2.1 Animals and ethics
In this study, 42 male Wistar rats, 6-8 weeks old and weighing an average of 230 ± 30 gr, were used and housed under standard laboratory conditions: temperature 25 ± 2°C, humidity 65–70%, and 12-h light/12-h dark cycle. The animals were supplied with acidified water and standard pelleted diet (Lactamin R36, Stockholm, Sweden) ad libitum throughout the entire experiment. All procedures in this study were approved by the Guidelines of Animal Ethics Committee of Bushehr University of Medical Sciences and institutional ethics committee with approval code IR.BPUMS.REC.1391.209, and were conducted in accordance with the Guide for the Care and Use of Laboratory Animals published by the National Research Council Committee.
2.2 Plant material
The leaves of plant P. Aculeata were collected from the garden of Agriculture, Bushehr University of Medical Sciences, Bushehr, Iran, during May 2007. After identification, the scientific name of the plant was approved by the Department of Pharmacognosy, Faculty of Pharmacy, Bushehr University of Medical Sciences.
2.3 Preparation of aqueous extract
All parts of the plant were cleaned and chopped in to small pieces and dried under shade. The dried plant material was powdered. Powder of dried aerial parts of P. Aculeata (500 g) were macerated with sterile distilled water for 24 hours and was heated 12 hours at 40°C. The supernatants obtained of extracts were centrifuged and concentrated using a rotary evaporator (Rotavapor, BÜCHI Labortechnik AG, Flawil, Switzerland). The extract was stored at −20°C until use.
2.4 Ethylene glycol induced nephrolithiasis model and experimental design
Formation of kidney stone was induced in rats using 0.75% ethylene glycol in drinking water [15]. A total of 42 rats were randomly divided into six equal groups with seven rats in each group:
Group A (Healthy control group): received distilled water for the first 30 days;
Group B (Ethylene glycol control group): received 1% v/v ethylene glycol in distilled water for the first 30 days;
Group C (Low dose preventive group): concurrently received 1% v/v ethylene glycol in distilled water for the first 30 days, along with aqueous extract of P. aculeata 100 mg/kg body weight via oral gavage for the first 30 days;
Group D (Moderate dose preventive group): concurrently received 1% v/v ethylene glycol in distilled water for the first 30 days, along with aqueous extract of P. aculeata 200 mg/kg body weight via oral gavage for the first 30 days;
Group E (High dose preventive group): concurrently received 1% v/v ethylene glycol in distilled water for the first 30 days, along with aqueous extract of P. aculeata 300 mg/kg body weight via oral gavage for the first 30 days;
Group F (Treatment group): received 1% v/v ethylene glycol in distilled water for the first 30 days, along with aqueous extract of P. aculeata 300 mg/kg body weight via oral gavage from 15th to 30th days.
2.5 Biochemical assays
Ethylene glycol was purchased from Merck (Darmstadt, Germany). All urine and serum biochemical parameters were measured with the use of an automatic biochemical analyzer (Hitachi, Ltd., Tokyo, Japan) and enzyme-based kits (Alpha Laboratories, Hampshire, United Kingdom).
2.6 Collection and analysis of urine
All rats were individually housed in metabolic cages (Tecniplast, Buguggiate, Varese, Italy) and had free access to drinking water during the urine collection period. 24-hour urine samples were collected on the 30th day of the calculi induction treatment period. The urinary volume and pH were measured. Urinary parameters include calcium, phosphate, uric acid, and creatinine were analyzed using Mindray BS-200 Chemistry Analyzer machine (Mindray, Shenzhen, China). Urinary crystals were examined by a light microscope (Olympus Optical Co., Lake Success, NY, USA).
2.7 Collection and analysis of serum
At the end of the study period, rats were anesthetized with diethyl ether and 5 mL of venous blood was collected from the retro-orbital sinus. Serum was separated by centrifugation at 10,000 × g for 10 min and analyzed for creatinine, phosphate, calcium, uric acid, albumin, and urea using Mindray BS-200 Chemistry Analyzer machine (Mindray, Shenzhen, China).
2.8 Histopathological studies
After blood collection, all rats were sacrificed and the abdomen was opened by midline incision and both kidneys were quickly removed, weighed and fixed at 10% neutral buffered formalin. Thin slices with a thickness of 5 µm were taken and stained with haematoxylin-eosin (H&E) and evaluated under a light microscope (Olympus Optical Co., Lake Success, NY, USA). Then in each section, 20 microscopic fields with magnification of 40×10 were randomly selected in equal numbers in the cortex and medulla, and numbers of calcium oxalate crystals (number of tubules containing calcium oxalate deposits) were counted.
2.9 Statistical analysis
The statistical analysis of the data was done using the SPSS software for Windows, version 21 (SPSS Inc., Chicago, IL, United States). Statistical evaluation was done using one-way analysis of variance (ANOVA) test followed by Tukey-Kramer multiple comparison test as data were normally distributed. Charts were drawn using Microsoft Excel 2015 software. The results were expressed as means ± standard deviations. A P-value of < 0.05 was considered statistically significant.