Effects of chitooligosaccharide-zinc on mice ovarian function in premature ovarian failure via regulating the sestrin2/nrf2 signaling pathway

The chitosan oligosaccharide-zinc (COS·Zn) is also a powerful antioxidant and anti-aging scavenger, whose anti-oxidative ability immensely exceeds that of vitamin C. Therefore, this study aimed to investigate the protective effects and potential mechanism by which COS·Zn improves ovarian function and delays aging in POF. The female KM adult mice and mice were divided into treated and prevention groups; these or COS·Zn (150mg/kg·d, for 21 respectively. treated groups, we analyzed the prevention groups (300mg/kg.d COS·Zn-treated group) using HE staining. The results showed a decrease in antral


Introduction
Premature ovarian failure (POF), as the main cause of ovogenic infertility, refers to the development of amenorrhea due to the cessation of ovarian function before the age of 40 years [1 -3]. A wide spectrum of pathogenic mechanisms may lead to the development of POF, including chromosomal, genetic, autoimmune, metabolic (galactosemia), infectious (mumps) and iatrogenic (anticancer treatments) causes [4][5][6]. In the degenerative process, mitochondrial dysfunction causes the overproduction of reactive oxygen species (ROS), which results in oxidative damage. Oxidative stress may play a role in the degeneration process of POF, which could be more pronounced in the POF population and could impair antioxidant defenses and increase susceptibility to oxidative stress and damage [1, 7,8]. Therefore, oxidative damage is considered inevitable in POF, and the theory of oxidative-stress-related ovarian damage has attracted increasing attention.
Nowadays, no effective therapies for POF are available. Chitosan oligosaccharide (COS) molecules contain a large number of active amino and hydroxyl groups [9], and can be chelated with inorganic zinc to produce an organic form of chitooligosaccharide-zinc (COS·Zn) by controlling the reaction conditions [10, 11]. 5 As a basic amino oligosaccharide, COS·Zn is also a powerful antioxidant and antiaging scavenger [12], whose anti-oxidative ability immensely exceeds that of vitamin C. Cumulative evidence suggests that COS·Zn possesses biological properties against oxidative stress in different organs and tissues [13][14][15]. One study has reported that COS·Zn effectively reduced the content of MDA in Dgalactose-treated mice and significantly increased the activities of SOD, T-AOC, GSH-Px and CAT in the serum, kidney and liver of the mouse model; in particular, the effect of protective enzymes in serum is better than in the COS, ZnSO 4 and COS+ZnSO 4 mixture groups [13].
Moreover, some studies have reported that COS·Zn is associated with regulation of the growth and development of the reproductive system in mice, and plays a major role in improvement of the tissue structure of reproductive organs/fertility in female and male mice. Another study has indicated that the relationship between COS·Zn and mammalian ovarian function is closely related [16]. The COS·Zn-treated group could obviously improve the female fertility rate, resulting in an increased number of ovarian follicles and endometrial thickness, an abundance in the uterine glands, increased egg vitality and fertilization [8]. However, in the process of POF, the oxidative damage to ovaries could not be avoided, so we included COS·Zn in our histological study to determine the effect of oxidative damage on POF, in consideration of the potential of COS·Zn in therapies for POF.
In mammals, the Sestrin family comprises three members (Sestrin 1-3), which can be induced by oxidative stress and DNA damage in a p53-dependent manner [17][18][19][20]. Sestrin2 (Sesn2), a highly conserved protein, is an essential member of the Sestrin family, which is also expressed in endothelial and macrophage cells, where it displays a protective role [21,22]. Our research has also shown that Sestrin is 6 strongly expressed in ovarian tissue; however, the exact mechanism for ovarianprotective effects have not been clearly elucidated. To date, the cellular function of Sestrin2 (SESN2) has been well recognized; however, the effect on ovarian function has not been fully determined.
As an important endogenous antioxidant factor, NF-E2-related factor 2 (NRF2) is an essential part of the oxidative stress response [23][24][25]. In oxidative stress injury, NRF2 is separated from Keap1 (Kelch-like ECH-associated protein 1) and enters the nucleus [26][27][28][29]. When combined with antioxidant response elements (AREs), the expression of phase II detoxification enzymes and antioxidants is regulated by multiple signaling pathways, such as sulfiredoxin1 (Srx1) and thioredoxin1 (Trx1) [30]. Studies have indicated that NRF2 is mainly regulated by Keap1 [31], and that NRF2 plays an important role in regulating ovarian dysfunction and treating ovarian cancer [32][33][34][35]. Further studies have clarify that SESN2 could both directly and indirectly participate in the regulation of NRF2 in the process of metabolism [19,36]. However, despite NRF2 being shown to be involved in many signaling pathways, the specific mechanism and functional effects are not completely understood.
The protective effects of COS·Zn against oxidative damage have not been clarified in POF, and there are no similar studies available. Therefore, this study was undertaken to demonstrate the protection against oxidative damage in POF to determine whether COS·Zn would, in part, ameliorate ovarian function. If COS·Zn shows protective effects in POF, dietary COS·Zn could serve as a medicine against oxidative damage.

