Effect of eugenol on lipid profile, oxidative stress, sex hormone, liver injury, ovarian failure, and expression of COX-2 and PPAR-α genes in a rat model of diabetes

Diabetes is among the leading causes of reproductive system failure and infertility in both women and men. Inflammation and oxidative stress have a main role in the development of diabetes. Eugenol or clove oil is a phenolic monoterpenoid with antioxidant and anti-inflammatory properties. Here, the effects of eugenol on diabetes features and ovarian function were investigated. Streptozotocin-induced diabetes rats were treated with 12 and 24 mg/kg of eugenol for 4 weeks. The biochemical and histological assay was done to evaluate the effects of eugenol on ovary and pancreas function, liver injury, oxidative status, sex hormones, lipid profile, and mRNA levels of cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor alpha (PPAR-α) genes. Streptozotocin increased levels of serum glucose, total cholesterol, triglyceride, low-density lipoprotein, aspartate transaminase, alanine transaminase, alkaline phosphatase, malondialdehyde, pancreas necrosis and inflammation, COX-2 expression, ovarian cystic, and anovulation. It decreased the levels of insulin, high-density lipoprotein, Superoxide dismutase, estradiol, progesterone, testosterone, luteinizing hormone, follicle-stimulating hormone, and PPAR-α expression. Eugenol administration ameliorated diabetes features through the improvement of lipid profile, oxidative status, insulin and glucose levels, sex hormone levels, liver markers, COX-2 and PPAR-α expression, and pancreas histology. It had no effect on ovarian cystic and follicular development. Therefore, eugenol may be useful for ameliorating some adverse features of diabetes and used as an adjunct treatment or protective agent accompany by other chemicals in diabetes patients.


Introduction
Diabetes refers to a group of metabolic disorders in which either the pancreas is unable to produce enough insulin or the cells cannot properly consume glucose. This condition causes the blood glucose to rise which leads to extensive damage to many of the tissues [1]. Previous studies have revealed that high glucose levels in diabetic individuals lead to diabetic complications via activation of the NF-kB inflammatory responses by increasing oxidative stress and reactive oxygen species (ROS) [2][3][4]. In fact, oxidative stress has been suggested as one of the main causes of diabetes development and its complication.
Diabetes is among the leading causes of reproductive system failure and infertility [5]. Both women and men with diabetes mellitus are likely to suffer from fertility disturbances compared with those without diabetes. Diabetes can promote ovarian senescence and reduce fertility rates. It can be the cause of irregular or absent periods and premature ovarian failure in women. Diabetes can influence the age of menarche and menopause. It can accelerate the onset of earlier menopause and decrease the store of the ovarian follicle at a lower age in diabetic women compared with those without diabetes [5].
Over the past years, the use of herbal remedies as one base of alternative therapy has grown exponentially worldwide for primary health care because of its cost-effectiveness, reduced side effects, and easier to obtain [6]. Polyphenolic compounds have received increased attention due to their anti-hyperglycemic potential and positive effects on diabetes management [7,8]. Eugenol or clove oil is a phenolic monoterpenoid that is related to the allylbenzene class of chemical agents found in various plants such as cinnamon, clove, and bay leaves [9]. It applies some biological properties including anti-inflammatory activity by suppression of the NF-κB signaling pathway and antioxidant activity which made it an attractive molecule for the development of new pharmacological compounds [9,10]. In the present study, the therapeutic effects of eugenol have been searched on the reproductive system, lipid profile, liver function, oxidative stress, and expression of cyclooxygenase-2 (COX-2) and peroxisome proliferator-activated receptor alpha (PPAR-α) genes in a streptozotocin-induced model of diabetes in Wistar rats. COX-2 is the main target for anti-inflammatory therapy because of its key role in inflammation responses. Its expression increases in oxidative stress conditions and its elevated expression have been reported following the activation of the NF-κB signaling pathway as well [11,12]. PPAR-α mediates inflammatory reactions by decreasing the activated forms of NF-κB, too [13]. It also has a role in the regulation of fat and sugar metabolism [13].

