Part 2: 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

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Results

Effects of hypoxia on GC-1 cells

To determine the effects of hypoxia on germ cells, we first examined the changes in cell viability 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-67staining 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 hypobaric hypoxic environment [8, 20]. Hence, endogenous ROS levels 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 causes marked DNAimpairment, which in turn causes cell apoptosis pathway activation and might be the major etiological factor for the increasing risk of male infertility [21, 22]. Next, we detected the apoptotic activation effect of hypoxia on GC-1 cells by TUNEL staining. 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.

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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-1cells in the control group. These findings 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 μMwas 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, 0.2μM 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 the 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-C, and Cis-H treatment significantly improved the Ki-67-positive cell ratio of hypoxia-induced GC-1 cells in vitro (Figure 2C).

Figure 2. Cis restored hypoxia-induced GC-1 cell viability.

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 Figure 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 DMSO groups. 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.

Figure 3. The mechanism by which Cis protects GC-1 cells from hypoxia-induced damage.

Effects of Cis on reproduction in hypobaric hypoxia-induced rats.

To determine the effects of hypobaric hypoxia on male rats, we first tested morphological alterations of testes in hypobaric hypoxia-induced rats. The results of HE staining showed that in the control group, normal spermatogenic cells at various stages were arranged in an orderly manner from the basement membrane to the lumen, and mature sperm were visible in tubule lumens (Figure 4A). Compared with the controls, pathological alterations of testicular tissue were observed in the model group, the basement membrane of testicular epithelial cells was arranged loosely, the spermatogenic epithelium was extremely thin, and the level and number of germ cells were markedly reduced (Figure 4A). However, treatment with Cis remarkably improved the histology of hypo- baric hypoxia-induced testicular damage in vivo(Figure 4A). We also measured body weight, testes weight, epididymis weight, and seminal vesicle gland weight, which led to the reproductive organ index (the reproductive organ/body weight ratio) being calculated. As shown in Figure 4B-D, the reproductive organ index (testes, epididymis, and seminal vesicle gland) was markedly lower in the model group (P < 0.01) than in the control group. However, the effect of hypobaric hypoxia on the reproductive organ index of rats was reversed with Cis treatment (Figure 4B-D).

Next, the acrosome enzyme activity and the live sperm rate of male rat sperm were also measured to elucidate testicular function damage. As shown in Figure 4E, 4F, acrosome enzyme activity and sperm motility were lower in the model group rats than that in the control group (P < 0.01). However, compared with rats in the model group, acrosome enzyme activity was restored in rats treated with 8 mg/kg/d Cis(P < 0.05) (Figure 4D). Moreover, as shown in figure 4F, treatment with Cis also enhanced the live sperm rate; the rats treated with 8 mg/kg/d Cis all showed a significantly increased live sperm rate (55.83 ± 6.03%, P < 0.05; 69.00 ± 2.29%, P < 0.01; 52.33 ± 3.40%, P < 0.05; and 53.67 ± 2.25%, P < 0.05 respectively) when compared with the model rats (43.83 ± 4.01%).

Taken together, these results suggested that the hypobaric hypoxic environment led to testicular morphological alterations, reproductive organ weight loss, and testicular function damage in male rats, and Cis could effectively protect the reproductive organs from hypoxia-induced damage.

Figure 4. Effects of Cis on the reproductive system of rats exposed to hypobaric hypoxia.

Effects of Cis on OS in the testes of hypobaric hypoxia-induced rats.

The ROS and LPO levels in the testes of rats were measured to analyze the effects of Cis on hypobaric hypoxia-induced OS. ROS analysis revealed that compared to the control group, ROS levels in the testes in the model group were significantly increased (P < 0.01 Figure 5A). Conversely, LPO was dramatically elevated in the testes (P < 0.01) under hypobaric hypoxia compared with normoxic conditions (Figure 5B). However, Cis treatment altered the above changes (P < 0.05), in which Cis-B exerted better effects than other Cis (Figure 5A, 5B). Cis seemed to protect the testes by reducing OS under hypobaric hypoxic conditions in vivo.

Additionally, apoptosis analyses were performed to further evaluate the mechanism by which Cis protected against hypobaric hypoxia-induced testicular function injury. The results of TUNEL staining (Figure 5C) showed that significant apoptosis existed in the model group compared to the control group. However, after Cis(8 mg/kg/d) treatment, fewer apoptotic cells occurred (P < 0.05) (Figure 5C). The Western blot data also showed that hypoxia and hypobaric treatment resulted in activation of Caspase-3 and PARP and an increased Bax/Bcl-2 ratio in testicular tissue, indicating an increase in apoptosis (Figure 5D). In addition, different types of Cis treatments significantly reduced apoptosis in testicular tissue (Figure 5D). Similarly, the IHC analysis of testicular tissue showed similar results (Supplementary Figure 1).

