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. 2023 Jan 23;61(1):213–227. doi: 10.1080/13880209.2023.2165114

Guilu-Erxian-Glue alleviates Tripterygium wilfordii polyglycoside-induced oligoasthenospermia in rats by resisting ferroptosis via the Keap1/Nrf2/GPX4 signaling pathway

Jin Ding a,b,c,*, Baowei Lu a,c,*, Lumei Liu a,c, Zixuan Zhong a,c, Neng Wang a,c, Bonan Li a,c, Wen Sheng a,c,✉, Qinghu He a,c,d,✉
PMCID: PMC9873281  PMID: 36688426

Abstract

Context

Guilu-Erxian-Glue (GLEXG) is a traditional Chinese formula used to improve male reproductive dysfunction.

Objective

To investigate the ferroptosis resistance of GLEXG in the improvement of semen quality in the oligoasthenospermia (OAS) rat model.

Materials and methods

Male Sprague-Dawley (SD) rats were administered Tripterygium wilfordii polyglycoside, a compound extracted from Tripterygium wilfordii Hook F. (Celastraceae), at a dose of 40 mg/kg/day, to establish an OAS model. Fifty-four SD rats were randomly divided into six groups: sham, model, low-dose GLEXG (GLEXGL, 0.25 g/kg/day), moderate-dose GLEXG (GLEXGM, 0.50 g/kg/day), high-dose GLEXG (GLEXGH, 1.00 g/kg/day) and vitamin E (0.01 g/kg/day) group. The semen quality, structure and function of sperm mitochondria, histopathology, levels of oxidative stress and iron, and mRNA levels and protein expression in the Keap1/Nrf2/GPX4 pathway, were analyzed.

Results

Compared with the model group, GLEXGH significantly improved sperm concentration (35.73 ± 15.42 vs. 17.40 ± 4.12, p < 0.05) and motility (58.59 ± 11.06 vs. 28.59 ± 9.42, p < 0.001), and mitigated testicular histopathology. Moreover, GLEXGH markedly reduced the ROS level (5684.28 ± 1345.47 vs. 15500.44 ± 2307.39, p < 0.001) and increased the GPX4 level (48.53 ± 10.78 vs. 23.14 ± 11.04, p < 0.01), decreased the ferrous iron level (36.31 ± 3.66 vs. 48.64 ± 7.74, p < 0.05), and rescued sperm mitochondrial morphology and potential via activating the Keap1/Nrf2/GPX4 pathway.

Discussion and conclusions

Ferroptosis resistance from GLEXG might be driven by activation of the Keap1/Nrf2/GPX4 pathway. Targeting ferroptosis is a novel approach for OAS therapy.

Keywords: Guilu-Erxian-Glue, oligoasthenospermia, Keap1/Nrf2/GPX4 signaling pathway, ferroptosis, Tripterygium wilfordii polyglycoside

Introduction

Infertility, which refers to the inability to conceive after at least one year of unprotected, regular sexual intercourse (Zegers-Hochschild et al. 2017), remains a major challenge affecting 8–12% couples of reproductive ages, of which males account for approximately 50% (Agarwal et al. 2021). Global epidemiological studies have consistently described decreased spermatozoa counts and motility in a persistent trend (Kilchevsky and Honig 2012; Barratt et al. 2017). Additionally, a decline in semen quality among young men in China has been observed between 2001 and 2015, especially in terms of total sperm counts and progressive motility (Huang et al. 2017). Male infertility, which remains poorly understood, is influenced by several factors including genetics, endocrinopathy, presence of varicocele, lifestyle, and adverse environmental exposure (Jensen et al. 2017; Krausz and Riera-Escamilla 2018; Kumar et al. 2019; Krzastek et al. 2020). Idiopathic oligoasthenospermia (OAS) accounts for 60–75% of cases of male infertility (Barratt et al. 2017).

