Research Article | Open Access
Ali Alsarhan, Kawther Faisal Amawi, Inas Saleh Al-Mazari, Hashem Abu Hurirah, Ahed J. Alkhatib, "The Compound Expression of HSP90 and INOS in the Testis of Diabetic Rats as Cellular and Pathologic Adverse Effects of Diabetes", Analytical Cellular Pathology, vol. 2020, Article ID 3906583, 7 pages, 2020. https://doi.org/10.1155/2020/3906583
The Compound Expression of HSP90 and INOS in the Testis of Diabetic Rats as Cellular and Pathologic Adverse Effects of Diabetes
Introduction. Diabetes is increasingly prevalent at global level and associated with various impacts including the male reproductive system. Aims. This research is aimed at investigating the influence of diabetes on the localization and expression of HSP90 and iNOS in the testicular tissue of diabetic rats. Methods. A diabetic model was developed through a single injection of alloxan monohydrate intraperitoneally (purchased from Sigma-Aldrich) 120 mg/kg body weight following fasting for 12 hrs. The experiment involved two groups, the control and diabetic groups with 10 albino rats in each group. Diabetes was considered if glucose concentration was ≥200 mg/dl. The experiment duration was for one month. After the experiment had finished, all rats were terminated and prepared for routine histological and immunohistochemical examination. Results. The results revealed that diabetes caused morphological changes at histological level in testicular tissue. Immunohistochemical examination showed that diabetes significantly upregulated the expression of both HSP90 and iNOS in the testicular tissue of diabetic rats as compared with that of the control group (). Conclusion. Diabetes may induce adverse health effects on the male reproduction through upregulation of HSP90 and iNOS in the testicular tissue of diabetic rats.
Diabetes is not a disease, but it is rather a sum of diseases known as metabolic diseases resulting from increased glucose levels (hyperglycemia) due to insulin insufficiency as in terms of either secretion or function or both [1, 2].
There is a loss of insulin secretion resulting in insulin deficiency associated with type 1 diabetes . The autoimmune attack by T-cells to pancreatic beta cells is believed to be the reason beyond type 1 diabetes [4, 5].
Autoimmunity is thought to cause type 1 diabetes that leads to insufficient production of insulin by pancreatic beta cells [4, 6]. Insulin insufficiency impacts the whole metabolic activities of the body . There are various complications associated with bad management of type 1 diabetes such as cardiovascular diseases, diabetic neuropathy, and diabetic retinopathy [8–12]. Type 1 diabetic patients who are under well management can suffer from damage in peripheral organs related to diabetic complications [13, 14].
There are adverse effects of type 1 diabetes against the testicular function on cellular level and the production of testosterone . However, among patients with diabetes, the development of oxidative stress has been shown to deteriorate the testicular function .
The main function of the testis is to produce testosterone and sperm. These functions work to retain the characteristics of male and species preservation . The testis of mammals is lined by a capsule composed of a fibrous tissue known as the tunica albuginea for protective purposes . Reactive oxygen species (ROS) disturbs performance of the testicular functions [22, 23].
Heat shock protein (HSP) 90 exists in cells as a chaperone that acts in controlling cellular changes such as protein refolding, removing denatured proteins through the process of the proteasome . HSP90 participates in making what is called the machine of chaperone . Various types of stress can induce the production of HSP90 including cancer and diabetes . Several studies pointed that the overexpression of HSP90 has negative impacts such as lowering cytoprotective induction of other HSPs including HSP40 and HSP70 through interaction with heat shock factor 1 (HSF-1) [25–28].
Nitric oxide (NO) is known by its ability to induce signals in several tissues and works to control a variety of physiological and cellular processes. Overproduction of NO may occur in some clinical conditions including the cardiovascular system as well as diabetes that impacts the vascular functions [29, 30].
1.1. Study Aims
The main aim of his study was to explore both the localization and expression of HSP90 and iNOS in the testicular tissue of diabetic rats.
2. Methods and Materials
2.1. Preparation of Animals and Diabetic Model
Twenty rats (males, albino) were selected in randomization approaches and subdivided to either the control group () or the diabetic group (). We conducted this study at the Department of Biology that is hosting animal house unit in Yarmouk University, Jordan. According to the instructions of the university, IRB gave the approval of this study. Animals were bought from the animal unit. Animals were weighted before the initiation of the experiment. The mean of animal’s weight was grams. A private room in the animal house was hosted for this study in which animals were placed in cages. One week prior to the conduction of research, all rats were treated as the same and were exposed to the same environmental conditions for acclimatization purposes.
