Exploration of Possible Effects of Cannabis sativa Seed Oil Applied in Different Doses in Ulcerative Colitis Model

Exploration of Possible Effects of Cannabis sativa Seed Oil Applied in Different Doses in Ulcerative Colitis Model | 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 Exploration of Possible Effects of Cannabis sativa Seed Oil Applied in Different Doses in Ulcerative Colitis Model İzel Kavak, Ferhat Şirinyıldız This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8751706/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 6 You are reading this latest preprint version Abstract BACKGROUND Ulcerative colitis is a chronic inflammatory disease characterized by extensive mucosal damage and insufficient treatment options. Cannabis sativa seed oil, distinguished by its rich bioactive lipid profile, offers significant potential in modulating inflammation and oxidative stress. Given that its dose-dependent efficacy remains largely unexplored, this study investigates the therapeutic effects of varying doses of Cannabis sativa seed oil to establish a novel intervention strategy for ulcerative colitis. MATERIALS AND METHODS 24 Wistar albino rats were distributed into four groups: control, TNBS-induced colitis, and two treatment groups receiving Cannabis sativa seed oil via intragastric gavage at doses of 1 ml/kg (CS1) and 3 ml/kg (CS3). Following a 72-hour protocol, colon tissues were harvested to evaluate oxidative stress markers (SOD, GPx, MDA, MPO, CAT) and pro-inflammatory cytokines (TNF-α, IL-1β). RESULTS Cannabis sativa seed oil administration significantly attenuated apoptotic and inflammatory signaling while restoring antioxidant enzyme homeostasis. Both biochemical and histopathological analyses confirmed that the treatment conferred robust protection against colitogenic damage (p < 0.05). CONCLUSION Our findings demonstrate that Cannabis sativa seed oil effectively ameliorates ulcerative colitis. Notably, the 3 ml/kg dose exhibited superior efficacy, yielding results comparable to the healthy control group, which underscores its potential as a novel pharmacological agent for inflammatory bowel diseases. Cannabis sativa Seed oil Ulcerative colitis İnflammation Oxidative stress Figures Figure 1 Figure 2 1. INTRODUCTION Inflammatory bowel disease (IBD), primarily classified as Crohn’s disease (CD) and ulcerative colitis (UC), is a chronic gastrointestinal condition ( 1 ). Globally, approximately 11.2 million individuals are affected, with the highest prevalence in Europe and North America ( 2 ). The disease typically manifests between ages 10–35 and occurs more frequently in females ( 1 , 2 ). Notably, 20% to 30% of ulcerative colitis patients show an inadequate response to medical therapy, often necessitating surgical intervention or colostomy ( 3 ). Long-term pharmacological management is essential in chronic diseases, yet complications and poor adherence often compromise patient outcomes. Reported non-adherence rates for IBD treatments vary significantly, ranging from 9.6–65% for aminosalicylic acid to 3–64.7% for thiopurines ( 4 , 5 ). Such non-adherence severely impairs symptom control and increases the risk of surgical interventions ( 6 ) Consequently, identifying natural phytochemicals to enhance treatment response and quality of life has become a key research focus, offering promising strategies to target oxidative stress and proinflammatory cytokines ( 7 ). Ulcerative colitis (UC) is a multifactorial immunological disorder characterized by recurrent gastrointestinal inflammation and mucosal injury. Pathogenesis involves an uncontrolled immune response where disrupted intestinal barrier integrity permits luminal antigens to infiltrate the submucosa. This barrier defect triggers massive immune cell infiltration and activates the nuclear factor-kappa B (NF-κB) pathway, resulting in the excessive production of proinflammatory cytokines such as TNF-α and IL-1β ( 8 ). This cytokine dysregulation accelerates tissue destruction and intensifies oxidative stress via reactive oxygen species (ROS) production. Oxidative stress promotes lipid peroxidation (increased MDA) and depletes endogenous antioxidants like SOD and GPx, escalating mucosal damage to a transmural scale ( 9 ). Growing interest in bioactive natural components stems from the adverse effects of conventional IBD therapies. Cannabis sativa seed oil emerges as a therapeutic agent due to its optimal 3:1 Omega-6/Omega-3 ratio and rich polyphenolic content. Its anti-inflammatory capacity primarily involves components like ALA and GLA, which suppress proinflammatory pathways via PPAR-γ activation ( 10 ). Furthermore, tocopherols within the oil directly neutralize ROS, protecting the mucosal barrier from oxidative assault and reducing immune cell infiltration through the modulation of MPO activity ( 11 ). This mechanistic synergy positions CS seed oil as a potent natural adjuvant strategy that repairs the intestinal barrier and mitigates the cytokine storm. Used since 3000 BC in traditional Chinese medicine, Cannabis sativa has long treated fatigue, rheumatism, and inflammatory disorders ( 12 ). Cannabis sativa seed oil (CSSO) is a unique plant-derived source of vitamin D ( 13 ). Beyond nutrition, its essential fatty acids contribute to anti-inflammatory, anticancer, and neuroprotective effects ( 14 ). CSSO lacks psychoactive properties as it bypasses cannabinoid 1 receptors. Instead, it exerts potent antioxidant and immune-modulating effects via vitamin E and cannabinoid 2 receptors ( 14 ). Inflammation is a fundamental biological defense mechanism that protects tissues from microorganisms and toxins but may also result in tissue damage ( 15 ). This process is mediated by blood vessels, leukocytes, and plasma proteins. Inflammation can also be triggered by tissue necrosis, toxins, and chemical agents. Recent studies have significantly associated ulcerative colitis with systemic indicators, specifically the platelet-to-lymphocyte and neutrophil-to-lymphocyte ratios ( 16 , 17 ). Ulcerative colitis pathophysiology involves diffuse mucosal inflammation extending from the rectum to proximal segments of the colon ( 18 , 19 ). This study aimed to evaluate the therapeutic potential of Cannabis sativa seed oil as a natural adjuvant for ulcerative colitis, addressing the limitations of current pharmacological interventions. Our objective was to investigate whether its rich antioxidant and anti-inflammatory profile could effectively mitigate mucosal damage and oxidative stress in an experimental colitis model. 2. MATERİALS AND METHODS 2.1. Study Design and Setting This study was conducted in accordance with the principles outlined in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health and was approved by the Ethics Committee for Animal Experiments of Aydın Adnan Menderes University. The clinical trial number is 64583101/2023/06, Approval date 19/01/2023. 2.2. Experimental Animals and Conditions A total of 24 male Wistar albino rats, weighing between 250–400 g and aged 10–12 weeks, were obtained from the Animal Production Center of Aydın Adnan Menderes University Faculty of Medicine. The animals were housed under semi-controlled laboratory conditions with a temperature of 22 ± 1°C, relative humidity of 40–50%, and a 12/12-hour light/dark cycle. Standard laboratory chow and tap water were provided ad libitum until 8–12 hours before the experimental procedures. 2.3. Characterization and Standardization of Cannabis sativa Seed Oil The Cannabis sativa seed oil used in this study was a standardized, commercially available product (Mecitefendi, Turkey) manufactured under ISO 9001 and ISO 22000 quality management systems (The fat content was analyzed using chromatographic methods and submitted to the official authorities). To ensure the reproducibility and scientific validity of the results, the chemical profile of the specific batch utilized was documented, and a single, uniform batch was used for all experimental groups to maintain consistency. The oil was extracted through a controlled cold-pressing process (below 40°C) and is characterized by a high concentration of polyunsaturated fatty acids (~ 80–90%), a significant tocopherol content (800–1100 mg/kg), and a specific 3:1 Omega-6 to Omega-3 ratio ( 20 ). This rigorous standardization ensures that the observed therapeutic effects are consistently attributable to the established phytochemical profile of high-quality hemp seed oil. 2.4. Experimental Design The rats were randomly allocated into four experimental groups (n = 6 per group): a healthy Control group, a Colitis group (TNBS-induced, untreated), and two treatment groups receiving Cannabis sativa seed oil at doses of 1 mL/kg (CS1) and 3 mL/kg (CS3). In the treatment arms, the oil was administered daily via intragastric gavage for five consecutive doses, with the initial dose starting six hours post-induction. Seventy-two hours after the induction of colitis, all rats were sacrificed under anesthesia, and colon tissues were harvested for subsequent biochemical and histopathological analyses. 2.5. Induction of Experimental Colitis Experimental colitis was induced by intracolonic administration of TNBS. The rats were fasted for 24 hours prior to induction, with free access to water. Under anesthesia with ketamine (75 mg/kg) and xylazine (8 mg/kg) administered intraperitoneally, a polyethylene cannula (6 Fr diameter) was gently inserted 8 cm into the anus. A 1 mL solution containing 25 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 37% ethanol was slowly injected. The rats were maintained in the Trendelenburg position for 30 seconds to ensure uniform distribution of the solution, then returned to their cages for recovery. 