Reactive oxygen species (ROS) are excessively active oxidative free radicals. Sperm is vulnerable to ROS due to its limited antioxidant stress damage capacity and limited DNA damage monitoring and repair mechanisms (De Iuliis et al. 2009). Moreover, supraphysiological ROS levels can damage sperm DNA, RNA transcripts, telomere (Tamburrino et al. 2012) and sperm plasma membrane lipid peroxidation (Aitken et al. 1989), which is the main cause of functional defects of sperm. Therefore, oxidative stress injury of the male reproductive system is the main cause of OAS, male infertility and spontaneous abortion (Bisht et al. 2017; Villaverde et al. 2019; Ritchie and Ko 2021). Ferroptosis has recently been discovered as an iron-dependent form of regulated cell death resulting from accumulating lipid peroxidation production and lethal ROS originating from iron metabolism (Dixon et al. 2012; Stockwell et al. 2017). Glutathione peroxidase (GPX4) is a significant antioxidant enzyme in mammals that modulates ferroptotic cell death by protecting cells from lipid peroxidation (Yang et al. 2014). Additionally, GPX4 is highly present in testes and spermatozoa. A decrease in GPX4 levels in spermatozoa has been reported in approximately 30% of infertile men with OAS (Imai et al. 2001). Studies have also indicated that the modification of the Keap1/Nrf2 pathway could inhibit oxidative stress in rats to attenuate testicular injury (He et al. 2018; Zhang et al. 2019), and ferroptosis may be involved due to the inhibition of the Keap1/Nrf2 pathway (Sun et al. 2016). Additionally, suppressed Nrf2 levels in the spermatozoa are significantly linked to OAS (Chen et al. 2012; Yu et al. 2013).

Currently, drug strategies against OAS are lacking. Coenzyme Q10, L-carnitine, vitamin E, and other drug treatments can improve semen quality; however, rigorous experimental design and high-level randomized clinical trial evidence are lacking (Omar et al. 2019). The indications and efficacy of surgical treatments are also limited (Velasquez and Tanrikut 2014). Recently, advances in assisted reproductive technology have mitigated fertility problems. However, this technology harbors disadvantages, in terms of its cost, genetic risk, and medical ethics (Hansen et al. 2013; Inhorn and Patrizio 2015). Traditional Chinese medicine (TCM) provides alternative treatment options, and its curative effects on male infertility have been demonstrated. For instance, Liuwei-Dihuang decoction, Wuzi-Yanzong formula, Jingui-Shenqi pill, and also acupuncture (Jiang et al. 2017; Zhou et al. 2019). Among them, Guilu-Erxian-Glue (GLEXG) is a TCM formula used in the treatment of infertility, which was first recorded in Yibian, an ancient book of TCM. This formula is extremely effective for male and female infertility in long term, especially for those who are suffering from a deficiency of kidney essence.

The inhibition of ferroptosis by GLEXG treatment in those with OAS has not been studied. Here, we established an OAS rat model induced by Tripterygium wilfordii polyglycoside (GTW), a compound extracted and purified from Tripterygium wilfordii Hook F. (Celastraceae), and evaluated the role of GLEXG in improving semen quality. We also determined the expression and role of the Keap1/Nrf2/GPX4 signaling pathway and evaluated the ferroptosis inhibition by GLEXG treatment in the OAS rat model. The results may provide an experimental basis for the potential application of GLEXG in the treatment of OAS.

Materials and methods

GLEXG preparation

GLEXG is referenced by the Chinese Pharmacopoeia (2020) (Xu et al. 2021) and consists of Cervi Cornus Colla [Cervus elaphus Linnaeus (Cervidae)] (9 g, batch No., 200102), Testudinis Carapacis et Plastri Colla [Chinemys reevesii (Geoemydidae)] (4.5 g, batch No., 2020002), Lycium barbarum L. (Solanaceae) (3 g, batch No., A210616), and Panax ginseng C.A.Mey (Acanthaceae) (3 g, batch No., 2104001). These herbs were purchased from the same batch at Kangmei Pharmaceuticals Co., Ltd. (Guangzhou, China), Prof. Na Yu from the Hunan University of Chinese Medicine authenticated all the herbs. The authenticated voucher specimens are kept in the College of Integrated Traditional Chinese and Western Medicine, Hunan University of Chinese Medicine. The GLEXG was further prepared and decocted according to the Chinese Pharmacopoeia (2020). The final GLEXG decoction was stored in a refrigerator at 4 °C for no more than 3 months. Then, a rotary evaporator was applied to prepare GLEXG at final concentrations of 0.125, 0.25, and 0.50 g/mL for further study.

Animals

Male Sprague-Dawley (SD) rats (eight weeks old, weighing 250 ± 20 g) were obtained from Hunan SJA Laboratory Animal Co. Ltd. (Hunan, China). All rats were housed in standard animal cages with a humidity of 60% and temperature of 20 °C with12 h light/dark cycles with free access to water and food. Approval for this study was granted by the Ethical Committee of Hunan University of Chinese Medicine (Hunan, China) (Approval Number: LL2021071403). The protocol followed the Care and Use of Laboratory Animals of the National Institutes of Health Guide.