Animal model
Female Wistar rats (200 ± 20 g) were obtained from Pasteur Institute (Tehran, Iran). All animals were freely supplied with typical chow and water ad libitum and were acclimatized at least for two weeks in the Razi animal facility of Islamic Azad University, Science and Research Branch (Tehran, Iran) under controlled laboratory conditions (temperature: 22 °C, relative humidity: 60%, light and dark cycles of 12 h) trough out the study. Housing and all procedures were approved by the Animal Care and Use Committee of Islamic Azad University (Ethical permission number: IR.IAU.SRB. REC.1396.51) and were in accordance with the Guide for the Care and Use of Laboratory Animals [14]. To model diabetes, an intraperitoneal (i.p.) injection of a single streptozotocin (60 mg/kg body weight; dissolved in 0.01 M sodium citrate buffer at pH 4.5) was done. After 2 days, animals with high plasma sugar levels (≥ 300 mg/dl) were considered diabetic and selected for the subsequent experiments.
Animals were divided into five experimental groups (n = 6 per group). The animals in group 1 were receiving a normal diet and were considered the control group (C).
The animals in group 2 were diabetic and were receiving a normal diet without any extra treatment throughout the study and were considered as the diabetic group (D). The animals in group 3 were diabetic and were receiving tween (80%, i.p.) as eugenol solvent for 4 weeks (for the purpose to remove solvent effects) and were considered the sham operation group. The animals in groups 4 and 5 were diabetic and were treated (i.p.) with eugenol (Sigma Chemical Co, St. Louis, MO, USA) at the dose of 12 and 24 mg/kg body weight for 4 weeks and were considered diabetes-12 (D12) and diabetes-24 (D24) groups; respectively.

Biochemical measurements
Ovary, liver, pancreas tissues, and serum samples, were collected the day after the final dosing of the eugenol as described previously [15]. Briefly, all animals were deeply anesthetized with ketamine and xylazine (0.8 and 0.5 mg/ kg, i.p.; respectively) and blood samples were gathered in a heparinized tube. After 2 h of incubation in the room, the serum was prepared by centrifuging (2500 rpm, 5 min) and maintained at -20 ℃ until further tests. Quantitative enzymatic photometric kits (Parsazmun Company, Karaj, Iran) were used for measuring the serum levels of total cholesterol (TC), high-density lipoprotein (HDL), low-density lipoprotein (LDL), triglyceride (TG), glucose, aspartate transaminase (AST) enzyme, alanine transaminase (ALT) enzyme, and alkaline phosphatase (ALP) enzyme and following the manufacturer's recommendations. Rat ELISA kits from Monobind Co. (USA) were used to determine the serum estradiol and progesterone content. Rat/mouse ELISA test kits from Cosmo Bio Co. Ltd. (Japan) were used to determine the serum insulin, testosterone, luteinizing hormone (LH), and follicle-stimulating hormone (FSH). That are immunoassays designed for the quantitative determination of hormones in serum samples of rodents. The assay system utilizes a polyclonal antibody for solid-phase immobilization and a mouse antibody in the antibody-enzyme (horseradish peroxidase) conjugate solution.
The activity of Superoxide dismutase (SOD) and malondialdehyde (MDA) enzymes were measured in liver homogenate using Nasdox and NalondicELISA kits, respectively (Navandsalamat Co., Urmia, Iran), following the manufacturer's recommendations. For this purpose, liver tissues were minced and homogenized in 0.15 M ice-cold KCl (3 ml/g liver tissue) and prepared by centrifuging (10,000 rpm, 20 min, 4 ℃). Supernatants were separated and stored at -70 ºC until evaluating time.

Ovary and pancreas histology
For light microscopic analysis, the liver and pancreas tissues were fixed in paraformaldehyde (10%), embedded in paraffin after dehydration and clearing by alcohol and xylene solutions, sectioned at 7 μm, mounted on glass slides, and stained with Hematoxylin and Eosin (H&E) as described previously [15]. The number of Primary, secondary (or antral follicle), Graafian follicles, Corpus luteum, and follicular cysts were counted in ovaries samples according to Erickson's classification [16]. Images of islets were taken in pancreas samples using a light microscope, too. Numbers of disturbed islands, necrotic islands, and inflammation cells were counted in pancreas samples.