2.6. Histopathological Evaluation and Scoring Colonic tissue samples were fixed in 10% neutral buffered formalin, dehydrated through a graded series of ethanol, and embedded in paraffin blocks. Sections of 5-µm thickness were obtained via microtome and stained with Hematoxylin and Eosin (H&E). The stained sections were examined under a light microscope to evaluate mucosal architecture and inflammatory infiltration. Histological damage was quantified using a validated semi-quantitative scoring system based on established literature. Parameters including epithelial loss, crypt architectural distortion, inflammatory cell infiltration, submucosal edema, and the presence of hemorrhage or necrosis were graded on a scale (typically 0–3 or 0–4). The total histological score, representing the sum of these individual parameters, was calculated to provide a comprehensive assessment of colonic mucosal injury. 2.7. Tissue Sampling and Processing At the end of the 72-hour experimental period, anesthesia was induced with xylazine (8 mg/kg) and ketamine (75 mg/kg) intraperitoneally. The abdominal cavity was opened under sterile conditions for macroscopic inspection of the colon to evaluate perforation or adhesion. Tissue samples obtained for biochemical analysis were stored at − 80°C until further processing. Portions of the colon designated for histopathological evaluation were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5 µm thickness, and stained with hematoxylin–eosin (H&E) for microscopic examination. 2.8. Biochemical Analysis Colon tissue samples were homogenized in 50 mM phosphate buffer (w/v = 1/10) at 0–4°C. The homogenates were centrifuged at 500 × g for 15 minutes at 4°C, and the supernatants were collected for analysis. The activities and concentrations of myeloperoxidase (MPO), catalase (CAT), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were determined using commercially available ELISA kits (BioVision and ScienceCell, USA) according to the manufacturers’ instructions. 2.9. Statistical Analysis Statistical analyses were performed using GraphPad Prism (version 7.04, GraphPad Software Inc., San Diego, USA). The normality of data distribution was assessed using the Shapiro-Wilk test. For normally distributed data, statistical differences between groups were evaluated using one-way analysis of variance (ANOVA), followed by Tukey’s multiple comparisons test for post-hoc analysis. Data are expressed as Mean ± Standard Error of the Mean (SEM), and a p-value of < 0.05 was considered statistically significant. 3. RESULTS 3.1. Histopathological Findings Histopathological findings are summarized in Table 1. No tissue damage was observed in the control group. In the colitis group, severe mucosal disruption, hemorrhage, necrosis, submucosal edema, crypt distortion, epithelial damage, reduced Goblet cell count, and wall thichening were evident. In the CS1 group, moderate necrosis, hemorrhage, and inflammation were observed. In contrast, the CS3 group exhibited no necrosis or crypt distortion, and the Goblet cell count remained unaltered. Only mild edema and inflammation were detected in this group. Microscopic examination of Hematoxylin-Eosin (H&E, x10) stained sections revealed no structural alterations in the control group (Fig. 1 A). In contrast, the TNBS group exhibited severe histopathological changes in all evaluated parameters (Fig. 1 C). The CS1 group showed improvements in submucosal edema, hemorrhage, wall thickening, epithelial damage, crypt distortion, necrosis, inflammation, and Goblet cell count relative to the TNBS group (Fig. 1 B). Similarly, the CS3 group demonstrated marked reductions in edema, hemorrhage, inflammation, necrosis, crypt distortion, Goblet cell loss, and wall thickening compared to the TNBS group (Fig. 1 D). 3.2. Biochemical Findings Comparisons among the study groups revealed significant differences in several biochemical parameters (Table 2, Fig. 2 ). TNBS administration successfully induced oxidative damage, evidenced by the marked elevation of MDA (355.8 ± 23.36) and the significant increase in MPO levels (618.0 ± 33.76), reflecting extensive lipid peroxidation and neutrophil infiltration, respectively. Furthermore, the TNBS group exhibited severe inflammation, demonstrated by the substantial rise in pro-inflammatory cytokines TNF-alpha (1549 ± 70.24) and IL-1 beta (1406 ± 67.31). This pathological state was further underscored by the dysregulation of the endogenous antioxidant system, shown by the diminished levels of CAT (277.7 ± 19.76) and GPx (619.3 ± 47.72) and the increased SOD activity (40.17 ± 1.682). Conversely, treatment with Cannabis sativa seed oil significantly mitigated these pathological changes in a dose-dependent manner. Specifically, the highest dose (CS3) significantly reduced the levels of MDA (220.5 ± 23.48) and MPO (400.5 ± 36.48) compared to the TNBS group (p < 0.05). Similarly, CS treatment markedly suppressed the inflammatory response; specifically, TNF-α (1234 ± 54.72 in CS3) and IL-1β (1081 ± 49.69 in CS3) levels demonstrated a significant restoration toward baseline values. The intervention also restored the antioxidant capacity, as evidenced by the significant elevation of CAT (380.7 ± 15.07 in CS3) and GPx (856.3 ± 23.19 in CS1) compared to the TNBS group. These findings collectively affirm the potent anti-inflammatory and antioxidant properties of Cannabis sativa in the experimental colitis model. Tablo 1 Shows the tissue histopathology scores (p < 0.05). Control Colitis CS1 CS3 Submucosal edema 0.22 2.87 2.25 1.88 Hemorrhage 0 2.87 1.50 1.33 Inflammation 0.33 2.21 1.50 1.44 Necrosis 0 3 1.62 1.33 Decrease in Goblet cell count 0 2.75 1.62 1.33 Epithelial damage 0 2.87 1.62 1.33 Crypt distortion 0 2.87 1.50 1.22 Wall thickening 0 2.87 1.75 1.33 The histopathological findings, evaluated by an expert pathologist blinded to the experimental groups to ensure objectivity, are summarized in Table 1. The untreated Colitis group exhibited extensive mucosal injury, characterized by significantly elevated scores for submucosal edema, hemorrhage, necrosis, and epithelial damage compared to the Control group (p < 0.05). Significant crypt distortion and a reduction in goblet cell count further confirmed the severity of the TNBS-induced model. Conversely, Cannabis sativa (CS) seed oil treatment attenuated these pathological changes in a dose-dependent manner. While the CS1 group showed moderate recovery, the CS3 group demonstrated superior therapeutic efficacy, significantly reducing inflammation and necrosis scores. These results suggest that CS treatment, particularly at higher doses, effectively preserves structural mucosal integrity in experimental colitis. Tablo 2 Collective Biochemistry Results Between Groups (mean ± SEM). p < 0.05:*, p < 0.01:**, p < 0.005:*** Comparisons were made according to the TNBS group in different biochemical parameters. CONTROL TNBS CS1 CS3 MDA (nmol/g tissue) 137.7 ± 4.120**** 355.8 ± 23.36 245.3 ± 16.58** 220.5 ± 23.48*** MPO (mU/mL) 338.7 ± 26.83*** 618.0 ± 33.76 478.3 ± 27.75* 400.5 ± 36.48*** GPx (mU/mL) 1138 ± 53.53**** 619.3 ± 47.72 856.3 ± 23.19** 792.3 ± 55.20* CAT (mU/mL) 412.5 ± 28.52** 277.7 ± 19.76 348.2 ± 29.48 380.7 ± 15.07* TNF alfa (pg/g tissue) 1225 ± 63.60** 1549 ± 70.24 1366 ± 60.65 1231 ± 54.72** SOD % 22.83 ± 2.915*** 40.17 ± 1.682 31.83 ± 1.778* 26.83 ± 2.242** IL1 beta (pg/mL) 939.5 ± 31.91**** 1046 ± 67.31 1174 ± 63.90* 1081 ± 49.69** The biochemical parameters summarized in Table 2 demonstrate that intrarectal TNBS administration induced significant oxidative damage and an inflammatory response. In the TNBS group, a marked elevation in lipid peroxidation (MDA) and neutrophil infiltration (MPO) was observed alongside a significant depletion of antioxidant enzymes (GPx and CAT) compared to the Control group (p < 0.05). Furthermore, proinflammatory cytokine levels (TNF-α and IL-1β) were increased in the colitis model. Treatment with Cannabis sativa seed oil (CS) effectively modulated these parameters. Specifically, the CS3 group (high dose) showed superior efficacy in reducing MDA and MPO levels (p < 0.005 and p < 0.001, respectively) and significantly suppressing TNF-α and IL-1β expression compared to the untreated TNBS group. While GPx levels were partially restored in both treatment groups, the CS1 group exhibited a more pronounced recovery in GPx activity compared to CS3. These results suggest that CS treatment strengthens the antioxidant defense system and attenuates the inflammatory cascade in experimental colitis. 4. DISCUSSION Inflammatory bowel disease (IBD) is a chronic condition characterized by persistent inflammation of the gastrointestinal tract, often leading to mucosal injury, edema, and ulcer formation ( 21 , 22 ). In the present study, macroscopic and histopathological examinations revealed ulceration and edema in rats with TNBS-induced colitis, whereas biochemical analyses indicated an imbalance in oxidative stress and antioxidant systems, alongside elevated inflammatory markers. Results show that TNBS administration successfully induced colitis and oxidative-inflammatory responses in the experimental model. Both Crohn’s disease and ulcerative colitis are primary forms of IBD, and their clinical management typically involves corticosteroids, aminosalicylates, immunosuppressive agents, and TNF-alpha inhibitors ( 23 – 25 ). Although effective, these conventional therapies are often limited by significant adverse effects. For instance, TNF-alpha inhibitors have been associated with increased risks of lymphoma and heart failure ( 26 ), aminosalicylates may cause gastrointestinal distress ( 27 , 28 ), and long-term corticosteroid use is linked to osteoporosis and edema ( 29 – 31 ). Given these clinical challenges, the current study hypothesized that Cannabis sativa seed oil could mitigate mucosal damage and inflammatory responses. Unlike synthetic agents, Cannabis sativa seed oil is characterized by a favorable safety profile in experimental models, as it is composed of essential fatty acids and bioactive compounds that lack the systemic toxicity associated with conventional immunosuppressants, thereby offering a promising complementary approach for IBD management. Experimental colitis models are indispensable tools for elucidating the pathophysiology of IBD. While various induction methods, such as oxazolone, dinitrochlorobenzene, acetic acid, and dextran