Experimental design and drug administration

After one week of adaptive feeding, the OAS rat model was subjected to GTW by gavage at a dose of 40 mg/kg/day for four weeks. The model was evaluated based on the histopathological features of the testicular tissue and the sperm concentration and motility (Chen et al. 2020; Li et al. 2020). Then, we stratified the model rats at random into five groups (n = 9) as follows: (1) model group: model rats administered distilled water; (2) low-dose GLEXG group (GLEXGL): model rats were subjected to GLEXG by gavage at a dose of 0.25 g/kg/day; (3) moderate-dose GLEXG group (GLEXGM): model rats were subjected to GLEXG by gavage at a dose of 0.50 g/kg/day; (4) high-dose GLEXG group (GLEXGH): model rats were subjected to GLEXG by gavage at a dose of 1.00 g/kg/day; (5) vitamin E group (VE, NO., H20073374, Hangzhou Yipin Xinwufeng Pharmaceutical Co., Ltd): model rats were subjected to VE by gavage at a dose of 0.01 g/kg/day. Additionally, the sham group (n = 9) was treated with distilled water. The rats were administered GLEXG, VE, or distilled water for four weeks. Then, they were anesthetized with ketamine (0.5 g/kg, i.p.). The testes and epididymis were removed for sperm quality assay or flow cytometry, and the rest testes and epididymis were frozen in liquid nitrogen or fixed in 4% paraformaldehyde (PFA) solution for further detection.

Chemical compounds of GLEXG using liquid chromatography to quadrupole/time-of-flight mass spectrometry (UPLC-Q/TOF-MS)

The chemical compounds in the alcohol extracts of GLEXG were characterized using an ACQUITY UPLC I-Class Plus UPLC system coupled with a XEVO TQ-XS Q/TOF-MS (Waters, USA) in positive and negative ion mode. Chromatography was performed on a Waters HSS T3 column (100.0 mm × 2.1 m, 1.7 μm) with a gradient elution of 0.1% formic acid aqueous solution (A) and acetonitrile (0–10 min, 0.2–20%; 10–20 min, 20–40%; 20–25 min, 40–50%; 25–33 min, 50–98%; 33 min, 50–98% (B). The mass spectrometry conditions were electrospray ionization with positive and negative ion mode scanning. For the negative ion mode, the ion source collision voltage was −4.5 kV, the ion source temperature was 100 °C, the dissolvent gas temperature was 550 °C, the sample and extraction cone voltages were 80 and 10 kV, respectively, and the ion source gas1 and gas2 were 55 Psi. For the positive ion mode, the ion source collision voltage was 5.5 kV, and the rest of the parameters were the same as in the negative ion mode. SCIEX OS (AB SCIEX, US) software was used for data acquisition and spectral processing, and the mass-to-charge ratio scan range was from 60 to 1000 m/z.

Epididymal sperm assay

Sperm concentration, motility, and the number of mobile sperm were detected as previously described (Chang et al. 2021). The cauda epididymis was minced into small pieces and incubated in PBS (37 °C, pH 7.4) for 15 min. Then, we transferred a drop of diluted sperm suspension into a glass slide for analysis using a computer-aided sperm analysis platform (Hamilton Thorn, MA, US) linked to a light microscope (Motic, Xiamen, China). In each rat, ≥10 fields were selected and captured, and the sperm quality index was judged by concentration and motility.

Total, ferric and ferrous iron assay

The total and ferrous iron content of testicular tissues were evaluated as described by the manufacturer. Testicular tissues were introduced to the buffer and homogenized on ice. They were then centrifuged, and the supernatant was obtained. We inoculated the supernatant with an iron probe as well as a microplate reader adopted to read OD values at 593 nm. The total and ferrous iron levels of testicular tissues were determined using an iron colorimetric assay kit as described by the manufacturer (DOJINDO, Beijing, China, I291). Ferric iron was obtained by subtracting ferrous iron from total iron.

Hematoxylin-eosin (H&E) staining

The testes were fixed in 4% PFA for 48 h. They were then dehydrated as they were transferred through a series of mixtures of alcohol and water, and then they were paraffin-embedded. The testes were then sliced into 5 μm segments stained with H&E solution. Thereafter, slides were observed by light microscopy (Motic, Xiamen, China), and micrographs were acquired.

MDA, GPX4, and GSH assays

The testes were taken from liquid nitrogen for preparation of tissue homogenates, and spun at 14,000 g for 10 min. The supernatant was collected and utilized to measure MDA (Beyotime, Shanghai, China, S0131M), GPX4 (Beyotime, Shanghai, China, AF7020), and GSH (Beyotime, Shanghai, China, S0053) using commercial ELISA kits as described by the manufacturers.