qRT-PCR
At the end of the experiment, ovarian tissue was frozen at − 75 ℃. Total RNA was extracted using a High Pure RNA Isolation kit from Roche (Germany) per the manufacturer's protocol. RNA concentrations and integrity were assessed using Nanodrop (Thermo Scientific, USA) at 260 and 280 nm and by ethidium bromide staining after agarose gel electrophoresis, respectively. cDNA was synthesized using Prime ScriptTM 1st strand cDNA Synthesis kit from Takara (Japan) in a final reaction volume of 20 µl and per manufacturer's recommendations. Synthetized cDNA was treated with DNase I. Real-time RT-PCR was applied using a StepOnePlus Real-Time PCR (Applied Biosystems, USA) using SYBR green PCR master mix from Takara (Japan) per manufacturer's recommendations. The real-time PCR program was as follows: initialization at 94 ºC for 30 s, amplification for 40 cycles with denaturation at 95ºC for 5 s, and annealing at 60 ºC for 30 s. 2 −ΔΔCt method and GraphPad Prism 6 software from GraphPad Software Inc. (SanDiego, USA) was used to quantify the expression data.

Statistical analysis
Data were analyzed using SPSS statistical software (version 17) with One-Way ANOVA (one-way analysis of variance) with a t-test to compare significant differences between the different model group means. Data are shown as the means ± SEM. A significance level of P < 0.05 was considered for all analyses.

Results of biochemical assays
The glucose, Insulin, and lipid levels of the control and diabetic groups are summarized in Table 1. The glucose, TG, TC, and LDL levels were markedly higher in diabetes (D) and sham rats compared with the control group, while the level of HDL and insulin were significantly lower. However, their level improved following eugenol administration in D12 and D24 groups compared to the D and sham rats (except for TG in the D12 group). Furthermore, the LDL level was significantly lower in the D12 group compared with the control one, while the HDL level was significantly higher. Increasing the concentration of eugenol to 24 mg/kg caused a further decrease in LDL levels in the D24 group in comparison to the control rats.
The LH, FSH, testosterone, progesterone, and estradiol levels were significantly decreased in D and sham rats compared with the control group (Table 1). However, their level improved following eugenol administration in D12 and D24 groups compared to the D and sham rats (except for FSH hormone in the D12 group and testosterone hormone in the D12 and the D24 group; P ≥ 0.05). Furthermore, the LH level was significantly higher in the D24 group compared with the control one (P ≤ 0.001).
The SOD level significantly decreased, while the MDA, AST, ALT, and ALP levels significantly increased in the D and sham animals in comparison to the control ones (Table 1). However, their level improved following eugenol administration in D12 and D24 groups compared to the D and sham rats (except for SOD in the D12 and D24 groups, MDA and AST in the D12 group; P ≥ 0.05).

COX-2 and PPAR-α mRNA expression levels
The ovarian COX2 mRNA expression level of diabetes (D) and sham groups was markedly higher than that of the control animals (Fig. 1). In contrast, the ovarian PPAR-α mRNA expression level of D and sham groups was markedly lower than that of the control group. Administration of eugenol at the concentration of 24 mg/kg improved the mRNA expression of COX2 and PPAR-α genes in the D24 group. In contrast to D24, the COX2 and PPAR-α mRNA expression levels were not markedly different between the D, sham, and D12 groups.

Histological observation of ovaries and pancreas
The animals in the control group showed normal ovarian morphology (Fig. 2). In contrast, in the diabetic group, the ovarian morphology was polycystic, and multiple cystic appeared. Histopathological changes along with structural abnormalities were observed in the animals with diabetes. The number of primary follicles, Graafian follicles, and corpus luteum significantly decreased in the D and sham rats when compared with the control group (Fig. 1). In contrast, the number of preantral follicles and the number of degenerating corpus luteum increased in the D and sham rats. Treatment with eugenol improved the histological features in the D12 and D24 groups in comparison to the control group, but these improvements were not significant. Administration of STZ had no effects on the number of primordial follicles. Figure 3 shows the transverse section of rat pancreatic tissue stained with hematoxylin and eosin (H&E). Samples of the control rats showed regular, well-organized, and well-defined islets. Islets were observed as faintly stained areas surrounded by hyperintense peripherally located pancreatic acini. The endocrine glucagon-secreting alpha cells with pale pink nuclei and insulin-secreting beta cells with euchromatin nuclei were observed. The size of the islets was larger compared to the other diabetic groups. The exocrine area showed regular and well-packed acini with normal morphology and structure (Fig. 3). Based on the obtained results (Fig. 1), the rate of destruction of beta cells and necrotic cells was 5%. Regarding the samples from sham and D animals, the amount of cell density in islets and the size of islets were significantly reduced. They appeared obviously shrunken, hypocellular with marked nuclear shrinkage and pyknosis. Most destroyed cells were seen in the center of the islets. The rate of destruction of beta cells and necrotic cells was 60% (Fig. 1). Tissue cohesion was less and in some areas, a lack of staining in the cells was observed, which is due to the change in the structure of the cells. In general, the number of inflammation cells, necrotic cells, and disturbed islets was increased in the D and sham groups compared to the control animals. Treatment with eugenol showed a marked improvement in islets' size and histological structure along with the reduction of necrotic cells, inflammation cells, and disturbed islets in the D12 and D24 groups compared to the sham and D groups. Also, tissue cohesion was somewhat improved compared to the D and sham groups. However, the lack of staining of cells was observed in some areas of the tissue.

