The effects of gallic acid and metformin on male reproductive dysfunction in diabetic mice induced by methylglyoxal: An experimental study

Abstract Background Diabetes mellitus is a disease that has reached a dangerous point. Today, nearly 500 million men and women around the world live with diabetes. Gallic acid (Gal) affects diabetes. Objective To evaluate the effects of Gal and metformin (met) on the levels of glucose, insulin, testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), sperm count, antioxidant status, and histological changes in the testes of diabetic mice induced by methylglyoxal (MGO). Materials and Methods In this experimental study, 50 male adult NMRI mice, weighting 25-30 gr, aged 3-4 months were randomly divided into five equal groups (n = 10/each). (i) Control (vehicle, normal saline), (ii) MGO (600 mg/kg/d) orally for 28 days, (iii) Gal (50 mg/kg/d), (iv) MGO+Gal, and (v) MGO+met (200 mg/kg/d). Gal and met were administered orally for 21 consecutive days after the induction of diabetes. Blood samples were taken at 24 hr after the latest doses of treatment. Histological assessment of the testis was done, and the epididymis sperm count was obtained. Antioxidant indices, glucose, insulin, LH, FSH, and testosterone levels were measured. Results In the MGO group compared to the control group, insulin, glucose (p = 0.001), LH (p = 0.04) and malondialdehyde (p = 0.001) were increased. However, the level of testosterone (p = 0.001), seminiferous tubule diameters, epithelial height, sperm count, superoxide dismutase activity (p = 0.02), and testis volume (p = 0.01) were decreased. The results indicated that Gal and met ameliorated the MGO effects. Conclusion These findings suggested that the animals receiving MGO became diabetic. According to the results, Gal and met can effectively prevent MGO-induced diabetes. The effect of Gal was equivalent and sometimes better than metformin.


Introduction
Diabetes mellitus (DM) is a disorder that is widely studied in terms of impaired glucose regulation, thus providing insights into glucose metabolism. Diabetes is a group of chronic and metabolic diseases that are caused by defects in insulin secretion or insulin function. DM has a negative effect on several organs of the body, and can harm the cardiovascular system, nerves, and kidneys, eyes, and male reproductive system, leading to degenerative changes in the testicles and epididymis, decrease in semen volume, sperm count and sperm motility, and changes in sperm  (4). Dhar and colleagues reported that intravenous injection of MGO caused the induction of type-2 diabetes and pancreatic β-cells dysfunction in their study (5). Also, MGO is used for the induction of type-2II diabetes in rodents. Oxidative stress is involved in the pathophysiology and reproductive disorders of men associated with diabetes (6).
Gallic acid (Gal) a type of polyphenol and is commonly present in gallnuts, sumac, tea leaves, oak bark, different berries, and grapes.
The chemical formula of Gal is C6H2(OH)3COOH.
Pharmacological studies have suggested that Gal has anti-inflammatory, anti-coagulant, and antitumor effects (7). Gal, as a potent antioxidant, has antidiabetic properties (8). Oyagbemi and colleagues showed that GA can have a beneficial effect on testis cyclophosphamide toxicity in rats and can improve reproductive parameters (9).
Since the effects of Gal on reproductive function in MGO-induced diabetic rats have not been previously reported, the aim of this study was to investigate the effect of Gal on the testicular tissue, testosterone, luteinizing hormone (LH), follicle-stimulating hormone (FSH), and oxidative stress index in diabetic male mice.

Drugs
All chemicals were of analytical grade. MGO, Gal, and met were obtained from Sigma Company (St. Louis, MO), and the Ketamine, and xylazine from Alfasan (the Netherlands).

Analysis of tissue
Following an overnight fasting, the animals were anesthetized with ketamine (60 mg/kg) and xylazine (10 mg/kg), blood was taken from the heart, the testis region was cut, and the epididymis was removed for sperm count. The blood samples

Sperm assessment
The

Histology assessment
The left testis of each animal was removed and kept in a 10% formalin solution. Six microscopic slides per animal were evaluated for histological features such as vacuolization (empty spaces within the germinal epithelium) and sloughing the germinal epithelium. The average percentage of normal and damaged tubules was also estimated (3). We also considered 150 tubules for each mouse to determine the seminiferous tubules diameters, and the lumen diameter using a Mutic Images software (Plus 3.0).

Statistical analysis
Data are expressed as mean ± SE. Statistical analysis was completed using SPSS software version 17 (Statistical Package for the Social Sciences, Inc., Chicago, II, USA). Kruskal-Wallis test was used to determine the normality of the variables. Also, the variance was tested for homogeneity. A comparison of the data between the groups was achieved using a oneway analysis of variance (ANOVA) followed by post hoc least significant difference tests. P < 0.05 were considered statistically significant.  (Table I).

