Protective properties of Rydingia persica in reproductive complications induced by diabetes in male rats: An experimental study

Abstract Background In Iranian traditional medicine, Rydingia persica (R.P) is commonly used to treat diabetes mellitus (DM). Objective We assessed the protective effects of R.P against testis and epididymis oxidative stress and the hormonal changes induced by DM. Materials and Methods Forty male Wistar rats (12 wk old) weighing 230-270 gr were divided into five groups (n = 8/each): 1. Control (C); 2. diabetic (D); 3. diabetic + R.P200 (D+R200); 4. diabetic + R.P400 (D+R400); and 5. diabetic + R.P600 (D+R600). Groups C and D received 2 ml of normal saline orally daily for two wk and groups D+R200, D+R400, and D+R600 received 200, 400, and 600 mg/kg body weight of R.P powder, respectively, orally daily for two wk. DM was induced by a single intraperitoneal injection of streptozotocin at 60 mg/kg body weight. We assessed malondialdehyde, glutathione peroxidase, glutathione reductase, superoxide dismutase, catalase, hydrogen peroxide, and glutathione in both the testis and epididymis and also the histological changes of the testis. Results Diabetic rats showed a significantly increased and decreased level of oxidant and antioxidant factors, respectively, and a significantly lower level of serum testosterone and luteinizing hormone than the control group. In the histological study of the testis, deteriorations were observed. Treatment with R.P reversed these changes toward the state of the control group with the highest effectiveness shown by group D+R600. Conclusion The data obtained suggest that R.P powder has antioxidant effects on testis and epididymis tissues in diabetic rats and that it improves histological testicular structure in diabetics. It can also correct testosterone and luteinizing hormone changes induced by DM


Introduction
Diabetes mellitus (DM) is diagnosed based on a high serum glucose level due to a lack of insulin secretion (1), absence of insulin receptor responsiveness, or both. Overall, DM is one of the most important causes of death in developed countries and is responsible for 5% of deaths worldwide (2). DM can lead to many structural and functional effects such as neuropathy, nephropathy, retinopathy, and also disorders of the reproductive system (3,4). Clinical and experimental investigations have revealed major changes in semen volume, spermatogenesis, sperm count, sperm motility, penile erection, ejaculation, serum luteinizing hormone (LH), follicle stimulating hormone, and testosterone levels as well as onset of puberty in diabetic subjects (5)(6)(7).
Oxidative stress (OS) is an important factor induced by hyperglycemia at the beginning and progression of DM complications. One of the main factors affecting the reproductive system in DM is the increased production of reactive oxygen species (ROS), which leads to protein and lipid oxidation (8). The balance between ROS and their scavengers is controlled tightly under physiological conditions. Defects in this balance can occur in metabolic disorders such as DM causing OS in the male reproductive system which can lead to a range of problems. Testicular oxidative damage can lead to biochemical and hormonal changes followed by a decrease in sexual libido and behavior (9)(10)(11).
In recent decades, some studies have shown the therapeutic effects of medicinal plants to control DM by modulating carbohydrate metabolism, repairing the pancreatic beta cell function and release of insulin, improving glucose utilization, and through their antioxidant properties (10,12).

Scheen & V.A.Albert (R.P) -also known as
Otostegia persica -have long been used as a medicinal herb in Iran and elsewhere in the Arab world and wider Asia as a way to alleviate various disorders (13). R.P is a medicinal plant containing polyphenolic compounds. These compounds are extensively distributed in different parts of the plant (14). R.P has some biological effects such as antioxidative, antidiabetic, anticancer, antiarthritic, and antimalarial effects (15,16). Aerial parts of R.P contains components such as flavonoids, steroids, tannins, triterpenoids, and some essential mineral This plant has been used for many varieties of disorders, including DM, but no studies have been done on its use for the reproductive complications of DM. We therefore decided to investigate the protective properties of the aerial parts of R.P against reproductive damage induced by DM by assessing the oxidative status and hormonal and histopathological changes in male rats.