Ethical approval
All procedures with animals were approved by the Institutional Animal Care and Use Committee of Nanchang, and were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.

The preparation of COS·Zn
COS·Zn was prepared under optimal reaction conditions, which were determined in the study of Jin Yue [11]. COS and zinc sulfate were weighed with a mass ratio of 2:1 and respectively dissolved in two beakers with equal amounts of distilled water.
Then the two solutions were mixed in equal volumes and the pH was slowly adjusted to 7 with 1% ammonium hydroxide solution. The mixture was then chelated at 40°C for about 30 minutes, and anhydrous ethanol at three times the volume of the mixture was slowly added to it. After rinsing with anhydrous ethanol, the product was dried in a 60°C oven for several hours to obtain a yellow powder, which was just COS·Zn. The zinc content of the COS·Zn prepared with this method was 10%.

Mice and treatments
Female KM mice aged 6 weeks (weighed 20-25g), purchased from the Laboratory Animal Center of Nanchang University, were allowed to adapt to the facility for at least 7 days before the experiment started. Food and water were abundant at all times. The temperature was maintained at 22±1°C. Each mouse received a 12h cycle of brightness and darkness, and mice were randomly divided into the treatment and prevention groups, with 6 mice in each.
Each group of mice, except the control group and COS·Zn+CY/BUS group, was administered CY/BUS for 21 days to build the models of premature ovarian failure (POF). The mice in the COS·Zn+CY/BUS group were prophylactically administered COS·Zn (300mg/kg.d) for 21 days. No special operations were offered to the control group. 8 The mass ratio of COS to Zn in the COS·Zn synthesized in our laboratory is 9:1, and all of the concentrations of COS·Zn mentioned in this article were measured by COS ( Fig. 1). Based on the literature review and our actual observation results, it was determined that the daily food intake of the experimental mice was about 6g, which means that every mouse in the COS·Zn (150mg/kg.d) group requires 1mg COS·Zn per day and 2mg COS·Zn per day in the COS·Zn (300mg/kg.d) group. We took 0.04g COS·Zn powder, dissolved in 8mL normal saline, shaken and mixed, and added 2mL sodium carboxymethyl cellulose to prevent the deposition of COS·Zn powder; this was shaken and mixed again, to make a 4mg/mL COS·Zn solution. After the establishment of POF models, the mice underwent the following procedures: (1)

Histological analysis of ovarian tissue and ovarian follicle count
Mice were killed by cervical dislocation. The ovaries were collected, and fixed in 4% paraformaldehyde, before they were embedded in paraffin, as described previously [37]. The paraffin-embedded ovaries were cut into serial sections and stained using hematoxylin and eosin (HE). The ovarian follicles were counted as described previously [38,39]. Immunostaining of the ovaries from mice from the treated and prevention groups was performed according to previously published procedures [40].

Tunnel assay
The apoptotic rates of ovarian sections were detected by terminal deoxynucleotidyl transferase mediated dUTP-biotin nick end labeling (TUNEL), and the procedure was performed using an Apoptosis Detection Kit (Solarbio Biotech Co., Ltd., Beijing, China) according to the manufacturer's instructions. After being treated with TUNEL reaction mixture for 1 h, the sections were washed twice with PBS for 10 min and counterstained with 4',6-diamidino-2-phenylindole (DAPI) (Sigma-Aldrich) for 5 min.
Washing was then performed three times with ddH 2 O. A confocal laser scanning microscope (Leica, TSP8) was used to photograph sections, and the apoptotic rate was analyzed using IPP 6.0 Software.

SOD and GSH detection
Superoxide dismutase (SOD): SOD was assessed using the WST-1 method. Blank wells, standard wells, determination wells and control wells were prepared according to the experimental requirements. Serum from mice was incubated at 37°C for 20 min. The absorbance value of each well at 450 nm was detected by a microplate reader.
Malondialdehyde (GSH): GSH was determined using the thiobarbituric acid method.
According to the instructions provided by Shanghai GeneChem Biotechnology Co., Ltd. (Shanghai, China), blank wells, standard wells, determination wells and control wells were prepared. The absorbance value of each well at 450 nm was detected by a microplate reader (1 cm of optical path, set zero by distilled water).