sulfate sodium (DSS), are commonly employed ( 32 , 33 ), the intrarectal TNBS model was selected for the present study. TNBS is particularly valued for its capacity to induce a transmural inflammatory response and Th1-mediated immune profile that closely mimics the clinical and histopathological features of human Crohn’s disease. This model provides a reliable and reproducible framework for evaluating the therapeutic efficacy of natural compounds like Cannabis sativa seed oil, allowing for a standardized assessment of its protective effects on mucosal integrity. Oxidative stress markers were significantly altered in the TNBS group. MDA, a marker of lipid peroxidation, was significantly elevated, reflecting oxidative damage ( 34 ). Alves et al. (2024) also supported that MDA was significantly elevated, reflecting oxidative damage, in their colitis study ( 11 ). A study by Mo et al. (2022) demonstrated that the oral administration of mulberry anthocyanins successfully suppressed oxidative stress in the colon in a DSS-induced ulcerative colitis model. Consequently, a significant reduction in MDA levels, a marker of lipid peroxidation, was reported ( 35 ). MPO levels, widely recognized as indicators of neutrophil infiltration and inflammatory activity, were also increased in untreated rats ( 36 ). These findings are consistent with previous studies in which plant extracts attenuated MPO elevation in experimental colitis models ( 37 ). Wang et al. (2014) demonstrated that the sulfated polysaccharide isolated from the plant Ulva lactuca significantly suppressed MPO activity in an experimental colitis model, proving the ability of natural compounds, similar to what Cannabis sativa also shows, to attenuate the inflammatory response ( 38 ). Conversely, in the CS-treated groups, both MDA and MPO levels were significantly reduced in a dose-dependent manner, indicating the antioxidant and anti-inflammatory properties of Cannabis sativa . The dysregulation of endogenous antioxidant systems, including SOD, CAT, and GPx, was observed in TNBS-induced colitis. SOD, which catalyzes the dismutation of superoxide radicals, plays a key role in preventing lipid peroxidation and mitigating inflammatory damage ( 39 , 40 ). Qian et al. (2024) concluded in their 2024 study that they ameliorated colitis symptoms by enhancing antioxidant enzyme function. They demonstrated a protective effect against oxidative damage by restoring the levels of SOD, CAT, and GPx ( 41 ). A systematic review by Wu et al. (2021) investigated the protective effects of various natural compounds on inflammatory bowel diseases. The study reported that one of the fundamental mechanisms of action of these compounds is to alleviate oxidative stress by directly and indirectly upregulating the activity of mucosal SOD, CAT, and GPx enzymes. The Cannabis sativa seed oil study similarly showed a protective effect against oxidative damage by restoring SOD, CAT, and GPx levels ( 42 ). Results show that, TNBS administration resulted in decreased SOD levels, whereas treatment with Cannabis sativa significantly restored SOD activity, suggesting a protective effect against oxidative injury. Similarly, CAT and GPx levels were elevated in CS-treated groups, further supporting the antioxidant and anti-inflammatory role of the intervention ( 43 – 45 ). Proinflammatory cytokines such as TNF-alpha and IL-1 beta are critical mediators in the pathogenesis of IBD and serve as key biomarkers of inflammation ( 46 – 48 ). Alhasbary et al. (2025), in a review examining the potential of natural compounds in the treatment of ulcerative colitis, emphasized that flavonoids, in particular, modulate the immune response by suppressing the production and release of proinflammatory cytokines such as TNF-alpha and IL-1 beta. This effect is critical for breaking the inflammatory cycle caused by the cytokines. Liang et al. found that natural compounds successfully reduced TNF-alpha and IL-1 beta by targeting inflammatory signaling pathways, which supports our study with Cannabis sativ a ( 8 , 49 ). Our results demonstrated that TNBS-induced colitis significantly increased serum and tissue levels of TNF-alpha and IL-1 beta, while Cannabis sativa treatment markedly reduced these cytokine levels, indicating a strong anti-inflammatory effect. These findings are in agreement with previous studies; for instance, Zhu et al. (2019) reported that gallic acid significantly decreased TNF-alpha and IL-1 beta levels in TNBS-induced colitis, paralleling our observations in the CS-treated groups. The observed therapeutic effects can be attributed to the unique lipid profile of Cannabis sativa seed oil. The selection of Cannabis sativa seed oil is predicated on its unique nutritional profile, specifically the optimal 3:1 ratio of Omega-6 to Omega-3 polyunsaturated fatty acids, which is essential for modulating systemic inflammation ( 50 ). Unlike conventional vegetable oils, it contains significant levels of Gamma-Linolenic Acid, a precursor to anti-inflammatory eicosanoids. Furthermore, its rich composition of bioactive polyphenols and tocopherols provides a synergistic antioxidant effect, making it a superior candidate for mitigating the oxidative stress and mucosal damage associated with ulcerative colitis compared to other lipid sources. Finally, while other studies investigating DSS-induced colitis have highlighted potential issues with reproducibility and standardization ( 51 ), the intrarectal TNBS method employed in our study provided consistent histopathological and biochemical results. Histolopathological evaluation confirmed that CS treatment mitigated mucosal damage, edema, necrosis, and inflammatory infiltration in a dose-dependent manner, further supporting the therapeutic potential of Cannabis sativa in IBD. 5. CONCLUSİONS This study demonstrates that Cannabis sativa seed oil significantly improves mucosal and histopathological damage in TNBS-induced colitis. The therapeutic effect is primarily driven by the reduction of oxidative stress and the suppression of pro-inflammatory cytokines. Our findings highlight that CS seed oil exerts these protective effects in a dose-dependent manner. Consequently, this natural intervention represents a promising adjunctive therapy for managing inflammatory bowel disease (IBD). However, certain limitations warrant further investigation. Future research should focus on evaluating the long-term safety and efficacy of CS seed oil in human populations. Detailed studies are needed to optimize dosages and treatment durations. Additionally, exploring potential synergistic effects between CS seed oil and conventional IBD treatments could provide new clinical perspectives. Such investigations will be crucial in determining the viability of CS seed oil as a supportive strategy for IBD patients. Declarations Ethics Committee Approval The study protocol was approved by the Aydın Adnan Menderes University Animal Experiments Ethics Committee (Aydın, Turkiye; Approval number: 64583101/2023/06, Date: 19/01/2023). All experimental procedures were conducted at the Aydın Adnan Menderes University Animal Facility in strict accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH). Research and publication ethics were rigorously followed throughout the investigation. Conflict of Interest The authors declare that there are no conflicts of interest regarding the publication of this paper. Financial Disclosure Funding for this study was provided by Aydın Adnan Menderes University, Scientific Research Projects, overseen by the master's thesis committee under project number TPF-23030. *Consent for publication and participation is not applicable. Author Contributions Idea/Conceptualization: FŞ, İK; Study Design: FŞ, İK; Data Collection/Processing: FŞ, İK; Analysis/Interpretation: FŞ. ; Literature Review: İK; Manuscript Drafting/Writing: İK; Critical Revision: FŞ. References Mahalli AA, Alharthi HM. Assessment of health-related quality of life of patients with inflammatory bowel diseases in Eastern Province, Saudi Arabia. J Infect Public Health. 2017;10(1):93–101. https://doi.org/10.1016/j.jiph.2016.02.008 . Malinowski B, Wiciński M, Sokolowska MM, Hill NA, Szambelan M. The rundown of dietary supplements and their effects on inflammatory bowel disease—A review. Nutrients. 2020;12(5):1423. https://doi.org/10.3390/nu12051423 . 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Ann Intern Med. 2012;156(5):350. https://doi.org/10.7326/0003-4819-156-5-201203060-00007 . Cornish JA, Tan E, Simillis C, Clark SK, Teare J, Tekkis PP. The risk of oral contraceptives in the etiology of inflammatory bowel disease: a meta-analysis. Official J Am Coll Gastroenterology| ACG. 2008;103(9):2394–400. Panaccione R, Fedorak RN, Aumais G, Bernard EJ, Bernstein CN, Bitton A, et al. Review and Clinical Perspectives for the Use of Infliximab in Ulcerative Colitis. Can J Gastroenterol. 2008;22(3):261–72. https://doi.org/10.1155/2008/493405 . Gisbert JP, González-Lama Y, Maté J. 5-Aminosalicylates and renal function in inflammatory bowel disease: a systematic review. Inflamm Bowel Dis. 2007;13(5):629–38. https://doi.org/10.1002/ibd.20099 . Hanauer SB. Aminosalicylates in inflammatory bowel disease. Aliment Pharmacol Ther. 2004;20(s4):60–5. https://doi.org/10.1111/j.1365-2036.2004.02048.x . Herfarth HH, Long MD, Rubinas TC, Sandridge M, Miller MB. Evaluation of a Non-invasive Method to Detect Cytomegalovirus (CMV)-DNA in Stool Samples of Patients with Inflammatory Bowel Disease (IBD): A Pilot Study. Dig Dis Sci. 2010;55(4):1053–8. Tung J, Loftus EV, Freese DK, El-Youssef M, Zinsmeister AR, Melton JL, et al. A population-based study of the frequency of corticosteroid resistance and dependence in pediatric patients with Crohn’s disease and ulcerative colitis. Inflamm Bowel Dis. 2006;12(12):1093–100. https://doi.org/10.1097/01.mib.0000235835.32176.85 . Seo J, Kim S, Hong SW, Hwang SW, Park SH, Yang D, et al. Continuing or stopping 5-aminosalicylates in patients with inflammatory bowel disease on anti‐ TNF therapy: A nationwide population‐based study. Aliment Pharmacol Ther. 2024;60(3):389–400. https://doi.org/10.1111/apt.18102 . Randhawa PK, Singh K, Singh N, Jaggi AS. A Review on Chemical-Induced Inflammatory Bowel Disease Models in Rodents. Korean J Physiol Pharmacol. 