Part2：Cistanoside Of Cistanche Herba Ameliorates Hypoxia-induced Male Reproductive Damage Via Suppression Of Oxidative Stress

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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 dam-age. 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.

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 com

pared 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 treatment 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.

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.

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. 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 overproduction 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 in 2013 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-neurodegenerative 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.

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.

Acknowledgments 

This study was supported by grants from the National Natural Science Foundation of China (Nos. 81672535; 82002686) and from the People’s Liberation Army of China (Medical Science Researching Fund; No. 16QNP114). Disclosure of conflict of interest None.

References 

[1] Bracke A, Peeters K, Punjabi U, Hoogewijs D and Dewilde S. A search for molecular mechanisms underlying male idiopathic infertility. Reprod Biomed Online 2018; 36: 327-339. 

[2] Agarwal A, Mulgund A, Hamada A, and Chyatte MR. A unique view on male infertility around the globe. Reprod Biol Endocrinol 2015; 13: 37. 

[3] Jankovic Velickovic L and Stefanovic V. Hypoxia and spermatogenesis. Int Urol Nephrol 2014; 46: 887-894. 

[4] Farias JG, Bustos-Obregon E, Orellana R, Bucarey JL, Quiroz E and Reyes JG. Effects of chronic hypobaric hypoxia on testis histology and round spermatid oxidative metabolism. Andrologia 2005; 37: 47-52. 

[5] Liao W, Cai M, Chen J, Huang J, Liu F, Jiang C, and Gao Y. Hypobaric hypoxia causes deleterious effects on spermatogenesis in rats. Reproduction 2010; 139: 1031-1038. 

[6] Paul C, Teng S, and Saunders PT. A single, mild, transient scrotal heat stress causes hypoxia and oxidative stress in mouse testes, which induces germ cell death. Biol Reprod 2009; 80: 913-919. 

[7] Gasco M, Rubio J, Chung A, Villegas L and Gonzales GF. Effect of high altitude exposure on spermatogenesis and epididymal sperm count in male rats. Andrologia 2003; 35: 368- 374. 

[8] Zepeda AB, Figueroa CA, Calaf GM and Farias JG. Male reproductive system and antioxidants in oxidative stress-induced by hypobaric hypoxia. Andrologia 2014; 46: 1-8. 

[9] Quindry J, Dumke C, Slivka D and Ruby B. Impact of extreme exercise at high altitude on oxidative stress in humans. J Physiol 2016; 594: 5093-5104. 

[10] Otasevic V, Stancic A, Korac A, Jankovic A, and Korac B. Reactive oxygen, nitrogen, and sulfur species in human male fertility. A crossroad of cellular signaling and pathology. Biofactors 2020; 46: 206-219. 

[11] Bui AD, Sharma R, Henkel R, and Agarwal A. Reactive oxygen species impact on sperm DNA and its role in male infertility. Andrologia 2018; 50: e13012.

[12] Bisht S, Faiq M, Tolahunase M, and Dada R. Oxidative stress and male infertility. Nat Rev Urol 2017; 14: 470-485. 

[13] Fu Z, Fan X, Wang X, and Gao X. Cistanches Herba: an overview of its chemistry, pharmacology, and pharmacokinetics property. J Ethnopharmacol 2018; 219: 233-247. 

[14] Wang T, Chen C, Yang M, Deng B, Kirby GM, and Zhang X. Cistanche tubulosa ethanol extract mediates rat sex hormone levels by induction of testicular steroidogenic enzymes. Pharm Biol 2016; 54: 481-487. 

[15] Wong HS and Ko KM. Herba Cistanches stimulates cellular glutathione redox cycling by reactive oxygen species generated from mitochondrial respiration in H9c2 cardiomyocytes. Pharm Biol 2013; 51: 64-73. 

[16] Wang N, Ji S, Zhang H, Mei S, Qiao L, and Jin X. Herba cistanches: anti-aging. Aging Dis 2017; 8: 740-759. 

[17] Gu C, Yang X, and Huang L. Cistanches Herba: A Neuropharmacology Review. Front Pharmacol 2016; 7: 289. 