Discussion
The present study was designed to evaluate the antidiabetic effects of four-week eugenol administration on the pathological changes of diabetes rats induced by streptozotocin (STZ) including lipid profiles, hormonal alterations, oxidative status, ovarian toxicity, pancreas toxicity, and COX-2 and PPAR-α expression. Data showed that STZ induction resulted in impaired profiles of glucose, insulin, and lipids. Therefore, the diabetes animals were successfully induced by STZ injection. STZ is a unique diabetogenic agent that is specifically destroyed insulin-producing cells of the pancreas (beta cells) which leads to an increase in glucose levels and a decrease in insulin production [17]. It C control group, D diabetic group, Sham diabetic animals + tween as eugenol solvent for 4 weeks, D12 diabetic animals + eugenol 12 mg/ kg, D24 diabetic animals + eugenol 24 mg/kg is widely used to develop an experimental model of diabetes in rodents. Here, treatment with STZ destroyed the beta cells, reduced the size of islets, and increased the number of necrotic cells and disturbed islets. Also, serum analysis confirmed the establishment of diabetes by increasing glucose and decreasing insulin amounts. Glucose metabolism is associated with fat metabolism in many ways [18]. It is demonstrated that individuals with diabetes represent dyslipidemia [18], which was also observed in this study such as elevated TG, TC, and LDL, and low HDL.
Here, STZ treatment increased the number of inflammation cells in the pancreatic tissues, too. Also, it dysregulated the activity of SOD and MDA, the main biomarkers of oxidative stress. Increasing evidence demonstrates that inflammation and oxidative stress have a main role in the development of diabetes [19]. Hyperglycemia can induce oxidative stress, the formation of reactive oxygen species (ROS), and the generation of inflammatory factors, and inflammation in turn can increase oxidative stress [2-4, 19, 20]. In the present study, STZ increased the levels of MDA and decreased the amount of SOD which indicates an increase in oxidative stress. These findings are in line with the previous study by Gilani et al. correlating STZ treatment rats with increased oxidative stress and impaired lipid profile, glucose metabolism, and insulin secretion [21].
Evidence has suggested the prevalence of liver dysfunction in individuals with diabetes is higher than in non-diabetic ones [22,23]. The liver is the largest gland in metabolism with a critical role in maintaining healthy blood sugar levels [24]. Hyperglycemia can dysregulate the metabolism pathway in the liver and cause non-alcoholic fatty liver disease (NAFLD) [22], which can progress to cirrhosis [25] and hepatocellular carcinomas [26] in the end in individuals with diabetes. Liver injury in diabetes patients may be the result of oxidative stress induced by hyperglycemia [27,28]. Increased levels of liver enzymes including ALT, AST, and ALP are an indicator of liver injury, which is linked to diabetes incidence [29]. Islam et al. have shown that the levels of liver enzymes (ALT, AST, and ALP) were significantly higher in diabetes people compared to non-diabetes people in Bangladeshi adults [23]. Our data confirmed previous studies [22,23]. We showed that the concentration of serum ALT, AST, and ALP was significantly increased in the STZ-induced diabetes rats compared to the control animals, which suggested liver injury in diabetic animals.
Diabetes is among the leading causes of reproductive system failure and infertility in both women and men [5]. It can promote ovarian senescence, irregular or absent periods, and the onset of earlier menopause [5]. Research also suggests that there is a link between polycystic ovary syndrome (PCOS) and an increased risk for the development of diabetes, which is the result of insulin resistance [30]. In the current study, STZ treatment increased cystic follicles and abnormality in follicular development, which suggests that PCOS developing. STZ treatment significantly reduced the number of primary follicles, Graafian follicles, and corpus D diabetic group, Sham diabetic animals + tween as eugenol solvent for 4 weeks, D12 diabetic animals + eugenol 12 mg/kg, D24 diabetic animals + eugenol 24 mg/kg luteum in diabetes animals, which indicated anovulation. In contrast, the number of preantral follicles and the number of degenerating corpus luteum increased in diabetic animals, which