Part1：Cistanoside Of Cistanche Herba Ameliorates Hypoxia-induced Male Reproductive Damage Via Suppression Of Oxidative Stress

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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 

1Department of Urology, Tang Du Hospital, The Fourth Military Medical University, Xi’an 710038, Shaanxi,  China; 

2Urology and Nephrology Hospital, Xi’an People’s Hospital, Xian 710100, Shaanxi, China; 

3Institute of  Basic Medical Science, The Fourth Military Medical University, Xi’an 710032, Shaanxi, China; 

4Department of  Radiotherapy, Tang Du Hospital, The Fourth Military Medical University, Xi’an 710038, Shaanxi, China. *Equal contributors. Received September 1, 2020;

Accepted January 18, 2021; Epub May 15, 2021; Published May 30, 2021

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 on reproductive 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 the viability 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

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) pro-

duction [8, 9].ROS plays an important role in the male reproductive system. At low levels, they are required for sperm capacitation, the acro-some reaction, and spermatozoa-0ocyte 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 con-tents of Cistanches Herba, PhGs have been regarded as the main active component. To date, 34 PhGs have been isolated from Cistanches 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 from Cistanches 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.

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 ug/mL) at 37°C in a humidified incubator with 5%CO, 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-8 assay(CCK-8). In brief, GC-1 cells were seeded into 96-well plates at1.5×103 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 (Dojindo Japan). 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).

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 (FACS Calibur, 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 for 72 h. After fixing all cells with 4% paraformaldehyde, they were incubated with the 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 (1.0 μg/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 with 10 μM DCFH-DA (Beyotime). ROS contents were then determined by fluorescence-activated cell sorting on a Beckman Coulter Flow Cytometry System 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 1 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 (PO.:20%; air pressure:101.3 kPa), while the rats in the model and Cis treated 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; PO: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)

t) ×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 uL 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).

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.

Results

Effects of hypoxia on GC-1 cells

To determine the effects of hypoxia on germ cells, we first examined the changes in cells via-ability after hypoxia treatment with different oxygen concentrations (20%,15%,10%,5%) for 1, 3,5, and 7 days, respectively. The CCK-8 assay results showed that, compared with the control group (20% oxygen concentration), cells exposed to hypoxia exhibited a significant decrease in viability (P<0.01; Figure 1A). Moreover, their survival rate was inversely proportional to the oxygen concentration and further decreased with induction time. To avoid an excessive cytotoxic effect, a 10% oxygen concentration and 3-day induction time were selected as the hypoxic model criteria for subsequent in vitro experiments.

Subsequently, FCM and immunofluorescence staining was performed to further evaluate the proliferation alteration of GC-1 cells under hypoxia treatment. The results showed that hypoxia could induce GC-1 cell arrest in the G1 phase, thereby reducing cell entry into the S phase and inhibiting DNA replication. Thus, hypoxia significantly reduced the proliferation index of GC-1 cells (P<0.01; Figure 1B). Positive Ki-67 staining is another specific biomarker of proliferating cells. Therefore, we also examined the ratio of Ki-67-positive cells with or without hypoxia treatment. Compared with the control group, hypoxia treatment remarkably reduced Ki-67-positive cells, as shown in Figure 1C.

Next, we aimed to investigate the mode of GC-1 cell viability inhibition induced by hypoxia. As reported in the literature, the level of ROS increased as rats were exposed to a hypo-baric hypoxic environment [8,20]. Hence, endogenous ROSlevels in GC-1 cells were measured using the FCM assay. The results showed higher ROS levels under hypoxia in comparison to the normal oxygen group (P< 0.01; Figure 1D). Accumulated ROS cause marked DNA impairment, which in turn causes cell apoptosis pathway activation and might be the major etiological factor for the increased risk of male infertility [21,22]. Next, we detected the apoptotic activation effect of hypoxia on GC-1 cells by TUNELstaining. As shown in Figure 1E, treatment with hypoxia resulted in an increase in TUNEL fluorescence compared with the control group, indicating an increase in apoptosis in the model group.

Since ROS-induced cell damage is usually caused by OS, we further tested the OS of GC-1 cells. As presented in Figure 1F, the LPO levels of GC-1 cells in the model group were markedly

increased compared to the LPO levels of GC-1 cells in the control group. These findings are suggested that hypoxia-induced GC-1 cell injury might be related to OS, which is induced by ROS accumulation.

