and eosin (H\u0026amp;E) staining to assess the presence of inflammation.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. mRNA and miRNA expressions\u003c/h2\u003e \u003cp\u003eTotal RNA was purified from samples using miRNeasy Tissue/cell Advanced Kit (#217604) following manifacturer\u0026rsquo;s instruction. Quantitative real time polymerase chain reaction (qPCR) were performed on a Qiagen Rotor-Gene Q device (Qiagen Hilden, Germany).\u003c/p\u003e \u003cp\u003eFor mRNA synthesis, cDNA was sythesized using QuantiTect Reverse Transcription kit (#205310). TNF-α and IL-10 expressions were quantified with QuantiNova LNA PCR assays TNF-α, (#SBM0788439-200) and IL-10 (#SBM1004151-200). The 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method was employed to quantify mRNA expression levels, which were normalized against GAPDH as an internal control.\u003c/p\u003e \u003cp\u003eFor miRNA synthesis, cDNA was sythesized using miRCURY LNA RT Kit; (#YP02119464). For miR-155-3p and miR-378a-3p expressions, miR-155-3p and miR-378a-3p miRCURY LNA\u0026trade; miRNA PCR Assays were used (#YP02104818 and #YP00204179, respectively). The expression levels of the miRNAs were calculated using the 2\u003csup\u003e\u0026minus;ΔΔCt\u003c/sup\u003e method. The analytical data were calibrated against U6 snRNA expression levels, which served as an internal control.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e3.1. EAE clinical score\u003c/h2\u003e \u003cp\u003eClinical symptoms manifested around day 10, peaking between days 13 and 14 with a maximum clinical score of 2, followed by a period of moderate remission (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The clinical scores of the EAE and EAE\u0026thinsp;+\u0026thinsp;LO groups were of a similar degree.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Histological assessment\u003c/h2\u003e \u003cp\u003eThe control and DMSO groups exhibited similar morphology to normal histology while the LO group displayed characteristics that were relatively close to normal histology. While inflammatory infiltration was observed in the EAE group, the number of inflammatory cells was declined in the EAE\u0026thinsp;+\u0026thinsp;LO group in contrasted to the EAE group (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e, original magnification H\u0026amp;E x200).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.3. mRNA expressions of TNF-α and IL-10\u003c/h2\u003e \u003cp\u003eNo discernible alterations were observed in TNF-α mRNA levels among the experimental groups. There was an increasing pattern in IL-10 expression in the EAE group compared to the control group, but it did not reach significance level. IL-10 expression in EAE\u0026thinsp;+\u0026thinsp;LO group was higher than EAE group. In addition, when LO was used alone, it increased the IL-10 mRNA expression compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.4. miR-155 and miR-378 expression\u003c/h2\u003e \u003cp\u003emiR-155 and miR-378 expressions were similar between the control and DMSO groups. They were found to be elevated in the EAE group in comparison with control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e). miR-378 expression was higher in the EAE\u0026thinsp;+\u0026thinsp;LO group than in EAE group. In addition, when LO was applied alone, the miR-378 expression increased compared to the control (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eSignificant correlation between IL-10 mRNA and miR-378 was found (r\u0026thinsp;=\u0026thinsp;0.651, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eMS represents a widespread neurodegenerative disorder, with a global prevalence exceeding one million individuals [\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e]. The etiology of MS is hypothesized to stem from a multifaceted interaction between genetic susceptibility and environmental triggers that precipitate immune system activation. For several decades, the EAE model has served as the primary paradigm for investigating the underlying mechanisms and developmental stages of the disease [\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e]. In one of the most prevalent experimental models, EAE is elicited through the subcutaneous administration of an encephalitogenic peptide, commonly MOG\u003csub\u003e35\u0026ndash;55\u003c/sub\u003e or (PLP) \u003csub\u003e139\u0026minus;151\u003c/sub\u003e. This peptide is formulated as an emulsion in CFA (comprising mineral oil and Mycobacterium tuberculosis H37RA), followed by supplementary i.p. injections of pertussis toxin [\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. In our study, EAE was induced by this model as depicted by Dias et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] and Tan et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e]. Dias et al. [\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e] have reported that clinical symptoms initially emerged around day 10, reaching peak severity by day 19 (mean clinical score 4.8) and continued with fluctuations until day 58. Tan et al. [\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e] found that clinical symptoms appeared on day 10, reached maximum values on day 16 (clinical score 2\u0026ndash;3) and lasted until day 28. Similar to their results, in our study the emergence of clinical symptoms occured around day 10, and peak clinical score attained on day 13\u0026ndash;14 (clinical score 2). Although the clinical scores showed a remission on day 28, inflammatory infiltration was observed in the brain tissue, histopathologically.