Part 1: Cistanoside Of Cistanche Herba Ameliorates Hypoxia-induced Male Reproductive Damage Via Suppression Of Oxidative Stress
Mar 26, 2022
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Fengqi Yan1*, Xiaoliang Dou1*, Guangfeng Zhu1*, Mingyuan Xia1, Yahui Liu3, Xiaozi Liu3, Guojun Wu2, He Wang1, Bo Zhang1, Qiuju Shao4, Yong Wang1
Abstract: Increasing evidence shows that hypoxia is a cause of male infertility, and hypoxia may be related to oxidative stress (OS). Cistanoside (Cis) is a phenylethanoid glycoside compound that can be extracted from Cistanches Herba and possesses various biological functions. This study aimed to investigate the protective effects of Cis onreproductive damage induced by hypoxia and explore the specific underlying mechanisms. Cell and animal hypoxia experimental models were constructed, and the protective effects of different subtypes of Cis on the male reproductive system were assessed both in vitro and in vivo. The results indicated that hypoxia significantly reduced theviability of GC-1 cells through cell cycle arrest and apoptosis activation, which were associated with increased OS. Moreover, Cis showed strong antioxidative effects both in vitro and in vivo, significantly restoring antioxidant enzyme activities and downregulating reactive oxygen species (ROS) levels while increasing cell viability and decreasing apoptosis. Importantly, the Cis subtypes (Cis-A, Cis-B, Cis-C, and Cis-H) studied herein all showed certain antioxidant effects, among which the effects of Cis-B were the most significant. This study demonstrates that Cis markedly attenuates the harmful effects of hypoxia-induced OS by affecting antioxidant enzyme activities in testes and GC-1 cells.
Keywords: Cistanoside, hypobaric hypoxia, male infertility, oxidative stress, reproductive protection
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Introduction
Currently, approximately 48.5 million (15%) couples of reproductive age worldwide are affected by infertility [1], among which 40-50% of the cases are ascribed to male infertility [2], a condition that is strongly associated with environmental and lifestyle factors. Evidence suggests that susceptibility of the mammalian testis to low oxygen pressure is a causative factor of some forms of male infertility [3]. As demonstrated in previous studies, spermatogenesis is impaired and reduced upon exposure to hypobaric hypoxia [4-7].
To explore the underlying mechanism, studies have shown that exposure to hypobaric hypoxia increases reactive oxygen species (ROS) production [8, 9]. ROS plays an important role in the male reproductive system. At low levels, they are required for sperm capacitation, the acrosome reaction, and spermatozoa-oocyte fusion [10]. However, excessive ROS can induce sperm nuclear/mitochondrial DNA damage and plasma membrane peroxidative damage, which in turn are major etiological factors for the increased risk of male infertility [11, 12]. Thus, the accumulation of hypoxia-induced ROS might be one cause of male infertility.
Cistanches Herba, a perennial parasitic medicinal plant, is widely distributed in arid areas [13] and used widely due to its pharmacological activities [14-17]. Among all the effective contents of Cistanches Herba, PhGs have been regarded as the main active component. To date, 34 PhGs have been isolated fromCistanches plants [13]. Cistanoside (Cis), an active PhG isolated from Cistanches Herba, has received attention for its antioxidant effects.
Considering the antioxidant effects of Cis and the role of ROS in hypoxia-induced male infertility, Cis is considered a potential drug candidate for the treatment of hypoxia-induced male infertility. However, few reports have addressed the antioxidant effects of Cis extracted fromCistanches Herba in the treatment of hypoxia-induced male infertility or the signaling pathways involved. In this study, in vitro, and in vivo hypoxia experimental models were constructed and the effects components of different Cis were assessed.
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Materials and methods
Cell culture and reagent
The mouse spermatogonia cell line GC-1spg (GC-1) was purchased from the American Type Culture Collection (ATCC) and cultured in DMEM (Invitrogen, USA) supplemented with 10% fetal bovine serum, 1% L-glutamine (100 mM), penicillin (100 U/mL), and streptomycin (100 μg/ mL) at 37°C in a humidified incubator with 5%CO2. Cis (Cis-A, B, C, H) was purchased from Chengdu Gelipu Biotechnology Co., Ltd. (China).
Cell viability assay
Cell viability was tested by cell counting kit-8assay (CCK-8). In brief, GC-1 cells were seeded into 96-well plates at 1.5×103 per well and cultured at 37°C for 24 h. Then, the cells were treated with different concentrations (20%, 15%, 10%, 5%) of oxygen or different concentrations (2 μM, 0.2 μM, 0.02 μM) of Cis (Cis-A, Cis-B, Cis-C, Cis-H) for the required time. Then, the supernatant was discarded, and cell viabilities were detected using a CCK-8 kit (DojindoJapan). The absorbance of each well was measured at 450 nm using a microplate reader (BioRad USA). Finally, the cell viabilities were calculated according to the following formula:
Cell viability = (ODexperimental group - ODblank group)/ (ODcontrol group - ODblank group) × 100%.