Flow cytometry assay

The assay of sperm mitochondrial membrane potential was performed with the lipophilic cationic dye using a JC-1 kit (Beyotime, Shanghai, China, C2006) as described by the manufacturer. Concisely, 1 × 106 spermatozoa were incubated for 15 min in a JC-1 solution (37 °C; 5 μM). Subsequently, spermatozoa were flushed and analyzed using flow cytometry. Further cytometric experiments were conducted on a flow cytometer (Beckman, CA, US), and the debris was gated out according to light scattering measurements. Flow cytometry acquisition of stained spermatozoa was performed using FL1 and FL2 fluorescence; ≥10,000 spermatozoa were detected for each analysis. The content of testicular tissue ROS production was detected using a DCFH-DA dye based on the ROS kit (Beyotime, Shanghai, China, S0033S) as described by the manufacturer. The testes were sliced into 5 μm sections and transferred to a centrifuge tube. Then, 3 mL collagenase II was added, digested at 37 °C for 40 min, filtered to obtain the cell suspension at 500 g, and spun for 5 min. Subsequently, 5 mL red blood cell lysate, which was added to resuspend the cells, lysed at RT (room temperature) for 5 min, and spun at 500 g for 5 min. The supernatant was discarded, and the cells were rinsed twice using PBS and cultured for 30 min in DMEM medium enriched with 10% FBS along with DCFH-DA dye. We digested the cells and analyzed them with a flow cytometer (Beckman, CA, US) to determine the intensity of DCFH-DA fluorescence.

Ultrastructural observation using transmission electron microscopy

Glutaraldehyde was slowly injected into fresh epididymis obtained from the rat using a syringe. Hardened testes were sliced (1 × 1 × 1 mm) and then pre-fixed with glutaraldehyde at 4 °C for 24 h. They were then, rinsed in 0.1 mol/L phosphate buffer and finally fixed in 1% osmic acid. Thereafter, gradient dehydration was performed, followed by embedment of the tissue with epoxy resin, which was processed into ultra-thin slices (1 μm). Finally, we dyed the sections using saturated acetate uranium and employed transmission electron microscopy (Hitachi, Tokyo, Japan) to observe the images.

Immunohistochemistry and immunofluorescence assay

The testes were fixed in 4% FPA overnight and sectioned into slices of 2–3 μm. The sections were rinsed three times in PBS, and endogenous peroxidase was blocked using 3% H2O2 at RT for 15 min. Thereafter, sections were blocked using 5% BSA in 0.3% Triton X-100 in PBS for 1 h at RT. Subsequently, we inoculated the sections overnight with anti-GPX4 antibody (Proteintech, Wuhan, China, 14432-1-AP, 1:1,000 dilution) at 4 °C. Then, we rinsed the sections three times in PBS and inoculated them with a secondary antibody for 1 h at RT. Positive immunoreactivity was performed using DAB and assessed by microscopy (Olympus, Tokyo, Japan). Images were analyzed using Image-Pro Plus 6.0 (Media Cybernetics, MD, US).

For fluorescent staining, the sections were blocked with 3% BSA for 30 min and incubated overnight with anti-GPX4 antibody (14432-1-AP, 1:100, Proteintech Group, Inc) and anti-FPN1 antibody (Cincinnati, OH, US, DF13561) at 4 °C. The following morning, the sections were rinsed three times in PBS and incubated with fluorescent secondary antibodies (SA00013-3, Proteintech, Wuhan, China) for 1 h in the dark at RT. Subsequently, the sections were rinsed in PBS, counterstained with DAPI (Wellbio, Guangzhou, China) and mounted using Prolong Gold anti-fade (Servicebio, Wuhan, China). The samples were then evaluated using a microscope (Olympus, Tokyo, Japan).

Reverse transcription quantitative polymerase chain reaction (qRT-PCR)

The TRI Reagent (Thermo Scientific, CA, US) was used to isolate the total RNA (tRNA), and cDNA was generated from the tRNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, CA, US). Then, qRT-PCR was conducted using 25 μL of reaction volume consisting of dilute cDNA, specific primers (10 μM) and UItraSYBR Mixture (ComWin, Beijing, China) on the QPCR Platform (Agilent, Shenzhen, China), with β-actin acting as the endogenous standard. Findings are given as fold changes relative to the control group which was set to 100%. Table 1 shows the primers used in this study.

Table 1.

Primary primers for real-time PCR.

