Effect of antioxidant treatment with n-acetylcysteine and swimming on lipid expression of sebaceous glands in diabetic mice

The sebaceous gland (SG) is involved in different inflammatory, infectious and neoplastic processes of the skin and can be related to specific diseases, e.g., diabetes mellitus. Sometimes, the histological diagnosis requires complementary tests due to the ability of diseases to mimic other tumors. We evaluated the sebaceous gland density in Non-obese diabetic mice to analyze the N-acetylcystein effects and swimming exercise treatment in sebaceous glands healing, using specific staining in histochemistry and immunohistochemistry reactions in the identification of the lipid expression in the sebaceous gland. We investigated the intracytoplasmic lipid expression and analysis of gland density from SG in dorsal skin samples from the Non-obese diabetic (NOD mice) and diabetic animals submitted to antioxidant treatment and physical exercise. For histological analysis of the sebaceous glands, specific staining in histochemistry with sudan black and immunohistochemistry reaction with adipophilin were used in the evaluation. Statistical analysis showed significant proximity between the values of the control group and the diabetic group submitted to the swimming exercise (DS group) and similar values between the untreated diabetic group (UD group) and diabetic group treated with the antioxidant N-acetylcysteine (DNa group), which did not prevent possible differences where p < 0.01. Adipophilin (ADPH) immunohistochemistry permitted more intense lipid staining in SGs, the preservation of the SG in the control group, and a morphological deformed appearance in the UD and DNa groups. However, weak morphological recovery of the SG was observed in the DS-Na group, being more expressive in the DS group. In conclusion, the groups submitted to physical exercises showed better results in the recovery of the analyzed tissue, even being in the physiological conditions caused by spontaneous diabetes.

www.nature.com/scientificreports/ in the tumor mass, which are often scarce 14 . Previous studies have reported the challenge in the diagnosis of SG diseases, as well as in their treatment [15][16][17] . Different alternatives exist for the treatment of diabetes, such as daily insulin application and glucose monitoring, diet changes, use of therapeutic drugs like N-acetylcysteine (NAC), and regular physical activity [18][19][20] . NAC is an antioxidant that is able to stimulate the synthesis of reduced glutathione (GSH), the enzyme responsible for the antioxidant defense system. Inside the cell, acetylcysteine is deacetylated to l-cysteine, an amino acid that is essential for the GSH 19,21 . GSH is an important tripeptide for cell integrity that acts as an intracellular defense against exogenous and endogenous oxidant radicals and against cytotoxic substances 19 . Furthermore, GSH induces an increase in CD4 and CD8 T lymphocytes infiltration, promoting the disease's development. The association of a specific antibody is necessary to reduce CD4 and CD8 T cell infiltration in the pancreas, preventing the destruction of beta cells [21][22][23][24][25] .
Physical activity is an important tool for glycemic control 26,27 . Regular physical activity increases energy expenditure and contributes to weight loss 27 . Exercise-mediated energy expenditure assists in glycemic control since it promotes consumption of glucose stored in blood 26 . In addition to reducing glucose levels, physical exercise improves insulin sensitivity, stimulates calorie burning, and induces the release of hormones such as estrogen 7,8 , cortisol and testosterone, among other benefits 26,27 . This physiological response during the treatment provides the morphological revitalization. Histopathological analyzes are essential to assist in the diagnosis of the lesion, as well as in the evaluation of the tissue after treatment 16,17 .
One method frequently used for the detection of lipids in the pathological diagnosis of inflammatory and neoplastic processes involving SGs is histochemical staining with sudan 3,7,28,29 , characteristic to stain lipids in fresh tissues 30 ; however, its use is limited due to the difficulty level of this technique in allowing sebaceous differing and its application to paraffin-embedded materials 30,31 . As a consequence of these limitations, other markers are explored as alternatives, for example, antibodies used to detect the immunohistochemistry expression of lipids [32][33][34] .
Immunohistochemistry allows the tracking of specific proteins [32][33][34] . Furthermore, it is superior to protein identification methods based on the demonstration of amino acids since it can detect molecules such as specific tissue antigens 34 . This procedure is therefore important for the diagnosis of inflammatory and infectious diseases, as well as for the identification of prognostic factors for skin lesion treatment 4,34-36 . These markers of lipid expression, can be used in specific clinical situations that alter the histophysiology of this gland, such as sebaceous hyperplasia, folliculitis, steatocystoma, sebaceous adenocarcinoma, sebaceous carcinoma, and skin disorders caused by diabetes mellitus 35,37,38 .
The objective of the study was to evaluate the sebaceous gland density in Non-obese diabetic mice and healthy mice to analysis the effects of the N-acetylcystein and swimming exercise treatment in Sebaceous Glands recuperation. The study also evaluated the efficacy of histochemistry coloring with Sudan Black stain markers to evaluate the sensibility of the immunohistochemistry with Adipophil-in (ADPH) antibody in identification of the lipidic expression in sebaceous gland.