Diabetes in rats was developed in rats using one dose of alloxan monohydrate (Sigma-Aldrich) through intraperitoneal injection (120 mg/kg) following 12-hour fasting. Blood glucose was measured daily to assure that animals were hyperglycemic (≥200 mg/dl) using a commercial device (Glucocheck, HomeMed (Pty) Ltd.). All animals were terminated at the end of the study (study duration was 1 month). The testes were removed, washed in normal saline, and fixed in 10% formalin for 24 hrs; then they were processed and stained for hematoxylin and eosin for routine histological examination, and other sets were stained for immunohistochemistry to examine the localization, expression, and immunoreactivity of HSP90 and iNOS. We have developed our protocols for immunohistochemistry in our laboratory and previously published them [31–33].
2.2. Immunohistochemistry Protocols
Testis tissue samples were processed, sectioned, and mounted on charged slides. Sections were then passed from deparaffinization till water. Immunohistochemistry staining protocols started by placing sections in a container containing a solution of 1% hydrogen peroxide for 20 minutes to neutralize internal cellular activity of peroxidase enzyme. A washing step by phosphate-buffered saline (PBS) (-7.4) was followed, and then 1% bovine serum albumin (BSA) was used to overcome nonspecific binding. After washing with PBS, primary monoclonal antibody solution (iNOS, 1 : 100/HSP90, 1 : 100; Santa Cruz Biotechnology) was incubated with sections for 1 hr using a humid chamber. A washing step by PBS was performed, and then incubation with secondary biotinylated antibodies for 20 minutes was followed, then washed with PBS and incubated with streptavidin conjugated with horseradish peroxidase enzyme for 20 minutes, followed by washing with PBS carried out. Immunohistochemical reactions were viewed through the reaction with DAB (diaminobenzidine) until the reaction had developed (brown color), and the reaction was finished by washing with tap water. Hematoxylin was used as a counterstain for 30 seconds; then sections were dehydrated and mounted with mounting medium.
2.3. Explanation of the Results
We evaluated the expression of HSP90 and iNOS based on the reading of output of Adobe Photoshop Software version 7.2. Micrographs representing immunostained sections were interpreted in terms of pixels. The pixels represented the colors of the biomarker (brown) and the color of the remaining tissue (blue). The number of pixels representing the color of the biomarker is divided by the whole number of pixels (the sum of brown and blue) to give a ratio of expression.
2.4. Statistical Analysis
We used the software SPSS version 21.0 to analyze the data. Differences in the means were computed using an independent -test. Significance was considered if . Data representing iNOS and HSP90 for each group were used as the .
3.1. Biochemical Findings of Study
Table 1 shows that the mean of glucose in the control group was , while that in the diabetic group was . The difference in means was statistically significant (). The mean of insulin was in the control group, and in the diabetic group, the insulin mean was . The difference in means was statistically significant ().
3.2. Histological Changes of Rat Testicular Tissue in the Control and Diabetic Groups
As seen in Figure 1, normal histology of rat testicular tissue is presented. There are a blood vessel (green arrow), Leydig cells (red arrow), and seminiferous tubules (yellow arrow).
As demonstrated in Figure 2, diabetes induced some histological changes in the testicular tissue such as the existence of inflammatory cells (blue arrows), congestion (yellow arrows), and transforming changes in seminiferous tubules from being oval to being elongated (red arrow).
3.3. Immunohistochemical Studies
We studied the localization and expression of HSP90 and iNOS in the study groups.
3.3.1. The Level of HSP90 in Study Groups
As seen in (Table 2), the level of HSP90 in the control group was , and diabetes significantly increased the level of HSP90 () ().
3.3.2. The Immunohistochemical Expression of HSP90 in Study Groups
As demonstrated in Figure 3, the expression of HSP90 was located in the nucleus as indicated by the arrow in seminiferous tubules. Diabetes led to significantly increased expression of HSP90 in testicular tissue and seminiferous tubules (Figure 4) as compared to the control group (). Moreover, the expression of HSP90 is obvious in both the cytoplasm and nucleus due to the diabetic effects.