Primer Forward Reverse Length
Keap1 5′-CCCATGCAGCCCGAACCCAA-3′ 3′- GTCACCTCCGCCTTGCACTCC-5′ 131bp
Nrf2 5′-ACGGCTAAAACTTCCTACTGTGA-3′ 3′- ACACTTACACAGAAACTAGCCCAA-’5 193bp
HO1 5′-TTGTTATTTCCCCAGTTCTACCAG-3′ 3′-CAAAAGACAGCCCTACTTGGTT’5 87bp
FPN1 5′-GCTTTGCTGTTCTTTGCCTTAGT-3′ 3′- GTGTGAGGAACCGGAGATAGC-5′ 90bp
β-actin 5′-ACATCCGTAAAGACCTCTATGCC-3′ 3′-TACTCCTGCTTGCTGATCCAC-’5 223bp
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Western blot

Testicular tissue preserved in liquid nitrogen was thawed, added to a grinder with the lysate, ground and lysed on ice for 15 min, transferred to a centrifuge tube, and span at 4 °C for 15 min at 14,000 g in an ultrafast freezing centrifuge. We collected the supernatant in an Eppendorf tube, total proteins were quantified using the BCA Kit (ComWin, Beijing, China, CW2011), and the protein sample was mixed with protein electrophoresis loading buffer, boiled for 10 min and set aside. Membranes were inoculated overnight with primary antibodies (Proteintech, Wuhan, China), including anti-Keap1 (10503-2-AP, 1:5,000 dilution), anti-Nrf2 (16396-1-AP, 1:2,000 dilution), anti-HO1 (10701-1-AP, 1:3,000 dilution), anti-FPN1(26601-1-AP, 1:1,000 dilution), anti-GPX4 (14432-1-AP, 1:1,000 dilution), anti-PCNA (60097-1-Ig, 1:6,000 dilution) and anti-β-actin (66009-1-Ig, 1:5,000 dilution) at 4 °C. They were then rinsed three times with PBST, and then inoculated for 2 h with HRP-linked goat anti-rabbit IgG (66009-1-Ig, 1:5,000 dilution). They were then rinsed three more times with PBST for 10 min each time and developed using ECL chemiluminescence. The hybridized bands were scanned for grey values via the Image J software (Bethesda, MDUS). The results are presented as the ratio of the grey values of the protein bands to that of the β-actin or PCNA bands and were corrected and normalized for β-actin or PCNA.

Statistical analysis

All statistical analyses were conducted in GraphPad Prism 8.01 software (La Jolla, CA, USA). The data are given as the mean ±standard deviation (SEM). One-way analyses of variance (ANOVA) followed by a post hoc least significant difference (LSD) test was adopted to calculate the statistical significance between groups, and p < 0.05 was considered to indicate statistical significance.

Results

Chemical compounds of GLEXG

The chemical compounds of GLEXG were analyzed using UPLC-Q/TOF-MS, and the ion diagrams are illustrated in Figure 1(A–B)) and identified compounds are shown in Table 2. Ultimately, 122 compounds in positive ion mode and 66 in negative ion mode were identified in GLEXG.

Figure 1.

Figure 1.

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Ion flow diagram of GLEXG detected by UPLC-Q/TOF-MS. Positive ion mode (A) and negative ion mode (B).

Table 2.

Mass spectrometry data and elemental composition of compounds GLEXG by UPLC-Q/TOF-MS analysis.