indicates a decrease in ovulation, again. Ovarian follicular degeneration and development abnormality in diabetes disease is consistent with findings from other reports [31,32]. These alterations may be due to hormonal dysregulation. Diabetes animals had markedly lower concentrations of sex hormones (LH, FSH, estradiol, progesterone, and testosterone). FSH and LH are necessary for the development of follicles, maturation of oocytes, and for normal ovulation. FSH is necessary for the proliferation of granulosa cells, the production of estrogen hormone, and the evolution of follicles [33]. LH plays the main role in androgen synthesis, oocyte meiosis, the development of the corpus luteum, and ovulation [34]. Progesterone is mainly secreted by the corpus luteum in the ovary. It plays an important role in the menstrual cycle, maintaining the early stages of pregnancy, and embryogenesis [34]. Therefore, pathophysiological features of ovaries may be the result of the dysregulation of sex hormones in diabetes animals. The decreased levels of LH, FSH, and estradiol in diabetes animals are consistent with findings from other studies [35][36][37], which may be partly due to hyperglycemia-induced hypothalamic dysfunction [38]. Changes in ovaries may be correlated with the dysregulation of lipid profiles, too [39].
We also found that STZ treatment caused alterations in the expression of the COX -2 and PPAR-α genes in diabetes animals. COX-2 is the main target for anti-inflammatory therapy because of its key role in inflammation responses. Its expression increases in oxidative stress conditions and its elevated expression have been reported following the activation of the NF-κB signaling pathway as well [11,12]. PPAR-α mediates inflammatory reactions by decreasing the activated forms of NF-κB, too [13]. It also has a role in the regulation of fat and sugar metabolism [13]. Increased COX-2 and decreased PPAR-α in diabetes rats can confirm the induction of oxidative stress, inflammation, and dyslipidemia. Similar results were reported by Miao et al. and Hu et al.,too [40,41].
In the present study, we searched the therapeutic effects of eugenol on pathophysiological features of diabetes including hyperglycemia, dyslipidemia, hypoinsulinemia, ovulation improvement, liver function, and COX-2 and PPAR-α genes expression in an STZ model of diabetes rats. In recent years, polyphenolic compounds have received increased attention due to their anti-hyperglycemic potential and positive effects on diabetes management [7,8]. Eugenol is a phenolic monoterpenoid that is related to the allylbenzene class of chemical agents found in various plants such as cinnamon, clove, and bay leaves [9]. It applies some biological properties including anti-inflammatory activity by suppression of the NF-KB signaling pathway and antioxidant activity [9,10]. Present data showed that eugenol treatment can attenuate adverse features of diabetes by improving the biochemical parameters of the serum and histological alterations in the pancreas. Administration of eugenol decreased the serum levels of glucose in the D12 and D24 groups, suggesting an anti-hyperglycemic potential of eugenol which is in line with previous reports [7,8]. Hypoglycemic effects of eugenol may be due to the reducing the key enzymes of glucose metabolism and regulation of the hepatic production of glucose [42,43]. Also, obtained data showed that the levels of insulin were restored in eugenol-treated diabetes rats. Therefore, the hypoglycemic potential of eugenol may be partly due to the improvement of insulin secretion. Previous studies demonstrated that ROS can decrease the secretion of insulin from pancreatic beta cells and develop insulin resistance [19,20]. Therefore, the regulation of insulin secretion may be partly the result of the antioxidant potential of eugenol.
Here, eugenol was shown to reduce serum amounts of MDA and increase the amounts of SOD, which confirmed the protective potential of eugenol against oxidative stress. The histological finding also confirmed the biochemical results and the improvement of insulin secretion in the D12 and D24 groups. According to obtained data, eugenol improved islet size and reduced the number of necrotic cells, inflammation cells, and disturbed islets in pancreatic samples of treated animals compared to non-treated rats. The reduction of inflammation cells in pancreas tissue of D12 and D24 animals emphasizes the anti-inflammatory properties of eugenol.
