Influence of 3-hydrazinylquinoxaline-2-thiol hydrogel on skin wound healing process in diabetic rats

Animals

Forty-two healthy adult male Wister albino rats participated in the in vivo experimental wound healing study. These rats, weighing between 150 and 200 g, were obtained from the animal facility of the Faculty of Pharmacy, King Abdul Aziz University (KAU), Jeddah, Saudi Arabia. The rats were housed in clean cages with a regulated light-dark cycle and a chamber temperature between 22 and 25 °C. The room relative humidity was set at 55%, and the rats had unrestricted access to water and food. “The study protocol was approved by the Research Ethics Committee of the Faculty of Pharmacy, KAU (King Abdul Aziz University) (reference number “PH-1443-76”), and all methods were performed in accordance with the Animal Experimentation Guidelines and ARRIVE guidelines.

Diabetes mellitus induction

To induce diabetes in the rats, they were given a single intraperitoneal injection of streptozotocin (STZ). Freshly prepared STZ with citrate buffer (pH 4.00) was administered to the rats at a dosage of 55 mg/kg after an overnight fast.^18,21After 72 hours, the presence of type 1 diabetes mellitus (T2DM) was confirmed by measuring fasting glucose levels with an ACCU-CHEK.^® Active glucose meter. The diabetic state was additionally confirmed by the presence of polyuria, polydipsia, polyphagia and weight loss. For subsequent experiments, only rats with blood glucose levels above 250 mg/dL were selected.

Model of an excision wound

Two weeks after confirmation of diabetes mellitus (DM), rats were anesthetized by intraperitoneal administration of ketamine (90 mg/kg) and xylazine (10 mg/kg). Before inflicting wounds, the dorsal fur of the rats was shaved with an electric clipper and disinfected with 70% ethanol. Circular 1 cm pieces of full-thickness skin were excised from pre-determined areas on the dorsal surface of the rats using a sterile biopsy punch. Animals were regularly monitored for signs of infection, and any rats showing signs of infection were excluded from the experiment and replaced with new subjects.

Test protocol

After wound excision, animals were placed in individual cages and then randomly assigned to three equal-sized groups, each group consisting of 14 animals (n = 14). This number was determined for each group to ensure their availability until the end of the experiment and to minimize the risk of STZ toxicity. Group I: Non-diabetic control rats treated daily with white petrolatum topically. Group II: Diabetic rats receiving daily vehicle alone. Group III: Diabetic animals treated with a 0.2% hydrogel containing 3-hydrazinylquinoxaline-2-thiol and specifically applied to the excision wounds. Important points are shown in the graphical summary (Fig. 1).

Graphical abstract: Testing the wound healing effect of a 3-hydrazinylquinoxaline-2-thiol hydrogel on the skin at a full-thickness excisional wound in rats with streptozotocin-induced type 2 diabetes.

Estimation of wound contraction

Daily measurement of the gradual reduction in wound area was done by delineating the wound area using transparent graph paper sheets. The process of wound contraction was quantified by expressing it as a proportion of the total healing process. A picture of the wound was taken using a digital camera to assess the progress of wound closure. The percentage of wound contraction was then calculated using the following formula²²:

Preparation of the test compound

The compound tested, 3-hydrazinylquinoxaline-2-thiol, was purchased from Sigma Aldrich D184977 in Taufkirchen, Germany. To ensure sterility and prevent contamination, all procedures were performed in a sterile environment according to a previously described protocol.²⁰Briefly, a 2% (w/w) hydroxypropylmethylcellulose (HPMC) gel was prepared by dissolving 3 g of HPMC in distilled water until the total weight reached 100 g. The mixture was then covered, gently stirred with a magnetic stirrer, and allowed to stand for 24 hours to allow complete water uptake and gel formation. To prepare the hydrogel (0.2%), 0.2 g of the drug was dissolved in 2 g of dimethyl sulfoxide (DMSO). The solution was then gently mixed with 3% HPMC gel (98 g) with constant stirring to ensure even distribution of the drug within the gel matrix. A similar procedure was followed to prepare the control sample. The gel was transferred to collapsible tubes and stored in a cool, dry place. The pH of the hydrogel was determined using a digital pH meter. One gram of the prepared hydrogel was dissolved in 25 ml of distilled water and the pH was measured at room temperature using a Thermo Scientific Expert pH meter. The pH measurement was performed in triplicate and the average value was calculated. The viscosity of the samples at 25 °C was measured using a Brookfield digital viscometer. The hydrogel was applied gradually from the outer edges towards the core to ensure comprehensive treatment of all affected areas.

