Contributed equally
Hydrogen has been reported to exert a therapeutic effect in several diseases due to its antioxidative, anti-inflammatory and anti-apoptotic properties. The aim of the present study was to investigate whether hydrogen-rich saline treatment could attenuate ovarian damage induced by cisplatin. A total of 240 adult, virgin, female Sprague Dawley rats, weighing 180–220 g, were randomly divided into four groups (n=60 per group): Control (Con), control + hydrogen-rich saline (Con + H2), cisplatin-induced ovarian injury (OI) and cisplatin-induced ovarian injury + hydrogen-rich saline (OI + H2). Cisplatin was diluted in saline immediately before use. In the OI and OI + H2 groups, the rats were administered a dose of cisplatin on the 1st and 7th days. The rats in the Con + H2 and OI + H2 groups were intraperitoneally injected with hydrogen-rich saline (10ml/kg body weight) once a day over a 2-week period. On the 14th, 28th and 42nd days (T1, T2 and T3) after the cisplatin injection, femoral vein blood was collected. At the end of the experiment, ovarian homogenates were prepared, and the samples were used for estrogen (E2), follicle-stimulating hormone (FSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) examination. In addition, rats (n=10 per group) were sacrificed for bilateral ovary removal; one was fixed in formalin for follicle-counting analysis, while the other was used for nuclear factor erythroid 2-related factor 2 (Nrf2) detection by western blotting. Hydrogen-rich saline attenuated the FSH release, elevated the level of E2, improved the development of follicles, and reduced the damage to the ovarian cortex at T1, T2 and T3 in the OI + H2 rats. Cisplatin induced oxidative stress by increasing the levels of oxidation products and attenuating the activity of antioxidant enzyme, which could be reversed by hydrogen-rich saline treatment. Furthermore, hydrogen-rich saline regulated the Nrf2 protein expression in rats with ovarian damage. In conclusion, hydrogen-rich saline exerts a protective effect against cisplatin-induced ovarian injury by reducing MDA and increasing SOD and CAT activity. Ovarian injury induced by chemotherapy involves the activation of Nrf2.
In excess of 678,000 women were diagnosed with cancer in 2007 (
Cisplatin (
Hydrogen has been reported to exert a therapeutic antioxidant effect, by selectively reducing cytotoxic ROS, and reduce levels of inflammation and apoptosis in several diseases (
Adult, virgin, female Sprague Dawley rats, weighing 180–220 g, were obtained from the Laboratory Animal Center of the Academy of Military Medical Sciences (Beijing, China). The animals were housed at 20–22°C with a 12-h light/dark cycle and fed standard chow and water
The hydrogen-rich saline was prepared as previously described (
A total of 240 animals were randomly divided into four groups (n=60 per group): Control (Con), control + hydrogen-rich saline (Con + H2), cisplatin-induced ovarian injury (OI) and cisplatin-induced ovarian injury + hydrogen-rich saline (OI + H2). The animal model was established using a previously described method (
In the Con + H2 and OI + H2 groups, the rats were intraperitoneally injected with hydrogen-rich saline (10 ml/kg body weight) once a day between the 1st and the 14th day. Vaginal smears were obtained from each rat at 8:00 a.m. each day for ≥5 days after cisplatin injection to ensure the ovarian injury model was successful.
On the 14th, 28th and 42nd days (T1, T2 and T3) after cisplatin injection, femoral vein blood was collected from the rats (n=10 per group) and centrifuged at 10,000 × g, 4°C for 10 min. At the same time, ovarian tissue was collected, and homogenates were prepared via centrifugation, using the aforementioned conditions. These sample were stored at −80°C until the estrogen (E2), follicle-stimulating hormone (FSH), superoxide dismutase (SOD), catalase (CAT) and malondialdehyde (MDA) examination. At each time-point, another 10 rats (n=10 per group) were sacrificed for bilateral ovary removal; one was fixed in formalin for follicle-counting analysis, while the other was used for nuclear factor erythroid 2-related factor 2 (Nrf2) detection by western blotting.
Ovarian follicle populations were counted using previously described methods (
The serum E2 and FSH levels were measured using ELISA kits from R&D Systems (Minneapolis, MN, USA). The assays were performed according to the manufacturer's instructions. The absorbance was read on a microplate reader (Denley Dragon Wellscan MK 3; Thermo Fisher Scientific, Vantaa, Finland), and the concentrations were calculated based on a standard curve. All standards and samples were run in duplicate.