Effect of MGO, Gal, and met on the testicular morphology
Although the testis width (p = 0.007) and volume (p = 0.01) in the MGO group were significantly smaller than inthe control group, no significant differences were noted in the weight and width of testes between the groups (p > 0.05). Administration of Gal and MGO + Gal was associated with a large width (p = 0.03) and volume of the testis (p = 0.04) compared to the MGO group, while a smaller relationship was not found for the met administration (Table II).

Discussion
The results showed that diabetes is well induced by MGO, as the measured blood glucose and plasma insulin levels clearly indicate this, in agreement with the study of Lee and colleagues (10). According to the present findings, Gal and met administration can cause an improvement in diabetes outcomes. Our findings demonstrated the antidiabetic effect of Gal.
In this study, MGO administration was associated with smaller seminiferous tubule diameters, epithelial height, sperm count, level of testosterone, SOD activity, and testis volume, while the level of LH and malondialdehyde were higher and there was no significant differences in the levels of FSH. Studies on diabetic rats and diabetic men have also confirmed a decline in male reproductive ability (4)(5)(6). These studies also showed that male diabetic mice can have diminished testis volume and a lower epididymis sperm count (1,10). In a study of the oxidative balance of the male reproductive system, it was shown that hyperglycemia can cause oxidative stress and apoptosis (14). Diabetic patients may experience sexual dysfunction (15). Plasma MGO levels are 1.3 times higher than in healthy people (4). Glucose metabolism in testicular cells has unique properties that cause these cells to be damaged in diabetic patients (1,16). Also, reactive oxygen species produced through hyperglycemia are an important mechanism by which DM affects male reproductive health (1). It was showed that intravenous injection of MGO, inducing type-2 diabetes, can lead to dysfunction of beta cells (5).
It was also reported that the incidence of diabetes, hyperglycemia, and pancreatic damage due to MGO in mice (17).
In the present study, Gal administration was associated with larger sperm count, seminiferous tubule diameters, epithelial height, testosterone, and LH level. These findings indicated positive effects of Gal on the reproductive system in diabetic male mice. In regard to the antioxidant effect of Gal, it was concluded that this effect may be related to the antioxidant properties of Gal. In this study, Gal and met seemed to ameliorate the MGO effects. These results are consistent with at study of Gal administration in diabetic rat models that reported an increase in the ability of the male reproductive system (7,8). In our study, the effect of Gal was equivalent and sometimes better than met; for example GA was associated with a higher testis volume and improved sperm count compared to the MGO group, but met did not demonstrate similar effects. It was reported an effect of met on MGO in patients with type-2 diabetes (18). It was also reported that metformin was effective in reducing glycation (19). Studies have shown that Gal can improve beta cells, and can also be used as a drug to increase insulin secretion (20,21). In agreement with our study, it was reported that Gal can have antidiabetic potential in Emblica officinalis and can be effective in regulating pAkt, PPAR-γ, and Glut4 through its antidiabetic activity. Therefore, they recommended E. officinalis as a dietary supplement to protect against diabetes (22). As a result, Gal could be a candidate for diabetes-related changes in the male reproductive system. Yousuf and colleagues reported that advanced glycation end products/receptors for AGEs downstream signaling stimulates reactive oxygen species and decrease the antioxidant status that collectively synthesizes collagen and fibrosis. They showed that concomitant treatment of Gal with glyoxal inhibited these changes and improved the fibrotic effects of glyoxal in rats (23). It was showed that Gal can improve insulin resistance in type-2 diabetic rats. Gal also increased the glucose uptake and translocation of insulin-stimulated glucose transporters, decreases FBG, and improved the glucose tolerance test (24). Gal may be of benefit in reproductive health protection by decreasing diabetic-based testicular damage (25).
Surprisingly, the lower testis weight in the MGO group compared to the control group was not statistically significant, but the testis volume in the MGO group was much lower than in the control group. Decreased TV is associated with lower testicular function (< 12 cm 3 ). It was reported that the conventional sperm parameters (concentration, motility, and morphology) and endocrine function (gonadotropins and testosterone serum concentrations) were altered with reduced TV (26). Daily sperm production and TV are significantly reduced in older men (27). Therefore, TV represents an androgen index that is even better than a single measurement of testosterone (28).
This study had some limitations: first, we did not have enough budget to investigate cell signaling pathways, and second, we did not conduct any genetics experiments. Further research is needed on genetic and cellular evaluations.

Conclusion
The study found that MGO induced DM by promoting oxidative stress, reducing the abilities of the male reproductive system and fertility. Gal and met were associated with higher SOD activity and testosterone levels and lower LH and MDA levels, which improve the male reproductive system. Gal and met also appeared to have a positive effect on the tissue of the testis. Thus, it seems from the results that Gal and met improved the diabetesrelated changes in the reproductive system by inhibiting oxidative stress in the MGO-induced diabetic model, and stopping the production of free radicals. The study also found that the GA effect was equal to and even better than met; for example, Gal was associated with larger TV and sperm count compared to the MGO group, but met was not. The study's findings suggest that Gal could be a candidate for reducing diabetes-related changes in the male reproductive system.