Chemoprotective Effect of Syringic Acid on Cyclophosphamide Induced Ovarian Damage via Inflammatory Pathway

: Cyclophosphamide (CP) is very well-known anticancer drug and commonly used against various cancers. CP therapy is related to female ovarian cancer and causes female infertility. The ovarian cancer associated with the increase oxidative stress and inflammatory reaction. Syringic acid (SA) is very well phyto-constituent and already proof antioxidant and anti-inflammatory effects on various diseases. We investigated the chemoprotective impact of SA on CP mediated ovarian damage, and the underlying mechanism. CP (75 mg/kg) was used to cause ovarian damage and rats were randomly divided into separate groups and received a different dose of SA for 14-day. Body weight, food and water intake were determined. Ovarian weight and tumor index was measured. Antioxidant parameters were determined in the serum and ovarian tissue. Pro-inflammatory cytokines, apoptosis parameters and inflammatory mediators were estimated in the serum. Hormonal parameters and Histomorphometry were estimated. Dose dependently treatment of SA significantly ( p < 0.001) decreased the levels of biochemical parameter such as nitric oxide (NO), myeloperoxidase (MPO) and augmented the antioxidant parameters include catalase (CAT), glutathione (GSH), glutathione peroxidase (GPx), superoxide dismutase (SOD) and reduced malondialdehyde (MDA) level in serum and ovarian tissue. SA treatment significantly ( p < 0.001) suppressed the level of luteinizing hormones (LH), anti-mullerian hormone (AMH), estradiol (E2) and follicle-stimulating hormone (FSH) as well as ovarian follicles. SA significantly ( p < 0.001) down-regulated cytokines, inflammatory mediator and caspase-3 parameters. Taken altogether, we conclude that SA considerably reduced ovarian damage via reduced oxidative stress and inflammatory reaction.

The reproductive system contains numerous signaling molecules responsible for oocyte maturation and follicle development 11,12 . Nuclear protein plays an important role in the cell cycle and is recognized as nuclear cell antigen PCNA . In strong antral follicles, increase immunoreaction of PCNA suggests the fast development of granulosa and theca cells during follicular enlargement 13 . During the development of follicles, A super family member such as TGF-b, GDF-9 Growth Differentiation Factor-9 is accountable for granulosa cell growth and differentiation. During follicular growth, they are discharged from the oocytes of mammals 14 . Literature has reported the negative impacts of CP on the tissue of the ovary and no research yet far have explored the effects of syringic acid SA on ovaries which was induced by drug CP and evaluating inflammatory pathway 14,15 .
SA is a benzoic acid in nature, which is obtained from fruits and vegetables 16 and utilized in the management of diabetes as a Ayurvedic Indian traditional medicine 16,17 . Several studies conducted in vitro and in vivo reported its beneficial function in different cancers and non-communicable diseases 18,19 . SA has several therapeutic activities for example anti-lipid peroxidative, anti-inflammatory, anticancer effects, anti-angiogenesis, antidiabetic, hepatic cancer, and neck cancer 16 18 . SA shows strong anti-proliferative activity in vivo in colorectal cancer and breast cancer via quenching its reactive oxygen species. Earlier research also revealed the inhibition property of SA in the development of exophytic tumor-induced HBPCs in DMBA entirely 16 18 .
In recent days, the usage of phytochemicals or bioactive compounds to manage diseases in humans has been of extensive community and scientific attention 20,21 . It has an efficient free radical scavenger and eases the signs of oxidative stress. At positions 3 and 5, SA s pharmacological property is ascribed via the occurrence of methoxy groups on the aromatic ring. SA s high antioxidant activity will bestow its valuable effects on the health of human beings 18,19 . SA can modulate the function of enzymes, the dynamics of proteins and various transcription factors implicated in diabetes, inflammation, cancer and angiogenesis 16,18,22 . Experimental evidence and histological studies during in vivo have illustrated the potential underlying mechanisms.

Animals
In the current experimental protocol, Swiss albino Wistar rats weight 150-180 g sex female used. The rats were collected from the animal laboratory and kept in the single cage under standard experimental conditions 21 3 temperature and 60 70 relative humidity with maintain the light and dark for 12 h cycle. The rats were free from food and water. The current experimental study was conducted according to the institutional animal protocol. The rats were acclimation for 10 days before the experimental study.

Preparation of toxicant
CP was used for the induction of ovarian damage. Single intraperitoneal injection of CP 75 mg/kg was dissolved in the saline and rats were treated once a week for next 3 weeks.