\u003c/p\u003e \u003cp\u003eThe precise mechanism for the initiation and progression of MS is still not clear, although intertwined mechanisms of innate and adaptive immunity are thought to be involved. The immunopathology of MS is characterized by the involvement of T helper (CD4+) lymphocytes and essential antigen-presenting cells (APCs), such as macrophages, B cells, dendritic cells, and CNS-resident microglia. As a consequence of the interaction between APCs with T lymphocytes Th cells differentiate into either Th1, Th2 or Th17 phenotypes. While Th1 and Th17 are phenotypes promoting inflammation, Th2 is anti-inflammatory one. Pro-inflammatory cytokines such as interferon-gamma and TNF-α are secreted by Th1 cells, whereas Th2 cells provide an anti-inflammatory response via IL-4, IL-10, and IL-13. Furthermore, the IL-17, IL-21, IL-22, and IL-26 cytokines are produced by the Th17 lineage of CD4\u0026thinsp;+\u0026thinsp;T cells. Th1 cytokines promote the CNS recruitment of Th17 subsets, thereby driving the neuro-inflammatory cascade that characterizes the EAE model [\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Destroying of the blood-brain barrier in EAE leads to the cerebral infiltration of autoreactive T cells and increases the pro-inflammatory cytokine levels. As consequence, initiated molecular mechanism produces the CNS immune self-attack, which is most prominent at the first phases of MS related with the microglia activation and macrophages infiltration promoting demyelination [\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eMany studies reported elevated TNF-α level in the CSF of MS patients, correlating with both disease severity and progression [\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e, \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. Since blocking TNF-α signaling has been established to attenuate EAE development [\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e], TNF-α inhibitors were suggested to be useful in MS treatment. Unfortunately, clinical trials demonstrated that MS patients treated with recombinant TNF receptor antibody experienced augmented disease pathology [\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e], suggesting that TNF-α exerts pleiotropic, cell-specific effects within the neuro-inflammatory landscape, including potential neuroprotective functions [\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. Conversely, IL-10 plays a fundamental role in EAE by modulating autopathogenic Th1 responses. Consistently, IL-10-deficient mice display exacerbated disease severity. [\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Moreover, virus-induced encephalitis is reported to respond to IL-10 treatment [\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] and significantly reduce EAE severity [\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. In our study, TNF-α mRNA expression did not change in the EAE group relative to the control, despite the presence of inflammatory infiltration in the brain tissue. In addition, IL-10 mRNA ​​showed an increasing pattern (28%, but not significant) in EAE animals at 28 day of immunization. Regarding to our clinical scores, we suggest that TNF-α mRNA and IL-10 mRNA expressions probably occurred on days 13\u0026ndash;14, when clinical symptoms were more prominent. During the remission period, probably a compensation appeared in the progression of the disease and the cytokine pattern reversed on day 28. De Waal Malefyt et al. [\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e] reported that IL-10 suppresses the expression of multiple inflammatory cytokines, including IFN-γ, TNF-α, IL-1, and IL-6, in monocytes and T cells ad the earlier stage of EAE. In our study, we suggest that elevated IL-10 inhibited the TNF-α synthesis in the course of the disease.