Effects of Cis on hypoxia-induced GC-1 cell viability in vitro.

To investigate whether Cis can prevent the inhibitory effects of hypoxia on GC-1 cell viability, a CCK-8 assay was performed. GC-1 cells were treated with different subtypes (Cis-A, B, C, H） and concentration ranges （0.02 μM， 0.2 μM， 2 μM） of Cis for 72 h. Comparison of the model group with the DMSO group showed that DMSO did not directly promote GC-1 cell viability(Figure 2A). However, cell viabilities were markedly restored (P< 0.05) with Cis treatments. Compared with the model group, Cis-A, Cis-B, Cis-C, and Cis-H all showed certain protective effects on hypoxia-induced damage to GC-1 cell viability, and Cis-B showed the most significant effect(Figure 2A). The protective effects of Cis at 0.2 μM were significantly higher than the protective effects of Cis at 0.02 μM， while the difference between 2 μM and 0.2 uM was not obvious, indicating that the restored GC-1 cell viability induced by Cis demonstrated a dose-dependent increase in the concentration range from 0.02-0.2 μM （Figure 2A）. Therefore, according to the experimental needs, 0.2 M Cis was selected as the optimal concentration in the following in vitro experiments. To further confirm whether germ cells were indeed protected by Cis, FCM, and Ki-67 staining were performed to assess the alteration of the proliferation of GC-1 cells after treatment with Cis. Upon Cis treatment, the proportion of GC-1 cells in the G1 phase was reduced. In contrast, more cells entered S-phase, suggesting that Cis-treatment could increase the germ cell proliferation index(P<0.01; Figure 2B). The statistics for the GC-1 cell cycle are shown in Figure 2Bb. The Ki-67 staining results also showed that Cis-A, Cis-B, Cis-Cand Cis-H treatment significantly improved the Ki-67-positive cell ratio of hypoxia-induced GC-1 cells in vitro(Figure 2C).

The mechanism of Cis protects germ cells from hypoxia in vitro.

To investigate whether the protective effects of Cis on GC-1 cells were related to the removal of excessive ROS, the fluorescent dye DCFH-DA was used to detect ROS levels in each group. As shown in Figures 3A,3B, treatment with DMSO did not change the intracellular ROS content or LPO level compared with the model group. However, ROS levels in GC-1 cells were markedly reduced in the Cis-treated groups (Figure 3A). Furthermore, a decrease in LPO was also observed in GC-1 cells subjected to Cis (Figure 3B).

To further explore the mechanism by which Cis protects germ cells from hypoxic injury, TUNEL staining and Western blot analyses were performed to evaluate apoptosis. TUNEL staining (Figure 3C) showed significant apoptosis in the model and DMSOgroups. However, fewer apoptotic cells were observed with Cis treatment, which indicated that Cis treatment reduced GC-1 cell apoptosis. Additionally, the expression of PARP, Caspase-3, Bax, and Bcl-2 was measured to corroborate the molecular mechanism. As presented in Figure 3D, Caspase-3 and PARP were activated in GC-1 cells under hypoxia, and this activation was inhibited by Cis treatment. In addition, the ratio of Bax/Bcl-2 was higher in the model group than in the control group, and Cis treatment reduced the ratio of Bax/Bcl-2(Figure 3D). These data indicated that Cis had a potential capacity to attenuate hypoxia-induced oxidant damage, and this protective effect might be achieved by reducing ROS accumulation and inhibiting Caspase-related apoptosis pathway activation.

The enzymatic mechanism inhibiting OS involves free radical scavengers such as glutathione reductase(GR), glutathione peroxidase (GPx), and superoxide dismutase (SOD)[23]. The enzymatic mechanism inhibiting OS plays an essential role in preventing oxidative damage in cells and tissues [23]. To further validate the potential mechanism of Cis inhibition of hypoxia-induced OS in GC-1 cells, the activities of GR, GPx and SOD were measured. The results revealed that GR, GPx, and SOD activities all significantly(P < 0.01, Figure 3E)decreased under hypoxia when compared to the control groups, and Cis treatment markedly restored their activities in GC-1 cells exposed to hypoxia (P< 0.05, Figure 3E), suggesting that these compounds could activate the powerful endogenous antioxidant system.

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