Experimental protocol
After successfully acclimation of animal 10 days , the rats were grouped as follow and each group contains the 6 rats. Group I: control receive only saline in the whole experimental period ; Group II: received CP 75 mg/kg ; Group III: received CP 75 mg/kg and SA 5 mg/kg ; Group IV: received CP 75 mg/kg and SA 10 mg/kg and Group V: received CP 75 mg/kg and SA 20 mg/kg , respectively. After 14 weeks, the rats were anesthetized using the thiopental sodium and blood samples were withdrawn by puncturing the retro-orbital and centrifuged at 10,000 rpm for 20 min and separate the plasma and serum and stored at the 80 for analysis of biochemical parameters. After that the rats were scarified using the cervical dislocation and immediately removed the ovarian tissue and processed for further analysis.

Biochemical assays
For the biochemical assay, ovarian tissue was homogenized using the Teflon glass homogenizer in KCl 150 mM at a dilution 1:10 w/v . The homogenates were centrifuged for 30 min at 18 k rpm at 4 .
Catalase CAT , glutathione GSH , superoxide dismutase SOD and malondialdehyde MDA and glutathione peroxidase GPx were estimated using ELISA kits.

Hormonal assays
Hormonal assays such as follicle-stimulating hormone FSH , estradiol E2 , anti-mullerian hormone AMH and luteinizing hormone LH were estimated in the serum of experimental rats using enzyme-linked immunosorbent assay ELISA kits Sunlong Biotech Co., Ltd., Zhejiang, China by using the manufacture instruction.

In ammatory and caspase parameters
Inflammatory mediators such as cyclooxygenase-2 COX-2 , prostaglandin E 2 PGE 2 and caspase parameters such as caspase-3 were estimated using the ELISA kits Sunlong Biotech Co., Ltd., Zhejiang, China using the manufacture instruction.

Histomorphometry
Female rats have a reproductive strategy, which allows ovulating and conceiving within 4-5 days. During the estrous cycle, a cohort of resting primordial follicles starts to expand into primary follicles and this process occurs until the formation of early tertiary follicles. A small number of tertiary follicles enter a preovulatory stage and converted into the Graafian follicle. Following the extrusion of the secondary oocyte from the Graafian follicle, the granulosa and thecal cells of the follicle remnant undergo hypertrophy and hyperplasia. This process is called luteinisation that occurs under the influence of luteinising hormone and prolactin. Luteinization results in mature corpus luteum.

Statistical analysis
For the statistical analysis, GraphPad Prism 5 USA was used. Data showed as mean standard deviation SD . All biochemical data were scrutinized using the one way analysis of variance ANOVA and Dennett s test was used. A significant value presented as p 0.001. Fig. 1 The effect of SA on the ovary weight of CP induced ovarian damage in rats. All the data presented SEM. *p 0.05; **p 0.01; ***p 0.001.

Ovarium weight
All rats groups ovary weight are illustrated in Fig. 1. CPinduced group rats showed decreased ovary weight and SA treatment significantly p 0.001 increased the ovary weight as dose dependent manner.

Follicular population
In Histomorphometrically study, no significant difference was observed among the groups regarding mean no. Of follicle count of normal and atretic size. The results of population of follicles are presented in Fig. 2. No statistically significant difference in the number of primordial or other follicles p 0.001 was found between the rat group. All the groups reported statistically significant differences p 0.001 in the values of corpus luteum and atretic follicles. Corpus luteum and atretic follicles achieved maximum levels in the CP group, whereas the minimum number of corpus luteum and atretic follicles was present in the SA lower dose group. Figure 3 illustrates the oxidant profile such as MDA and antioxidant enzymatic profile such as SOD, CAT, GPx, and GSH. CP induced ovarian damage shows up-regulation in the MDA levels and down-regulation in the GSH levels and SOD activity compared to the control group. There was a significant reduction observed in level of MDA and elevation in the level of GSH and SOD activity in the ovarian tissue of the animals treated with SA in dose dependent manner compared to negative controls.

Hormonal assays
Assessment of the activity of SA on the ovarian reserve markers are displayed in Fig. 4. Significantly reduction was observed in the levels of Serum FSH and LH in SA treated rats in diseases with ovarian damage in a dose dependent way. Although, the level of E2 and AMH in rats treated with SA were remarkably raised contrast with CP induced ovarian damage group. Not more significance difference was observed in the level of serum hormone of the rats group received CP SA 5 mg/kg bw and CP SA 10 mg/ kg bw Fig. 4 .