[18] Yan Q, He B, Hao G, Liu Z, Tang J, Fu Q and Jiang CX. KLF9 aggravates ischemic injury in cardiomyocytes through augmenting oxidative stress. Life Sci 2019; 233: 116641. 

[19] Xu F, Guo G, Zhu W, and Fan L. Human sperm acrosome function assays are predictive of fertilization rate in vitro: a retrospective cohort study and meta-analysis. Reprod Biol Endocrinol 2018; 16: 81. 

[20] Farias JG, Puebla M, Acevedo A, Tapia PJ, Gutierrez E, Zepeda A, Calaf G, Juantok C and Reyes JG. Oxidative stress in rat testis and epididymis under intermittent hypobaric hypoxia: protective role of ascorbate supplementation. J Androl 2010; 31: 314-321. 

[21] Saleh RA, Agarwal A, Nada EA, El-Tonsy MH, Sharma RK, Meyer A, Nelson DR and Thomas AJ. Negative effects of increased sperm DNA damage in relation to seminal oxidative stress in men with idiopathic and male factor infertility. Fertil Steril 2003; 79 Suppl 3: 1597-1605. 

[22] Aitken RJ and Baker MA. Causes and consequences of apoptosis in spermatozoa; contributions to infertility and impacts on development. Int J Dev Biol 2013; 57: 265-272. 

[23] Martin-Hidalgo D, Bragado MJ, Batista AR, Oliveira PF and Alves MG. Antioxidants and male fertility: from molecular studies to clinical evidence. Antioxidants (Basel) 2019; 8: 89. 

[24] Drevet JR. The antioxidant glutathione peroxidase family and spermatozoa: a complex story. Mol Cell Endocrinol 2006; 250: 70-79. 

[25] Aitken RJ, Clarkson JS, Hargreave TB, Irvine DS, and Wu FC. Analysis of the relationship between defective sperm function and the generation of reactive oxygen species in cases of oligozoospermia. J Androl 1989; 10: 214-220. 

[26] Agarwal A, Sharma RK, Nallella KP, Thomas AJ, Jr, Alvarez JG and Sikka SC. Reactive oxygen species as an independent marker of male factor infertility. Fertil Steril 2006; 86: 878- 885. 

[27] Zepeda A, Aguayo LG, Fuentealba J, Figueroa C, Acevedo A, Salgado P, Calaf GM and Farias J. Blueberry extracts protect testis from hypobaric hypoxia induced oxidative stress in rats. Oxid Med Cell Longev 2012; 2012: 975870. 

[28] Yang S, Zhang W, Xuan LL, Han FF, Lv YL, Wan ZR, Liu H, Ren LL, Gong LL and Liu LH. Akebia Saponin D inhibits the formation of atherosclerosis in ApoE(-/-) mice by attenuating oxidative stress-induced apoptosis in endothelial cells. Atherosclerosis 2019; 285: 23-30. 

[29] Saxena DK. Effect of hypoxia by intermittent altitude exposure on semen characteristics and testicular morphology of male rhesus monkeys. Int J Biometeorol 1995; 38: 137- 140. 

[30] Verratti V, Berardinelli F, Di Giulio C, Bosco G, Cacchio M, Pellicciotta M, Nicolai M, Martinotti S and Tenaglia R. Evidence that chronic hypoxia causes reversible impairment on male fertility. Asian J Androl 2008; 10: 602-606. 

[31] Henkel RR. Leukocytes and oxidative stress: the dilemma for sperm function and male fertility. Asian J Androl 2011; 13: 43-52. 

[32] Tremellen K. Oxidative stress and male infertility--a clinical perspective. Hum Reprod Update 2008; 14: 243-258. 

[33] Saradha B and Mathur PP. Induction of oxidative stress by lindane in the epididymis of adult male rats. Environ Toxicol Pharmacol 2006; 22: 90-96. 

[34] Aitken RJ and Roman SD. Antioxidant systems and oxidative stress in the testes. Adv Exp Med Biol 2008; 636: 154-171. 