Western blot analysis
Harvested cells or tissues were homogenized in RIPA buffer to extract proteins (RIPA Beyotime China; Cocktail Roche Switzerland). Supernatants were collected, and the concentration of proteins was tested by the BCA method(Beyotime). Approximately 40 μg of the extracted proteins from each sample were separated by SDS-PAGE and electrotransferred onto nitrocellulose (NC) filter membrane (Beyotime China). NC filter membranes were blocked with 5% nonfat milk for 1.5 h and incubated with specific antibodies (anti-PARP 1:1000, anti-Caspase-3 1:1000, anti-Bcl-2 1:1000, anti-Bax 1:1000, anti-GAPDH 1:1000; all antibodies were purchased from Cell Signaling Technology, USA) overnight at 4°C. Then, all NC membranes were incubated with the corresponding horseradish peroxidase-conjugated secondary antibody for 1.5 h at room temperature and imaged with an imaging system (Tannon, China).
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Cell cycle detection
A flow cytometric assay (FCM) was performed to analyze the cell cycle. Cells were treated under different conditions for 72 h and harvested. The cells were then washed with PBS, fixed in 75% ethanol, and stained with propidium iodide (PI). For each sample, 1×104 cells were collected and analyzed by flow cytometry (FACSCalibur, BD Biosciences). Then, the proportion of G1/S/G2 phase cells and the proliferation index [Phase(S+G2)/Phase(G1+S+G2) × 100%] were calculated.
Ki-67 staining
Cells were cultured in a confocal dish and treated with hypoxia or different subtypes of Cis for72 h. After fixing all cells with 4% paraformaldehyde, they were incubated with anti-Ki-67 anti-body (1:200, Cell Signaling Technology). Then, all the cells were incubated with a corresponding CY-3-conjugated anti-rabbit IgG antibody (1:200, Boster, China) and DAPI solution (1.0μg/mL, Beyotime). Fluorescence was observed with a Fluoview FV1000 confocal microscope (Olympus, Japan).
Detection of ROS
FCM was introduced to measure intracellular ROS levels using DCFH-DA. Suspended cells were seeded in 6-well plates and subjected to different treatments. After 72 h of treatment, cells were suspended in serum-free DMEM with10 μM DCFH-DA (Beyotime). ROS contents were then determined by fluorescence-activated cell sorting on a Beckman Coulter Flow CytometrySystem with an excitation wavelength of 488 nm and emission wavelength of 525 nm [18].
Determination of Lipid Peroxidation (LPO)
The thiobarbituric acid reactive substances (TBARS) assay was performed to detect LPO. All operating steps were performed in accordance with the instructions (Sigma USA). The concentrations were calculated using a molar extinction coefficient of 1.56×105/(M·cm), which was obtained utilizing malondialdehyde as a standard. The results are expressed as nmol of MDA equivalents/mg of protein.
Determination of enzyme activities
The enzyme activities were tested using assay kits including glutathione reductase (GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD). All operating steps were performed in accordance with the instructions supplied (Nan Jing Jian Cheng Bioengineering Institute China).
Animals and experimental protocol
Mature male Wistar rats (180-220 g, 8 w old) were obtained from the animal center of the Fourth Military Medical University, Xi’an, China. Permission for use of the animals was obtained from the University Ethics Committee (Reference number: 20190506). The animal experiment was carried out according to university guidelines for the care and use of laboratory animals.
All rats were allowed to adapt for approximately one week prior to the commencement of the experiment. After acclimation, the rats were randomly distributed into 6 groups with 5 animals in each group. The rats in the control group were raised under normal pressure (PO2: 20%; air pressure:101.3 kPa), while the rats in the model and Cistreated groups were raised in a low-pressure oxygen chamber (internal pressure of 61.6 kPa, equivalent to a height of 4000 meters above sea level; PO2: 14.55%) to simulate a high-altitude hypoxic environment. All rats had free access to food and water in plastic cages at 22 ± 2°C and humidity conditions with an automatic 12-h light/dark cycle. The rats in the model and Cis treated groups remained under hypobaric conditions but were transferred to normobaric conditions every 96 h, at which time food and water were provided to them and the cages cleaned. The duration of the transition from hypobaric to hypobaric was approximately 2 h. All treatment groups were treated with the corresponding Cis (8 mg/kg/d) via oral gavage for 8 weeks, whereas the control and model rats were treated with an equal volume of water.
After 8 weeks, rats were sacrificed under anesthesia. Testes, epididymis, and seminal vesicles were separated and weighed, and the organ index was calculated according to the following formula: (weight of organ/animal weight) × 100%. Subsequently, sperm in the epididymis were collected, and their motility and acrosome enzyme activity was tested. Similarly, testicular tissues were collected for histopathologic studies and ROS, LPO, and antioxidant enzyme activity detection.
Evaluation of live sperm rate
The epididymis was incised with surgical scissors, and the sperm suspension was prepared in normal saline. To evaluate the live sperm rate, 5 μL of the sperm suspension was carefully mixed with an equal volume of eosin-Y stain. The sperm were then counted under a light microscope. Live sperm rates were assessed by calculating both stained (dead sperm) and unstained sperm (live sperm).
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Determination of sperm acrosome enzyme activity
The sperm acrosome enzyme activity assay was utilized to evaluate sperm acrosome enzymes [19]. The results in each group were calculated using the following formula: Acrosome enzyme activity (μIU) = (ODExperiment group - ODBlank group)/(247.5×10) × 106.
Statistical analysis
All quantitative data are expressed as the mean ± SD and were analyzed using SPSS 22.0 software. Independent Student’s t-test was used to compare the data between two groups. *P < 0.05 and **P < 0.01 were considered statistically significant differences.