3.3.3. The Level of iNOS in the Control and Diabetic Groups
As indicated in (Table 3), the mean level of iNOS in the testicular tissue in the control group was , and that was significantly less than that in the diabetic group () ().
3.3.4. The Immunohistochemical Expression of iNOS in Study Groups
As seen in Figure 5, the expression of iNOS was not strong in terms of intensity and was localized mainly in the nucleus (blue arrow) and to a less extent in the cytoplasm of seminiferous tubules (red arrow).
As indicated in Figure 6, diabetes increased the expression of iNOS seminiferous tubules of testicular tissue. The localization of iNOS was prominent in the nucleus (blue arrow).
The results of the present study showed that the diabetic model was successfully induced as reflected by both the glucose level () and the insulin level (). As compared to control, the differences were statistically significant. These findings showed that the induction of the diabetic model permits the development of further changes in histological and immunohistological levels. These findings confirmed previous studies that were based on the induction of diabetes [1, 2, 4, 5]. On the histological level, the results of this study showed different types of changes including the development of inflammatory changes as reflected by eosinophilic changes, the lack of well preservation of seminiferous tubules, and the lack of well intact between seminiferous tubules. These changes are thought to affect the main functions of the testicular tissue such as the production of testosterone and sperms [20, 34–36].
The results of the present study showed that HSP90 was significantly expressed in the testicular tissue of diabetic rats compared with control group rats (), although HSP90 is potentially important in offering cytoprotection at physiological limits . But in the case where its upregulation is increased, it is expected to develop negative impacts through inhibition of production of other HSPs that lead to the cytoprotection [25–28].
The results of the present study showed that iNOS was significantly expressed in the testicular tissue of diabetic rats compared with the testicular tissue in rats of the control group. This implies that the testicular tissue of diabetic rats is impacted by events of the oxidation process that leads to the infertility problems. Such trends were discussed in other studies [37–40].
This study showed synergistic effects of both HSP90 and iNOS impacting the testicular tissue of diabetic rats. Appropriate therapeutic strategies for diabetes and infertility problems resulting from diabetes may be achieved through lowering the expression of HSP90 and iNOS.
Diabetes type 1 impacts testicular functions through synergistic actions of HSP90 and iNOS.
The experimental data used to support the findings of this study are included within the article.
Conflicts of Interest
All authors have no conflict of interest.
- M. S. Atta, E. A. Almadaly, A. H. El-Far et al., “Thymoquinone defeats diabetes-induced testicular damage in rats targeting antioxidant, inflammatory and aromatase expression,” International Journal of Molecular Sciences, vol. 18, no. 5, p. 919, 2017.
- H. Shaikh and K. S. Vinoy, “Mohammad A. Diabetes mellitus and impairment of male reproductive function: role of hypothalamus pituitary testicular axis and reactive oxygen species,” Iranian journal of Diabetes and Obesity, vol. 8, no. 1, pp. 41–50, 2017.
- American Diabetes Association, “Diagnosis and classification of diabetes mellitus,” Diabetes Care, vol. 33, Supplement 1, pp. S62–S69, 2009.
- A. L. Burrack, T. Martinov, and B. T. Fife, “T cell-mediated beta cell destruction: autoimmunity and alloimmunity in the context of type 1 diabetes,” Frontiers in Endocrinology, vol. 8, p. 343, 2017.
- K. I. Rother, “Diabetes treatment — bridging the divide,” The New England Journal of Medicine, vol. 356, no. 15, pp. 1499–1501, 2001.
- J. W. Yoon and H. S. Jun, “Autoimmune destruction of pancreatic β cells,” American Journal of Therapeutics, vol. 12, no. 6, pp. 580–591, 2005.
- N. Baldini and S. Avnet, “The effects of systemic and local acidosis on insulin resistance and signaling,” International Journal of Molecular Sciences, vol. 20, no. 1, p. 126, 2019.
- S. Tesfaye, A. J. Boulton, P. J. Dyck et al., “Diabetic neuropathies: update on definitions, diagnostic criteria, estimation of severity, and treatments,” Diabetes Care, vol. 33, no. 10, pp. 2285–2293, 2010.