Effect on caspase-3 activity
Caspase-3's most strong immunoreactivity was measured by semi-quantitative assay in rats treated with CP when contrast with the other groups. The administration of dif- ferent doses of SA in dose dependent manner alleviated the extent of caspase-3 immunoreactivity. Caspase-3 level of immunoreactivity in the control group was close to that of the SA treated group Fig. 5 .
3.6 Effects of SA on ovarian NF-κB, TNF-α, IL-1β and IL-6 levels The levels of NF-ÿB, TNF-α, IL-1β and IL-6 levels in the ovary of the CP-treated group were significantly elevated as evaluated by the control group. We observed a substantial difference between the CP-treated group and control group in the levels of NF-ÿB, TNF-α, IL-1β and IL-6. Nevertheless, these levels were significantly modulated by SA treatment 5, 10 and 20 mg/kg compared with the CP group. Not too much significance difference between the control group rats and in the SA treated group rats 20 mg/ kg bw of ovarian pro-inflammatory cytokine levels NF-ÿB, TNF-α, IL-1β and IL-6 Fig. 6 .

Effects of ZO on ovarian and uterine iNOS, PGE 2 and
COX-2 activities In contrast to the control group p 0.001 , iNOS, PGE 2 and COX-2 behaviors in ovarian damage were observed to be considerably bigger in the CP-treated group. Although, compared to the CP group, treatment with SA 5, 10 and 20 and 50 mg/kg significantly reduced iNOS, PGE 2 and COX-2 activities in dose dependent manner Fig. 7 .

Effect of SA on BcL-2 level
Compared to the control group p 0.001 , the Bcl-2 level in ovary was significantly decreased in the CP-treated group. significant elevation was observed in levels of ovarian Bcl-2 after the administration of SA in CP induced ovary damage group Fig. 8 .