Eugenol improved diabetic dyslipidemia induced by STZ, too. Serum TG, TC, and LDL amounts were found to significantly decrease with eugenol administration, while the amount of HDL increased following eugenol treatment. These findings are in line with the earlier suggested antihyperlipidemic properties of eugenol in diabetic animal models [43,44]. Consistent with the relation between glucose and lipid metabolism [18], these data may be partially explained by the hypoglycemic potential of eugenol.
Although eugenol administration had a beneficial impact on reproductive hormones, it had no effects on ovarian follicular development. Following eugenol treatment, the levels of LH, FSH, estradiol, progesterone, and testosterone hormones increased in D12 and D24 groups compared to diabetes and sham animals, which is in line with findings from previous studies [45,46]. Despite the regulation of glucose level, insulin level, oxidative status, lipid profile, and sex hormone level, eugenol could not improve ovarian follicle pools in diabetic rats. Eugenol treatment changed the number of primary follicles, Graafian follicles, corpus luteum, preantral follicles, and the number of degenerating corpus luteum in the D12 and D24 groups, but these changes were not significant. These results are contrary to those found in a previous study of PCOS rats [15]. In the previous study, data indicated that eugenol could restore follicular development in the estradiol valerate-induced PCOS model of rats. Administration of eugenol decreased the number of cysts, improved the number of primordial follicles, Graafian follicles, corpus luteum, and preantral follicles, and decreased the number of degenerating corpus luteum in PCOS animals [15]. This discrepancy may be the result of the different action mechanisms of estradiol valerate and streptozotocin in the induction of ovarian abnormality. However, further studies are needed to demonstrate the mechanisms of action of eugenol on ovarian function and follicular development.
Eugenol advantages were also observed in liver tissue and on liver AST, ALT, and ALP enzymes. The liver is an organ that is sensitive to changes in insulin and glucose metabolism [29]. Here, eugenol treatment markedly decreased the levels of liver AST, ALT, and ALP enzymes indicating that eugenol may preserve the liver tissue from STZ-induced damage in diabetic animals. These observations are in agreement with the findings of Srinivasan et al. (2014) who exhibited that eugenol regulated hepatic marker enzymes in streptozotocin-induced diabetic rats [42]. Regulation of liver markers may be partially explained by the hypoglycemia, hypolipidemia, and the antioxidant potential of eugenol.
Finally, we searched the therapeutic effects of eugenol on the expression of the COX -2 and PPAR-α genes in diabetes animals. Administration of eugenol improved the mRNA levels of COX -2 and PPAR-α genes in the D24 group compared to diabetes and sham animals. Decreased COX -2 and increased PPAR-α in treated rats can reconfirm hypolipidemia and the antioxidant potential of eugenol.

Conclusion
Overall, the present study suggests that the STZ-induced diabetic model of rats exhibited ovarian cysts formation, abnormality in follicular development, and decreased sex hormones. These features are accompanied by dyslipidemia, hypoinsulinemia, liver damage, inflammation and necrosis in the pancreas, and oxidative stress. Eugenol has given anti-diabetic properties by marked restoration of the serum insulin, sex hormones, liver markers, lipids profile, oxidative status, and beta cells of pancreatic tissue. Additionally, contrary to the results obtained from the effect of eugenol on the regulation of ovarian function in PCOS rats, it had no effect on the ovarian function in the STZ-induced diabetes rats. Therefore, further evaluation of obtained data and the mechanisms underlying them are warranted. Further studies are helpful to investigate the interaction of eugenol with other antidiabetic supplements and clarify the mechanism responsible for the reproductive disorders in diabetic individuals. Also, it would be better to measure the serum levels of insulin and sex hormones weekly or biweekly. In conclusion, eugenol may be useful for ameliorating some adverse features of diabetes and used as an adjunct treatment or protective agent accompany by other chemicals in diabetes patients.