DOI: 10.5061/dryad.7m0cfxq8r
title: Data from: Coenzyme Q10 ameliorates hepatoprotective effect of metformin in experimentally induced type 2 diabetes mellitus in rats: A functional and histological study

description: 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.
description: <strong>Ethical approval statement</strong>
 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 <em>Guide for the Care and Use of Laboratory
 Animals</em> (National Research Council).
 <strong>Experimental design</strong>
 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.  <strong>T2D group (untreated):</strong> diabetic
 rats received no treatment. 2.  <strong>Metformin
 group:</strong> diabetic rats were treated with metformin (250
 mg/kg/day, oral gavage) for six weeks (Salama et al., 2013).
 3.  <strong>Metformin + CoQ10 group:</strong>
 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.
 <strong>Induction of T2D associated with
 MASLD</strong> 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) <em>ad
 libitum</em> 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).
 <strong>Chemicals and drugs</strong>
 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. <strong>Biochemical
 analysis</strong> 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)] <em>/</em> 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. <strong>Histopathological
 examination</strong> <strong>Light
 microscopic study</strong> 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).
 <strong>Immunohistochemistry</strong>
 <strong>Bcl-2-associated X-protein
 (BAX)</strong> 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). <strong>Cluster
 of Differentiation 68 (CD68)</strong>
 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. <strong>Electron
 microscopic (EM) study</strong> 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).
 <strong>Morphometric study</strong>
 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. <strong>Statistical
 analysis</strong> 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.
description: # Data from: Coenzyme Q10 ameliorates hepatoprotective effect of metformin
 in experimentally induced type 2 diabetes mellitus in rats: A functional
 and histological study Dataset DOI:
 [10.5061/dryad.7m0cfxq8r](https://doi.org/10.5061/dryad.7m0cfxq8r) ##
 Description of the data and file structure The data was collected as part
 of an experimental study aimed at evaluation 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. ### Files
 and variables #### File: raw_data_-dryad.xlsx **Description:** excel file
 contain raw data ##### Variables * Group: T2D, metformin treated
 or metformin/CoQ10 treated - FBG: fasting blood glucose (mg/dl) -
 HbA1C: glycated hemoglobin level (%) - insulin (uIU/ml) - HOMA:
 Homeostatic Model Assessment for Insulin Resistance (HOMA-IR) index -
 cholesterol: serum cholesterol level (mg/dl) - TG: triglycerides (mg/dl) -
 HDL: high density lipoproteins-cholesterol (mg/dl) - LDL: low density
 lipoproteins-cholesterol (mg/dl) - ALT: alanine transaminase (IU/l) - AST:
 aspartate transaminase (IU/l) - ALP: alkaline phosphatase (IU/l) -
 adeponectine (ug/dl) - MDA: malondialdehyde (nmol/g.tissue) -
 SOD:superoxide dismutase (U/g.tissue) - Catalase (U/g.tissue) - GPx:
 glutathione peroxidase (U/g.tissue) - BAX: mean area % of Bax staining -
 CD68: mean intensity of CD68 immune reactivity - Masson: mean area % of
 collagen fibers deposition. - steatosis: mean scores of steatosis -
 inflammation: mean scores of inflammtion - Ballooning: mean scores of
 ballooning ## Access information Other publicly accessible locations of
 the data: * None Data was derived from the following sources: * None