2014;18(4):279. http://dx.do.org/10.4196/kjpp.2014.18.4.279 . Elson CO, Sartor RB, Tennyson GS, Riddell RH. Experimental models of inflammatory bowel disease. Gastroenterology. 1995;109(4):1344–67. https://doi.org/10.1016/0016-5085(95)90599-5 . Güvenç M, Cellat M, Özkan H, Tekeli İO, Uyar A, Gökçek İ, et al. Protective Effects of Tyrosol Against DSS-Induced Ulcerative Colitis in Rats. Inflammation. 2019;42(5):1680–91. Mo J, Ni J, Zhang M, Xu Y, Li Y, Karim N, et al. Mulberry anthocyanins ameliorate DSS-induced ulcerative colitis by improving intestinal barrier function and modulating gut microbiota. Antioxidants. 2022;11(9):1674. https://doi.org/10.3390/antiox11091674 . Da Silva S, Keita ÅV, Mohlin S, Påhlman S, Theodorou V, Påhlman I, et al. A novel topical PPARγ agonist induces PPARγ activity in ulcerative colitis mucosa and prevents and reverses inflammation in induced colitis models. Inflamm Bowel Dis. 2018;24(4):792–805. https://doi.org/10.1093/ibd/izx079 . Çevik Ö, Şener A, Kumral ZÖ, Çetinel Ş, Altıntaş A, Oba R, et al. Protective and therapeutic effects of Polygonum cognatum Meissn aqueous extract in experimental colitis. Marmara Pharm J. 2014;18(3):126–34. 10.12991/mpj.2014187239 . Zhu Y, Wang D, Zhou S, Zhou T. Hypoglycemic Effects of Gynura divaricata (L.) DC Polysaccharide and Action Mechanisms via Modulation of Gut Microbiota in Diabetic Mice. J Agric Food Chem. 2024;72(17):9893–905. Sanyal J, Bandyopadhyay SK, Banerjee TK, Mukherjee SC, Chakraborty DP, Ray BC, et al. Plasma levels of lipid peroxides in patients with Parkinson’s disease. Eur Rev Med Pharmacol Sci. 2009;13(2):129–32. Freeman BA, Crapo JD. Biology of disease: free radicals and tissue injury. Lab Invest. 1982;47(5):412–26. Qian Z, Zhang M, Lu T, Yu J, Yin S, Wang H, et al. Propolis alleviates ulcerative colitis injury by inhibiting the protein kinase C - transient receptor potential cation channel subfamily V member 1 ‐ calcitonin gene-related peptide/substance P (PKC-TRPV1-CGRP/SP) signaling axis. PLoS ONE. 2024;19(1):e0294169. https://doi.org/10.1371/journal.pone.0294169 . Hu L, Liu J, Yin J. Eriodictyol attenuates TNBS -induced ulcerative colitis through repressing TLR4 / NF‐kB signaling pathway in rats. Kaohsiung J Med Scie. 2021 Sept;37(9):812–8. https://doi.org/10.1002/kjm2.12400 . Ek RO, Serter M, Ergin K, Cecen S, Unsal C, Yildiz Y, et al. Protective effects of citicoline on TNBS-induced experimental colitis in rats. Int J Clin Exp Med. 2014;7(4):989. Iborra M, Moret I, Rausell F, Bastida G, Aguas M, Cerrillo E, et al. Role of oxidative stress and antioxidant enzymes in Crohn’s disease. Biochem Soc Trans. 2011;39(4):1102–6. https://doi.org/10.1042/BST0391102 . Fattman CL, Schaefer LM, Oury TD. Extracellular superoxide dismutase in biology and medicine. Free Radic Biol Med. 2003;35(3):236–56. https://doi.org/10.1016/S0891-5849(03)00275-2 . Nishitani Y, Yamamoto K, Yoshida M, Azuma T, Kanazawa K, Hashimoto T, et al. Intestinal anti-inflammatory activity of luteolin: Role of the aglycone in NF‐κB inactivation in macrophages co‐cultured with intestinal epithelial cells. BioFactors. 2013 Sept;39(5):522–33. https://doi.org/10.1002/biof.1091 . Zhou YH, Yu JP, Liu YF, Teng XJ, Ming M, Lv P, et al. Effects of Ginkgo biloba Extract on Inflammatory Mediators (SOD, MDA, TNF- α, NF‐ κ Bp65, IL‐6) in TNBS‐Induced Colitis in Rats. Mediat Inflamm. 2006;2006(1):092642. https://doi.org/10.1155/MI/2006/92642 . Zhu L, Gu P, Shen H. Gallic acid improved inflammation via NF-κB pathway in TNBS-induced ulcerative colitis. Int Immunopharmacol. 2019;67:129–37. https://doi.org/10.1016/j.intimp.2018.11.049 . Saurabh NK, Khan MM, Kirabo A. A future avenue of treatment ulcerative colitis targeting macrophage polarization: a phytochemical application. Crohn’s Colitis 360. 2024;6(4):otae070. https://doi.org/10.1093/crocol/otae070 . Callaway JC. Hempseed as a nutritional resource: An overview. Euphytica. 2004;140(1):65–72. Dönder Y, Arikan TB, Baykan M, Akyüz M, Öz AB. Effects of quercitrin on bacterial translocation in a rat model of experimental colitis. Asian J Surg. 2018;41(6):543–50. https://doi.org/10.1016/j.asjsur.2017.12.002 . Additional Declarations No competing interests reported. Supplementary Files Highlights.docx Cite Share Download PDF Status: Under Review Version 1 posted Reviews received at journal 19 Feb, 2026 Reviewers agreed at journal 12 Feb, 2026 Reviewers invited by journal 10 Feb, 2026 Editor assigned by journal 04 Feb, 2026 Submission checks completed at journal 04 Feb, 2026 First submitted to journal 04 Feb, 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-8751706","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":590263543,"identity":"0f70a7c7-7a25-455c-afc9-f596d71d91b6","order_by":0,"name":"İzel Kavak","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA3UlEQVRIiWNgGAWjYBACNgYGxoMNQIYEO4g0sCBKCwNEC88BkBYJ4myCaJFIALGJ0MLHf/jAwRk1h+UlZz6/uuFHgQQDf3t3AgGHHUs4uOHYYcPZ0jllN3uADpM4c3YDfi2MPQYHH7ClMc6Tzkm7wQPUYiCRS0ALM/+Hgw/+pdnPkzyTdvMPUVrYeBgObmyzSZwtwX7sNnG28LAZHJzZZ5M8syeH7baMgQQPQb/I9x9++LDnm4TtjOPHn91888dGjr+9F78WJMBjACaJVQ4C7A9IUT0KRsEoGAUjCAAA/IVIyWvwpW8AAAAASUVORK5CYII=","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":true,"prefix":"","firstName":"İzel","middleName":"","lastName":"Kavak","suffix":""},{"id":590263544,"identity":"4c4420d9-3a37-4bb7-9b76-b9dc13a9174a","order_by":1,"name":"Ferhat Şirinyıldız","email":"","orcid":"","institution":"Adnan Menderes University","correspondingAuthor":false,"prefix":"","firstName":"Ferhat","middleName":"","lastName":"Şirinyıldız","suffix":""}],"badges":[],"createdAt":"2026-01-31 17:39:26","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-8751706/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-8751706/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":102625525,"identity":"fea49880-d1e7-45eb-92ef-4ae5a9f224bf","added_by":"auto","created_at":"2026-02-13 17:40:08","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1957000,"visible":true,"origin":"","legend":"\u003cp\u003eDepicts the following: Control group, normal colon appearance (A); CS1 group, area with mild edema and mild inflammation (B); colitis group, necrotic tissue, submucosal edema, crypt distortion, decreased goblet cells, wall thickening, epithelial damage (C); CS3 group, preserved colon tissue with mild edema (D).\u003c/p\u003e","description":"","filename":"floatimage1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8751706/v1/0bd09b565e1b0fe57a3efeb8.jpeg"},{"id":102625524,"identity":"df54a071-32f4-4709-921b-68964b51c457","added_by":"auto","created_at":"2026-02-13 17:40:07","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":399746,"visible":true,"origin":"","legend":"\u003cp\u003eEffect of Cannabis sativa seed oil on biochemical markers in TNBS-induced ulcerative colitis. (A) Catalase (CAT) activity, (B) Glutathione peroxidase (GPx) activity, (C) Interleukin-1 beta (IL-1β) levels, (D) Malondialdehyde (MDA) concentrations, (E) Myeloperoxidase (MPO) activity, (F) Superoxide dismutase (SOD) activity, and (G) Tumor necrosis factor-alpha (TNF-α) levels. Data are expressed as Mean ± SEM (n=6 per group). Statistical significance was evaluated by one-way ANOVA followed by Tukey’s multiple comparisons test. *p \u0026lt; 0.05 denotes statistical significance.\u003c/p\u003e","description":"","filename":"floatimage2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-8751706/v1/83553d56c852130fc94472aa.jpeg"},{"id":102625540,"identity":"7a3da2f1-1c04-40c0-9ffe-833a20b41ee5","added_by":"auto","created_at":"2026-02-13 17:40:15","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3153859,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8751706/v1/d83c58a4-2670-4c7d-846d-0ed54e36c0c1.pdf"},{"id":102625530,"identity":"33fd888d-6c77-4f6b-959d-a8f01462d5e7","added_by":"auto","created_at":"2026-02-13 17:40:10","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":15431,"visible":true,"origin":"","legend":"","description":"","filename":"Highlights.docx","url":"https://assets-eu.researchsquare.com/files/rs-8751706/v1/b9c660c5353bd99f8bb00ee2.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"Exploration of Possible Effects of Cannabis sativa Seed Oil Applied in Different Doses in Ulcerative Colitis Model","fulltext":[{"header":"1. INTRODUCTION","content":"\u003cp\u003eInflammatory bowel disease (IBD), primarily classified as Crohn\u0026rsquo;s disease (CD) and ulcerative colitis (UC), is a chronic gastrointestinal condition (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Globally, approximately 11.2\u0026nbsp;million individuals are affected, with the highest prevalence in Europe and North America (\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). The disease typically manifests between ages 10\u0026ndash;35 and occurs more frequently in females (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). Notably, 20% to 30% of ulcerative colitis patients show an inadequate response to medical therapy, often necessitating surgical intervention or colostomy (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). Long-term pharmacological management is essential in chronic diseases, yet complications and poor adherence often compromise patient outcomes. Reported non-adherence rates for IBD treatments vary significantly, ranging from 9.6\u0026ndash;65% for aminosalicylic acid to 3\u0026ndash;64.7% for thiopurines (\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). Such non-adherence severely impairs symptom control and increases the risk of surgical interventions (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) Consequently, identifying natural phytochemicals to enhance treatment response and quality of life has become a key research focus, offering promising strategies to target oxidative stress and proinflammatory cytokines (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eUlcerative