No. Compounds Formula Library score Retention time (min) Precursor mass (m/z) Area Height
  Positive ion mode
1 γ-Glutamate-cysteine C8H14N2O5S 89.9 0.84 3.99E + 05 1.07E + 05 248.962
2 Histidine C6H9N3O2 97.6 0.88 8.29E + 04 3.19E + 04 154.064
3 L-Arginine C7H16N4O2 99.7 0.88 3.27E + 05 1.19E + 05 173.106
4 Aspartic acid C4H7NO4 98.3 0.9 1.61E + 05 5.31E + 04 132.032
5 D-(+)-Mannose C6H12O6 68.4 0.96 8.11E + 06 1.32E + 06 215.035
6 Gluconic acid C6H12O7 48.9 0.97 2.40E + 06 4.96E + 05 195.053
7 Saccharate C6H10O8 99.5 0.98 1.09E + 06 2.04E + 05 209.032
8 D-Tagatose C6H12O6 72.8 1 1.30E + 06 2.27E + 05 179.057
9 Isomaltose C12H22O11 84.9 1.04 3.89E + 06 8.97E + 05 377.087
10 Quinic acid C7H12O6 98.2 1.1 1.25E + 06 3.74E + 05 191.058
11 Malate C4H6O5 99.7 1.27 1.45E + 06 4.27E + 05 133.015
12 N-Acetylglutamate C7H11NO5 97.3 1.51 1.48E + 05 2.02E + 04 188.058
13 Lactate C3H6O3 98.9 1.59 3.54E + 05 2.91E + 04 89.025
14 Oxoproline C5H7NO3 93.5 2.37 1.14E + 06 1.26E + 05 128.037
15 Methylmalonate C4H6O4 99.5 2.7 2.55E + 05 2.26E + 04 117.02
16 cis-Aconitic acid C6H6O 96.4 2.9 1.81E + 05 1.24E + 04 173.01
17 N-Acetylalanin C5H9NO3 98.2 3.03 8.61E + 04 9.59E + 03 130.088
18 Xanthine C5H4N4O2 99.2 3.06 1.13E + 05 1.21E + 04 151.027
19 Tyrosine C9H11NO3 96.9 3.13 3.56E + 05 3.40E + 04 180.068
20 (S)-2-Hydroxybutanoic acid C4H8O3 100 3.4 5.66E + 04 5.23E + 03 103.041
21 Uridine C9H12N2O6 89.1 3.42 2.63E + 05 2.28E + 04 243.064
22 Perfluorooctane sulfonate (PFOS) C8HF17O3S 68.8 3.99 7.53E + 04 6.18E + 03 499.132
23 Adenosine 2′,3′-cyclic phosphate C10H12N5O6P 98.6 4.18 3.74E + 05 3.02E + 04 328.047
24 Guanosine monophosphate C10H14N5O8P 99 4.23 1.13E + 05 7.30E + 03 362.052
25 L-Tryptophanamide C11H13N3O 94 4.44 1.62E + 05 7.33E + 03 202.11
26 Neohesperidin C28H34O15 35.5 5.04 2.18E + 04 6.06E + 03 609.077
27 Acadesine C9H14N4O5 45 5.05 5.69E + 04 1.19E + 04 337.079
28 Citric acid C6H8O7 99.2 5.12 4.38E + 05 4.82E + 04 191.021
29 Inosine C10H12N4O5 100 5.15 9.97E + 04 3.21E + 04 267.074
30 3,4,5-Trimethoxybenzoic acid C10H11O5 63.8 5.2 3.29E + 05 6.00E + 04 211.074
31 Resibufogenin C24H32O4 53.3 5.27 1.51E + 05 2.36E + 04 429.162
32 Phenylalanine C9H11NO2 96.7 5.3 3.36E + 05 7.71E + 04 164.073
33 Secalonic acid D C32H30O14 74 5.34 1.04E + 05 2.04E + 04 637.298
34 Heteroclitin D C27H30O8 42.5 5.35 1.63E + 05 2.29E + 04 481.207
35 Typhaneoside C34H42O20 84.6 5.39 2.32E + 05 6.00E + 04 769.352
36 Pedunculoside C36H58O10 60.7 5.42 1.62E + 05 3.10E + 04 695.207
37 Trehalose C12H22O11 60.3 5.43 9.80E + 04 2.45E + 04 341.085
38 Calceorioside B C23H26O11 85.6 5.46 2.23E + 05 4.90E + 04 477.126
39 D-Pantothenic acid C9H17NO5 97.3 5.48 6.57E + 04 1.84E + 04 218.104
40 Adipate C6H10O4 93.2 5.77 4.65E + 04 1.34E + 04 145.051
41 L-Tryptophan C11H12N2O2 97.6 5.83 4.28E + 05 1.20E + 05 203.084
42 2-Chloro-L-phenylalanine C9H10ClNO2 96.4 5.87 1.60E + 05 4.72E + 04 198.034
43 2-Hydroxyphenylacetate C8H8O3 96.3 5.96 1.03E + 05 3.00E + 04 181.052
44 Sibiricose A5 C22H30O14 78.5 6.11 1.03E + 05 1.87E + 04 517.157
