Vitamin D3 Prevents the Deleterious Effects of Testicular Torsion on Testis by Targeting miRNA-145 and ADAM17: In Silico and In Vivo Study

Testicular torsion (TT) is the most common urological emergency in children and young adults that can lead to infertility in many cases. The ischemia-reperfusion (IR) injury due to TT has been implicated in the pathogenesis of testicular damage. The main pathological mechanisms of contralateral injury after ipsilateral TT are not fully understood. In the presented study, we investigated the molecular and microscopic basis of ipsilateral and contralateral testicular injury following ipsilateral testicular torsion detorsion (T/D) and explored the possible protective role of vitamin D3. The biochemical analysis indicated that IR injury following T/D significantly decreased the activity of testicular glutathione peroxidase (GPx) enzyme, level of serum testosterone, serum inhibin B, and expression of testicular miRNA145, while increased the activity of testicular myeloperoxidase (MPO) enzyme, level of testicular malondialdehyde (MDA), level of serum antisperm-antibody (AsAb), and expression of ADAM-17. The histological and semen analysis revealed that torsion of the testis caused damages on different tissues in testis. Interestingly, administration of vitamin D3 prior to the IR injury reversed the deterioration effect of IR injury on the testicular tissues as indicated by biochemical and histological analysis which revealed normal appearance of the seminiferous tubules with an apparent decrease in collagen fiber deposition in both ipsilateral and contralateral testes. Our results revealed that the protective effect of vitamin D3 treatment could be attributed to target miRNA145 and ADAM17 protein. To further investigate these findings, we performed a detailed molecular modelling study in order to explore the binding affinity of vitamin D3 toward ADAM17 protein. Our results revealed that vitamin D3 has the ability to bind to the active site of ADAM17 protein via a set of hydrophobic and hydrophilic interactions with high docking score. In conclusion, this study highlights the protective pharmacological application of vitamin D3 to ameliorate the damages of testicular T/D on the testicular tissues via targeting miRNA145 and ADAM17 protein.Peer Reviewe

Physiological Performance of Rabbits Administered Buffalo Milk Yogurts Enriched with Whey Protein Concentrate, Calcium Caseinate or Spirulina platensis

The present study examines the impacts of supplementing yogurt with 1% whey protein concentrate (WPC), Ca-caseinate (Ca-CN) and Spirulina platensis on the physiological performance of V-line rabbits receiving diets containing yogurt (at a dose of 5 g/kg body weight/day) and the different meat quality aspects. The results show that fat content was highest (p < 0.05) in yogurt fortified with Spirulina powder, but protein (%) was highest in yogurt enriched with WPC. Yogurt containing Spirulina powder showed a significant (p < 0.05) increase in total antioxidant activity. The final live body weight for G1 was higher than the other groups. However, additives affected the saddle, hind legs, liver and neck percentages significantly (p < 0.05). There were not significant differences for all groups in the forelegs, lung and heart percentages. LDL-cholesterol, total protein, globulin, albumin, creatinine and immunoglobulin M values were lowest (p < 0.05) in the WPC group. Significant improvements appeared in the small intestinal wall, microbiology, growth performance, serum biochemistry, organ histology and meat quality of the group receiving enriched yogurt. Yogurts enriched with WPC, Ca-CN and Spirulina platensis can be used as functional foods

DJ-1 Protects Pancreatic Beta Cells from Cytokine- and Streptozotocin-Mediated Cell Death

<div><p>A hallmark feature of type 1 and type 2 diabetes mellitus is the progressive dysfunction and loss of insulin-producing pancreatic beta cells, and inflammatory cytokines are known to trigger beta cell death. Here we asked whether the anti-oxidant protein DJ-1 encoded by the Parkinson’s disease gene <i>PARK7</i> protects islet cells from cytokine- and streptozotocin-mediated cell death. Wild type and DJ-1 knockout mice (KO) were treated with multiple low doses of streptozotocin (MLDS) to induce inflammatory beta cell stress and cell death. Subsequently, glucose tolerance tests were performed, and plasma insulin as well as fasting and random blood glucose concentrations were monitored. Mitochondrial morphology and number of insulin granules were quantified in beta cells. Moreover, islet cell damage was determined <i>in vitro</i> after streptozotocin and cytokine treatment of isolated wild type and DJ-1 KO islets using calcein AM/ethidium homodimer-1 staining and TUNEL staining. Compared to wild type mice, DJ-1 KO mice became diabetic following MLDS treatment. Insulin concentrations were substantially reduced, and fasting blood glucose concentrations were significantly higher in MLDS-treated DJ-1 KO mice compared to equally treated wild type mice. Rates of beta cell apoptosis upon MLDS treatment were twofold higher in DJ-1 KO mice compared to wild type mice, and <i>in vitro</i> inflammatory cytokines led to twice as much beta cell death in pancreatic islets from DJ-1 KO mice versus those of wild type mice. In conclusion, this study identified the anti-oxidant protein DJ-1 as being capable of protecting pancreatic islet cells from cell death induced by an inflammatory and cytotoxic setting.</p></div

DJ-1 islet cell-autonomously protects beta cells from cytokine-induced apoptosis.