To investigate the mechanism associated with the effects of hydrogen-rich saline, the levels of antioxidant enzymes and oxidation products in the serum and ovarian tissue were measured. The activities of SOD, CAT and MDA were measured using commercial kits purchased from Cayman Chemical Company (Ann Arbor, MI, USA). According to the manufacturer's instructions, total SOD activity was assayed at 450 nm and CAT at 540 nm. Spectrophotometric readings of SOD and CAT were performed using a DU-640B spectrophotometer (Beckman Coulter, Miami, FL, USA), while readings for MDA were obtained using a microplate reader (CA94089; Molecular Devices, Sunnyvale, CA, USA). All standards and samples were run in duplicate.
At each time-point, ovarian tissue was collected to lyse on ice in 100 µl radioimmunoprecipitation assay buffer [50 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, 1 mmol/l phenylmethanesulfonyl fluoride, 1 mmol/l ethylenediaminetetraacetic acid, 1% Triton X-100, 0.5% sodium deoxycholate and 0.1% sodium dodecyl sulfate]. The lysates were cleared by centrifugation at 15,000 × g for 10 min, and the supernatant protein samples were denatured at 100°C for 5 min, separated on 10% acrylamide gels and then electrotransferred to polyvinylidene fluoride membranes. Following the blocking of the membranes with 5% non-fat dried milk in Tris buffer saline tween 20 buffer at room temperature for 2 h, primary rabbit polyclonal antibodies against Nrf2 (1:200; ab92946; Abcam, Cambridge, MA,USA) and β-actin (1:2,000; ab129348; Abcam) were added for incubation overnight at 4°C. The immunoblots were subsequently incubated with horseradish peroxidase-conjugated goat anti-rabbit IgG (ab175773; Abcam, Cambridge, MA,USA and detected using enhanced chemiluminescence reagent (Merck Millipore, Molsheim, France). Images of the immunoblots were visualized and captured using the Quantity One® quantitative gel analysis system (Bio-Rad, Tokyo, Japan). All western blot analyses were carried out ≥3 times.
Differences between the groups were analyzed using one-way analysis of variance followed by the Tukey comparison. Results are expressed as the mean ± standard deviation of ≥3 independent experiments, and P<0.05 was considered to indicate a statistically significant difference between groups.
Following the injection of two single doses of 5 mg/kg cisplatin in the rats, abnormal levels of the sex hormones appeared in the serum. Compared with group Con, no significant difference in the release of FSH was found in group Con + H2 (P>0.05,
All developmental stages of the follicles, and the structure of the ovarian cortex and medulla, could be clearly seen in group Con. Compared with group Con, few follicles were observed in group OI; furthermore, the group OI rats exhibited significant damage to the cortex, which could not be distinguished from the medulla. Compared with group OI, the development of the follicles in group OI + H2 was much more regular; all developmental stages of the follicles could be seen, and the damage to the cortex was less severe (P<0.05,
To determine the underlying mechanism of the effects observed, antioxidant enzymes and oxidation products were detected in the serum and ovarian tissue of the rats at the T1, T2 and T3 time-points. As shown in
Nrf2 is the key molecule of the Nrf2/antioxidant response element (ARE) signaling pathway, which is a relatively conservative and important endogenous antioxidative system (
In the present study, cisplatin stimulation was used to establish a model of ovarian injury in young female rats. Compared with group OI, it was found that hydrogen-rich saline treatment regulated the release of sex hormones and markedly improved the pathological condition and antral follicle counts. Hydrogen-rich saline additionally improved the production of antioxidant enzymes and attenuated the levels of oxidation products following cisplatin injection. These results indicate that hydrogen-rich saline treatment exerts a protective effect against ovarian injury in rats by regulating the balance of the redox system.
Cisplatin is a commonly used chemotherapeutic agent that exerts a therapeutic effect in various types of cancer and is often used to treat female patients at a reproductive age; however, despite its therapeutic effect, the side effects of cisplatin have also received considerable attention. The side effects of cisplatin are associated with an excessive production of free radicals and ROS, such as superoxide anions or H2O2, in different kidney cells (
It has previously been found that hydrogen exerts protective effects against various diseases. Hydrogen selectively alleviates hydroxyl radicals, other ROS and oxidation products and has been shown to improve the activity of antioxidants in a number of disease models, including shock, multiple organ dysfunction syndrome and ischemia-reperfusion (
It has previously been shown that the follicle microenvironment consists of a dynamic balance of oxidation and antioxidation, including antioxidant enzymes in the follicular fluid (such as SOD and CAT), which has a close association with follicle maturation (
Nrf2, as a member of the capncollar basic leucine zipper subfamily of leucine zipper transcription factors, is one of the key factors involved in initiating the endogenous protective effect against oxidative stress. Following stimulation by chemical toxicity, carcinogenesis and pathological processes, Nrf2 and its cytoplasmic binding protein, Kelch-like ECH-associated protein 1, become uncoupled, and Nrf2 is transferred into the nucleus. Nrf2 combines with ARE to induce two sets of target genes: Phase II detoxification enzymes and antioxidant enzymes, which play an essential role in cellular protection (
There were several limitations in the present study. First, the changes in oxidation product and antioxidative enzymes were measured
In conclusion, exposure to certain harmful factors can lead to the destruction of ovarian follicles in animal models. Cisplatin induces abnormal sex hormone secretion in a rat model of ovarian injury. Hydrogen-rich saline exerts a protective effect against cisplatin-induced ovarian injury by reducing MDA and increasing SOD and CAT activity. Furthermore, high Nrf2 expression is associated with ovarian injury.