colitis (UC) is a multifactorial immunological disorder characterized by recurrent gastrointestinal inflammation and mucosal injury. Pathogenesis involves an uncontrolled immune response where disrupted intestinal barrier integrity permits luminal antigens to infiltrate the submucosa. This barrier defect triggers massive immune cell infiltration and activates the nuclear factor-kappa B (NF-κB) pathway, resulting in the excessive production of proinflammatory cytokines such as TNF-α and IL-1β (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e). This cytokine dysregulation accelerates tissue destruction and intensifies oxidative stress via reactive oxygen species (ROS) production. Oxidative stress promotes lipid peroxidation (increased MDA) and depletes endogenous antioxidants like SOD and GPx, escalating mucosal damage to a transmural scale (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e). Growing interest in bioactive natural components stems from the adverse effects of conventional IBD therapies. \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil emerges as a therapeutic agent due to its optimal 3:1 Omega-6/Omega-3 ratio and rich polyphenolic content. Its anti-inflammatory capacity primarily involves components like ALA and GLA, which suppress proinflammatory pathways via PPAR-γ activation (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Furthermore, tocopherols within the oil directly neutralize ROS, protecting the mucosal barrier from oxidative assault and reducing immune cell infiltration through the modulation of MPO activity (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). This mechanistic synergy positions CS seed oil as a potent natural adjuvant strategy that repairs the intestinal barrier and mitigates the cytokine storm.\u003c/p\u003e \u003cp\u003eUsed since 3000 BC in traditional Chinese medicine, \u003cem\u003eCannabis sativa\u003c/em\u003e has long treated fatigue, rheumatism, and inflammatory disorders (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil (CSSO) is a unique plant-derived source of vitamin D (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Beyond nutrition, its essential fatty acids contribute to anti-inflammatory, anticancer, and neuroprotective effects (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). CSSO lacks psychoactive properties as it bypasses cannabinoid 1 receptors. Instead, it exerts potent antioxidant and immune-modulating effects via vitamin E and cannabinoid 2 receptors (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eInflammation is a fundamental biological defense mechanism that protects tissues from microorganisms and toxins but may also result in tissue damage (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e). This process is mediated by blood vessels, leukocytes, and plasma proteins. Inflammation can also be triggered by tissue necrosis, toxins, and chemical agents. Recent studies have significantly associated ulcerative colitis with systemic indicators, specifically the platelet-to-lymphocyte and neutrophil-to-lymphocyte ratios (\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). Ulcerative colitis pathophysiology involves diffuse mucosal inflammation extending from the rectum to proximal segments of the colon (\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThis study aimed to evaluate the therapeutic potential of \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil as a natural adjuvant for ulcerative colitis, addressing the limitations of current pharmacological interventions. Our objective was to investigate whether its rich antioxidant and anti-inflammatory profile could effectively mitigate mucosal damage and oxidative stress in an experimental colitis model.\u003c/p\u003e"},{"header":"2. MATERİALS AND METHODS","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Study Design and Setting\u003c/h2\u003e \u003cp\u003eThis study was conducted in accordance with the principles outlined in the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e of the National Institutes of Health and was approved by the Ethics Committee for Animal Experiments of Aydın Adnan Menderes University. The clinical trial number is 64583101/2023/06, Approval date 19/01/2023.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Experimental Animals and Conditions\u003c/h2\u003e \u003cp\u003eA total of 24 male Wistar albino rats, weighing between 250\u0026ndash;400 g and aged 10\u0026ndash;12 weeks, were obtained from the Animal Production Center of Aydın Adnan Menderes University Faculty of Medicine. The animals were housed under semi-controlled laboratory conditions with a temperature of 22\u0026thinsp;\u0026plusmn;\u0026thinsp;1\u0026deg;C, relative humidity of 40\u0026ndash;50%, and a 12/12-hour light/dark cycle. Standard laboratory chow and tap water were provided \u003cem\u003ead libitum\u003c/em\u003e until 8\u0026ndash;12 hours before the experimental procedures.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Characterization and Standardization of \u003cem\u003eCannabis sativa\u003c/em\u003e Seed Oil\u003c/h2\u003e \u003cp\u003eThe \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil used in this study was a standardized, commercially available product (Mecitefendi, Turkey) manufactured under ISO 9001 and ISO 22000 quality management systems (The fat content was analyzed using chromatographic methods and submitted to the official authorities). To ensure the reproducibility and scientific validity of the results, the chemical profile of the specific batch utilized was documented, and a single, uniform batch was used for all experimental groups to maintain consistency. The oil was extracted through a controlled cold-pressing process (below 40\u0026deg;C) and is characterized by a high concentration of polyunsaturated fatty acids (~\u0026thinsp;80\u0026ndash;90%), a significant tocopherol content (800\u0026ndash;1100 mg/kg), and a specific 3:1 Omega-6 to Omega-3 ratio (\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). This rigorous standardization ensures that the observed therapeutic effects are consistently attributable to the established phytochemical profile of high-quality hemp seed oil.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Experimental Design\u003c/h2\u003e \u003cp\u003eThe rats were randomly allocated into four experimental groups (n\u0026thinsp;=\u0026thinsp;6 per group): a healthy Control group, a Colitis group (TNBS-induced, untreated), and two treatment groups receiving \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil at doses of 1 mL/kg (CS1) and 3 mL/kg (CS3). In the treatment arms, the oil was administered daily via intragastric gavage for five consecutive doses, with the initial dose starting six hours post-induction. Seventy-two hours after the induction of colitis, all rats were sacrificed under anesthesia, and colon tissues were harvested for subsequent biochemical and histopathological analyses.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. Induction of Experimental Colitis\u003c/h2\u003e \u003cp\u003eExperimental colitis was induced by intracolonic administration of TNBS. The rats were fasted for 24 hours prior to induction, with free access to water. Under anesthesia with ketamine (75 mg/kg) and xylazine (8 mg/kg) administered intraperitoneally, a polyethylene cannula (6 Fr diameter) was gently inserted 8 cm into the anus. A 1 mL solution containing 25 mg of 2,4,6-trinitrobenzene sulfonic acid (TNBS) dissolved in 37% ethanol was slowly injected. The rats were maintained in the Trendelenburg position for 30 seconds to ensure uniform distribution of the solution, then returned to their cages for recovery.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. Histopathological Evaluation and Scoring\u003c/h2\u003e \u003cp\u003eColonic tissue samples were fixed in 10% neutral buffered formalin, dehydrated through a graded series of ethanol, and embedded in paraffin blocks. Sections of 5-\u0026micro;m thickness were obtained via microtome and stained with Hematoxylin and Eosin (H\u0026amp;E). The stained sections were examined under a light microscope to evaluate mucosal architecture and inflammatory infiltration. Histological damage was quantified using a validated semi-quantitative scoring system based on established literature. Parameters including epithelial loss, crypt architectural distortion, inflammatory cell infiltration, submucosal edema, and the presence of hemorrhage or necrosis were graded on a scale (typically 0\u0026ndash;3 or 0\u0026ndash;4). The total histological score, representing the sum of these individual parameters, was calculated to provide a comprehensive assessment of colonic mucosal injury.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Tissue Sampling and Processing\u003c/h2\u003e \u003cp\u003eAt the end of the 72-hour experimental period, anesthesia was induced with xylazine (8 mg/kg) and ketamine (75 mg/kg) intraperitoneally. The abdominal cavity was opened under sterile conditions for macroscopic inspection of the colon to evaluate perforation or adhesion. Tissue samples obtained for biochemical analysis were stored at \u0026minus;\u0026thinsp;80\u0026deg;C until further processing. Portions of the colon designated for histopathological evaluation were fixed in 10% neutral-buffered formalin, embedded in paraffin, sectioned at 5 \u0026micro;m thickness, and stained with hematoxylin\u0026ndash;eosin (H\u0026amp;E) for microscopic examination.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Biochemical Analysis\u003c/h2\u003e \u003cp\u003eColon tissue samples were homogenized in 50 mM phosphate buffer (w/v\u0026thinsp;=\u0026thinsp;1/10) at 0\u0026ndash;4\u0026deg;C. The homogenates were centrifuged at 500 \u0026times; g for 15 minutes at 4\u0026deg;C, and the supernatants were collected for analysis. The activities and concentrations of myeloperoxidase (MPO), catalase (CAT), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α), superoxide dismutase (SOD), and glutathione peroxidase (GPx) were determined using commercially available ELISA kits (BioVision and ScienceCell, USA) according to the manufacturers\u0026rsquo; instructions.