\u003c/p\u003e \u003cp\u003eMany physiological processes such as neurogenesis, neuronal differentiation, synaps formation and myelinisation are regulated by miRNAs. Therefore disequilibrium between miRNAs expressions may result to various neurological pathologies, neurodegenerative and autoimmune processes [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. miR-155 and miR-378 are important in brain inflammation and immune responses [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e, \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Many studies demonstrated a link between miR-155 expression and T-cell differentiation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e]. miR-155 was among the first miRNAs implicated in CD4\u0026thinsp;+\u0026thinsp;T cells activation [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e, \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] and unveiled to be upregulated in Th1 and Th17 cells concurrent with EAE [\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e]. Moreover, in mice deficient in miR-155, increased numbers of Th2 cells and resistance to EAE was reported [\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e, \u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. Also, miR-155 deficiency has been shown to modulate the differentiation of Th1 cells [\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. With regard to miR-378, it has been shown that this small molecule supports cell survival and angiogenesis [\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e] and suppress neuro-inflammation [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. On the other hand, pro-inflammatory mediators, specifically TNF-α and IL-6 lead to upregulation of miR-378 expression [\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e]. In our study, miR-155 and miR-378 expressions were found to be boosted in the brain tissue of EAE mice. Based on our current understanding, miR-378 expression was analysed for the first time in an EAE model. In addition, a correlation between miR-378 and IL-10 mRNA expressions was found. The elevation of both miR-378 and IL-10 mRNA probably is a compensatory mechanism against inflammation seen in EAE. It can be suggest that along with anti-inflammatory properties miR-378 and IL-10 mRNA may have also neuroprotective effects on CNS.\u003c/p\u003e \u003cp\u003eAromatic plants and their essential oils (EOs) have long been utilized for their antioxidant, antimicrobial, anticancer, and anti-inflammatory properties [\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e]. Today, there are various studies suggesting that some EOs may also be effective on epigenetic mechanisms. For instance, it has been shown that miRNAs (such as miR-155, miR-146a, miR-16, miR-21) may be implicated in inflammation, autophagy and apoptosis by causing changes in their expressions [\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e]. LO is believed to exert antibacterial, antifungal properties and effective for burns and insect bites [\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. The anti-inflammatory effect of LO has been shown in former reports in experimental renal ischemia [\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e], thrombosis [\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e], and stroke [\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e] studies. Therefore, the aim of this study was to delineate the influence of LO on EAE. Although LO had no noticeable efficacy on the clinical score of EAE, in histopathological examination, brain leukocyte infiltration was observed to be less severe in the EAE\u0026thinsp;+\u0026thinsp;LO group compared to the EAE group. While there was no change in TNF-α mRNA expression in the EAE\u0026thinsp;+\u0026thinsp;LO group relative to EAE group, a notable increase in IL-10 mRNA expression was found. Although there was no statistically significant deviation in miR-155 expression between EAE and EAE\u0026thinsp;+\u0026thinsp;LO, an increase in miR-378 expression was observed in EAE\u0026thinsp;+\u0026thinsp;LO. When LO was used alone, it increased in IL-10 mRNA and miR-378 expressions compared to the control. Furthermore, a correlation between IL-10 and miR-378 mRNA expressions was found. This finding suggested that increased IL-10 and miR-378 may have neuroprotective impacts on the CNS along with anti-inflammatory functions. Indeed, Sun et al. [\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e] reported that overexpression of miR-378a-5p attenuated neuronal damage, whereas knockdown of miR378a-5p reversed this neuroprotective effect. Our study supports their findings.\u003c/p\u003e \u003cp\u003eIn the current study, the observations and pathogenetic mechanisms of the LO administration were assessed in EAE model. It was observed that LO per se increase the anti-inflammatory and immunomodulatory IL-10 mRNA and miR-378 levels, therefore we suggest that these RNAs contribute to anti-inflamatory effects of LO. In addition, the rise of IL-10 mRNA and miR-378 leves in EAE\u0026thinsp;+\u0026thinsp;LO and the presence of the correlation between them suggest that these RNAs, together with anti-inflammarory function, may have also neuroprotective efects on CNS. Additional research is warranted to substantiate our current observations.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eData availability statement\u0026nbsp;\u003c/strong\u003eThe authors confirm that data supporting the findings of this study are available upon request.