In this experimental study, 49 healthy male BALB/C mice, weighing about 22±1g and 5-weeks-old (each group 8 samples) were selected. As an adaptation period, animals were kept for a week at a temperature of 19 ± 2°C with a 12-hour dark-light cycle. In order to provide a convenient environment during the experiment period, cage floors were covered with fresh and soft sawdust, and every three days the cages were cleansed to maintain the hygiene. The animals were prepared with commercial mouse pellet food containing essential vitamins required for the body besides free access to water. The commercial mouse pellet diet was purchased from Experimental Animal Center in Tehran, Iran. All ethical regulations and protocols on laboratory animal's research were considered and approved by the Animal Rights Committee of the Shahrekord Veterinary Faculty.
Preparation of ZnO Nanoparticles
ZnO nanoparticles gained from US Research Nanomaterials, Inc., (Houston, TX, 77084, USA). This nanoparticle (ZnO) harvest itself was a white powder with amount of purity ≥99 + %. The actual characteristics of ZnO nanoparticles refer to its basic physical properties. Nanoparticles of ZnO had dimensions of 10-30 nm in size (average 20 nm), as listed in Table 1, and used in concentrations of 0.1 and 0.5 mg/kg (figure 1) .
Grouping and Sampling
After a week of adaptation to the laboratory environment, the mice were randomly divided into 7 groups of 7 individuals each. The diabetes was induced in the experimental mice (groups 3 to 7) by using a single intraperitoneal injection (IP) at a dose of 220 mg/kg body weight of alloxan monohydrate (Sigma-Aldrich, St. Louis, MO, USA) in PBS soluble (pH=7). Seventy-two hours later, a few mice were randomly selected from diabetic mice and their fasting blood glucose was measured. Mice with a blood glucose level of above 200 mg/dl  were considered diabetic mice. Also, the effects of diabetes were assessed by biopsy of the pancreas with the approval of a pathologist in some induced diabetes specimens as definitive confirmation.
With diabetes confirmed, ZnO nanoparticles were injected into the diabetic mice at two doses of 0.1 and 0.5 mg/kg  and thiamine injected at a dose of 5 mg/100 g. Finally, all mice were divided into the following groups: All the injections were intraperitoneal (IP).
group I: Control group; distilled water administered.
group II: Thiamine (5 mg/100 g)
group III: Diabetic group (injection of alloxan; 220 mg/kg)
group IV: Diabetes + ZnO nanoparticles 0.1 mg/kg
group V: Diabetes + ZnO nanoparticles 0.5 mg/kg
group VI: Diabetes + ZnO nanoparticles 0.1 mg/kg along with thiamine (5 mg/100 g)
group VII: Diabetes + ZnO nanoparticles 0.5 mg/kg along with thiamine (5 mg/100 g)
On the 20th day after the last injection, the mice were anesthetized (with fasting) by chloroform (Merck KGaA, Index No: 2806-18-9), and the blood samples were withdrawn directly from the ventricle of the heart. Blood was centrifuged (Hetich-Germany) for 10 min at 3000 r/min and 4 oC, the supernatant solution was reserve at -70 oC until biochemical analysis.
The skin samples at the three areas mid-dorsal, mid-ventral and lateral were considered. After shaving the long hair, the skin from the mentioned regions was excised and trimmed into small-size pieces, fixed in a 10% buffered formalin (Merck) solution for histo-morphometric examinations. Tissue sections of about 5μm were serially obtained, stained with H&E and …., and evaluated under the light microscope.
According to the table of random numbers, in the interval between the 50 sections the first section, or primary section (e.g., 10th section), and the second section that is reference section were selected. Sampling in the distance between sections was so that each 50th section was sampled (the primary sections). Therefore, for the primary and reference sections, e.g. 10, 12, 60, 62 were sampled in the entire skin; as detailed in our previous research (figure 1) .
To evaluate the volume densities of tissue collagen bundles and fibroblast, a network of uniformly and permanently spaced points (point grid) was superimposed at random over the image of each predicted section. Collagen bundles that were hit by a point were sampled and subjected to volume densities estimation according to the number of points that hitting with the bundles. The total volume of collagen and fibroblasts were calculated using the following formula :
Where, Asec is the area of cut surfaces (1st -nth slice) of systematic-random sections through the structure, and t, the thickness of the sections.
Measurement of serum glucose
Serum glucose levels were measured using a glucose analysis kit (Pars Azmoon Co, Tehran, Iran). The principle was based on the bi-enzymatic assay comprise enzymatic oxidation of glucose to gluconic acid, yielding hydrogen peroxide and then the reaction of hydrogen peroxide with 4-aminoantipyrine and phenol to yield the colorimetric product which is measured at 500 nm. The results were reported as mg/dl.
Estimation of MDA Level
Malondialdehyde (MDA) as the end product of lipid peroxidation reacts with Thiobarbituric acid (TBA) and yields colored complex which can be measured spectrophotometrically . In this study, 2 ml of TBA reagent containing 0.375% TBA, 15% TCA and 0.25 mol/L HCl was added to 1 ml of serum from all groups. The mixture was placed in boiling water for 50 min, cooled to room temperature and centrifuged at 1000 rpm for 10 min. Thereafter, the absorbance of the supernatant was read at the wavelength of 535 nm against the blank reference. The results were calculated by using a molar extinction coefficient for MDA of 1.56×105/M/cm. The concentration of MDA was expressed as nmol/ml .
Nitric oxide assay
Briefly, 1 ml of serum was deproteinized by adding ten microliters of 1.5 g/mL ZnSO4 solution. The mixture was vortexed and centrifuged at 10000 × g for 10 min at 4 °C. Then, 100 ul of the supernatant was added to a 96-well ELISA plate, and each well was submitted to 100 μL of vanadium (III) chloride (8mg/ml) which converts nitrate to nitrite. After the addition of 100 ul of Griess reagent (equal mixture of 1% sulphanilamide in 5% phosphoric acid and 0.1% N-(1-naphthyl) ethylenediamine hydrochloride in distilled water), the plates were maintained at room temperatures for 30 minutes and the nitrite production was quantified calorimetrically at 540 nm. The NO levels were calculated using various concentrations of sodium nitrite (0.1-100 μm) as a standard and expressed as μmol/ml .
Measurement of serum BUN and Cr levels
Serum BUN was measured using a commercial kit (Pars Azmoon Co., Tehran, Iran) based on the enzymatic hydrolysis of urea to ammonia by urease. Then, in a parallel reaction, the ammonia is converted to glutamate by glutamate dehydrogenase and this reaction was monitored at 340 nm. The Cr level was measured by the Jaffe method (Pars. Azmoon Kit, Tehran, Iran) in which creatinine reacts with picric acid to form a reddish complex .
Gamma-glutamyl transferase (GGT) activity was measured by using the Pars Azmoon kit (Tehran, Iran, according to the manufacturer). The reaction is evaluated based on transferring of γ-glutamyl group from γ-glutamyl-p-nitroanilide to acceptor glycylglycine and production of para-nitroaniline which can be determined calorimetrically at 405 nm; the results were reported as U/l.
Statistical analyses of volume, histo-morphometric and biochemical data were carried out using the SPSS statistical software package version 23.0 (SPSS Inc., Chicago, IL) for Windows. Data are declared as mean ± standard deviation (SD) and statistical variations were tested by One-way ANOVA. LSD post-hoc test was applied and the values confirming p<0.05 were considered significant compared to the rest.