Management of metabolic dysfunction–associated steatotic liver disease (MASLD) remains a significant clinical challenge. This study evaluated the efficacy of Coenzyme Q10 (CoQ10) as an adjuvant therapy to metformin in the treatment of MASLD using an experimentally induced type 2 diabetes (T2D) rat model. T2D was induced in 18 rats through a high-fat diet combined with a low dose of streptozotocin. The diabetic rats were then randomly allocated into three equal groups: untreated, metformin-treated, and metformin/CoQ10-treated. An additional six rats maintained on a normal chow diet served as the control group. Development of MASLD was confirmed through biochemical assays and histopathological analyses. Liver histology and electron microscopy were performed, along with immunohistochemistry for BAX and CD68 markers. Our results demonstrated that T2D rats exhibited impaired glucose and lipid profiles, elevated liver enzymes, and reduced adiponectin levels. These alterations were accompanied by hepatic oxidative stress, histopathological features of MASLD, fibrosis, and increased expression of BAX and CD68. Treatment with either metformin monotherapy or metformin/CoQ10 combination therapy significantly ameliorated the biochemical and histopathological manifestations of MASLD, as well as reduced the expression of BAX and CD68 in the liver. Importantly, combined metformin/CoQ10 therapy exerted a stronger hepatoprotective effect than metformin alone.

Ethical approval statement

The study protocol was reviewed and approved by the Research Ethics Committee of the Faculty of Medicine, Menoufia University (IRB approval No. 3/2025 PHYS19). All experimental procedures were conducted in accordance with the Guide for the Care and Use of Laboratory Animals (National Research Council).

Experimental design

Twenty-four adult male Wistar rats (weighing 180–200 g) were housed under standard laboratory conditions with a natural light–dark cycle. They had free access to a normal chow diet and water and were acclimatized to the environment for one week before the start of the experiment.

Six rats were maintained on a normal chow diet and served as the control group. Type 2 diabetes (T2D) was induced in 18 rats using a high-fat diet (HFD) combined with a low-dose streptozotocin (STZ) injection, as described previously (Srinivasan et al., 2005). Sample size was calculated using the resource equation method (Arifin and Zahiruddin, 2017).

After successful model establishment, the diabetic rats were randomly allocated into three equal groups (n = 6 per group) using simple randomization:

1.  T2D group (untreated): diabetic rats received no treatment.

2.  Metformin group: diabetic rats were treated with metformin (250 mg/kg/day, oral gavage) for six weeks (Salama et al., 2013).

3.  Metformin + CoQ10 group: diabetic rats received metformin as above, in addition to CoQ10 (10 mg/kg, oral gavage) administered 30 minutes after metformin for six weeks. The hepatoprotective dose of CoQ10 was selected based on previous studies (Elshazly et al., 2020; Rashied et al., 2020).

All diabetic rats were maintained on an HFD until the end of the experiment.

At the end of the study, rats were fasted for 12 hours, and retro-orbital blood samples were collected into two tubes under light ether anesthesia. The first tube contained EDTA for the measurement of glycated hemoglobin (HbA1c), while the second was plain for serum separation. Samples were stored at −80 °C until biochemical analysis.

Subsequently, the animals were sacrificed by decapitation following anesthesia with ketamine (100 mg/kg) and xylazine (10 mg/kg, intraperitoneally). Liver tissues were immediately dissected and prepared for histopathological examination.

Induction of T2D associated with MASLD

Type 2 diabetes (T2D) was experimentally induced as previously described by Srinivasan et al. (2005). Rats were fed a high-fat diet (HFD) containing 58% fat, 25% protein, and 17% carbohydrate (as a percentage of total kcal) ad libitum for two weeks. They were then injected intraperitoneally with streptozotocin (35 mg/kg) dissolved in cold 0.1 M citrate buffer. Control group rats received an equivalent volume of citrate buffer intraperitoneally.

Fasting blood glucose (FBG) was measured 72 hours after streptozotocin injection. Rats with FBG levels ≥300 mg/dl were considered diabetic. This model has been widely established to mimic MASLD in humans (Dwivedi and Jena, 2020).

Chemicals and drugs

Streptozotocin was purchased from Sigma-Aldrich (USA). Metformin (500 mg/tablet) was obtained from Amoun Pharmaceutical Company (Egypt), and CoQ10 was obtained from Arab Company for Pharmaceuticals and Medicinal Plants (Egypt). All other chemicals were of analytical grade and purchased from commercial sources.

Biochemical analysis

Fasting blood glucose (FBG) was measured from the tail vein using a glucometer (CLEVER CHECK TD-4231, Taiwan). Glycated hemoglobin (HbA1c) levels were determined using kits obtained from Spectrum Diagnostics, Egypt (Catalog No. 255-000). Serum insulin concentrations were measured using an ELISA kit (DRG Instruments GmbH, Marburg, Germany). Insulin resistance was calculated using the Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) according to the following formula:

 (HOMA-IR) index = [Fasting serum insulin (μU/ml) X FBG (mg/dl)] / 405 (Matthews et al., 1985).