Histological examination

Before sacrifice and sample collection, animals were euthanized by intraperitoneal injection of sodium pentobarbital (800 mg/kg).²³. Histological examinations were performed based on histological assessments at 0, 14, and 21 days post-injury. Representative tissues from the wound areas were removed and fixed in 10% buffered formalin solution. Skin samples were then subjected to the standard dehydration process in a series of increasing ethanol concentrations for 24 hours. Tissues were then embedded in paraffin and cut into 5 µm thick sections. Sections were stained with hematoxylin and eosin (H&E) and examined by light microscopy at 100x magnification. Wound tissue sections were classified based on the degree of healing, fibroblast proliferation, angiogenesis, and the presence of inflammatory cells. All images were taken using an Olympus microscope (BX-51; Olympus, Tokyo, Japan).

Determination of proinflammatory cytokines and NF-κB

Skin tissue homogenate was tested for inflammatory markers (TNF-α, IL-6, IL-1β) and NF-κB, cluster of differentiation 68 (CD68), and collagen-1 on days 3, 7, 14, and 21 post-injury using specific ELISA kits and the manufacturer’s instructions.

Biochemical analyses

The following biochemical parameters (oxidative stress) were investigated in skin tissue homogenate: glutathione (GSH), superoxide dismutase (SOD) and catalase (CAT) using biochemical ELISA kits according to the manufacturer’s instructions on days 3, 7, 14 and 21 after injury.

Preparation of tissue homogenate

The tissue was thoroughly washed with ice-cold saline and then gently pressed between filter papers. The samples were then homogenized in phosphate buffered saline (PBS) to obtain 10% w/v tissue homogenates. The prepared homogenate was then centrifuged at 3000 rpm for 20 minutes at 4°C. Later, the supernatants were analyzed to estimate each specific parameter.

In silico activity

Prediction of ADME properties, molecular targets and CYP P450 enzyme inhibition profile for 3-hydrazinoquinoxaline-2-thiol

In this study, we used several online computational tools to predict various properties of 3-hydrazinoquinoxaline-2-thiol. We used Simplified Molecular Input Line Entry System (SMILES) as input (Table 1) to predict properties based on chemical structure. Swiss ADME web server helped us predict pharmacokinetic parameters such as molecular weight, lipophilicity, solubility, blood-brain barrier (BBB) ​​permeability, oral (gastrointestinal/GI) absorption, and skin penetration. To further analyze the molecule, we constructed a pharmacokinetic radar chart and molecular target pie chart using Swiss ADME and Swiss Target Prediction. We also evaluated the inhibition profile of CYP isoenzymes including CYP1A2, CYP2C19, CYP2C9, CYP2D6, and CYP3A4.^24,25,26.

Predictions of the organ and endpoint toxicity profile for the chemical structure of 3-hydrazinoquinoxaline-2-thiol

For a chemical scaffold to enter the drug market, it is crucial to evaluate its toxicity and safety. For this purpose, we have used two web servers, Protox II and Predherg. These tools provide us with a comprehensive overview of various toxicity parameters, such as organ-specific toxicities like liver toxicity and cardiac toxicity, as well as endpoints such as immunotoxicity, cytotoxicity, carcinogenicity and mutagenicity. By using these web servers, we have gained valuable insights into the toxicity profile of small molecules, thus facilitating drug development.^27,28.

Acute skin irritation test

To evaluate the skin tolerance of the prepared hydrogel, six Wister rats with flawless skin were selected. The backs of these rats were carefully shaved and 200 mg of the prepared hydrogel was applied to the shaved area. Then, the treated area was covered with a cotton bandage for 24 hours. An amount of 500 mg of the test samples was applied to the selected test site. Observations for signs of morbidity and toxicity and for adverse skin reactions, including pruritus, edema, scabbing and erythema, were made initially within the first 24 hours and then over a period of 21 days.²⁹.

Statistical analysis

Data were analyzed using Graph Pad Prism 10.1.1 and statistical significance values ​​were set at P ≤ 0.05. Biomarkers were calculated for each group and then compared using a one-way or two-way ANOVA with multiple comparisons. Data representing three or more biological replicates are presented as mean ± SD. Statistical significance is indicated as follows: