# Resveratrol Mitigates Diabetic Testicular Dysfunction, Endocrine Deficits, and Insulin Resistance via Suppression of Sperm-Endocrine Aberrations and Oxidative Inflammation in Rats

Authors: Hany Elsawy; Ademola C. Famurewa; Azza Sedky
Journal: Andrologia
 (2023)

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## Abstract

Diabetes mellitus (DM) provokes reproductive impairments through endocrine disturbance, sperm deficits, and testicular oxidative inflammation. The study investigated the reproductive protective effects of resveratrol (RSV) against testicular oxidative inflammation, sperm/endocrine deficits, and insulin resistance in streptozotocin- (STZ-, 65 mg/kg) induced DM rat model. Male rats were randomly divided into 4 groups (n=6): control, DM, RSV (150 mg/kg bw, orally), and RSV+DM group (21 days). The nontreated DM rats showed marked decreases in serum insulin, reproductive hormones (T, LH, and FSH), and lipid profile levels compared to control. The homeostatic index of insulin resistance (HOMA-IR) and the quantitative insulin sensitivity check index (QUICKI) were adversely modulated. Sperm count and motility were profoundly decreased, whereas sperm abnormality was significantly increased. The testicular activities of catalase (CAT), glutathione peroxidase (GPx), superoxide dismutase (SOD), and malondialdehyde (MDA) level, along with inflammatory cytokines (TNF-α, IL-6, IL-4, and IL-10) were significantly dysregulated. The DM induced histopathological lesions compared to control rats. Interestingly, the RSV administration to DM rats attenuated the altered reproductive parameters, restored antioxidant mechanism, and anti-inflammatory responses with improved insulin resistance. RSV could prevent DM-induced reproductive deficits and insulin resistance via modulating oxidative stress-mediated testicular inflammation in rats.


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## Body

## 1. Introduction

Diabetes is a global health challenge in various countries of the world with prevalence estimated 463 million adults [1]. It has been projected that, by 2045, the endocrine disease may affect 700 million adults [1, 2]. The pathophysiology of the frequently diagnosed type 2 diabetes mellitus is complicated and defined by 50% pancreatic cell loss at diagnosis, reduced insulin sensitivity of target tissues, and decreased insulin production. As a consequence, chronic hyperglycemia promotes reactive oxygen species (ROS) accumulation, oxidative damage, and impaired systemic antioxidant mechanism leading to organ damage [3, 4]. During hyperglycemic milieu, self-oxidation of glucose is accelerated provoking excessive production of ROS and lipid peroxidation resulting into oxidative stress mechanism [5]. The chronic hyperglycemia-mediated oxidative stress mechanism adversely affects reproductive apparatus leading to spermatogenesis decline and testicular damage [6, 7]. Diabetic reproductive impairment therefore is a major complication which has been reported in a considerable number of diabetic patients. In the testis oxidative stress, ROS impairs cell membrane and DNA integrity and activates overexpression of genes related to regulatory cascades of testicular inflammation, including cytokines and cyclooxygenases [5]. Given the role of oxidative stress in the pathogenesis of diabetic testicular complication, spermatogenic cells may undergo natural apoptosis, structural damage, and decrease in sperm count and motility [7, 8]. Furthermore, studies have reported the deleterious effect of diabetes on endocrine physiology, steroidogenic signaling, and lipid metabolism [9–11]. For example, remarkable decreases in the concentrations of testosterone and gonadotropins-follicle-stimulating hormone (FSH), gonadotropin releasing hormone (GnRH), and luteinizing hormone (LH) have been reported in published papers [10, 12, 13].However, the pharmacological management of diabetes mellitus (DM) is currently unsatisfactory and is associated with some considerable side effects [14]. Natural products have been recognized as an effective and safer approach for therapeutics development. Accumulating evidence suggests that natural compounds could improve glucose homeostasis in DM and consequently mitigate development and progression of diabetic complications, including DM testicular damage [15–18]. Hence, the search for antioxidant natural products is to scavenge ROS and curtail hyperglycemia-mediated reproductive impairments.Resveratrol (3,5,4′-trihydroxy-trans-stilbene) is a bioactive natural compound belonging to the group of polyphenolic derivatives of stilbene. Resveratrol (RSV) is abundant in grape, grape juice, peanuts, wine, mulberries, and blackcurrants [19]. Among many stilbene compounds, RSV has distinctively demonstrated several beneficial biological activities like antioxidant, anti-inflammatory, antiobesity, anticancer, neuroprotective, hepatoprotective, and cardioprotective effects in various models as well as mitigation against diabetic nephropathy and organ toxicities [14, 19–22]. Although there are reports that RSV may improve glucose homeostasis and reverse insulin resistance in DM, the reported data are inconsistent [23]. In addition, the molecular effect of RSV particularly on DM-induced testicular damage and endocrine dysfunction remains to be profoundly elucidated [24]. The current study was therefore investigated to elucidate the role RSV in DM-induced testicular oxidative inflammation, endocrine deficits, sperm characteristics, and insulin resistance in rats.


## 2. Materials and Methods

### 2.1. Chemicals

Resveratrol, STZ, buffered saline solution, and other reagents were procured from Sigma-Aldrich Chemical, Shanghai, China. The standard lipid peroxide kits for ELISA assays for MDA (Cat No: MD2528, Bio-Diagnostic, Egypt), CAT (Cat No: CA2517), SOD (Cat. No: SD2521), and GPx (Cat. No: GP2529) activities were obtained from Bio-Diagnostics, Giza, Egypt. Commercial kits for determination of luteinizing hormone (LH: Cat. No. LH-010) and follicle-stimulating hormone (FSH: Cat. No. E-El-M0511) were purchased from Shibayagi Co., Ltd., Shibukawa-Gunma, Japan, and testosterone (Cat. No: KT29533) was purchased from K-assay, WA, USA. Rat ELISA kit (Cat. No: E-EL-R3034) for serum insulin was purchased from Elabscience, Texas, USA, while glucose assay kit (Cat No: ab65333) was from Abcam, UK.


### 2.2. Animals

Twenty-four (24) adult male rats weighing 180-200 g (8 weeks old) were purchased from the animal house of the Faculty of Science, King Faisal University, Saudi Arabia. Ethical statement—all experimental procedures were done according to the Institutional Animal Care and Use Committee (IACUC) of the King Faisal University (Reference number: KFU-REC-2022-OCT-ETHICS196). The rats were housed in plastic cages, floored with soft wood shaving that was changed three times per week. The animals were acclimatized for 2 weeks prior the study and were maintained under 12 h light/dark cycle at (25°C±2°C), with free access to water and rat chow.


### 2.3. Induction of Diabetes Mellitus

Healthy male rats were fasted overnight and a single dose of streptozotocin (STZ) (65 mg/kg bw), newly prepared in citrate buffer, pH 4.5, was injected by intraperitoneal route to induce type 2 diabetes mellitus. Diabetes was confirmed 72 hours after STZ injection via estimating plasma glucose levels from blood samples (3 ml each) collected through lateral tail vein. Rats with blood glucose levels of 250 mg/dl or higher were considered diabetic and used for this study. Rats that appeared unhealthy were identified and separated out from the study.


### 2.4. Experimental Design and Treatment Protocols

After the two-weeks acclimatization, all rats were randomly divided into 4 groups with six rats in each group. Group 1 was the control administered only water solution of carboxymethyl cellulose (CMC, 2 ml/kg bw) by gavage for 21 days. Group 2 rats were diabetic rats and were administered CMC by gavage for 21 consecutive days. Group 3 contained nondiabetic rats administered only RSV (150 mg/kg) by gavage for 21 consecutive days [25]. RSV was dissolved in water solution of CMC. Group 4 contained diabetic rats administered RSV (150 mg/kg) by gavage for 21 consecutive days. After the experimental treatment, rats were fasted overnight, anesthetized, dissected, and both trunk blood and testis samples were collected for biochemical and histological studies. The animals were euthanized by anesthetic exsanguination using the combination of 10 mg/kg xylazine and 100 mg/kg ketamine HCl. Whole blood samples were collected in clean dry centrifuge tubes, containing no anticoagulation factors and allowed to clot for a minimum of 30 minutes at 37°C before centrifuged to obtain serum (769 × g for 15 minutes) and stored at -20°C until the analysis. The testes were weighed and homogenized (1 : 5 w/v) with a Potter-Elvehjem homogenizer attached to a Teflon plunger in ice cold phosphate buffer (50 mM, pH 7.5). The homogenates were centrifuged to obtain supernatant samples (11,000 × g for 20 minutes). The supernatant samples divided into aliquots stored at -20°C for different assays.


### 2.5. Biochemical Estimations

#### 2.5.1. Estimation of Fasting Serum Insulin and Glucose Concentration

Fasting serum insulin levels were measured by an Insulin ELISA Kit (Cat. No: E-EL-R3034) according to the manufacturer’s procedures. Serum glucose was determined using the glucose oxidase method [26].


#### 2.5.2. Homeostatic Index of Insulin Resistance (HOMA-IR)

The homeostatic index of insulin resistance (HOMA-IR) was estimated as a surrogate of insulin sensitivity [27]. HOMA-IR was calculated by using the homeostasis model assessment equation below
(1)HOMA‐IR=fastingglucosemg/dl×fastinginsulinμIU/ml÷405


#### 2.5.3. Quantitative Insulin Sensitivity Check Index (QUICKI)

Quantitative insulin sensitivity check index (QUICKI) was determined as a surrogate of insulin sensitivity [28]. QUICKI was calculated by using the inverse of the sum of the logarithms of fasting insulin and fasting glucose as follows:
(2)QUICKI=1÷logfastingglucosemg/dl+logfastinginsulinμIU/ml


### 2.6. Determination of Serum Lipid Profile Parameters

Total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) levels were determined by using commercial kits (TC, Cat. No: CH 1220; TG, Cat. No: TR 2030, HDL-C, Cat. No: CH 1230; Bio-Diagnostics, Giza, Egypt). Very-low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) were calculated using Friedewald’s formula [29].


### 2.7. Determination of Reproductive Hormones

Serum testosterone levels were determined using the K-Assay Testosterone ELISA Kit (Cat No. KT29533) following Sakuma [30] method. Luteinizing hormone levels were estimated with the Japanese Shibayagi Company Rat ELISA Kit (Cat No. LH-10) according to Shioya and Wakabayashi [31] method, while follicle-stimulating hormones were assessed using commercial kits (Cat No. E-EI-M0511) based on the method of Teerds et al. [32] protocols from BioVendor, Tokyo, Japan.


### 2.8. Determination of Sperm Count, Motility and Abnormality

The suspension from testis epididymis was used for assessing sperm quality. Sperm count was done using hemocytometer following the method of Freud and Carol (1964). The technique of Evans and Maxwell [33] was adopted for sperm abnormality study. Whereas, the assessment of sperm motility was done according to Morrissey et al. (1988) as stated in our recent paper by Alturki et al. [34].


### 2.9. Determination of Testicular Oxidative Stress Markers

Supernatant homogenate samples were used for the oxidative stress analysis. Testis tissues were homogenized (1 : 5 w/v) with a Potter-Elvehjem homogenizer attached to a Teflon plunger in ice cold phosphate buffer (50 mM, pH 7.5). The homogenates were centrifuged to obtain supernatant samples (11,000 × g for 20 minutes). Malondialdehyde levels were measured following the method of Ohkawa et al. [35]. Testicular catalase activity was determined by Aebi [36]. The activity of SOD was determined by Masayasu and Hiroshi [37]. Glutathione peroxidase (GPx) activity was estimated by the method of Paglia and Valentine [38].


### 2.10. Quantification of Testicular Proinflammation and Anti-Inflammation

The testicular levels of TNF-α (Cat No: EA100365), IL-4 (Cat No: MBS162452), IL-10 (Cat No: MBS764911), and IL-6 (Cat No: MBS726707) were measured with rat standard-ELISA kits procured from OriGene Technologies Inc., Rockville, MD and MyBioSource, Inc., San Diego, USA, following the manufacturers’ directions.


### 2.11. Testicular Histopathological Analysis

Testis tissue samples were removed and immediately fixed in 10% buffered formalin. Dehydration was carried out with graded ethanol and embedded in paraffin. Testis sections were stained with haematoxylin and eosin (H and E) for microscopic histopathological lesions according to Bancroft and Gamble [39]. The prepared slides were examined under light microscope. Testicular lesions were semiquantitatively graded as follows: 0: intact histoarchitecture (normal), 1: mild lesions, 2: moderate lesions, and 3: severe damage [34].


### 2.12. Statistical Analyses

Data were compared using one-way ANOVA followed by LSD multiple range test. Significant differences were obtained atP<0.05. Statistical tests were performed using SAS statistical software (SAS v.9.2, SAS Institute, Inc.).


## 2.1. Chemicals

Resveratrol, STZ, buffered saline solution, and other reagents were procured from Sigma-Aldrich Chemical, Shanghai, China. The standard lipid peroxide kits for ELISA assays for MDA (Cat No: MD2528, Bio-Diagnostic, Egypt), CAT (Cat No: CA2517), SOD (Cat. No: SD2521), and GPx (Cat. No: GP2529) activities were obtained from Bio-Diagnostics, Giza, Egypt. Commercial kits for determination of luteinizing hormone (LH: Cat. No. LH-010) and follicle-stimulating hormone (FSH: Cat. No. E-El-M0511) were purchased from Shibayagi Co., Ltd., Shibukawa-Gunma, Japan, and testosterone (Cat. No: KT29533) was purchased from K-assay, WA, USA. Rat ELISA kit (Cat. No: E-EL-R3034) for serum insulin was purchased from Elabscience, Texas, USA, while glucose assay kit (Cat No: ab65333) was from Abcam, UK.


## 2.2. Animals

Twenty-four (24) adult male rats weighing 180-200 g (8 weeks old) were purchased from the animal house of the Faculty of Science, King Faisal University, Saudi Arabia. Ethical statement—all experimental procedures were done according to the Institutional Animal Care and Use Committee (IACUC) of the King Faisal University (Reference number: KFU-REC-2022-OCT-ETHICS196). The rats were housed in plastic cages, floored with soft wood shaving that was changed three times per week. The animals were acclimatized for 2 weeks prior the study and were maintained under 12 h light/dark cycle at (25°C±2°C), with free access to water and rat chow.


## 2.3. Induction of Diabetes Mellitus

Healthy male rats were fasted overnight and a single dose of streptozotocin (STZ) (65 mg/kg bw), newly prepared in citrate buffer, pH 4.5, was injected by intraperitoneal route to induce type 2 diabetes mellitus. Diabetes was confirmed 72 hours after STZ injection via estimating plasma glucose levels from blood samples (3 ml each) collected through lateral tail vein. Rats with blood glucose levels of 250 mg/dl or higher were considered diabetic and used for this study. Rats that appeared unhealthy were identified and separated out from the study.


## 2.4. Experimental Design and Treatment Protocols

After the two-weeks acclimatization, all rats were randomly divided into 4 groups with six rats in each group. Group 1 was the control administered only water solution of carboxymethyl cellulose (CMC, 2 ml/kg bw) by gavage for 21 days. Group 2 rats were diabetic rats and were administered CMC by gavage for 21 consecutive days. Group 3 contained nondiabetic rats administered only RSV (150 mg/kg) by gavage for 21 consecutive days [25]. RSV was dissolved in water solution of CMC. Group 4 contained diabetic rats administered RSV (150 mg/kg) by gavage for 21 consecutive days. After the experimental treatment, rats were fasted overnight, anesthetized, dissected, and both trunk blood and testis samples were collected for biochemical and histological studies. The animals were euthanized by anesthetic exsanguination using the combination of 10 mg/kg xylazine and 100 mg/kg ketamine HCl. Whole blood samples were collected in clean dry centrifuge tubes, containing no anticoagulation factors and allowed to clot for a minimum of 30 minutes at 37°C before centrifuged to obtain serum (769 × g for 15 minutes) and stored at -20°C until the analysis. The testes were weighed and homogenized (1 : 5 w/v) with a Potter-Elvehjem homogenizer attached to a Teflon plunger in ice cold phosphate buffer (50 mM, pH 7.5). The homogenates were centrifuged to obtain supernatant samples (11,000 × g for 20 minutes). The supernatant samples divided into aliquots stored at -20°C for different assays.


## 2.5. Biochemical Estimations

### 2.5.1. Estimation of Fasting Serum Insulin and Glucose Concentration

Fasting serum insulin levels were measured by an Insulin ELISA Kit (Cat. No: E-EL-R3034) according to the manufacturer’s procedures. Serum glucose was determined using the glucose oxidase method [26].


### 2.5.2. Homeostatic Index of Insulin Resistance (HOMA-IR)

The homeostatic index of insulin resistance (HOMA-IR) was estimated as a surrogate of insulin sensitivity [27]. HOMA-IR was calculated by using the homeostasis model assessment equation below
(1)HOMA‐IR=fastingglucosemg/dl×fastinginsulinμIU/ml÷405


### 2.5.3. Quantitative Insulin Sensitivity Check Index (QUICKI)

Quantitative insulin sensitivity check index (QUICKI) was determined as a surrogate of insulin sensitivity [28]. QUICKI was calculated by using the inverse of the sum of the logarithms of fasting insulin and fasting glucose as follows:
(2)QUICKI=1÷logfastingglucosemg/dl+logfastinginsulinμIU/ml


## 2.5.1. Estimation of Fasting Serum Insulin and Glucose Concentration

Fasting serum insulin levels were measured by an Insulin ELISA Kit (Cat. No: E-EL-R3034) according to the manufacturer’s procedures. Serum glucose was determined using the glucose oxidase method [26].


## 2.5.2. Homeostatic Index of Insulin Resistance (HOMA-IR)

The homeostatic index of insulin resistance (HOMA-IR) was estimated as a surrogate of insulin sensitivity [27]. HOMA-IR was calculated by using the homeostasis model assessment equation below
(1)HOMA‐IR=fastingglucosemg/dl×fastinginsulinμIU/ml÷405


## 2.5.3. Quantitative Insulin Sensitivity Check Index (QUICKI)

Quantitative insulin sensitivity check index (QUICKI) was determined as a surrogate of insulin sensitivity [28]. QUICKI was calculated by using the inverse of the sum of the logarithms of fasting insulin and fasting glucose as follows:
(2)QUICKI=1÷logfastingglucosemg/dl+logfastinginsulinμIU/ml


## 2.6. Determination of Serum Lipid Profile Parameters

Total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) levels were determined by using commercial kits (TC, Cat. No: CH 1220; TG, Cat. No: TR 2030, HDL-C, Cat. No: CH 1230; Bio-Diagnostics, Giza, Egypt). Very-low-density lipoprotein cholesterol (VLDL-C) and low-density lipoprotein cholesterol (LDL-C) were calculated using Friedewald’s formula [29].


## 2.7. Determination of Reproductive Hormones

Serum testosterone levels were determined using the K-Assay Testosterone ELISA Kit (Cat No. KT29533) following Sakuma [30] method. Luteinizing hormone levels were estimated with the Japanese Shibayagi Company Rat ELISA Kit (Cat No. LH-10) according to Shioya and Wakabayashi [31] method, while follicle-stimulating hormones were assessed using commercial kits (Cat No. E-EI-M0511) based on the method of Teerds et al. [32] protocols from BioVendor, Tokyo, Japan.


## 2.8. Determination of Sperm Count, Motility and Abnormality

The suspension from testis epididymis was used for assessing sperm quality. Sperm count was done using hemocytometer following the method of Freud and Carol (1964). The technique of Evans and Maxwell [33] was adopted for sperm abnormality study. Whereas, the assessment of sperm motility was done according to Morrissey et al. (1988) as stated in our recent paper by Alturki et al. [34].


## 2.9. Determination of Testicular Oxidative Stress Markers

Supernatant homogenate samples were used for the oxidative stress analysis. Testis tissues were homogenized (1 : 5 w/v) with a Potter-Elvehjem homogenizer attached to a Teflon plunger in ice cold phosphate buffer (50 mM, pH 7.5). The homogenates were centrifuged to obtain supernatant samples (11,000 × g for 20 minutes). Malondialdehyde levels were measured following the method of Ohkawa et al. [35]. Testicular catalase activity was determined by Aebi [36]. The activity of SOD was determined by Masayasu and Hiroshi [37]. Glutathione peroxidase (GPx) activity was estimated by the method of Paglia and Valentine [38].


## 2.10. Quantification of Testicular Proinflammation and Anti-Inflammation

The testicular levels of TNF-α (Cat No: EA100365), IL-4 (Cat No: MBS162452), IL-10 (Cat No: MBS764911), and IL-6 (Cat No: MBS726707) were measured with rat standard-ELISA kits procured from OriGene Technologies Inc., Rockville, MD and MyBioSource, Inc., San Diego, USA, following the manufacturers’ directions.


## 2.11. Testicular Histopathological Analysis

Testis tissue samples were removed and immediately fixed in 10% buffered formalin. Dehydration was carried out with graded ethanol and embedded in paraffin. Testis sections were stained with haematoxylin and eosin (H and E) for microscopic histopathological lesions according to Bancroft and Gamble [39]. The prepared slides were examined under light microscope. Testicular lesions were semiquantitatively graded as follows: 0: intact histoarchitecture (normal), 1: mild lesions, 2: moderate lesions, and 3: severe damage [34].


## 2.12. Statistical Analyses

Data were compared using one-way ANOVA followed by LSD multiple range test. Significant differences were obtained atP<0.05. Statistical tests were performed using SAS statistical software (SAS v.9.2, SAS Institute, Inc.).


## 3. Results

### 3.1. Effect of RSV on Testes of Rats

The diabetic status of rats led to a drastic reduction in testis weights. The percentage of testes weight loss was 59.68% in the DM group compared to the standard control group (P<0.05). On the other hand, RSV at the administered dose considerably increased the testis weights compared with the DM group (P<0.05; Table 1).Table 1
Effect of RSV on testis weight of rats.
GroupTestis weight (g)Control3.77±0.03DM1.52±0.08∗RSV3.74±0.11DM+RSV2.33±0.09#Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant when compared to DM in the same column.


### 3.2. Effect of RSV on Serum Glucose, Insulin, and Insulin Resistance Markers

Table2 presents the effect of RSV on serum glucose, insulin, HOMA-IR, and QUICKI in STZ-induced diabetic rats. In DM rats, serum glucose level and homeostatic index of insulin resistance (HOMA-IR) were significantly increased compared to control (P<0.05), whereas insulin level and quantitative insulin sensitivity check index (QUICKI) were significantly decreased compared to control (P<0.05). In contrast, the administration of RSV to DM rats markedly decreased glucose level and suppressed insulin resistance as shown by reduced HOMA-IR compared to DM group. Also, the insulin level significantly increased as well as insulin sensitivity check index (QUICKI) in comparison to the DM group.Table 2
Effect of RSV on serum glucose, insulin, and markers of insulin resistance in DM rats.
GroupGlucose (mg/dl)Insulin (μIU/ml)HOMA-IRQUICKIControl81.32±0.2310.38±0.122.08±0.020.34±0.01DM373.58±0.32∗4.20±0.01∗3.89±0.03∗0.31±0.01RSV80.62±0.2111.02±0.092.19±0.010.34±0.03DM+RSV175.70±0.24#6.38±0.06#2.76±0.02#0.33±0.02Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant when compared to DM in the same column. HOMA-IR: homeostatic index of insulin resistance; QUICKI: quantitative insulin sensitivity check index.


### 3.3. Effect of RSV on Serum Lipid Profile

Table3 presents the effect of RSV on serum levels of TC, TG, HDL-C, and LDL-C in diabetic rats. It was observed that the diabetic condition of the rats considerably increased serum levels of TC, TG, and LDL-C, whereas HDL-C reduced compared to the nondiabetic control group (P<0.05). In DM+RSV group, the levels of the lipid profiles were reversed significantly (P<0.05) when the levels were compared to the DM group.Table 3
Effect of RSV on serum lipid profile in DM rats.
GroupTC (mg/dl)TG (mg/dl)HDL-C (mg/dl)LDL-C (mg/dl)Control54.6±0.2236.2±0.2029.2±0.2017.1±0.20DM114.7±0.20∗76.2±0.20∗17.6±0.11∗56.5±0.14∗RSV54.7±0.1236.5±0.2128.9±0.1017.4±0.21DM+RSV66.2±0.30#48.7±0.13#20.3±0.10#29.6±0.13#Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant when compared to DM in the same column.


### 3.4. Effect of RSV on Reproductive Hormones

The results of levels of serum reproductive hormones are presented in Table4. The levels of T, LH, and FSH were prominently declined in nontreated DM group in comparison to nondiabetic control group (P<0.05). However, the RSV administration in DM+RSV group markedly increased the hormones appreciably compared to DM group in this study.Table 4
Effect of RSV on serum reproductive hormones in DM rats.
GroupT (pg/ml)LH (ng/ml)FSH (ng/ml)Control8.72±0.121.50±0.050.87±0.01DM4.91±0.01∗0.69±0.01∗0.36±0.01∗RSV8.72±0.091.40±0.000.89±0.00DM+RSV6.38±0.06#0.90±0.02#0.57±0.01#Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant compared to DM in the same column. T: Testosterone; LH: leutenizing hormone; FSH: follicle-stimulating hormone.


### 3.5. Effect of RSV on Sperm Count, Motility, and Abnormality

Table5 depicts the effect of RSV on the sperm count, sperm motility, and abnormality of rats with STZ-induced DM. In DM group, it was found that the sperm count, motility, and abnormality were increased significantly compared to control (P<0.05). Interestingly, in the DM+RSV group, the count, motility, and abnormality were profoundly prevented by RSV in comparison to DM group.Table 5
Effect of RSV on sperm parameters in DM rats.
GroupSperm count (106 cells/ml)Motility (%)Abnormality (%)Control97.4±0.8083.4±0.838.0±0.44DM65.4±0.72∗57.2±1.01∗42.2±1.02∗RSV97.2±0.9185.2±0.848.2±0.42DM+RSV74.0±1.00#79.6±0.74#23.8±0.71#Data are displayed asmean±SEM of n=5 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant compared to DM in the same column.


### 3.6. Effect of RSV on Testicular Oxidative Stress

Figures1–4 show the effect of RSV and diabetes on testicular antioxidant enzyme activities and lipid peroxidation marker, MDA, in diabetic rats. In nontreated diabetic rats, testicular activities of the antioxidant enzymes, CAT, SOD, and GPx were significantly reduced, whereas the level of MDA markedly increased (P<0.05) compared to control. Contrarily, the administered RSV in DM+RSV group significantly enhanced the enzyme activities and reduced MDA level in comparison to DM group in this study.Figure 1
Effect of RSV on testicular superoxide dismutase activity in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 2
Effect of RSV on testicular catalase activity in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 3
Effect of RSV on testicular glutathione peroxidase activity in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 4
Effect of RSV on testicular malondialdehyde level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).


### 3.7. Effect of RSV on Testicular Inflammation

In the current study, we evaluated proinflammatory and anti-inflammatory markers in the testis of diabetic rats. Figures5–8 present the effect of RSV on testicular proinflammatory markers (TNF-α and IL-6) and anti-inflammatory markers (IL-4 and IL-10). In DM group, the levels of TNF-α and IL-6 increased significantly while IL-4 and IL-10 levels decreased significantly in comparison to control. However, it was interesting to observe that the RSV administration to the DM rats reduced TNF-α and IL-6 levels and increased IL-4 and IL-10 levels significantly compared to DM group.Figure 5
Effect of RSV on testicular tumor necrosis factor-alpha level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 6
Effect of RSV on testicular interleukin-6 level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 7
Effect of RSV on testicular interleukin-4 level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 8
Effect of RSV on testicular interleukin-10 in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).


### 3.8. Effect of RSV on Testis Histopathology

The histopathological lesions observed in the testis samples were presented in Figure9. Control group with normal seminiferous tubules (indicated by the star) with complete spermatogenic series and separated by interstitial cells indicated by arrow. The DM group revealed wide interstitial space (arrow) between irregular/distorted tubules (ST) and depletion of sperms (asterisks). RSV group showed normal architecture in which tubules were separated by narrow interstitial tissue (circle) with aggregation of spermatozoa in the lumen (S). RSV+DM group showed mild improvement in the seminiferous tubules (ST) and restoration of spermatogenesis (asterisks).Figure 9
Photomicrographs of DM rat testis administered with RSV (H&E stain). Control (a) DM (b) RSV (c), and RSV+DM (d). Control group with normal seminiferous tubules (star) with interstitial cells (arrow). The DM group revealed interstitial space (arrow), irregular/distorted tubules (ST), and depletion of sperms (asterisks). RSV group showed normal tubules, separated by narrow interstitial tissue (circle) with aggregation of spermatozoa in the lumen (S). RSV+DM group showed mildly damaged seminiferous tubules (ST) and restoration of spermatogenesis (asterisks). Values are expressed asmean±SEM (n=3). ∗Significant when compared to control (P<0.05); #significant compared to DM group.


## 3.1. Effect of RSV on Testes of Rats

The diabetic status of rats led to a drastic reduction in testis weights. The percentage of testes weight loss was 59.68% in the DM group compared to the standard control group (P<0.05). On the other hand, RSV at the administered dose considerably increased the testis weights compared with the DM group (P<0.05; Table 1).Table 1
Effect of RSV on testis weight of rats.
GroupTestis weight (g)Control3.77±0.03DM1.52±0.08∗RSV3.74±0.11DM+RSV2.33±0.09#Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant when compared to DM in the same column.


## 3.2. Effect of RSV on Serum Glucose, Insulin, and Insulin Resistance Markers

Table2 presents the effect of RSV on serum glucose, insulin, HOMA-IR, and QUICKI in STZ-induced diabetic rats. In DM rats, serum glucose level and homeostatic index of insulin resistance (HOMA-IR) were significantly increased compared to control (P<0.05), whereas insulin level and quantitative insulin sensitivity check index (QUICKI) were significantly decreased compared to control (P<0.05). In contrast, the administration of RSV to DM rats markedly decreased glucose level and suppressed insulin resistance as shown by reduced HOMA-IR compared to DM group. Also, the insulin level significantly increased as well as insulin sensitivity check index (QUICKI) in comparison to the DM group.Table 2
Effect of RSV on serum glucose, insulin, and markers of insulin resistance in DM rats.
GroupGlucose (mg/dl)Insulin (μIU/ml)HOMA-IRQUICKIControl81.32±0.2310.38±0.122.08±0.020.34±0.01DM373.58±0.32∗4.20±0.01∗3.89±0.03∗0.31±0.01RSV80.62±0.2111.02±0.092.19±0.010.34±0.03DM+RSV175.70±0.24#6.38±0.06#2.76±0.02#0.33±0.02Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant when compared to DM in the same column. HOMA-IR: homeostatic index of insulin resistance; QUICKI: quantitative insulin sensitivity check index.


## 3.3. Effect of RSV on Serum Lipid Profile

Table3 presents the effect of RSV on serum levels of TC, TG, HDL-C, and LDL-C in diabetic rats. It was observed that the diabetic condition of the rats considerably increased serum levels of TC, TG, and LDL-C, whereas HDL-C reduced compared to the nondiabetic control group (P<0.05). In DM+RSV group, the levels of the lipid profiles were reversed significantly (P<0.05) when the levels were compared to the DM group.Table 3
Effect of RSV on serum lipid profile in DM rats.
GroupTC (mg/dl)TG (mg/dl)HDL-C (mg/dl)LDL-C (mg/dl)Control54.6±0.2236.2±0.2029.2±0.2017.1±0.20DM114.7±0.20∗76.2±0.20∗17.6±0.11∗56.5±0.14∗RSV54.7±0.1236.5±0.2128.9±0.1017.4±0.21DM+RSV66.2±0.30#48.7±0.13#20.3±0.10#29.6±0.13#Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant when compared to DM in the same column.


## 3.4. Effect of RSV on Reproductive Hormones

The results of levels of serum reproductive hormones are presented in Table4. The levels of T, LH, and FSH were prominently declined in nontreated DM group in comparison to nondiabetic control group (P<0.05). However, the RSV administration in DM+RSV group markedly increased the hormones appreciably compared to DM group in this study.Table 4
Effect of RSV on serum reproductive hormones in DM rats.
GroupT (pg/ml)LH (ng/ml)FSH (ng/ml)Control8.72±0.121.50±0.050.87±0.01DM4.91±0.01∗0.69±0.01∗0.36±0.01∗RSV8.72±0.091.40±0.000.89±0.00DM+RSV6.38±0.06#0.90±0.02#0.57±0.01#Data are displayed asmean±SEM of n=6 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant compared to DM in the same column. T: Testosterone; LH: leutenizing hormone; FSH: follicle-stimulating hormone.


## 3.5. Effect of RSV on Sperm Count, Motility, and Abnormality

Table5 depicts the effect of RSV on the sperm count, sperm motility, and abnormality of rats with STZ-induced DM. In DM group, it was found that the sperm count, motility, and abnormality were increased significantly compared to control (P<0.05). Interestingly, in the DM+RSV group, the count, motility, and abnormality were profoundly prevented by RSV in comparison to DM group.Table 5
Effect of RSV on sperm parameters in DM rats.
GroupSperm count (106 cells/ml)Motility (%)Abnormality (%)Control97.4±0.8083.4±0.838.0±0.44DM65.4±0.72∗57.2±1.01∗42.2±1.02∗RSV97.2±0.9185.2±0.848.2±0.42DM+RSV74.0±1.00#79.6±0.74#23.8±0.71#Data are displayed asmean±SEM of n=5 rats/group. ∗P<0.05: significant compared to control in the same column. #P<0.05: significant compared to DM in the same column.


## 3.6. Effect of RSV on Testicular Oxidative Stress

Figures1–4 show the effect of RSV and diabetes on testicular antioxidant enzyme activities and lipid peroxidation marker, MDA, in diabetic rats. In nontreated diabetic rats, testicular activities of the antioxidant enzymes, CAT, SOD, and GPx were significantly reduced, whereas the level of MDA markedly increased (P<0.05) compared to control. Contrarily, the administered RSV in DM+RSV group significantly enhanced the enzyme activities and reduced MDA level in comparison to DM group in this study.Figure 1
Effect of RSV on testicular superoxide dismutase activity in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 2
Effect of RSV on testicular catalase activity in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 3
Effect of RSV on testicular glutathione peroxidase activity in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 4
Effect of RSV on testicular malondialdehyde level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).


## 3.7. Effect of RSV on Testicular Inflammation

In the current study, we evaluated proinflammatory and anti-inflammatory markers in the testis of diabetic rats. Figures5–8 present the effect of RSV on testicular proinflammatory markers (TNF-α and IL-6) and anti-inflammatory markers (IL-4 and IL-10). In DM group, the levels of TNF-α and IL-6 increased significantly while IL-4 and IL-10 levels decreased significantly in comparison to control. However, it was interesting to observe that the RSV administration to the DM rats reduced TNF-α and IL-6 levels and increased IL-4 and IL-10 levels significantly compared to DM group.Figure 5
Effect of RSV on testicular tumor necrosis factor-alpha level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 6
Effect of RSV on testicular interleukin-6 level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 7
Effect of RSV on testicular interleukin-4 level in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).Figure 8
Effect of RSV on testicular interleukin-10 in DM rats. Data are expressed asmean±SEM (n=6). ∗Significant when compared to control (P<0.05); #significant compared to DM group (P<0.05).


## 3.8. Effect of RSV on Testis Histopathology

The histopathological lesions observed in the testis samples were presented in Figure9. Control group with normal seminiferous tubules (indicated by the star) with complete spermatogenic series and separated by interstitial cells indicated by arrow. The DM group revealed wide interstitial space (arrow) between irregular/distorted tubules (ST) and depletion of sperms (asterisks). RSV group showed normal architecture in which tubules were separated by narrow interstitial tissue (circle) with aggregation of spermatozoa in the lumen (S). RSV+DM group showed mild improvement in the seminiferous tubules (ST) and restoration of spermatogenesis (asterisks).Figure 9
Photomicrographs of DM rat testis administered with RSV (H&E stain). Control (a) DM (b) RSV (c), and RSV+DM (d). Control group with normal seminiferous tubules (star) with interstitial cells (arrow). The DM group revealed interstitial space (arrow), irregular/distorted tubules (ST), and depletion of sperms (asterisks). RSV group showed normal tubules, separated by narrow interstitial tissue (circle) with aggregation of spermatozoa in the lumen (S). RSV+DM group showed mildly damaged seminiferous tubules (ST) and restoration of spermatogenesis (asterisks). Values are expressed asmean±SEM (n=3). ∗Significant when compared to control (P<0.05); #significant compared to DM group.


## 4. Discussion

Diabetes mellitus is a metabolic disease that affects about 8.4% of world population [40]. The hallmark of DM pathogenesis is hyperglycemia and impairment in insulin secretion and/or action. Reproductive damage is a debilitating complication of DM. Resveratrol is a natural polyphenol associated with health-promoting effects in both animals and humans [22]. In the present study, we have reported the effect of RSV on DM-induced testicular damage and endocrine dysfunction.The dose of STZ (65 mg/kg bw) used for DM induction has been widely reported in experimental models of DM. It is a cytotoxic destroyer of pancreaticβ cells via free radical oxidative damage and attack on GLUT-2 receptors which are abundant on the β cells. This damage results in β cell deficient capacity to secrete insulin and thus hyperglycemia [3]. This underscores the observation of significantly elevated levels of serum glucose in the DM group in comparison to control (Table 2). The rats in RSV group showed glucose levels comparable to control in this study. The administration of RSV in RSV+DM group significantly reduced the glucose level when compared to DM. By implication, RSV possesses antihyperglycemic effect in agreement with the existing reports [14, 23, 41]. The antioxidant property of RSV may play a role in this effect via exerting therapeutic effect on the pancreatic β cells for insulin secretion. In addition, RSV might have enhanced the transport of blood glucose molecules across the cell membranes of peripheral tissues. The resultant antihyperglycemic effect of RSV could be associated with the marked increase levels of insulin in RSV+DM group. In the first instance, the STZ-induced DM (DM group) caused a considerable reduction in insulin levels. Expectedly, the cytotoxic attack of STZ as well as the glucotoxicity of hyperglycemia in DM exerted β-cell damage and impairment of insulin secretion [3]. Therefore, it could be suggested that RSV treatment in RSV+DM group provoked insulin secretion through its protection for the active β-cells that produced insulin and ameliorated the glucotoxicity [42]. Moreover, the antidiabetic action of RSV in the present study was further evident by the improved insulin resistance (HOMA-IR) and/or insulin sensitivity (QUICKI) indices. These indices are known reliable clinical and epidemiological tools for assessment of insulin resistance [43]. The HOMA-IR index value prominently increased in the nontreated DM group, whereas the QUICKI index decreased insignificantly compared to control. Insulin action is crucial to the maintenance of blood glucose level; it initiates the transmembrane signaling cascades via glucose receptors. Insulin resistance index is a calculated value from fasting serum glucose and insulin to reveal the resistance of the peripheral tissue to membrane influx of glucose. Thus, the values for HOMA-IR and QUICKI show that insulin resistance developed in the experimental groups. Our findings herein are in consonance with the reports of previous studies [18, 42, 43]. In contrast, RSV administration to the DM rats evidently restored the insulin action and sensitivity demonstrated by the considerably reduced HOMA-IR and insignificantly increased QUICKI values. Hence, present results indicate that RSV may have improvement effects on DM in relation to β-cell function, insulin sensitivity, insulin secretion, and reduction of insulin resistance de novo [23, 42].Besides hyperglycemia, hyperlipidemia is a critical metabolic derangement in DM. Glucolipid metabolism is complicated, and usually triggered by pancreaticβ-cell deficiency in optimal insulin secretion [44]. The clinical characteristics of diabetic dyslipidemia are elevated serum LDL-C, TG, and depressed HDL-C levels which are biochemical precursors to hypertension, hepatic steatosis, and cardiovascular diseases [18, 45]. In our study herein, the experimental DM-induced dyslipidemia evident by considerable elevation in TC, TG, and LDL-C serum levels, while HDL-C level was markedly decreased compared to control (Table 3) [9, 46, 47]. However, on the contrary, the RSV mitigated the dyslipidemia and restored the lipid profiles comparable to control.In the present study, DM-induced endocrine disruption observed by the altered levels of T, LH, and FSH. Literature has shown that DM is an endocrine disruptor that impairs spermatogenesis and steroidogenesis [10, 11]. It was observed in the DM group that T, LH, and FSH serum levels prominently reduced in comparison to control (Table 4). The depressed levels of these reproductive hormones suggest deficits in testicular testosterone synthesis which can be endocrinologically associated with dysregulations in the gonadotrophic hormones found in this study [34]. Furthermore, insulin resistance, as observed in the DM rats has been linked to reduced secretion of T by Leydig cells [7]. Consequent upon the hormonal deficits in DM rats, the sperm count, mobility, and structural integrity were adversely affected (Table 5) as reported in the earlier systematic publications [24, 41, 48]. The reduction in sperm count was due to reduced spermatogenesis which could be ascribed to reduction in serum T level and/or DM-induced ROS generation. In consonance with the published reports therefore, our study also confirms that DM can disrupt endocrine axis and spermatogenesis [41]. Interestingly, RSV mediated the alterations and caused restoration in the status of the hormones and sperm characteristics. The LH, T, and FSH levels along with sperm count, mobility, and abnormality were significantly restored in RSV+DM group in comparison to DM group. Studies indicate RSV potential to modulate diabetic complications and preserve the testicular integrity [24, 48]. In the study [24], RSV improved the sperm count, motility, viability, and sperm abnormalities as found in our study herein. The antioxidant protection of RSV on spermatocytes and its capacity to increase T levels could contribute to the observed beneficial effects.Robust body of evidence has implicated elevated ROS generation in the pathophysiology of DM-induced testicular damage [5–7]. The status of imbalance between ROS generation and antioxidant defense results into oxidative stress. The impaired glucose metabolism in DM is a critical mechanism to induce oxidative stress. Herein, DM produced oxidative stress leading to impaired antioxidant milieu in the testes of rats. This was demonstrated by considerable diminution in the testicular activities of CAT, SOD, and GPx, while MDA level conspicuously increased (Figures 1–4) compared to control. The antioxidant enzymes, CAT, SOD, and GPx are the guardian of cellular antioxidant homeostasis. Physiologically, SOD detoxifies superoxide radicals; CAT catalyzes the reaction that drives the lysis of H2O2 to H2O and molecular O2. Whereas, GPx, a tripeptide peroxidase mediates the neutralization of peroxyl radicals through the reducing action of its glutathione (GSH) moiety [49]. Therefore, DM-induced depression in the testicular activities of SOD, CAT, and GPx would consequently result into testicular oxidative stress [24, 47] and degeneration of testicular ultrastructure which might have contributed to decreased testis weight herein (Table 1). And the elevated level of MDA in the testis unarguably reveals the oxidative stress-mediated damage in the testis of DM rats [8, 47, 50]. The histology of testis confirmed the oxidative damage. The histopathological lesions of the testis were consistent with wide interstitial space, irregular/distorted tubules, and depletion of spermatogenesis in seminiferous tubules, which were earlier reported by Aly [24]. However, the oxidative deterioration in the testis triggered proinflammatory process which caused marked elevation in IL-6 and TNF-α in comparison to control (Figures 5–8). Intriguingly, the significantly reduced levels of anti-inflammatory cytokines, IL-4, and IL-10 (Figures 7 and 8), further confirmed the DM-induced oxidative stress-mediated testicular proinflammation in the current study. DM and ROS are known triggers of nuclear transcription of a number of inflammatory genes that translate into several cytokine proteins, including TNF-α, IL-6, and IL-1β [4, 51]. Thus, it can be concluded that the induced inflammation was associated with DM oxidative stress in the testicular tissue. RSV is a bioactive antioxidant polyphenol and its potency to combat oxidative stress has been documented [19–22]. We confirm in this study of the antioxidant prowess of RSV in that it significantly elevated the SOD, CAT, and GPx activities in comparison to DM group rats. Antioxidant effect of RSV might have scavenged and blocked ROS production, thereby spearing the enzymes from being consumed and/or overwhelmed by the ROS spurts; hence the markedly reduced MDA level. In consequence to this mechanism, the expressions of the inflammatory cytokines were abated and manifested in the testes as considerably modulated levels of IL-4, IL-6, IL-10, and TNF-α. However, the mild effect of oxidative stress was still observed in the histopathological tissue.In conclusion, the study findings suggest that RSV could combat redox imbalance, inflammation, sperm-endocrine dysfunctions, and insulin resistance via its antioxidant, antihyperglycemic, and anti-inflammatory mechanisms. Our results add to the existing paucity of data on the effect of RSV on DM-induced testicular complications.



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*Source: 6385767-2023-02-06.xml*