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was financially supported by the Research Fund of Istanbul University (Project No. TSA-2022-39004). \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical approval\u0026nbsp;\u003c/strong\u003eThis study was approved by the Animal Experimentaion Local Ethics Board of Istanbul Bezmialem Foundation University (27.05.2022-E.63382).\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflicts of Interest\u003c/strong\u003e No potential conflict of interest relevant to this article was reported.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConception/Design of study:\u0026nbsp;\u003c/strong\u003eAMB, PV\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Acquisition:\u0026nbsp;\u003c/strong\u003eAMB, IB, MST, SNG\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData Analysis/Interpretation:\u0026nbsp;\u003c/strong\u003eAMB, PV, IB, SDA \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp; \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDrafting Manuscript:\u0026nbsp;\u003c/strong\u003eAMB, PV\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCritical Revision of Manuscript:\u0026nbsp;\u003c/strong\u003eIB, SDA\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eLin HE, Gregory JH (2004) MicroRNAs: small RNAs with a big role in gene regulation. 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Brain Res 1548:56\u0026ndash;62. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.brainres.2013.12.019\u003c/span\u003e\u003cspan address=\"10.1016/j.brainres.2013.12.019\" targettype=\"DOI\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"Experimental autoimmune encephalomyelitis, IL-10 mRNA, miR-155, miR-378, TNF-α mRNA","lastPublishedDoi":"10.21203/rs.3.rs-9516476/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-9516476/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjective\u003c/h2\u003e \u003cp\u003eTo investigate the effect of lavender oil (LO) on expressions of tumor necrosis factor α (TNF-α), interleukin 10 (IL-10), microRNA-155 (miR-155) and miR-378 in brain tissue of experimental autoimmune encephalomyelitis (EAE).\u003c/p\u003e\u003ch2\u003eMaterials and Methods\u003c/h2\u003e \u003cp\u003eEAE was provoked by s.c. injection containing encephalitogenic peptide MOG35\u0026ndash;55 and Mycobacterium tuberculosis H37RA, followed by i.p. injection of pertussis toxin in C57BL/6 J mice. LO was administered i.p. daily for 28 days. mRNA expressions of pro-/anti-inflammatory cytokines (TNF-α, IL-10), and miR-155 and miR-378 expressions together with histopathology were investigated in the brain tissue.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eThe clinical manifestation of symptoms commenced around day 10, with peak intensity observed during days 13 and 14 (clinical score 2). Although the clinical scores showed a remission on day 28, inflammatory infiltration was observed in the brain tissue histopathologically. TNF-α mRNA expression not changed, while IL-10 mRNA had an increasing pattern in EAE. miR-155 and miR-378 expressions were elevated in EAE. When LO administered to EAE animals IL-10 mRNA and mir-378 expressions increased.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eOur results demonstrate that LO may have a neuroprotective effect in EAE, and exert this effect by increasing the expression of anti-inflammatory and immunomodulatory IL-10 mRNA and miR-378. Nevertheless, additional research is required to validate the results of this investigation.\u003c/p\u003e","manuscriptTitle":"The Effects of Lavender Oil on IL-10 mRNA, miR-378 in the Brain of Experimental Autoimmune Encephalomyelitis","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-05-07 17:07:59","doi":"10.21203/rs.3.rs-9516476/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewersInvited","content":"","date":"2026-04-29T23:55:58+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-04-28T09:12:29+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-04-28T09:12:25+00:00","index":"","fulltext":""},{"type":"submitted","content":"Molecular Biology Reports","date":"2026-04-24T11:00:12+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"molecular-biology-reports","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"mole","sideBox":"Learn more about [Molecular Biology Reports](https://www.springer.com/journal/11033)","snPcode":"11033","submissionUrl":"https://submission.nature.com/new-submission/11033/3","title":"Molecular Biology Reports","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"782f37fc-1877-4ef8-a540-0393b159a246","owner":[],"postedDate":"May 7th, 2026","published":true,"recentEditorialEvents":[{"type":"reviewersInvited","content":"12","date":"2026-04-29T23:55:58+00:00","index":"","fulltext":""}],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-05-07T17:07:59+00:00","versionOfRecord":[],"versionCreatedAt":"2026-05-07 17:07:59","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-9516476","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-9516476","identity":"rs-9516476","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}