45 Traumatic acid C12H20O4 83.7 6.13 8.91E + 04 2.36E + 04 227.105
46 2′-Deoxyguanosine-5′-diphosphate trisodium salt C10H12N5Na3O10P2 50 6.13 8.61E + 04 2.00E + 04 426.2
47 Asiatic acid C30H48O5 49.3 6.18 9.87E + 04 1.96E + 04 487.253
48 2-Isopropylmalic acid C7H12O5 100 6.25 6.87E + 05 1.72E + 05 175.063
49 Chlorogenic acid C16H18O9 100 6.27 1.56E + 05 4.14E + 04 353.089
50 3-Methyladipic acid C7H12O4 99 6.5 2.02E + 05 6.33E + 04 159.068
51 Raffinose C18H32O16 82.2 6.62 2.03E + 05 6.08E + 04 503.178
52 (S)-(-)-2-Hydroxyisocaproic acid C6H12O3 98.1 6.96 1.71E + 05 3.84E + 04 131.072
53 Ginsenoside-Ro C48H76O19 70.6 7.07 2.23E + 05 3.32E + 04 955.377
54 4-Coumarate C9H8O3 98.1 7.29 5.41E + 05 1.61E + 05 163.041
55 6-Phenyl-2-thiouracil C10H8N2OS 50.6 7.31 1.02E + 05 3.21E + 04 203.131
56 8-Hydroxyoctanoic acid C8H16O3 99.7 7.35 5.56E + 05 1.75E + 05 159.104
57 L-3-Phenyllactic acid C9H10O3 98.9 7.39 2.47E + 05 7.51E + 04 165.057
58 N-Acetyl-L-tryptophan C13H14N2O3 98.6 7.45 1.17E + 06 3.18E + 05 245.095
59 Decanoate C10H20O2 90.9 7.51 2.03E + 05 5.18E + 04 171.068
60 Isoferulic acid C10H10O4 100 7.53 1.81E + 05 5.42E + 04 193.052
61 Scopoletin C10H8O4 98.6 7.57 7.87E + 04 2.30E + 04 191.037
62 Capric acid C10H20O2 33.9 7.71 3.93E + 05 1.22E + 05 231.126
63 Notoginsenoside R1 C47H80O18 96.2 7.76 1.33E + 06 3.79E + 05 977.537
64 Ginsenoside Re C48H82O18 86.3 7.93 3.51E + 06 9.07E + 05 991.553
65 Azelate C9H16O4 97.3 7.94 4.79E + 06 1.37E + 06 187.099
66 Ginsenoside Rg1 C42H72O14 98.2 7.99 1.35E + 07 3.85E + 06 845.494
67 Suberate C8H14O4 54.1 8.1 4.11E + 05 1.39E + 05 173.12
68 L-Dihydroorotic acid C5H6N2O4 41 8.37 1.78E + 05 5.48E + 04 157.088
69 Adenosine diphosphate ribose C15H23N5O14P2 71.2 8.51 6.24E + 04 1.70E + 04 558.295
70 Agistatin B C11H18O4 33.5 8.54 3.18E + 04 9.17E + 03 213.114
71 Oxoadipic acid C6H8O5 91.9 8.58 8.13E + 05 2.37E + 05 159.104
72 Sebacate C10H18O4 99 8.64 1.21E + 06 3.48E + 05 201.115
73 Crocetin C20H24O4 38.2 8.75 1.21E + 05 2.56E + 04 327.219
74 10-Hydroxydecanoate C10H20O3 80.2 8.83 1.60E + 05 4.73E + 04 187.135
75 Astragaloside IV C41H68O14 96.8 8.85 3.94E + 04 9.63E + 03 829.499
76 10-Hydroxydec-2-enoic acid C10H18O3 72.3 8.89 4.11E + 05 1.17E + 05 185.119
77 Astragaloside I C45H72O16 90.8 8.96 6.94E + 04 2.06E + 04 913.52
78 4-Ethylbenzoic acid C2H5C6H4CO2H 100 8.98 7.47E + 04 2.40E + 04 149.062
79 Ginsenoside Rf C42H72O14 95.9 9.05 4.64E + 06 1.36E + 06 845.493
80 Ginsenoside Rb1 C54H92O23 40.1 9.1 1.44E + 06 4.10E + 05 1153.605
81 Ginsenoside Rc C53H90O22 87.5 9.25 4.56E + 05 1.35E + 05 1077.588
82 Ginsenoside Rb2 C53H90O22 98.1 9.26 1.37E + 06 4.20E + 05 1123.593
83 Undecanedioic Acid C11H20O4 99.6 9.3 4.58E + 05 1.31E + 05 215.13
84 Ginsenoside Rg2 C42H72O13 100 9.42 8.41E + 05 1.93E + 05 829.498
85 4-Bromophenylalanine C9H10BrNO2 77.8 9.53 3.45E + 05 1.09E + 05 242.177
86 20(R)-Ginsenoside Rh1 C36H62O9 98 9.55 7.28E + 05 2.40E + 05 683.439
87 11a-Hydroxyprogesterone C21H30O3 89.4 9.61 2.33E + 06 5.27E + 05 659.476