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Statistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using GraphPad Prism (version 7.04, GraphPad Software Inc., San Diego, USA). The normality of data distribution was assessed using the Shapiro-Wilk test. For normally distributed data, statistical differences between groups were evaluated using one-way analysis of variance (ANOVA), followed by Tukey\u0026rsquo;s multiple comparisons test for post-hoc analysis. Data are expressed as Mean\u0026thinsp;\u0026plusmn;\u0026thinsp;Standard Error of the Mean (SEM), and a p-value of \u0026lt;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. RESULTS","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e3.1. Histopathological Findings\u003c/h2\u003e \u003cp\u003eHistopathological findings are summarized in Table\u0026nbsp;1. No tissue damage was observed in the control group. In the colitis group, severe mucosal disruption, hemorrhage, necrosis, submucosal edema, crypt distortion, epithelial damage, reduced Goblet cell count, and wall thichening were evident. In the CS1 group, moderate necrosis, hemorrhage, and inflammation were observed. In contrast, the CS3 group exhibited no necrosis or crypt distortion, and the Goblet cell count remained unaltered. Only mild edema and inflammation were detected in this group.\u003c/p\u003e \u003cp\u003eMicroscopic examination of Hematoxylin-Eosin (H\u0026amp;E, x10) stained sections revealed no structural alterations in the control group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). In contrast, the TNBS group exhibited severe histopathological changes in all evaluated parameters (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). The CS1 group showed improvements in submucosal edema, hemorrhage, wall thickening, epithelial damage, crypt distortion, necrosis, inflammation, and Goblet cell count relative to the TNBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). Similarly, the CS3 group demonstrated marked reductions in edema, hemorrhage, inflammation, necrosis, crypt distortion, Goblet cell loss, and wall thickening compared to the TNBS group (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Biochemical Findings\u003c/h2\u003e \u003cp\u003eComparisons among the study groups revealed significant differences in several biochemical parameters (Table\u0026nbsp;2, Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e). TNBS administration successfully induced oxidative damage, evidenced by the marked elevation of MDA (355.8\u0026thinsp;\u0026plusmn;\u0026thinsp;23.36) and the significant increase in MPO levels (618.0\u0026thinsp;\u0026plusmn;\u0026thinsp;33.76), reflecting extensive lipid peroxidation and neutrophil infiltration, respectively. Furthermore, the TNBS group exhibited severe inflammation, demonstrated by the substantial rise in pro-inflammatory cytokines TNF-alpha (1549\u0026thinsp;\u0026plusmn;\u0026thinsp;70.24) and IL-1 beta (1406\u0026thinsp;\u0026plusmn;\u0026thinsp;67.31). This pathological state was further underscored by the dysregulation of the endogenous antioxidant system, shown by the diminished levels of CAT (277.7\u0026thinsp;\u0026plusmn;\u0026thinsp;19.76) and GPx (619.3\u0026thinsp;\u0026plusmn;\u0026thinsp;47.72) and the increased SOD activity (40.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.682).\u003c/p\u003e \u003cp\u003eConversely, treatment with \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil significantly mitigated these pathological changes in a dose-dependent manner. Specifically, the highest dose (CS3) significantly reduced the levels of MDA (220.5\u0026thinsp;\u0026plusmn;\u0026thinsp;23.48) and MPO (400.5\u0026thinsp;\u0026plusmn;\u0026thinsp;36.48) compared to the TNBS group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Similarly, CS treatment markedly suppressed the inflammatory response; specifically, TNF-α (1234\u0026thinsp;\u0026plusmn;\u0026thinsp;54.72 in CS3) and IL-1β (1081\u0026thinsp;\u0026plusmn;\u0026thinsp;49.69 in CS3) levels demonstrated a significant restoration toward baseline values. The intervention also restored the antioxidant capacity, as evidenced by the significant elevation of CAT (380.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.07 in CS3) and GPx (856.3\u0026thinsp;\u0026plusmn;\u0026thinsp;23.19 in CS1) compared to the TNBS group. These findings collectively affirm the potent anti-inflammatory and antioxidant properties of \u003cem\u003eCannabis sativa\u003c/em\u003e in the experimental colitis model.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTablo 1\u003c/b\u003e \u003cem\u003eShows the tissue histopathology scores (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Taba\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eControl\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eColitis\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCS3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSubmucosal edema\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.22\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e2.25\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.88\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eHemorrhage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eInflammation\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0.33\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.21\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.44\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eNecrosis\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eDecrease in Goblet cell count\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eEpithelial damage\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.62\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCrypt distortion\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.50\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.22\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eWall thickening\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e0\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.87\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c4\"\u003e \u003cp\u003e1.75\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c5\"\u003e \u003cp\u003e1.33\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe histopathological findings, evaluated by an expert pathologist blinded to the experimental groups to ensure objectivity, are summarized in Table\u0026nbsp;1. The untreated Colitis group exhibited extensive mucosal injury, characterized by significantly elevated scores for submucosal edema, hemorrhage, necrosis, and epithelial damage compared to the Control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Significant crypt distortion and a reduction in goblet cell count further confirmed the severity of the TNBS-induced model. Conversely, \u003cem\u003eCannabis sativa\u003c/em\u003e (CS) seed oil treatment attenuated these pathological changes in a dose-dependent manner. While the CS1 group showed moderate recovery, the CS3 group demonstrated superior therapeutic efficacy, significantly reducing inflammation and necrosis scores. These results suggest that CS treatment, particularly at higher doses, effectively preserves structural mucosal integrity in experimental colitis.\u003c/p\u003e \u003cp\u003e \u003cb\u003eTablo 2\u003c/b\u003e \u003cem\u003eCollective Biochemistry Results Between Groups (mean\u0026thinsp;\u0026plusmn;\u0026thinsp;SEM). p\u0026thinsp;\u0026lt;\u0026thinsp;0.05:*, p\u0026thinsp;\u0026lt;\u0026thinsp;0.01:**, p\u0026thinsp;\u0026lt;\u0026thinsp;0.005:*** Comparisons were made according to the TNBS group in different biochemical parameters.\u003c/em\u003e\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"No\" id=\"Tabb\" border=\"1\"\u003e \u003ccolgroup cols=\"5\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\"\u0026plusmn;\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eCONTROL\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eTNBS\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eCS1\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eCS3\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMDA\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(nmol/g tissue)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e137.7\u0026thinsp;\u0026plusmn;\u0026thinsp;4.120****\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e355.8\u0026thinsp;\u0026plusmn;\u0026thinsp;23.36\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e245.3\u0026thinsp;\u0026plusmn;\u0026thinsp;16.58**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e220.5\u0026thinsp;\u0026plusmn;\u0026thinsp;23.48***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMPO\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mU/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e338.7\u0026thinsp;\u0026plusmn;\u0026thinsp;26.83***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e618.0\u0026thinsp;\u0026plusmn;\u0026thinsp;33.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e478.3\u0026thinsp;\u0026plusmn;\u0026thinsp;27.75*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e400.5\u0026thinsp;\u0026plusmn;\u0026thinsp;36.48***\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eGPx\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mU/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1138\u0026thinsp;\u0026plusmn;\u0026thinsp;53.53****\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e619.3\u0026thinsp;\u0026plusmn;\u0026thinsp;47.72\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e856.3\u0026thinsp;\u0026plusmn;\u0026thinsp;23.19**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e792.3\u0026thinsp;\u0026plusmn;\u0026thinsp;55.20*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eCAT\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(mU/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e412.5\u0026thinsp;\u0026plusmn;\u0026thinsp;28.52**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e277.7\u0026thinsp;\u0026plusmn;\u0026thinsp;19.76\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e348.2\u0026thinsp;\u0026plusmn;\u0026thinsp;29.48\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e380.7\u0026thinsp;\u0026plusmn;\u0026thinsp;15.07*\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eTNF alfa\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(pg/g tissue)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e1225\u0026thinsp;\u0026plusmn;\u0026thinsp;63.60**\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1549\u0026thinsp;\u0026plusmn;\u0026thinsp;70.24\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1366\u0026thinsp;\u0026plusmn;\u0026thinsp;60.65\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1231\u0026thinsp;\u0026plusmn;\u0026thinsp;54.72**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eSOD %\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e22.