Effect of Trans-Aconi̇ti̇c Aci̇d on the li̇ver i̇n streptozotoci̇n (STZ)-induced type I di̇abetes model

Diabetes Mellitus (DM) is considered one of the most common endocrine disorders today due to its increasing prevalence and the systemic complications it causes, which makes research on its treatment a priority. Aconitic acid (propene-1,2,3-tricarboxylic acid) is a six-carbon organic acid that naturally accumulates at high concentrations in plants, particularly in sugarcane (Saccharum officinarum) and sweet sorghum (Sorghum bicolor), and possesses antioxidant and anti-inflammatory properties. Therefore, in this thesis, the potential therapeutic effects of trans-aconitic acid (TAA) against a Type I diabetes model were investigated through both in vitro and in vivo studies. İn vitro analyses, α-amylase and DPPH assays were performed, while in vivo analyses involved the induction of a Type I Diabetes Mellitus (TIDM) model using Streptozotocin (STZ), followed by evaluation of the therapeutic efficacy of different doses (30–60 mg/kg) of trans-aconitic acid (TAA) in comparison with acarbose. In this study, a total of 54 male Wistar albino rats were divided into six groups: Group I: control group, Group II: 60 mg/kg TAA-treated group, Group III: STZ-treated group, Group IV: STZ + 30 mg/kg TAA-treated group, Group V: STZ + 60 mg/kg TAA-treated group and Group VI: STZ + 20 mg/kg acarbose treated group. A single intramuscular dose of 50 mg/kg STZ was administered to the diabetic groups to induce TIDM. Five days after injection, fasting blood glucose levels were measured from tail blood using a glucometer, and values above 200 mg/dL were considered diabetic. For 28 days, the relevant groups received physiological saline, TAA and acarbose via gastric gavage. Body weight, food intake, and water intake of the rats were monitored daily. At the end of the experimental period, serum and liver tissues were collected vii for biochemical analyses. Serum Glucose (SG), Serum Insulin (SI), Glycated Hemoglobin (HbA1c), Alanine aminotransferase (ALT), Aspartate aminotransferase (AST), Malondialdehyde (MDA), Total Cholesterol (TC), and Triglyceride (TG) levels were measured from serum samples, while MDA, Total Superoxide Dismutase (SOD), and Catalase (CAT) activities were analyzed from liver tissue. For histological examinations, liver and pancreatic tissues were evaluated using Hematoxylin–Eosin, Periodic acid–Schiff, and Gomori's staining. Molecular analyses involved the assessment of Nuclear Factor kappa B p65 subunit (NF-κB p65 subunit), Tumor Necrosis Factor alpha (TNFα), Interleukin-6 (IL-6), and beta-Actin (Actβ) gene expression levels. Subsequently, Western blot analyses were performed to determine the protein levels of NF-κB p65, TNFα, and Actβ. İn vitro analyses, although the antioxidant activity of TAA was found to be lower than that of gallic acid in the DPPH assay, TAA inhibited α-amylase by 94.8% at a concentration of 2.5 mg/mL. İn vivo studies supported the potential antidiabetic effect identified in vitro. Biochemical analyses revealed that the 60 mg/kg TAA group showed a significant increase in SG, SI, and TG levels compared to the control group. Compared to the control group, the diabetic group showed a statistically significant increase in SG, HbA1c, ALT, and AST levels, while SI, Total SOD, and TG levels significantly decreased. When comparing the diabetic group with the Diabetic + 30 mg/kg TAA and Diabetic + 60 mg/kg TAA groups, significant decreases in HbA1c and AST levels were detected in the treatment groups. In the Diabetic + 20 mg/kg Acarbose group, compared to the diabetic group, SI levels significantly increased, while SG, HbA1c, AST, and TG levels significantly decreased. Histological analyses showed degeneration of hepatocytes, disruption of the localization of hepatocytes from the central vein toward the periphery, vacuole formation, and loss of hepatocyte boundaries in diabetic liver sections; in the pancreas, islet structures were narrowed and degeneration increased under diabetic conditions. Following TAA treatment, these histopathological alterations were observed to diminish, indicating a protective effect of TAA against TIDM-induced liver and pancreatic damage. No marked differences were observed between the TAA-treated and acarbose-treated groups histologically. In molecular analyses, while the increase in NF-κB p65 subunit gene expression levels was not significant between the control and diabetic groups, the increases in TNFα and IL-6 were significant. Additionally, in the TAA treatment groups, the decreases in IL-6 and TNFα gene expression levels were found to be significant compared to the control group. Western blot analyses showed that TNFα protein levels were consistent with gene expression findings, while for NF-κB, protein levels were more intense despite lower expression levels in the treatment groups. In conclusion, although TAA exhibited limited biochemical effects at doses of 30 and 60 mg/kg, histological and physical findings indicate its potential in ameliorating diabetic damage. Gene expression and protein analyses suggest a dose-dependent and heterogeneous response profile within inflammatory pathways.