Hormone level measurement
Blood was drawn from the orbital socket and serum was collected. The levels of E2 and FSH were measured by the biological company.

Statistical methods
All analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., San Diego, CA). The statistical comparisons between different groups were analyzed by Student's paired t-test. The threshold of p < 0.05 was considered significant; p < 0.01 and p < 0.001 were each considered extremely significant.

11
The bimodal C=O bond disappeared at about 1600 cm -1 , and the absorption peak obviously weakened; these data indicate that the C-O double bond chelates with the zinc ion in the COS molecule and forms "C.O-Zn". The results of infrared spectroscopy showed that COS chelates with the zinc ion and forms a stable chelate which is known as "COS·Zn" (Fig. 1a, b).

COS·Zn supplementation improves ovarian and follicular developmental defects caused by POF in mice
Ovarian morphology of the control and treatment groups (150, 300mg/kg·d COS·Zntreated groups) was determined by HE staining (Fig. 2a). The numbers of primary follicles, secondary follicles, corpus, and atretic follicles were counted (Fig. 2a). In the 150 mg/kg·d group, we found that the number of antral follicles in the COS·Zntreated groups was lower than that in the control group (p < 0.05), and their numbers were dramatically reduced in CY/BUS groups and the 300 mg/kg·d group (p < 0.05), but the 150 mg/kg·d and 300 mg/kg·d COS·Zn-treated groups were not significantly different. The numbers of primary follicles and secondary follicles were significantly higher in the 150 mg/kg·d and 300 mg/kg·d COS·Zn-treated groups compared with the control group (p < 0.01, p < 0.05). However, the number of primary follicles was significantly higher in the 300 mg/kg·d COS·Zn-treated group compared with the CY/BUS group (p < 0.05). Obviously, we found that the ovarian index (ovarian weight/body weight ratio), and levels of FSH and LH all increased notably in the 300 mg/kg·d COS·Zn-treated and CY/BUS groups (p < 0.05, p < 0.001, (Fig. 2b, c).
To further clarify the changes in the amounts of follicles and their proportion between different treated groups, we analyzed the prevention groups (300mg/kg.d COS·Zn-treated group) using HE staining. The results showed a decrease in antral follicles in the prevention group compared to the control group (Fig. 3a, p < 0.01).
We also found that the numbers of primary and secondary follicles increased in the prevention group and control group; however, for the prevention-ovary index, the level of FSH and LH were all dramatically increased in the prevention group compared with the control and CY/BUS groups, respectively (Figs. 3b, c, p < 0.01, p < 0.001). Collectively, the data suggest that COS·Zn (300mg/kg.d) improves the level of ovarian and follicle damage by POF.

COS·Zn supplementation improved ovarian function in the POF in mice
To evaluate changes in the ovarian functions of COS·Zn-treated mice, we first detected the expression of MVH and OCT4. The results revealed increased MVH and OCT4 levels in the 300 mg/kg·d COS·Zn-treated groups compared with the CY/BUS group (Fig.4a, c, p< 0.05, p < 0.001). The changes in MVH were observed with a significant increase in the COS·Zn-prevention groups (Fig.4b, e, p < 0.01). In addition, PCNA levels also increased during the 150 mg/kg.d.d and 300 mg/kg.d.d COS·Zn-treated groups compared with the CY/BUS group (Fig.4a,c, p < 0.05); this trend was also detected in the control and COS·Zn-prevention groups (Fig.4b,e, p < 0.05). Using the Tunnel assay, we found that the apoptotic signal was negative in granulosa cells and oocytes (Fig.4d, f). The number of apoptotic cells per section in the 300 mg/kg.d.d COS·Zn-treated groups was significantly lower than that in the control and CY/BUS groups.
Meanwhile, the protein levels of P53 and P16 were markedly down-regulated in the treated and prevention groups compared with CY/BUS, except for the expression of P53 in the prevention groups (Fig.5a, b).