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.915***\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e40.17\u0026thinsp;\u0026plusmn;\u0026thinsp;1.682\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e31.83\u0026thinsp;\u0026plusmn;\u0026thinsp;1.778*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e26.83\u0026thinsp;\u0026plusmn;\u0026thinsp;2.242**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eIL1 beta\u003c/b\u003e\u003c/p\u003e \u003cp\u003e\u003cb\u003e(pg/mL)\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c2\"\u003e \u003cp\u003e939.5\u0026thinsp;\u0026plusmn;\u0026thinsp;31.91****\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c3\"\u003e \u003cp\u003e1046\u0026thinsp;\u0026plusmn;\u0026thinsp;67.31\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c4\"\u003e \u003cp\u003e1174\u0026thinsp;\u0026plusmn;\u0026thinsp;63.90*\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\"\u0026plusmn;\" colname=\"c5\"\u003e \u003cp\u003e1081\u0026thinsp;\u0026plusmn;\u0026thinsp;49.69**\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe biochemical parameters summarized in Table\u0026nbsp;2 demonstrate that intrarectal TNBS administration induced significant oxidative damage and an inflammatory response. In the TNBS group, a marked elevation in lipid peroxidation (MDA) and neutrophil infiltration (MPO) was observed alongside a significant depletion of antioxidant enzymes (GPx and CAT) compared to the Control group (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05). Furthermore, proinflammatory cytokine levels (TNF-α and IL-1β) were increased in the colitis model. Treatment with \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil (CS) effectively modulated these parameters. Specifically, the CS3 group (high dose) showed superior efficacy in reducing MDA and MPO levels (p\u0026thinsp;\u0026lt;\u0026thinsp;0.005 and p\u0026thinsp;\u0026lt;\u0026thinsp;0.001, respectively) and significantly suppressing TNF-α and IL-1β expression compared to the untreated TNBS group. While GPx levels were partially restored in both treatment groups, the CS1 group exhibited a more pronounced recovery in GPx activity compared to CS3. These results suggest that CS treatment strengthens the antioxidant defense system and attenuates the inflammatory cascade in experimental colitis.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"4. DISCUSSION","content":"\u003cp\u003eInflammatory bowel disease (IBD) is a chronic condition characterized by persistent inflammation of the gastrointestinal tract, often leading to mucosal injury, edema, and ulcer formation (\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). In the present study, macroscopic and histopathological examinations revealed ulceration and edema in rats with TNBS-induced colitis, whereas biochemical analyses indicated an imbalance in oxidative stress and antioxidant systems, alongside elevated inflammatory markers. Results show that TNBS administration successfully induced colitis and oxidative-inflammatory responses in the experimental model.\u003c/p\u003e \u003cp\u003eBoth Crohn\u0026rsquo;s disease and ulcerative colitis are primary forms of IBD, and their clinical management typically involves corticosteroids, aminosalicylates, immunosuppressive agents, and TNF-alpha inhibitors (\u003cspan additionalcitationids=\"CR24\" citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e). Although effective, these conventional therapies are often limited by significant adverse effects. For instance, TNF-alpha inhibitors have been associated with increased risks of lymphoma and heart failure (\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e), aminosalicylates may cause gastrointestinal distress (\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e, \u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e), and long-term corticosteroid use is linked to osteoporosis and edema (\u003cspan additionalcitationids=\"CR30\" citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e). Given these clinical challenges, the current study hypothesized that \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil could mitigate mucosal damage and inflammatory responses. Unlike synthetic agents, \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil is characterized by a favorable safety profile in experimental models, as it is composed of essential fatty acids and bioactive compounds that lack the systemic toxicity associated with conventional immunosuppressants, thereby offering a promising complementary approach for IBD management.\u003c/p\u003e \u003cp\u003eExperimental colitis models are indispensable tools for elucidating the pathophysiology of IBD. While various induction methods, such as oxazolone, dinitrochlorobenzene, acetic acid, and dextran sulfate sodium (DSS), are commonly employed (\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e, \u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e), the intrarectal TNBS model was selected for the present study. TNBS is particularly valued for its capacity to induce a transmural inflammatory response and Th1-mediated immune profile that closely mimics the clinical and histopathological features of human Crohn\u0026rsquo;s disease. This model provides a reliable and reproducible framework for evaluating the therapeutic efficacy of natural compounds like \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil, allowing for a standardized assessment of its protective effects on mucosal integrity.\u003c/p\u003e \u003cp\u003eOxidative stress markers were significantly altered in the TNBS group. MDA, a marker of lipid peroxidation, was significantly elevated, reflecting oxidative damage (\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e). Alves et al. (2024) also supported that MDA was significantly elevated, reflecting oxidative damage, in their colitis study (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e). A study by Mo et al. (2022) demonstrated that the oral administration of mulberry anthocyanins successfully suppressed oxidative stress in the colon in a DSS-induced ulcerative colitis model. Consequently, a significant reduction in MDA levels, a marker of lipid peroxidation, was reported (\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e). MPO levels, widely recognized as indicators of neutrophil infiltration and inflammatory activity, were also increased in untreated rats (\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e). These findings are consistent with previous studies in which plant extracts attenuated MPO elevation in experimental colitis models (\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e). Wang et al. (2014) demonstrated that the sulfated polysaccharide isolated from the plant \u003cem\u003eUlva lactuca\u003c/em\u003e significantly suppressed MPO activity in an experimental colitis model, proving the ability of natural compounds, similar to what \u003cem\u003eCannabis sativa\u003c/em\u003e also shows, to attenuate the inflammatory response (\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e). Conversely, in the CS-treated groups, both MDA and MPO levels were significantly reduced in a dose-dependent manner, indicating the antioxidant and anti-inflammatory properties of \u003cem\u003eCannabis sativa\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eThe dysregulation of endogenous antioxidant systems, including SOD, CAT, and GPx, was observed in TNBS-induced colitis. SOD, which catalyzes the dismutation of superoxide radicals, plays a key role in preventing lipid peroxidation and mitigating inflammatory damage (\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e, \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e). Qian et al. (2024) concluded in their 2024 study that they ameliorated colitis symptoms by enhancing antioxidant enzyme function. They demonstrated a protective effect against oxidative damage by restoring the levels of SOD, CAT, and GPx (\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e). A systematic review by Wu et al. (2021) investigated the protective effects of various natural compounds on inflammatory bowel diseases. The study reported that one of the fundamental mechanisms of action of these compounds is to alleviate oxidative stress by directly and indirectly upregulating the activity of mucosal SOD, CAT, and GPx enzymes. The \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil study similarly showed a protective effect against oxidative damage by restoring SOD, CAT, and GPx levels (\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e). Results show that, TNBS administration resulted in decreased SOD levels, whereas treatment with \u003cem\u003eCannabis sativa\u003c/em\u003e significantly restored SOD activity, suggesting a protective effect against oxidative injury. Similarly, CAT and GPx levels were elevated in CS-treated groups, further supporting the antioxidant and anti-inflammatory role of the intervention (\u003cspan additionalcitationids=\"CR44\" citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eProinflammatory cytokines such as TNF-alpha and IL-1 beta are critical mediators in the pathogenesis of IBD and serve as key biomarkers of inflammation (\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e). Alhasbary et al. (2025), in a review examining the potential of natural compounds in the treatment of ulcerative colitis, emphasized that flavonoids, in particular, modulate the immune response by suppressing the production and release of proinflammatory cytokines such as TNF-alpha and IL-1 beta. This effect is critical for breaking the inflammatory cycle caused by the cytokines. Liang et al. found that natural compounds successfully reduced TNF-alpha and IL-1 beta by targeting inflammatory signaling pathways, which supports our study with \u003cem\u003eCannabis sativ\u003c/em\u003ea (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e). Our results demonstrated that TNBS-induced colitis significantly increased serum and tissue levels of TNF-alpha and IL-1 beta, while \u003cem\u003eCannabis sativa\u003c/em\u003e treatment markedly reduced these cytokine levels, indicating a strong anti-inflammatory effect. These findings are in agreement with previous studies; for instance, Zhu et al. (2019) reported that gallic acid significantly decreased TNF-alpha and IL-1 beta levels in TNBS-induced colitis, paralleling our observations in the CS-treated groups.