Materials and Methods
This experimental study was carried out in the Department of Biology, Shahid Bahonar University of Kerman in November 2018.

Preparation of Rydingia persica powder
The aerial parts of the R.P were collected from
The rats were divided into five groups (n = 8/each) and were fed orally for two wk: Group C: Control rats, animals received normal saline.
Group D: Diabetic rats, animals received normal saline.

OS markers measurement
To measure the malondialdehyde (MDA) concentration, 0.2 gr of the desired tissue was homogenized in 5 ml of 0.1% trichloroacetic acid and the obtained solution was centrifuged for 10 min at 10,000 g and 4°C. Then, 1 ml of the supernatant was added to 20% trichloroacetic acid solution with 0.5% thiobarbituric acid. Finally, the mixture was heated for 30 min at 95°C and then cooled quickly in ice, and again was centrifuged for 10 min at 4000 g. Finally, the absorbance at 532 nm was read (20).
The amount of reduced glutathione (GSH) was measured using the total and oxidized glutathione (GSSG). Half a gr of the target tissue was homogenized in 2 ml of metaphosphoric acid.
The resulting homogenate was centrifuged for 10 min at 10,000 g using a refrigerator centrifuge.

Preparation of testes for histological studies
For histology examination of the testis, the testis was promptly fixed in 10% formaldehyde, buffered by a solution containing 54 mM of NaH 2 PO 4 and 28 mM of Na 2 HPO 4 (pH = 7.4). To complete the tissue fixation, a transverse section was made through the middle of the testis while immersed in the fixator. Thereafter, the tissues were put in paraffin and cut in slices (6-7 micrometer thick) and placed on the glass slides precoated with albumin. The samples were then deparaffinized with xylol, and finally for histological examination by light microscopy (Nikon, Y-THM, Japan) they were stained with hematoxylin and eosin.
Based on the following criteria, a grading score was applied from 1 to 10 for each tubule crosssection. Complete spermatogenesis with perfect tubules = 10, many spermatozoa but disorganized spermatogenesis = 9, a few spermatozoa = 8, absence of spermatozoa but many spermatids = 7, a few spermatids = 6, absence of spermatozoa and spermatids but many spermatocytes = 5, a few spermatocytes = 4, only presence of spermatogonia = 3, absence of germ cells but presence of Sertoli cells = 2, absence of both germ and Sertoli cells = 1.

Ethical considerations
The study protocol and all animal procedures were approved by the educational assistant of Shahid Bahonar University of Kerman, Kerman, Iran Statistics were performed by one-way analysis of variance (ANOVA) and Tukey's post hoc test, and significance was considered at p < 0.05.

Glucose, testosterone and LH concentrations
The results showed that the glucose concentration was significantly higher in the experimental groups compared to in the C group.
The highest concentration was observed in group D and the lowest in group D+R600 ( Figure 1).

Figures 2 and 3 show that serum testosterone
and LH concentration were significantly lower in the experimental groups compared to in the C group. Table I shows that the testis weight in the D and D+R200 groups was significantly lower compared to in the C group. Also, the D+R400 and D+R600 groups showed a significantly higher weight compared to the D and D+R200 groups.

Effects in the testis tissue
The MDA concentration in the testes in the D, D+R200 and D+R400 groups was also significantly International Journal of Reproductive BioMedicine Ostovan et al. higher compared to in the C group (p < 0.05 and p < 0.01, respectively). However, the D+R600 group showed a significantly lower level compared to the D+R200 group.
The GSH concentration in the testes in the D, D+R200 and D+R400 groups was significantly lower compared to in the C group. Also, the D+R400 and D+R600 groups showed a significantly higher concentration compared to the D and D+R200 groups. Furthermore, the D+R600 group had a significantly higher concentration compared to the D+R400 group.
The GSSG concentration in the testes of the D and D+R200 groups was significantly higher compared to in the C group. Also, the D+R600 group showed a significantly lower concentration compared to the D and D+R200 groups. The D+R400 group had a significantly lower concentration compared to the D+R200 group.
GR activity in the testes of the rats in the D, D+R200, D+R400 and D+R600 groups was significantly lower compared to in the C group. Also, the D+R400 and D+R600 groups showed a significantly higher level compared to the D and D+R200 groups. The D+R600 group showed significantly higher activity compared to the D+R400 group.
GPx activity in the testes of the D, D+R200, D+R400 and D+R600 groups was significantly lower compared to in the C group. Also, the D+R600 rats showed a significantly higher level of activity compared to the D and D+R200 rats.
CAT activity in the testes of the D and D+R200 groups was significantly higher compared to in the C group. Also, the D+R200 group showed a significantly higher level compared to the D+R400 and D+R600 groups.
SOD concentration in the testes of the D, D+R200, D+R400 and D+R600 rats was significantly higher compared to in the C group. The D+R400 and D+R600 groups had a significantly lower concentration compared to the D and D+R200 groups.
H 2 O 2 concentration in the testes in the D, D+R200, D+R400 and D+R600 rats was significantly higher compared to in the C group. Moreover, the D+R600 group had a significantly lower concentration compared to the D, D+R200 and D+R400 groups. Table II shows that the epididymis weight in the D, D+R200 and D+R400 groups was significantly lower compared to in the C group. Also, the D+R600 group showed a significantly higher weight compared to the D and D+R200 groups. The D+R400 group had a significantly higher weight compared to the D and D+R200 groups.

Effects in the epididymis tissue
The MDA concentration in the epididymis of the D, D+R200 and D+R400 rats was significantly higher compared to in the C rats, and the D+R600 group's concentration was significantly higher compared to the C group. Also, the D+R600 group had a significantly lower concentration compared to the D, D+R200 and D+R400 groups.
The GSH concentration in the epididymis of the D, D+R200, D+R400 and D+R600 groups was significantly lower compared to in the C group.
Also, the D+R600 group showed a significantly higher concentration compared to the D, D+R200 and D+R400 groups.
The GSSG concentration in the epididymis in the D, D+R200, D+R400 and D+R600 groups was a significantly higher level compared to in the C group. Also, the D+R600 and D+R200 groups showed a significantly lower concentration in comparison with the D group.
GR activity in the epididymis of the D, D+R200, D+R400 and D+R600 rats was significantly lower International Journal of Reproductive BioMedicine Rydingia persica and reproductive complications compared to in the C group. Also, the D+R400 and D+R600 rats showed a significantly higher level of activity compared to the D and D+R200 groups, and the D+R600 group demonstrated a significantly higher level compared to the D+R200 group.
GPx concentration in the epididymis of the D, D+R200 and D+R400 groups was significantly lower compared to in the C group. Also, the D+R600 group showed a significantly higher concentration compared to the D, D+R200 and D+R400 groups. The D+R400 group also had a significantly higher concentration compared to the D+R200 group.
CAT activity in the epididymis in the D, D+R200, D+R400 and D+R600 groups was significantly higher compared to in the C group. Also, the D+R400 and D+R600 groups showed significantly higher activity compared to the D+R200 group.
The D+R200 and D+R600 groups had significantly lower activity compared to the D group.
SOD activity in the epididymis of the D, D+R200, D+R400 and D+R600 rats was significantly higher compared to in the C group. Also, the D+R600 group showed a significantly lower level compared to the D, D+R200 and D+R400 groups.
H 2 O 2 concentration in the epididymis of the D, D+R200 and D+R400 groups showed a significantly higher level compared to the C group. Also, the D+R600 group had a significantly lower concentration compared to the D, D+R200 and D+R400 groups.  Figure 4A).

Histopathology
In addition, examination of the D group showed testicular parenchyma with spermatogenic arrest (1+), seminiferous tubular atrophy (3+) and thickening of the basal lamina (2+). It also did not show Leydig cell hyperplasia or edema. The Johnson score was 5/10 basal on the morphological scoring system. The mean of the counted number of germ cells per same sized tubules was 190 per tubule ( Figure 4B).
The D+R200 group showed testicular parenchyma with spermatogenic arrest (1+), seminiferous tubular atrophy (2+) and thickening of the basal lamina (2+). It also did not show Leydig cell hyperplasia but moderate interstitial edema was observed (2+). The Johnson score was 5/10 basal on the morphological scoring system. The mean of the counted number of germ cells per same sized tubule was 185.1 per tubule ( Figure 4C).
Microscopic examination of the D+R400 group showed testicular parenchyma with spermatogenic arrest (1+), seminiferous tubular atrophy (2+) and thickening of the basal lamina (1+). In this group, Leydig cell hyperplasia was not observed but the examination did show mild interstitial edema (1+). The Johnson score was 6/10 basal on the morphological scoring system. The mean of the counted number of germ cells per same sized tubule was 197.7 per tubule ( Figure 4D). Furthermore, microscopic examination of the D+R600 group did not show spermatogenic arrest or thickening of the basal lamina, but showed atrophy of the seminiferous tubules, Leydig cell hyperplasia, and mild interstitial edema (1+). The Johnson score was 7/10 basal on the morphological scoring system. The mean of the counted number of germ cells per same sized tubule was 212.5 per tubule ( Figure 4E).

Discussion
In this study, the diabetic group showed increased serum glucose, decreased serum LH and testosterone, decreased testis and epididymis weight, and finally, it showed changes in all variables assayed related to OS, indicating DMinduced OS. Also, diabetic rats treated with R.P with a dose of 600 mg/kg, and in some cases the dose of 400 mg/kg, showed changes in measured variables toward the control group, indicating the protective effect of R.P against OS, testosterone and LH changes.
A previous study emphasized the connection between DM and OS and the resulting complications. DM-induced OS can be the result of increased glucose auto-oxidation from hyperglycemia and protein glycation followed by oxidative degradation of glycated proteins (28). It has been reported that the MDA level in the tissues and blood of diabetic rats is increased due to both lipid peroxidation and reduced antioxidant activity (29,30). In this study, testis and epididymis MDA levels were significantly higher in the diabetic group. Glutathione plays an important role in cellular defense against ROS. We saw a significant depletion of GSH in the testis and epididymis of diabetic rats, which is a sign of OS (31). In the present study, lower levels of GSH and higher levels of GSSG in the diabetic rats probably were related to the lower activity of GR in both the testis and epididymis of diabetic rats as also described in another study (31). We observed higher activity of SOD and CAT in the diabetic group. As has already been shown, SOD converts superoxide anion to H 2 O 2 which in turn is converted to H 2 O by CAT (32,33).
Increased activity of CAT in the heart and liver and decreased activity in the kidney of diabetic rats have already been shown (34). In another study, DM led to increased CAT and SOD activity in the liver and serum of diabetic rats (35). Also, it was shown that CAT activity increased in the testis, epididymis and liver of diabetic rats (33,36). In addition, increased SOD and CAT enzyme expression with hyperglycemia have been demonstrated (37). So, the increased activity of SOD and CAT observed in the present study can be interpreted as a compensatory response against hyperglycemia-induced OS.
We saw decreased serum testosterone in the diabetic group. Leydig cell membrane injury induced by OS can be one explanation for this (15). It can also be the result of steroidogenic defects in the Leydig cells in DM (16). Moreover, the mechanism by which testosterone is reduced in DM may be the direct effect of glucose or its metabolism. It has been reported that insulin stimulates the hypothalamus and hypophysis for secretion of follicle stimulating hormone and LH; therefore, DM leads to a reduction in these two hormones and thereby disturbs spermatogenesis and testosterone production (38). In the present study, diabetic rats treated with R.P with the dose of 400 or 600 mg/kg showed changes in the measured variables toward the control group, indicating the protective effect of R.P against OS.
Honey has been shown to reverse the reduced LH and testosterone in diabetic animals toward the control group (39). In our study, the D+R400 group did not display any effects on LH concentration, but showed increased testosterone concentration, while for the D+R600 group, effects both on LH and testosterone were observed. The reason for this could be that at a dose of 600 mg/kg, R.P could have some effect on hypophysis, but at dose of 400 mg/kg, R.P had its effect directly on the testis (40).
The histological examinations of the testis showed that in diabetic rats, spermatogenesis was impaired compared to in control rats. These histological changes included a decreased number of spermatogonia, atrophy of the seminiferous tubules, thickening of the basal lamina, hypoplasia of the Leydig cells, and presence of edema in the diabetic testes. In the present study, the Johnson scores of the diabetic rats were 5, indicating testicular damage. Our results are in agreement with a study that showed DM causes cell death through apoptosis leading to testicular dysfunction, seminiferous tubule atrophy, reduction in tubule diameter, and diminished spermatogenetic cell series (41). Basal lamina thickness has a crucial role in spermatogenesis and its increase results in diminished sperm production and decrease in the size of seminiferous tubules (42). In the histopathological examination of the epididymis, we observed a decrease in sperm density, and atrophy and vacuolation of the epithelium in the diabetic group, which is consistent with another study (43). However, treatment with R.P had a protective effect on the studied tissues, which is consistent with biochemical data. R.P is used widely by the people of Baluch who live in the southeastern part of Iran. It has some antioxidant, antidiabetic, antimicrobial, antiinflammatory and antimalarial properties (14). The hypoglycemic effect of R.P may be due to the presence of one or more antidiabetic components which demonstrate synergistic properties (44). Phytochemical screening of the R.P crude extract have revealed the presence of flavonoids, phenolic acids, and steroids/ triterpenoids (44,45).
Flavonoids have been shown to work as antidiabetic compounds, because they possess multiple characteristics that promote both glucoselowering actions (insulin-mimetic) and insulin secretion (insulin secretagogue) (46). Two flavonoids, morin and quercetin, both present in R.P, have been shown to have strong antioxidant activity, which is equal to BHA and stronger than alpha tocopherol (47). Quercetin strongly inhibits the transport of glucose and fructose through intestinal cell GLUT2 (48). Another study showed it potentiates insulin secretion induced by glucose and has a protective effect against beta-cell oxidative injury induced by H 2 O 2 (49). The effects of quercetin administrated intraperitoneally was analyzed (10 or 15 mg/kg body weight) for 10 days in control and diabetic rats which significantly decreased glucose levels in the diabetic rats. Quercetin increases the glucokinase activity, leading to this effect (50). Also it has been shown that quercetin has a similar effect as metformin (51).
The antioxidant activity of morin has been reported to be effective against OS in several disease profiles (52)(53)(54). Administration of morin has been reported to improve the pathological condition of hyperglycemia, glucose intolerance, lipid peroxidation, and insulin resistance (52,55). Improvement in insulin receptor signaling and reduction of hyperglycemia and lipid deposits in the liver of diabetic rats has been reported, as well as antidiabetic and antioxidant effects, which directly supports the notion of the antidiabetic activity of morin (56,57). In another study, morin administration was shown to increase insulin receptor activation and decrease gluconeogenesis potency (58). The effect of morin has been found to be similar to the conventionally used antidiabetic drug Glibenclamide. Moreover, monoterpene derivatives in the R.P flower are other compounds with antioxidant activity (59). Therefore, it can be suggested that the reason for the improvement in the hyperglycemic, oxidative, and antioxidant status observed in the rats treated with R.P powder was due to the insulin-like activity and antidiabetic properties of R.P.

Conclusion
Our findings showed that R.P powder in doses of 600 or 400 mg/kg body weight had protective