- V. Spallone, D. Ziegler, R. Freeman et al., “Cardiovascular autonomic neuropathy in diabetes: clinical impact, assessment, diagnosis, and management,” Diabetes/Metabolism Research and Reviews, vol. 27, no. 7, pp. 639–653, 2011.
- J. Fleischer, K. Yderstraede, E. Gulichsen et al., “Cardiovascular autonomic neuropathy is associated with macrovascular risk factors in type 2 diabetes,” Journal of Diabetes Science and Technology, vol. 8, no. 4, pp. 874–880, 2014.
- L. R. Tannus, K. R. Drummond, E. L. Clemente, M. F. da Matta, and M. B. Gomes, “Brazilian Type 1 Diabetes Study Group (BrazDiab1SG) predictors of cardiovascular autonomic neuropathy in patients with type 1 diabetes,” Frontiers in Endocrinology, vol. 5, p. 191, 2014.
- V. Spallone, “Update on the impact, diagnosis and management of cardiovascular autonomic neuropathy in diabetes: what is defined, what is new, and what is unmet,” Diabetes & Metabolism Journal, vol. 43, no. 1, pp. 3–30, 2019.
- R. Dimova, T. Tankova, V. Guergueltcheva et al., “Risk factors for autonomic and somatic nerve dysfunction in different stages of glucose tolerance,” Journal of Diabetes and its Complications, vol. 31, no. 3, pp. 537–543, 2017.
- C. L. Martin, J. W. Albers, R. Pop-Busui, and DCCT/EDIC Research Group, “Neuropathy and related findings in the diabetes control and complications trial/epidemiology of diabetes interventions and complications study,” Diabetes Care, vol. 37, pp. 31–38, 2013.
- H. Shoorei, A. Khaki, A. A. Khaki, A. A. Hemmati, M. Moghimian, and M. Shokoohi, “The ameliorative effect of carvacrol on oxidative stress and germ cell apoptosis in testicular tissue of adult diabetic rats,” Biomedicine & Pharmacotherapy, vol. 111, pp. 568–578, 2019.
- S.-H. Abtahi-Evari, M. Shokoohi, A. Abbasi, A. Rajabzade, H. Shoorei, and H. Kalarestaghi, “Protective effect of Galega officinalis extract on Streptozotocininduced kidney damage and biochemical factor in diabetic rats,” Crescent Journal of Medical and Biological Sciences, vol. 4, pp. 108–114, 2017.
- W. R. Rowley, C. Bezold, Y. Arikan, E. Byrne, and S. Krohe, “Diabetes 2030: insights from yesterday, today, and future trends,” Population Health Management, vol. 20, no. 1, pp. 6–12, 2017.
- R. A. Condorelli, S. La Vignera, L. M. Mongioì, A. Alamo, and A. E. Calogero, “Diabetes mellitus and infertility: different pathophysiological effects in type 1 and type 2 on sperm function,” Frontiers in Endocrinology, vol. 9, p. 268, 2018.
- M. Kanter, C. Aktas, and M. Erboga, “Protective effects of quercetin against apoptosis and oxidative stress in streptozotocin-induced diabetic rat testis,” Food and Chemical Toxicology, vol. 50, no. 3-4, pp. 719–725, 2012.
- M. Darbandi, S. Darbandi, A. Agarwal, D. D. PallavSengupta, R. Henkel, and M. R. Sadeghi, “Reactive oxygen species and male reproductive hormones,” Reproductive Biology and Endocrinology, vol. 16, no. 1, p. 87, 2018.
- L. D. Russell and L. R. França, “Building a testis,” Tissue and Cell, vol. 27, no. 2, pp. 129–147, 1995.
- P. Kavoussi, R. A. Costabile, and A. Salonia, Clinical Urologic Endocrinology: Principles for Men’s Health, Springer, London, 2012.
- J. L. Jameson, Harrison’s Endocrinology, McGraw-Hill Education, New York, 4 edition, 2016.
- J. Li, W. Xue, X. Wang et al., “HSP90 as a novel therapeutic target for posterior capsule opacification,” Experimental Eye Research, vol. 189, article 107821, 2019.