To verify the mechanism of Cis-reduced OS triggered by hypobaric hypoxia, we further tested the activities of GR, GPx, and SOD in testicular tissue. As shown in Figure 5E, compared with the control group, hypobaric hypoxia treatment significantly reduced GR, GPx, and SOD activities (P < 0.01). However, Cis treatment restored the enzyme activities (GR, GPx, and SOD) of testes tissue in rats treated with hypobaric hypoxia (P < 0.05). In conclusion, Cis seemed to protect the testes by activating a powerful endogenous antioxidant enzyme defense mechanism under hypobaric hypoxia conditions.

Figure 5. The Effect of Cis on OS in the testes of hypobaric hypoxia-induced rats.

Discussion

In high-altitude areas, hypobaric hypoxia is known to affect multiple systems in humans, including the male reproductive system [4, 20]. Recent experimental investigations are geared towards understanding the mechanisms of how hypobaric hypoxia impairs the male reproductive system. In this study, the therapeutic effect of Cis extract from Cistanches Herba on hypoxia-induced reproductive damage was investigated. The results demonstrated that Cis may protect the male reproductive system from hypoxic damage by reducing hypoxia-induced ROS accumulation and OS through enhancing the activity of endogenous antioxidant enzymes.

Cistanche enhances the antioxidant enzyme activity.

ROS are oxygen-derived free radicals that play a vital role in human physiology and pathology. Low doses of ROS are essential for sperm capacitation, the acrosome reaction, and spermatozoa-oocyte fusion [24, 25]. However, excessive accumulation of ROS often leads to damage to germ cells and stromal cells, resulting in male infertility [26]. ROS can easily damage cell membranes, nucleic acids, proteins, enzymes, and other biological macromolecules.

through peroxidation. Moreover, they also lead to potential cellular and DNA damage when they exceed the antioxidant carrying capacity. Accumulated evidence supports the pivotal role of ROS in the pathogenesis of male fertility [27, 28]. The production of ROS is regulated by oxygen tension. Under hypoxic conditions, the available oxygen in the environment decreases, and the blood viscosity increases, thereby affecting many oxygen-dependent metabolic processes in the organism [29, 30]. However, the lower atmospheric pressure at high altitudes causes poor venous return and a decrease in the quantity of oxygen transported by the bloodstream to all cells of the organism, which further increases the hypoxia of organs and cells [29, 30]. Thus, exposure to a high attitude gives rise to a series of hypoxic physiological responses, including the production and accumulation of ROS, when the demand for oxygen exceeds the vascular supply. As mentioned previously, the accumulation of ROS leads to a variety of intracellular effects, the most critical of which is to cause OS in cells.

OS refers to an imbalance between oxidation and reduction reactions, leading to the generation of excess oxidants or molecules that accept an electron from another reactant, which in turn produces ROS [31, 32]. OS is well understood to be able to be triggered by a series of endogenous and exogenous factors, including exposure to high altitudes. Spermatozoa are cells that are particularly susceptible to OS given their inadequate cell repair systems and high plasma membrane content of polyunsaturated fatty acids [33]. Testicular and epididymal tissues are not the exception, as the presence of severe OS has been observed in round spermatids in rats subjected to hypoxia [4]. OS affects the stability of DNA, thereby jeopardizing the integrity of the gamete genetic material [34-36]. However, a high level of DNA damage in male gametes has been confirmed to lead to activation of apoptosis signaling, which results in a reduction of epididymal sperm count and an increase in the percentage of defective cells [28, 37]. In the present study, hypoxia significantly reduced the viability of GC-1 cells through the induction of apoptosis and cell cycle arrest. More importantly, significantly increased ROS levels were shown by FCM analysis after hypoxia stimulation, with an increased apoptosis rate and higher activation of Caspase-3, PARP, and Bax/Bcl-2 ratio, indicating that ROS could activate apoptosis by activating the Caspase signaling pathway during hypoxia-induced fertility damage. The present findings demonstrated that hypoxia led to excessive ROS accumulation, causing oxidative damage to reproductive cells. Thus, it is meaningful to identify new antioxidants that can serve as an effective approach to alleviate hypoxia-induced fertility injury.

Cistanche alleviated fertility injury.