<p><b>(a-p)</b> TUNEL labelling of pancreatic islets exposed to IL-1β, IFN-γ, and TNF-α for 24 hours. Islet sections from male control <b>(a-d;i-l)</b> and DJ-1 KO mice <b>(e-h;m-p)</b> showing staining for cell nuclei <b>(</b>DAPI<b>, a,e,i</b> and <b>m)</b>, beta cells (insulin) and dead cells (TUNEL) <b>(b,f,j</b> and <b>n)</b>, and alpha cells (glucagon) and dead cells (TUNEL) <b>(c,g, k</b> and <b>o)</b>. Merged images are shown in <b>d,h,l</b> and <b>p</b>. Apoptotic cells are shown in red. Scale bar, 50 μm. <b>(q)</b> Quantification of TUNEL positive beta cells in pancreatic islets from control and DJ-1 KO mice after 24 hours exposure to IL-1β, IFN-γ, and TNF-α. The number of apoptotic beta cells per total number of beta cells is presented as percentage of untreated control. For one experiment, islets harvested from 3–5 mice per genotype were pooled, treated and quantified. Statistical significance was assessed using the means of n = 3 independent experiments. *p<0.05 (Two-way ANOVA followed by Tukey’s multiple comparison test). Data are expressed as means ± S.D.</p

DJ-1 is required to reduce loss of beta cell mass following treatment with MLDS.

<p><b>(a,d)</b> Representative fluorescence microscopy images of pancreatic sections from 16 weeks-old male control and DJ-1 KO mice following MLDS treatment, stained for cell nuclei (DAPI) and beta cells (insulin). Scale bars, 0.5 mm. (<b>b</b>,<b>c</b>,<b>e</b>,<b>f</b>) LSM images of pancreatic islets in sections of pancreata from male control <b>(b</b>,<b>c)</b> and DJ-1 KO mice <b>(e-f)</b> after MLDS treatment, stained for cell nuclei (DAPI) and beta cells (insulin) <b>(b,e).</b> Merged images <b>(c,f)</b> are also shown. Scale bars, 50 μm. <b>(g)</b> Morphometric analyses of relative beta cell area from DJ-1 KO and control mice calculated as insulin-positive area per total nuclei area of evenly spaced pancreatic sections. n = 3 mouse pancreata per experimental group. Beta cell area was quantified after four weeks of STZ treatment. For comparison, untreated control/wild type mice without STZ treatment were included. *p<0.05 (One-way ANOVA followed by Tukey´s multiple comparison test). Data are expressed as means ± S.D.</p

MLDS treatment induces a diabetic phenotype in the absence of DJ-1.

<p>Male control and DJ-1 KO mice at 12–13 weeks of age were treated with 40 mg STZ/kg body weight on five consecutive days. Blood glucose concentrations, glucose tolerance, and plasma insulin concentrations were determined. <b>(a,c)</b> Random <b>(a)</b> and fasting <b>(c)</b> blood glucose concentrations in control (black squares) and DJ-1 KO mice (grey squares). n = 6–8 mice per experimental group. (<b>b</b>,<b>d</b>) Corresponding areas under the curve (AUC) to (<b>a</b>,<b>c</b>) are shown for control (black columns) and DJ-1 KO (grey columns) mice each. <b>(e,f)</b> Glucose tolerance test <b>(e)</b> and its corresponding AUC <b>(f)</b> in 14–16 weeks-old control (black squares and black column) and DJ-1 KO mice (grey squares and grey column). The glucose tolerance test was performed after intraperitoneal administration of glucose (1 g/kg body weight). n = 8 mice per experimental group. <b>(g,h)</b> Relative fasting <b>(g)</b> and non-fasting <b>(h)</b> plasma insulin concentrations in 14–16 weeks-old control (black columns) and DJ-1 KO mice (grey columns) normalised to controls. n = 8 mice per experimental group in (<b>g</b>) and n = 5 in (<b>h</b>). *p<0.05 (Student’s t-test in <b>b, d, f-h</b>. Student’s t-test with Holm-Bonferroni correction in <b>a, c, e</b>). All values are means ± SD.</p

DJ-1 islet cell–autonomously protects from STZ-induced beta cell death in vitro.

<p><b>(a-p)</b> LSM live cell images of pancreatic islets from male control <b>(a-d;i-l)</b> and DJ-1 KO mice <b>(e-h;m-p)</b> stained with Hoechst <b>(a,e,i</b> and <b>m)</b>, calcein AM <b>(b,f, j</b> and <b>n)</b>, and ethidium homodimer-1 <b>(c,g,k</b> and <b>o)</b>. Merged images are shown in <b>d,h,l</b> and <b>p</b>. Scale bar, 50 μm. <b>(q)</b> Quantification of dead cells of pancreatic islets from control and DJ-1 KO mice after 24 hours exposure to STZ treatment. Data are presented as ethidium homodimer-1 positive area per total cell nuclei area. For one experiment, islets harvested from 3–5 mice per genotype were pooled, treated and quantified. Statistical significance was assessed using the means of n = 3 independent experiments. *p<0.05 (Two-way ANOVA followed by Tukey’s multiple comparison test). Data are expressed as means ± S.D.</p

DJ-1 is required for maintaining mitochondrial morphology and number of insulin secretory granules after MLDS treatment.

<p><b>(a,b)</b> Electron micrographs of islets from male control <b>(a)</b> and DJ-1 KO mice <b>(b)</b> after 4 weeks of MLDS treatment. <b>(c,d)</b> Image segmentation into mitochondria (red) and insulin secretory granules (green) using the trainable Weka Segmentation plugin for Fiji/ImageJ. Scale bar, 2 μm. <b>(e,f)</b> Quantification of total number of secretory granules <b>(e)</b> and mitochondrial area per section <b>(f)</b> in control and DJ-1 KO mice. n > 30 images per condition from n = 2 control and n = 3 DJ KO mice. Data are expressed as means ± SD.</p