Materials and methods
Experimental design. 25  Intervention. After the validation of hyperglycemic condition, the mice from the DS group were submitted to swimming exercises with an average duration of 30 min, five times a week for 21 days. The description of the swimming method was adjusted, we added the water temperature and the depth of the swimming pool used. The training was done in a similar method to the previously developed mice protocol, where proper aerobic and cardio physical training intensity, frequency and duration were carefully studied 19 . Mice from the DNa group received a daily intraperitoneal dose of 50 mg/kg NAC and a weekly intravenous dose of 25 µl anti-CD4 and anti-CD8 antibodies, to block the negative effects of NAC (mainly the lymphocyte stimulation) for 21 days. Previous group studies showed a reduction in lymphocyte activity in pancreatic beta cells submitted to treatment with anti-CD4 and anti-CD8, in the same interventions described in this work 23,24 . In animals from the DS-Na group, both treatments from the DS and DNa groups were applied.
Urinary glucose (mg/dL) levels were monitored daily for all subjects. Urine was collected daily by manual compression of the bladder, discarding the first drop. Next, the concentrated urine was analyzed with a reagent strip (MultistiK 10 SG Bayer) and the reference table provided by the manufacturer was used for glucose levels measurement.
The animals were classified as diabetic when glucose levels were higher than 300 mg/dL. To confirm and guarantee the reliability of the values indicating the hyperglycemic state of the animals, blood glucose levels were measured weekly with a Roche Accu-Chek Performa glucose meter and reagent strips (New York, USA). www.nature.com/scientificreports/ Skin samples collection. After the experimental period, the subjects were anesthetized with ketamine (Ketalar, Parke-Davis) and xylazine (Rompun, Bayer Animal Health) in doses of 0.02 g/ml for skin tissue samples removal. In the study, we conducted a pilot group to evaluate the areas where samples were collected, through the routine HE methodology, which presented us with the dorsal area near the skull region, with a good amount of SGs, with good cell volume. After this process, the animals were sacrificed with an anesthetic overdose according to ethical guidelines (Ethical Process Number: 351\2011).
Histological techniques. Tissue fixation. The samples collected were divided into two fragments submitted to different tissue fixation techniques. Both fragments were stained with hematoxylin-eosin, but one was submitted to histochemistry and the other to immunohistochemistry for lipid expression analysis in SGs.
The first fragment was prefixed in phosphate-buffered saline solution, then embedded in Tissue Tek OCT (optimal cutting temperature) compound, frozen in liquid nitrogen, and stored in a biofreezer at -80 °C for subsequent histochemical analysis. The second fragment was fixed in 10% formalin buffered with anhydrous dibasic and monobasic sodium phosphate. Next, the fragment was submitted to routine processing for dehydration and embedding in paraffin. Histological sections (4 µm) were cut for immunohistochemical analysis.
Histochemical staining. The samples were cut into 8-µm thick sections with a cryostat for staining with Sudan Black. For this purpose, the sections were rinsed in 70% alcohol, immersed in a saturated solution of Sudan Black for 2 h, and again rinsed in 70% alcohol to remove excess dye. The sections were washed in distilled water and dried at room temperature. The slides were counterstained with panoptic stain, washed in deionized water, and coverslipped.
Immunohistochemistry reaction. For immunohistochemistry, the slides were immersed in an antigen retrieval solution consisting of EDTA/Tris, pH 9.0, at 125 °C in an electric pressure cooker managed by a REP-TIDE microprocessor (Celerus). Nonspecific binding of the antibodies was blocked by incubation of the slide with hydrogen peroxide. Next, the slides were incubated with the primary ADPH antibody (clone AA5-27, LifeSpan) diluted 1/100. To amplify the antigen-antibody binding, two other antibodies were applied to the slides, post-primary (Novolink polymer Novocastra) and the polymer (Novolink polymer Novocastra). The reaction was developed with diaminobenzidine (DAB) as a chromogen (Liquid DAB + Substrate Chromogen System Kit, DakoCytomation, Carpinteria, CA, USA).
Microscopic analysis. The slides were analyzed and photographed under an Olympus BX-41 light microscope (Olympus, Japan), equipped with an image capture system. All observations were made with achromatic objectives of the 20×, 40× and 60× planes. These slides were also used for quantification of three-dimensional tissue allowing to analyze the density of SGs. The images were analyzed using the Sensus digital imaging software of the Olympus cell. The area and perimeter of five SGs of each staining method were measured per animal.
Stereological and statistical analysis. The SG area (µm 2 ) and perimeter (µm) were calculated in the groups studied by ADPH immunohistochemistry, since this was the best marker of this tissue. The results were analyzed by analysis of variance (ANOVA) using the BioEstat 5.3 software. When a difference was observed between the groups studied, the Tukey test was applied at a minimum significance level of 5% (p < 0.05).

Ethics approval. The study was approved by the Ethics Committee on Animal Experimentation of Jundiaí
Medical School, Jundiaí, Brazil (Protocol 351/2011). Experiments were performed in accordance with relevant guidelines and regulations.