Markers of liver function, including serum alanine transaminase (ALT), aspartate transaminase (AST), alkaline phosphatase (ALP), and albumin, as well as lipid profile parameters, including total cholesterol (TC), high-density lipoprotein cholesterol (HDL-C), and triglycerides (TG), were measured using kits from Biodiagnostic Company, Giza, Egypt. Low-density lipoprotein cholesterol (LDL-C) was calculated using the Sampson equation: LDL = TC - HDL-C - (TG/5) (Sajja et al., 2021).

Markers of oxidative stress and antioxidant status, including malondialdehyde (MDA), superoxide dismutase (SOD), catalase, and glutathione peroxidase (GPx), were measured in liver homogenates using colorimetric kits from Biodiagnostic Company. Serum adiponectin levels were determined using an ELISA kit from Chemi-Con International, USA. All biochemical assays were performed according to the manufacturers’ instructions. Histopathological examination

Light microscopic study

Liver tissues from all rats were fixed in 10% formalin, dehydrated in ascending grades of ethyl alcohol, cleared in xylene, and embedded in soft paraffin followed by hard paraffin. Sections of 5 μm thickness were prepared and used for histological examination. Routine histology was performed using Hematoxylin and Eosin (H&E) staining, while Masson’s Trichrome (MT) staining was employed to visualize collagen fibers (Jones et al., 2008).

Immunohistochemistry

Bcl-2-associated X-protein (BAX)

BAX immunostaining, a marker of apoptosis, was performed using a primary rabbit monoclonal antibody E63 to BAX (Catalog No. LS-B2510; Lab Vision Corp, NeoMarkers, Inc., Fremont, California, USA) at a 1:50 dilution. Positive staining was indicated by a brown coloration in the cytoplasm of hepatocytes (Biaggio et al., 2014).

Cluster of Differentiation 68 (CD68)

CD68, a highly glycosylated transmembrane protein primarily located in lysosomes, serves as a marker for stellate macrophages (Kupffer cells). CD68 immunostaining was performed using a primary mouse monoclonal antibody (Clone KP1; Lab Vision Corp., NeoMarkers, Inc., Fremont, California, USA). Positive staining was indicated by a brown coloration in the cytoplasm of stellate macrophages within the hepatic sinusoids (Saleh et al., 2022).

Immunohistochemical staining was performed on 4-μm-thick paraffin-embedded liver sections using the streptavidin–biotin complex technique (Hsu et al., 1981). Serial sections were deparaffinized on charged slides and incubated in 0.1% hydrogen peroxide for 30 minutes to block endogenous peroxidase activity. Sections were then incubated with primary antibodies overnight at room temperature for 60 minutes.

Following primary antibody incubation, slides were washed with diluted phosphate-buffered saline and incubated with the secondary anti-mouse antibody (universal kit) for 30 minutes at room temperature. Positive control slides included Hodgkin’s lymphoma tissue for BAX immunostaining and tonsil tissue for CD68 immunostaining, and control slides were included in each staining run. Negative controls were prepared by omitting the primary antibody.

Finally, sections were counterstained with Mayer’s hematoxylin, dehydrated through ascending grades of ethyl alcohol, cleared in xylene, and mounted in Canada balsam.

Electron microscopic (EM) study

Small liver specimens (1 × 1 mm²) were fixed in 2.5% glutaraldehyde, post-fixed in 1% osmium tetroxide, dehydrated, and embedded in epoxy resin. Semi-thin sections (1 μm) were stained with toluidine blue, while ultrathin sections (50–80 nm) were contrasted with lead citrate and uranyl acetate. All sections were examined using a transmission electron microscope (Seo, Russia) at the Electron Microscopy Unit, Faculty of Medicine, Tanta University (Glauert and Lewis, 2014).

Morphometric study

All measurements were performed on 10 non-overlapping, randomly selected sections per group at consistent magnification using the Leica Qwin 500 LTD image analysis system (Cambridge, England) at the Department of Histology, Faculty of Medicine, Menoufia University.

Histological scoring of MASLD was semi-quantitatively assessed using the NASH Clinical Research Network (NASH CRN) system. Steatosis (micro- or macrovesicular) was graded 0–3, lobular inflammation 0–3, and hepatocytic ballooning 0–2 (H&E ×200). Additionally, the percentage area of collagen fibers (Masson’s Trichrome ×200), color intensity of CD68 immunostaining (×400), and percentage of BAX-positive cells (×400) were quantified.

Statistical analysis

Data were analyzed using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). The normality of data distribution was assessed using the Shapiro–Wilk test, and all variables were found to be normally distributed. Inter-group differences were evaluated using one-way ANOVA followed by Tukey’s post hoc test. Results are presented as mean ± standard deviation (SD), and p ≤ 0.05 was considered statistically significant.