[35] Wagner H, Cheng JW and Ko EY. Role of reactive oxygen species in male infertility: An updated review of the literature. Arab J Urol 2018; 16: 35-43. 

[36] Agarwal A, Saleh RA and Bedaiwy MA. Role of reactive oxygen species in the pathophysiology of human reproduction. Fertil Steril 2003; 79: 829-843. 

[37] Redza-Dutordoir M and Averill-Bates DA. Activation of apoptosis signaling pathways by reactive oxygen species. Biochim Biophys Acta 2016; 1863: 2977-2992. 

[38] Zengerling F and Schmidt S. [Antioxidants for male subfertility]. Urologe A 2016; 55: 956- 959. 

[39] Esteves SC and Agarwal A. Novel concepts in male infertility. Int Braz J Urol 2011; 37: 5-15. 

[40] Pisoschi AM and Pop A. The role of antioxidants in the chemistry of oxidative stress: a review. Eur J Med Chem 2015; 97: 55-74.

[41] Tahvilzadeh M, Hajimahmoodi M, Toliyat T, Karimi M and Rahimi R. An evidence-based approach to medicinal plants for the treatment of sperm abnormalities in traditional Persian medicine. Andrologia 2016; 48: 860-879. 

[42] Yuce A, Turk G, Ceribasi S, Sonmez M, Ciftci M, and Guvenc M. Effects of cinnamon (Cinnamomum zeylanicum) bark oil on testicular antioxidant values, apoptotic germ cell, and sperm quality. Andrologia 2013; 45: 248-255. 

[43] Zhang L, Diao RY, Duan YG, Yi TH, and Cai ZM. In vitro antioxidant effect of curcumin on human sperm quality in leucocytospermia. Andrologia 2017; 49: e12760. 

[44] Heidary M, Vahhabi S, Reza Nejadi J, Delfan B, Birjandi M, Kaviani H, and Givrad S. Effect of saffron on semen parameters of infertile men. Urol J 2008; 5: 255-259. 

[45] Safarinejad MR, Shafiei N and Safarinejad S. A prospective double-blind randomized placebo-controlled study of the effect of saffron (Crocus sativus Linn.) on semen parameters and seminal plasma antioxidant capacity in infertile men with idiopathic oligoasthenoteratozoospermia. Phytother Res 2011; 25: 508-516. 

[46] Kolahdooz M, Nasri S, Modarres SZ, Kianbakht S and Huseini HF. Effects of Nigella sativa L. seed oil on abnormal semen quality in infertile men: a randomized, double-blind, placebo-controlled clinical trial. Phytomedicine 2014; 21: 901-905. 

[47] Jiao N, Chen Y, Zhu Y, Wang W, Liu M, Ding W, Lv G, Lu J, Yu B and Xu H. Protective effects of catalpol on diabetes mellitus-induced male reproductive damage via suppression of the AGEs/RAGE/Nox4 signaling pathway. Life Sci 2019; 116736. 

[48] Xiong Q, Kadota S, Tani T and Namba T. Antioxidative effects of phenylethanoids from Cistanche deserticola. Biol Pharm Bull 1996; 19: 1580-1585. 

[49] Shao SY, Feng ZM, Yang YN, Jiang JS, and Zhang PC. Forsythenethosides A and B: two new phenylethanoid glycosides with a 15-membered ring from Forsythia suspensa. Org Biomol Chem 2017; 15: 7034-7039. 

[50] Xue Z and Yang B. Phenylethanoid glycosides: research advances in their phytochemistry, pharmacological activity, and pharmacokinetics. Molecules 2016; 21: 991. 

[51] Chen W, Lin HR, Wei CM, Luo XH, Sun ML, Yang ZZ, Chen XY and Wang HB. Echinacoside, a phenylethanoid glycoside from Cistanche deserticola, extends the lifespan of Caenorhabditis elegans and protects them from Abeta-induced toxicity. Biogerontology 2018; 19: 47-65. 

[52] Xuan GD and Liu CQ. [Research on the effect of phenylethanoid glycosides (PEG) of the Cistanche deserticola on anti-aging in aged mice induced by D-galactose]. Zhong Yao Cai 2008; 31: 1385-1388.