To protect against OS, a complex antioxidant system exists in the body, mainly composed of enzymatic factors. Under physiological conditions, the ROS contents and antioxidant system maintain a certain balance. However, ROS over-production depletes the sperm antioxidant system, leading to OS, which causes sperm DNA damage and results in lower fertility and pregnancy rates [23]. Thus, to address ROS over-production and related deleterious effects at the cellular level in the male reproductive system, different antioxidant strategies have been tested [23]. Currently, the literature concerning the use of compounds with antioxidant activity and improvement of sperm function is extensive. Importantly, most reports describe an improvement in sperm parameters after oral antioxidant intake, including improvements in sperm concentration and motility or decreases in DNA damage [38]. Thus, a growing number of urologists are prescribing oral antioxidants for infertility due to OS-related problems [39]. These antioxidants include mainly carnitines, vitamins, zinc, melatonin, and natural compounds [23, 40]. Presently, with the development of drug extraction technology, an increasing number of TCM extracts are also being considered to mitigate male infertility because these antioxidants can reduce the destructive effects of OS [41]. Yüce A. et al. reported in2013 that cinnamon has beneficial effects on the oxidative and antioxidant balance in testes and sperm quality [42]. Zhang L et al. showed that curcumin significantly improves sperm motility in patients and decreases H2O2 [43]. In addition, a variety of other plant extracts such as blueberry, crocus sativus, pomegranate seeds, and green tea have also been shown to protect the reproductive system via antioxidant mechanisms [27, 44-47]. Cistanches Herba is an important TCM that possesses a favorable safety profile and broad medicinal functions for the treatment of infertility, among other conditions [13]. Modern pharmacological studies have shown that Cistanches Herba possesses various activities, such as antioxidative, anti-inflammatory, hepatoprotective, and anti-neuro-degenerative disease activities [13, 48]. Therefore, extracts, fractions, or compounds from Cistanches Herba may have potential antioxidant features for the treatment of infertility.

The active substances in plants that improve fertility include various chemical groups such as PhGs, saponins, oxygenated volatile compounds, and alkaloids [41]. Pharmacological activity studies of PhGs have demonstrated that PhGs exhibit a wide range of bioactivities, such as antioxidation, antiradiation neuroprotection, and sexual function enhancement [49, 50]. Among these activities, antioxidation is gradually attracting attention. Some single components or fractions of PhGs have been reported to inhibit germ cell apoptosis induced by various chemicals, and their antioxidation capabilities in vitro have also been demonstrated in vivo in several animal models [51, 52]. These results indicate that PhGs could be an attractive candidate for the treatment of male infertility. Cis is an active PhG that can be isolated from Cistanches Herba. In the present study, we explored the effects of Cis on hypoxia-treated cells or a rat model and investigated the underlying molecular mechanisms. Cis exhibited protective activities on decreases in hypoxia-induced viability and increases in apoptosis in GC-1 cells, and it also showed a protective effect on hypoxia-induced damage in the reproductive system of rats in vivo. A significant decrease in GR, GPx and SOD activities under hypoxia in comparison to normoxic groups was observed, while the specific activities of GR, GPx, and SOD significantly increased in testes or GC-1 cells treated with Cis. Cis seemed to protect the testes and GC-1 cells under hypoxic conditions by enhancing the activities of antioxidant enzymes.

Enzyme antioxidants function mainly by scavenging superoxide anions, thus preventing lipid peroxidation and DNA damage to prevent infertility. Enzymatic antioxidant mechanisms play a crucial role in preventing oxidative damage [23]. The enzymatic mechanism against OS comprises free radical scavengers and glutathione-dependent enzymes including GR, GPx, and SOD [12]. Antioxidant enzymes are well understood to be essential for the male reproductive system. In the current study, the effect of reduced antioxidant enzyme activities under hypobaric hypoxia was accompanied by increased ROS and LPO in the model group, which is consistent with previous reports [12]. However, Cis administration led to a recovery of antioxidant enzyme activities in GC-1 cells and the testes of rats, making it possible to generate strategies for administering Cistanches Herba to prevent hypobaric hypoxia-induced damage, as previously suggested. Although the present results showed that treatment with Cis partly decreased hypoxia-induced germ cell damage in rats, further investigations are needed to unravel the full picture of its reproductive protective effects. For example, the specific mechanism of Cis affects the activity of antioxidant enzymes. In addition, there is a question of whether any other mechanisms could also be pertinent as Cis only partially recovered the reproductive damage caused by hypoxia. Finally, whether Cis has a direct growth-promoting effect on germ cells should also be considered.

Cistanche has a direct growth-promoting effect on germ cells.

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Conclusions

In general, the findings of this study emphasize the potential of Cis as an antioxidant for the treatment of hypoxia-induced male reproductive damage. Cis can protect against hypoxia-induced male reproductive damage by restoring antioxidant enzyme activity, reducing ROS- induced OS, simultaneously increasing cell viability, and decreasing apoptosis. Importantly, the Cis subtypes (Cis-A, Cis-B, Cis-C, and Cis-H) studied in this study all showed a certain protective effect on the reproductive system, and Cis-B showed the most significant effect. Therefore, we speculate that Cis might be a good candidate antioxidant for the treatment of hypoxia-induced male reproductive damage, although the precise underlying mechanism requires further investigation.