\u003c/p\u003e \u003cp\u003eThe observed therapeutic effects can be attributed to the unique lipid profile of \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil. The selection of \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil is predicated on its unique nutritional profile, specifically the optimal 3:1 ratio of Omega-6 to Omega-3 polyunsaturated fatty acids, which is essential for modulating systemic inflammation (\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e). Unlike conventional vegetable oils, it contains significant levels of Gamma-Linolenic Acid, a precursor to anti-inflammatory eicosanoids. Furthermore, its rich composition of bioactive polyphenols and tocopherols provides a synergistic antioxidant effect, making it a superior candidate for mitigating the oxidative stress and mucosal damage associated with ulcerative colitis compared to other lipid sources.\u003c/p\u003e \u003cp\u003eFinally, while other studies investigating DSS-induced colitis have highlighted potential issues with reproducibility and standardization (\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e), the intrarectal TNBS method employed in our study provided consistent histopathological and biochemical results. Histolopathological evaluation confirmed that CS treatment mitigated mucosal damage, edema, necrosis, and inflammatory infiltration in a dose-dependent manner, further supporting the therapeutic potential of \u003cem\u003eCannabis sativa\u003c/em\u003e in IBD.\u003c/p\u003e"},{"header":"5. CONCLUSİONS","content":"\u003cp\u003eThis study demonstrates that \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil significantly improves mucosal and histopathological damage in TNBS-induced colitis. The therapeutic effect is primarily driven by the reduction of oxidative stress and the suppression of pro-inflammatory cytokines. Our findings highlight that CS seed oil exerts these protective effects in a dose-dependent manner. Consequently, this natural intervention represents a promising adjunctive therapy for managing inflammatory bowel disease (IBD).\u003c/p\u003e \u003cp\u003eHowever, certain limitations warrant further investigation. Future research should focus on evaluating the long-term safety and efficacy of CS seed oil in human populations. Detailed studies are needed to optimize dosages and treatment durations. Additionally, exploring potential synergistic effects between CS seed oil and conventional IBD treatments could provide new clinical perspectives. Such investigations will be crucial in determining the viability of CS seed oil as a supportive strategy for IBD patients.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics Committee Approval\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe study protocol was approved by the Aydın Adnan Menderes University Animal Experiments Ethics Committee (Aydın, Turkiye; Approval number: 64583101/2023/06, Date: 19/01/2023). All experimental procedures were conducted at the Aydın Adnan Menderes University Animal Facility in strict accordance with the ARRIVE guidelines and the Guide for the Care and Use of Laboratory Animals published by the National Institutes of Health (NIH). Research and publication ethics were rigorously followed throughout the investigation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConflict of Interest\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that there are no conflicts of interest regarding the publication of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFinancial Disclosure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eFunding for this study was provided by Aydın Adnan Menderes University, Scientific Research Projects, overseen by the master\u0026apos;s thesis committee under project number TPF-23030.\u003c/p\u003e\n\u003cp\u003e*Consent for publication and participation is not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIdea/Conceptualization: FŞ, İK; Study Design: FŞ, İK; Data Collection/Processing: FŞ, İK; Analysis/Interpretation: FŞ. ; Literature Review: İK; Manuscript Drafting/Writing: İK; Critical Revision: FŞ.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eMahalli AA, Alharthi HM. 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Asian J Surg. 2018;41(6):543\u0026ndash;50. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://doi.org/10.1016/j.asjsur.2017.12.002\u003c/span\u003e\u003cspan address=\"10.1016/j.asjsur.2017.12.002\" 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":"journal-of-cannabis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcan","sideBox":"Learn more about [Journal of Cannabis Research](https://jcannabisresearch.biomedcentral.com/)","snPcode":"42238","submissionUrl":"https://submission.springernature.com/new-submission/42238/3","title":"Journal of Cannabis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Cannabis sativa, Seed oil, Ulcerative colitis, İnflammation, Oxidative stress","lastPublishedDoi":"10.21203/rs.3.rs-8751706/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8751706/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBACKGROUND\u003c/h2\u003e \u003cp\u003eUlcerative colitis is a chronic inflammatory disease characterized by extensive mucosal damage and insufficient treatment options. \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil, distinguished by its rich bioactive lipid profile, offers significant potential in modulating inflammation and oxidative stress. Given that its dose-dependent efficacy remains largely unexplored, this study investigates the therapeutic effects of varying doses of \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil to establish a novel intervention strategy for ulcerative colitis.\u003c/p\u003e\u003ch2\u003eMATERIALS AND METHODS\u003c/h2\u003e \u003cp\u003e24 Wistar albino rats were distributed into four groups: control, TNBS-induced colitis, and two treatment groups receiving \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil via intragastric gavage at doses of 1 ml/kg (CS1) and 3 ml/kg (CS3). Following a 72-hour protocol, colon tissues were harvested to evaluate oxidative stress markers (SOD, GPx, MDA, MPO, CAT) and pro-inflammatory cytokines (TNF-α, IL-1β).\u003c/p\u003e\u003ch2\u003eRESULTS\u003c/h2\u003e \u003cp\u003e \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil administration significantly attenuated apoptotic and inflammatory signaling while restoring antioxidant enzyme homeostasis. Both biochemical and histopathological analyses confirmed that the treatment conferred robust protection against colitogenic damage (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e\u003ch2\u003eCONCLUSION\u003c/h2\u003e \u003cp\u003eOur findings demonstrate that \u003cem\u003eCannabis sativa\u003c/em\u003e seed oil effectively ameliorates ulcerative colitis. Notably, the 3 ml/kg dose exhibited superior efficacy, yielding results comparable to the healthy control group, which underscores its potential as a novel pharmacological agent for inflammatory bowel diseases.\u003c/p\u003e","manuscriptTitle":"Exploration of Possible Effects of Cannabis sativa Seed Oil Applied in Different Doses in Ulcerative Colitis Model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-02-13 17:39:29","doi":"10.21203/rs.3.rs-8751706/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"editorInvitedReview","content":"","date":"2026-02-19T13:02:23+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"263978280116627305636399433334475320407","date":"2026-02-12T12:17:54+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2026-02-10T11:57:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2026-02-05T01:18:35+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2026-02-04T08:24:45+00:00","index":"","fulltext":""},{"type":"submitted","content":"Journal of Cannabis Research","date":"2026-02-04T07:12:29+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"journal-of-cannabis-research","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"jcan","sideBox":"Learn more about [Journal of Cannabis Research](https://jcannabisresearch.biomedcentral.com/)","snPcode":"42238","submissionUrl":"https://submission.springernature.com/new-submission/42238/3","title":"Journal of Cannabis Research","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"BMC/SO AJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"9a0d1218-5503-4666-a4f6-7f53ea81bd86","owner":[],"postedDate":"February 13th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2026-02-13T17:39:29+00:00","versionOfRecord":[],"versionCreatedAt":"2026-02-13 17:39:29","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8751706","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8751706","identity":"rs-8751706","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}