Discussion
This research examined the potential protecting effect of SA in female rats against ovarian damage induced by CP. Anticancer agents such as CP, cisplatin, methotrexate, are employed in the management of various types of cancer like lung cancer, cancer of testes, ovarian cancer, which is metastatic in nature and in other solid tumors 23,24 . Con- Fig. 4 The effect of SA on hormonal parameters of CP induced ovarian damage in rats. a: estradiol, b: FSH, c: LH and d: AMH. All the data presented SEM. *p 0.05; **p 0.01; ***p 0.001. Where FSH Follicle stimulating hormone, AMH Anti-mullerian hormone; LH Luteinizing hormone. Fig. 5 The effect of SA on caspase-3 activity of CP induced ovarian damage in rats. All the data presented SEM. *p 0.05; **p 0.01; ***p 0.001. Fig. 6 The effect of SA on cytokines and inflammatory mediator of CP induced ovarian damage in rats. a: TNF-α, b: IL-6, c: IL-1β and d: NF-kB. All the data presented SEM. *p 0.05; **p 0.01; ***p 0.001. Where TNF-α Tumor necrosis factor-α, IL-6 Interleukin-6, IL-1β Interleukin-1β and NF-kB Nuclear Kappa B factor. Fig. 7 The effect of SA on inflammatory mediator of CP induced ovarian damage in rats. a: iNOS, b: COX-2 and c: PGE 2 . All the data presented SEM. *p 0.05; **p 0.01; ***p 0.001. Where iNOS inducible nitric oxide synthase, COX-2 Cyclooxygenase-2 and PGE 2 Prostaglandin E 2 . Fig. 8 The effect of SA on Bcl-2 activity of CP induced ovarian damage in rats. All the data presented SEM. *p 0.05; **p 0.01; ***p 0.001. Where Bcl-2 B-cell lymphoma-2.
versely, some of these substances lead to cause dysfunction in ovary in females and gonadotoxic and develop an imbalance of hormonal production, injury, cause a infertility, which may be temporary or permanent 24,25 . Ovarian cancer induced by CP is scrutinized by calculated levels of hormones FSH, E2, inhibin B and AMH in plasma 9,26 . Cytokines are proteins of low molecular weight mediating the communication between cells. They also control the proliferation, differentiation, cell survival, activation of immune cells, migration of cell and cell apoptosis. NF-κB is most significant transcription factor considered to be oxidative stress prone 7, 14 . This pathway is well known therapeutic objective as it plays a vital role in activating and regulating the genes of transcription factors for example, TNF-α, IL-1β, IL-6, COX-2 and iNOS. Hence, NF-κB inhibition can be helpful in reducing ovarian damage.
Furthermore, tumourigenicity inhibition has been observed in ovarian cancer cells in humans after blocking activity of NF-κB 27 . iNOS is an enzyme utilized in the formation of nitric oxide NO inside the body 28 . Surplus quantity of NO reacts to oxidize and nitrate macromolecules such as GSH, proteins, lipids and DNA with a superoxide anion to form the radical peroxynitrite producing damage to cell 29,30 . Furthermore, intracellular GSH is consumed by extreme NO, thus increased the oxidative stress sensitivity 5 . In several studies, the critical role of NF-κB and NO in showing the adverse reaction of therapy with CP is well mentioned in literature 31,32 . Enhanced levels of pro-inflammatory cytokines for example, NF-κB, TNF-α, IL-1β and IL-6, and COX-2 and iNOS activities were found in ovarian cancer induced by CP compared to the control group is recorded in this study. In contrast to the CP-induced group, treatment with SA observed to substantially declined certain values.
The protein family Bcl-2 was also studied for its function in apoptosis 33 . The family contains anti-apoptotic members e.g. Bcl-2, Bcl-XL, and Mcl-1 and pro-apoptotic members e.g., Bax, Bak, Bid, and Bad 32,33 . The anti-apoptotic Bcl-2 regulates several primary apoptosis steps involving the ion channel formation mediating cytochrome c release, homeostasis of mitochondria and membrane potential of mitochondria 32 . This study demonstrated thoughtful effects of CP on caspase-3 and Bcl-2. However, CP increases the expression of Caspase-3 and declined the level of Bcl-2. SA prevents the tissue by raising the Bcl-2 level and reducing the Caspase-3 expression proven the antioxidant properties.
Among all cell lines, SA hindered dose-dependent cell proliferation. The induction of cell apoptosis induction may lead to cell cycle attestation G1/S or G2/M. Yet it is unclear whether other mechanisms exist to explain the induction of apoptosis by SA. In an earlier study, Liu et al. suggested the of MLS-2384 anti-survival effect on different variety of cancer cells such as ovarian cancer 19 . Yu et al. reported 2Z, 3E -6-bromoindirubin-3 -oxime inhibition effect on ovarian cancer cell lines regarding proliferation, migration and cell invasion. We performed experiments on 2 separate lineages of ovarian cancer cells in this research. Our findings indicated that SA drastically repressed the viability cell and initiated apoptosis in cancer cells of the ovaries. Mustard phosphoramide lead to apoptosis by cross-linked DNA and acrolein, extremely reactive molecules provoking toxicity and interrupt the function of normal cells. More production of free radicals with DNA binding stimulates caspase-3 signals and facilitates cell death subsequently. This study revealed the presence less quantity of caspase-3 in the SA and CP group as compared CP induced ovarian damage group.
The outcomes of this study reveal the potential of SA strengthened markers of ovarian reserve after CP induced an insufficiency of ovaries. A significant elevation in AMH and E2 levels and substantial decline in levels of FSH and LH were observed in groups treated with different doses of SA as comparative to CP induced ovarian damage in rats. Various research paper has examined the impacts of ovarian harm and antioxidants caused by chemotherapy on markers of the ovarian reserve 9 . Özcan et al. reported the impact of resveratrol on oxidative damage caused by cisplatin to the ovarian reserve markers in animals. They observed that treatment with resveratrol up-regulated the amount of AMH considerably compared with the control group.
In this study, oxidative stress profile, MDA was observed to be raised and SOD activity was significantly reduced in the CP-induced rat ovary, indicating that administration of CP produces oxidative destruction the ovary component i.e. lipids and proteins 9,34 . The levels of MDA and SOD activity were reversed after the administration of SA in dose dependent manner and reflecting the protective effects of SA on the side effects of CP. SA declines the generation of peroxynitrite, which is a strong oxidant, formed during the reaction between nitric oxide with superoxide anion by quenching the activity of SOD with superoxide anion. Levels of Catalase were also improved by different doses of SA and it may be estimation of nonselective level of enzyme catalase that utilize hydrogen peroxide. This chemical is not only detached by catalase but some other antioxidant enzymes for example GPx, GSTs are also involved in it 9,34,35 . According to best knowledge, on the basis of biochemical assays, CP, produce a lipid peroxidation and antioxidant component in the ovary of the rats but administration of SA alleviates the lipid peroxidation induced by a CP and high level of SOD in the ovary of rats, which evidenced the preventive effects of SA in ovarian damage.