- A. Ali, S. Bharadwaj, R. O’Carroll, and N. Ovsenek, “HSP90 interacts with and regulates the activity of heat shock factor 1 in Xenopus oocytes,” Molecular and Cellular Biology, vol. 18, no. 9, pp. 4949–4960, 1998.
- N. Erekat, A. Al-Khatib, and M. Al-Jarrah, “Heat shock protein 90 is a potential therapeutic target for ameliorating skeletal muscle abnormalities in Parkinsons disease,” Neural Regeneration Research, vol. 9, no. 6, pp. 616–621, 2014.
- H. R. Kim, H. S. Kang, and H. D. Kim, “Geldanamycin induces heat shock protein expression through activation of HSF1 in K562 erythroleukemic cells,” IUBMB Life, vol. 48, no. 4, pp. 429–433, 1999.
- J. Zou, Y. Guo, T. Guettouche, D. F. Smith, and R. Voellmy, “Repression of heat shock transcription factor HSF1 activation by HSP90 (HSP90 complex) that forms a stress-sensitive complex with HSF1,” Cell, vol. 94, no. 4, pp. 471–480, 1998.
- M. Al-Jarrah, M. B. Ahmad, M. Maayah, and A. Al-Khatib, “Effect of exercise training on the expression of p53 and iNOS in the cardiac muscle of type I diabetic rats,” Journal of Endocrinology and Metabolism, vol. 2, no. 4-5, pp. 176–180, 2012.
- S. Habib and A. Ali, “Biochemistry of nitric oxide,” Indian Journal of Clinical Biochemistry, vol. 26, no. 1, pp. 3–17, 2011.
- A. Al-khatib, “Co-expression of iNOS and HSP70 in diabetes type 1 makes a rational hypothesis to explain the diabetic neuropathy,” European Scientific Journal, vol. 9, no. 3, pp. 145–156, 2013.
- A. AlKhatib, F. Laiche, M. Alkhatatbeh et al. et al., “Leaf extract of U. Pilulifera down regulates the expression of INOs in kidneys of diabetic rats,” European Scientific Journal, vol. 10, no. 21, pp. 302–309, 2014.
- L. A. Raffee, K. Z. Alawneh, A. J. Al-Khatib, and L. W. Al-Mehaisen, “Overexpression of HSP90 in skin of diabetic rats impacts wound healing process,” Research Journal of Biological Sciences, vol. 11, pp. 63–66, 2016.
- A. P. Rolo and C. M. Palmeira, “Diabetes and mitochondrial function: role of hyperglycemia and oxidative stress,” Toxicology and Applied Pharmacology, vol. 212, no. 2, pp. 167–178, 2006.
- T. Yu, J. L. Robotham, and Y. Yoon, “Increased production of reactive oxygen species in hyperglycemic conditions requires dynamic change of mitochondrial morphology,” Proceedings of the National Academy of Sciences of the United States of America, vol. 103, no. 8, pp. 2653–2658, 2006.
- M. F. Sönmez, E. Kılıç, D. Karabulut, K. T. Çilenk, E. Deligönül, and M. Dündar, “Nitric oxide synthase in diabetic rat testicular tissue and the effects of pentoxifylline therapy,” Systems Biology in Reproductive Medicine, vol. 62, no. 1, pp. 22–30, 2015.
- N. Asadi, M. Bahmani, A. Kheradmand, and M. Rafieian-Kopaei, “The impact of oxidative stress on testicular function and the role of antioxidants in improving it: a review,” Journal of Clinical and Diagnostic Research, vol. 11, no. 5, pp. IE01–IE05, 2017.
- H. Nasri and M. Rafieian-Kopaei, “Protective effects of herbal antioxidants on diabetic kidney disease,” Journal of Research in Medical Sciences : The Official Journal of Isfahan University of Medical Sciences, vol. 19, no. 1, pp. 82-83, 2014.
- M. Bahmani, M. Mirhoseini, H. Shirzad, M. Sedighi, N. Shahinfard, and A. Rafieian-Kopaei, “A review on promising natural agents effective on hyperlipidemia,” Journal of Evidence-Based Complementary & Alternative Medicine, vol. 20, no. 3, pp. 228–238, 2015.
- M. Rafieian-Kopaie and A. Baradaran, “Plants antioxidants: from laboratory to clinic,” Journal of Nephropathology, vol. 2, no. 2, pp. 152-153, 2013.
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