Results
Glycaemic levels. Diabetic subjects presented glucose levels average of 505 mg/dl. The average levels of control animals were 118 mg/dl.

Comparison of sebaceous gland morphology between groups.
In the control group, normal SGs with an alveolar shape were noted in the dermis. The glands contained voluminous sebaceous cells with welldefined nuclei and intracytoplasmic vesicles. This SG morphology was characterized by ADPH immunohistochemistry ( Figs. 1 and 2AI). Sudan staining only allowed lipid staining without defined sorting of the intracellular content (Figs. 1 and 3A).
In the UD group, immunohistochemistry permitted the observation of fragmented and deformed SGs with an irregular orientation in the dermis. Sebaceous cells' nuclei identification was challenging. However, intracytoplasmic vesicles staining was possible (Fig. 2BI). The Sudan Black staining allowed the SGs localization, but cellular content differentiation is not possible (Fig. 3B). Immunohistochemistry (Fig. 2CI) and Sudan staining (Fig. 3C) revealed the recovery of the SG morphology in the DS group, resembling that of the control group, with the observation of marked glandular volume. Opposite results were obtained for the DNa (Figs. 2DI and 3D) and DS-Na (Figs. 2EI and 3E) groups, in which the glands exhibited a deformed shape similar to the UD group. This demonstrates that the group of diabetic mice treated with the antioxidant NAC did not express sebaceous gland recovery.

Comparison of sebaceous gland morphology between the histological techniques used. ADPH
immunohistochemistry, which allowed the use of tissue sections obtained from paraffin-embedded samples used for HE staining, showed a good expression of intracytoplasmic lipids through brown staining of the membranes of intracytoplasmic vesicles, and the nuclei of sebaceous cells could be clearly identified. Histochemical staining with Sudan Black showed strong marking (Fig. 4).  (Fig. 2AII,III), 500,324 group UD (Fig. 2BII,III), 2868,100 group DS (Fig. 2CII,III), 522,782 group DNa (Fig. 2DII,III) and 925,71 DS-Na group (Fig. 2EII,III). Statistical analysis showed proximity between the values of the control and DS groups and between the UD and DNa groups, which did not prevent possible differences where p < 0.01.

Discussion
When analyzing the skin samples' morphology from the control group, we observed that SGs had an alveolar shape and contained round cells and cytoplasmic lipid accumulation. In the UD group, cellular atrophy of the SG was observed. The deleterious effects of diabetes on SGs are related to hyperglycemia, which induces cellular abnormalities through different metabolic pathways such as non-enzymatic protein glycosylation, activation of aldose reductase and diacylglycerol phosphatidyl kinase C, and reduced hydration through the inhibition of insulin signal transduction 1,3,4 . Tissue recovery of the SG was observed in animals submitted to swimming exercise (DS group), with the glands exhibiting characteristics similar to those seen in the control group and being even more voluminous in some cases. Stereology confirmed this result, with the observation of similar SG areas in the control and DS groups. This finding can be corroborated by studies that explain the fact that physical exercise promotes blood circulation and improves the body organs' oxygenation, including the skin 26,27 . Studies have shown that exercise induces a reduction in serum glucose levels by using energy sources that tend to release hormones such as estrogen, cortisol and testosterone 19,26,27 . Insulin, in conjunction with estrogen, can promote the management of glucose levels and the restructuring of tissues damaged by this disease 3,9,27 .
No morphological recovery of SGs was observed in the DNa group, which showed features similar to those seen in the UD group. The same was observed in the stereological analysis of gland volume. These findings agree with the results of other studies demonstrating the lack of NAC treatment efficacy for tissue recovery in diabetic animals 21 . In the DS-Na group, recovery of SG morphology and volume was observed in the animals, but was still less pronounced than that seen in the control and DS groups. These findings suggest that physical exercise promotes the recovery of SGs, but this process is slow due to the antagonistic action of NAC.
NAC is a potent antioxidant due to its capacity to stimulate the synthesis of GSH 22 , the enzyme responsible for the antioxidant defense system 25 . However, NAC can increase the infiltration of CD4 and CD8 T lymphocytes www.nature.com/scientificreports/ in the pancreas, causing the destruction of beta cells and consequently aggravating the effects of diabetes on tissues 23,24 . The morphological features in the SGs of diabetic and treated groups were better demonstrated by ADPH immunohistochemistry compared to Sudan staining 39,40 , the former showing an advantage in distinguishing diagnostics contribution 40 . According to Heid 35 and Muthusamy 33 , ADPH is a specific marker of lipid accumulation which stains the membranes of intracytoplasmic lipid vesicles in a brown color [32][33][34] .
It is well established that the histochemical staining by Sudan is ineffective in paraffin-embedded samples used for HE staining 3,7,28 . Consequently, Santos et al. 3 corroborate showing good lipid marking in histochemistry and the impossibility of differentiation of sebaceous cells with Sudan 3 .