This study was supported by the National Natural Science Foundation of China (grant nos. 81372033 and 81101409), the Natural Science Foundation of the Tianjin Science Committee (grant nos. 11JCYBJC12900 and 13JCQNJC11400) and the Foundation of Tianjin Bureau of Public Health (grant no. 2011KZ108).
catalase
estrogen
enzyme-linked immunosorbent assay
follicle stimulating hormone
malondialdehyde
nuclear factor erythroid 2-related factor 2
reactive oxygen species
superoxide dismutase
Time-dependent effects of hydrogen-rich saline on the release of sex hormones (E2 and FSH) during the process of chemotherapy-induced ovarian injury. Cisplatin was injected twice, each time at 5 mg/kg, in group OI. The same dose of saline was injected in group Con. The rats received intraperitoneal hydrogen-rich saline at 10 ml/kg body weight for 14 days. The levels of (A) FSH and (B) E2 were measured on the 14th, 28th and 42nd days (T1, T2 and T3) after the cisplatin injection. Results are presented as the mean ± standard deviation (n=10 per group). aP<0.05 compared with group Con; bP<0.05 compared with group OI. E2, estrogen; FSH, follicle-stimulating hormone; Con, control; Con + H2, control + hydrogen-rich saline; OI, cisplatin-induced ovarian injury; OI + H2, cisplatin-induced ovarian injury + hydrogen-rich saline.
Effect of hydrogen-rich saline on the different developmental stages of follicles during the process of chemotherapy-induced ovarian injury in rats (hematein eosin staining; magnification, ×100). Cisplatin was injected at 5 mg/kg in group OI. Rats received intraperitoneal hydrogen-rich saline at 10 ml/kg body weight for 14 days. The number of antral follicles was counted on the 14th, 28th and 42nd days (T1, T2 and T3) after the cisplatin injection. All levels of the follicles, as well as the clear structure of the cortex, could be observed in group Con. Few follicles could be seen in group OI. In group OI + H2 the development of the follicles was much more regular, all levels of follicles could be determined and the damage to the cortex was less severe. Results are presented as the mean ± standard deviation (n=10 per group). aP<0.05 compared with group Con; bP<0.05 compared with group OI. Con, control; Con + H2, control + hydrogen-rich saline; OI, cisplatin-induced ovarian injury; OI + H2, cisplatin-induced ovarian injury + hydrogen-rich saline.
Hydrogen-rich saline regulates cisplatin-induced oxidative stress by increasing the activity of SOD and CAT and reducing the level of MDA in the serum and ovarian tissue of rats. Cisplatin was injected at 5 mg/kg in group OI. Rats received intraperitoneal hydrogen-rich saline at 10 ml/kg body weight for 14 days. On the 14th, 28th and 42nd days (T1, T2 and T3) after the cisplatin injection, the (A-C) blood and (D-F) tissue samples were collected for the measurement of (A and D) SOD, (B and E) CAT and (C and F) MDA. Values are expressed as the mean ± standard deviation (n=10 per group). aP<0.05 compared with group Con; bP<0.05 compared with group OI. SOD, superoxide dismutase; CAT, catalase; MDA, malondialdehyde; Con, control; Con + H2, control + hydrogen-rich saline; OI, cisplatin-induced ovarian injury; OI + H2, cisplatin-induced ovarian injury + hydrogen-rich saline.
Hydrogen-rich saline increases Nrf2 expression in ovarian tissue on the 14th, 28th and 42nd days (T1, T2 and T3) after cisplatin injection. Ovarian tissue was collected to detect the Nrf2 and β-actin expression using western blotting. Values are expressed as the mean ± standard deviation (n=10 per group). aP<0.05 compared with group Con; bP<0.05 compared with group OI. Nrf2, nuclear factor erythroid 2-related factor 2; Con, control; Con + H2, control + hydrogen-rich saline; OI, cisplatin-induced ovarian injury; OI + H2, cisplatin-induced ovarian injury + hydrogen-rich saline.