COS·Zn causes oxidative stress status and anti-aging capacity to
increase by regulating the sestrin2-nrf2 signaling pathway in the POF mice 13 To investigate causes of the changes in ovarian function, we detected the oxidative stress status and anti-aging capacity in ovaries using COS·Zn-treated mice. In the treated groups, the protein level of NRF2 in the 150 mg/kg.d.d and 300 mg/kg.d.d COS·Zn-treated groups were markedly increase (Fig.6a, p < 0.05). Meanwhile, the SESN2 and SOD2 proteins were significantly higher in the 150 mg/kg.d.d compared with the CY/BUS groups (Fig.6a, p < 0.01, p < 0.001), but the 300 mg/kg.d.d and CY/BUS groups showed no significant difference in the level of SESN2. Similarly, the NRF2 and SESN2 proteins were up-regulated in the COS·Zn-prevention groups ( Fig.6c,p < 0.05), and the change in SOD2 exhibited no significant effect. We then analyzed changes in the GSH and SOD2 levels in different groups. The results revealed increased GSH and SOD2 levels in the 150 mg/kg.d.d and 300 mg/kg.d.d COS·Zn-treated groups compared with the CY/BUS group (Fig.6b), and the same trend was shown in the COS·Zn-prevention groups (Fig.6d).

Discussion
COS·Zn showed good anti-aging effects and enhanced immune function in previous studies, as documented by Hao et al. [13], who found that the scavenging effect of COS·Zn complexes was significantly better than that of COS, ZnSO 4 and COS+ZnSO 4 [13]. At the same time, with an increase in the concentrations of COS·Zn, the scavenging ability of DPPH free radicals gradually increased [41]. According to the references, the successful synthesis of COS·Zn was investigated by infrared spectroscopic analysis which showed that COS chelates with zinc ions and forms a stable chelate [41]. However, there are several studies devoted to the roles of COS·Zn, including the inhibitory activity of microorganisms, and improved organ index for the liver, thymus, spleen, and kidney. Notably, the ovary was the major target organ, so the aim of the present study was to analyze the anti-oxidative effect of COS·Zn on the ovarian function of POF mice.
In treated groups, our study showed that the numbers of primary and secondary follicles were significantly increased in the 300mg/kg·d COS·Zn-treated group, and atretic follicles were also decreased in the 300mg/kg·d COS·Zn groups. Furthermore, the ovarian index results further suggested that COS·Zn decelerated apoptosis in ovaries and advanced ovarian follicular development, which is consistent with previous studies [16]. Collectively, the data suggest that COS·Zn (300mg/kg·d) improved the level of ovarian and follicle damage in POF. Thus, the preparation groups were treated with 300mg/kg·d COS·Zn. The decrease in antral follicles in the prevention groups was like that in the treatment groups. We also found that the numbers of primary and secondary follicles increased in the prevention groups.
Meanwhile, regarding the prevention-ovary index, the levels of FSH and LH were all dramatically increased in the prevention groups compared with the control and CY/BUS groups, respectively. Therefore, the development of the ovary was promoted by COS·Zn treatment in POF mice. Follicular development-related genes may play certain roles in this process [42]. Surprisingly, the changes in MVH, PCNA and OCT4 observed a significant increase in the COS·Zn groups compared with normal and CY/BUS mice.
To further examine the mechanism of COS·Zn on the ovaries, we performed ovarian proteomic analysis. Sestrin2/NRF2 is probably the most important master of the expression of molecules that exert antioxidant functions within organs and cells. In the ovaries of POF mice, COS·Zn up-regulates the levels of Sestrin2, NRF2 and SOD2, which are responsible for the antioxidant effects. Taken together, COS·Zn improves ovarian and follicular developmental defects caused by regulating the sestrin2-nrf2 signaling pathway, and these results suggest a novel product that is effective a POF prevention and treatment drug.

Conclusion
In reproduction, no effective therapies for POF are available. COS·Zn is the basic and primary requirement for the growth and development of the reproductive system in mice [16], and plays a major role in improvement of the tissue structure of reproductive organs/fertility in female and male mice. Increasing the level of COS·Zn is an effective way to improve ovarian and follicular development caused by regulating the sestrin2-nrf2 signaling pathway, and these results suggest a novel product that is effective a POF prevention and treatment drug.

Availability of data and materials
The datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.

Ethics approval and consent to participate
Our study protocol was approved by the Institutional Animal Care and Use Committee of Nanchang, and were performed in accordance with the National Research Council Guide for the Care and Use of Laboratory Animals.

Consent for publication
Not applicable. The expression of P53/P16 genes during COS•Zn-treated ovaries in mice by Western blotting 27 Figure 6 The expression of NRF2/SESN2/SOD2 genes during COS•Znic-treated ovaries in mice. (a) Wes