88 Ginsenoside Rd C48H82O18 91 9.77 2.18E + 06 6.50E + 05 991.55
89 Gypenoside XVII C48H82O18 62.3 9.77 3.04E + 05 9.41E + 04 945.545
90 Kaempferol C15H10O6 92.5 9.85 2.41E + 05 5.48E + 04 285.208
91 Glyceraldehyde-3-phosphate C3H7O6P 47.2 9.9 9.28E + 04 2.59E + 04 169.088
92 Dodecanedioic acid C12H22O4 100 9.93 3.04E + 05 8.56E + 04 229.146
93 Notoginsenoside Ft1 C47H80O17 98.2 10.28 1.06E + 05 1.98E + 04 961.541
94 Arachidate C20H40O2 56.4 10.51 1.68E + 05 3.13E + 04 311.224
95 1,11-Undecanedicarboxylic acid C13H24O4 98.4 10.55 9.97E + 05 2.68E + 05 243.162
96 Estriol C18H24O3 70.1 10.94 2.10E + 05 5.63E + 04 287.224
97 Geranyl-PP C10H20O7P2 61.7 11.02 9.57E + 05 1.38E + 05 313.24
98 Chikusetsusponin Iva C42H66O14 100 11.09 2.90E + 05 7.32E + 04 793.44
99 Stachyose C24H42O21 97.4 11.23 2.49E + 05 7.13E + 04 665.429
100 20(R)-Ginsenoside Rg3 C42H72O13 97.9 11.47 3.27E + 05 8.85E + 04 829.498
101 Heptadecanoate C17H34O2 73.2 12.18 1.41E + 05 3.92E + 04 269.214
102 Isorhamnetin C16H12O7 84.4 12.47 1.71E + 05 4.73E + 04 315.255
103 2,4-Di-tert-butylphenol C14H22O 38.5 13.9 3.50E + 04 9.54E + 03 205.161
104 All-trans-retinoic acid C20H28O2 76.7 14.21 9.16E + 04 2.35E + 04 299.261
105 Nonadecanoic acid C19H38O2 80.5 14.51 5.03E + 04 1.44E + 04 297.244
106 Sulfamethazine C12H14N4O2S 100 15 4.27E + 05 1.14E + 05 277.219
107 1-Pentadecanol CH3(CH2)14OH 98.1 15.15 9.93E + 04 2.82E + 04 227.202
108 Shikimate-3-phosphate C7H11O8P 90.8 15.39 3.86E + 05 9.38E + 04 253.218
109 Arachidonic acid C20H32O2 99.8 15.51 1.37E + 05 3.78E + 04 303.234
110 Linoleic acid C18H32O2 100 15.67 1.31E + 06 3.34E + 05 279.234
111 Palmitate C16H32O2 92 16.26 9.61E + 05 2.37E + 05 255.234
112 Glycerophosphocholine C22H44NO6PS2 73.6 16.26 1.80E + 05 4.47E + 04 256.237
113 Vaccenic acid C18H34O2 89.3 16.42 1.51E + 06 3.68E + 05 281.25
114 1-Aminocyclopropanecarboxylate C4H7NO2 88.8 16.64 5.40E + 03 1.49E + 03 99.926
115 Estrone C18H22O2 90.1 16.82 4.89E + 04 1.24E + 04 269.25
116 Ethyl hexadecanoate1 CH3(CH2)14COOC2H5 100 17.42 8.38E + 05 1.66E + 05 283.266
117 Sulfadoxine C12H14N4O4S 97.2 17.52 6.31E + 04 1.27E + 04 309.281
118 N, N-Dimethyl-1,4-phenylenediamine C8H12N2 32.4 18.19 3.79E + 04 6.62E + 03 134.895
119 Valine C5H11NO2 63 19.54 2.82E + 04 1.30E + 04 115.921
120 Mesoxalate C3H2O5 100 19.87 5.29E + 05 4.31E + 04 116.929
121 Diethanolamine C4H11NO2 68.1 20.63 1.35E + 03 5.68E + 02 103.921
122 Aicar C9H15N4O8P 45 5.05 5.69E + 04 1.19E + 04 337.079
  Negative ion mode
1 Caprolactone C6H10O2 92.7 0.72 3.29E + 04 6.60E + 03 114.988
2 Arginine C6H14N4O2 89.7 0.89 4.71E + 04 2.05E + 04 349.231
3 Glutamine C5H10N2O3 99.7 0.92 9.45E + 04 2.82E + 04 147.076
4 Glycerophosphocholine C8H20NO6P 97.2 0.97 3.96E + 05 1.11E + 05 258.11
5 Betain C5H11NO2 36.2 1.01 4.91E + 05 1.30E + 05 118.086
6 Proline acid C5H9NO2 53 1.09 3.97E + 05 1.15E + 05 116.07
7 Tyrosin C9H11NO3 98.7 1.43 3.91E + 05 5.39E + 04 182.081
8 Vitamin C C6H8O6 98.9 2.08 2.20E + 05 2.59E + 04 177.039
9 Suberic acid C8H14O4 32.3 2.2 6.20E + 04 8.17E + 03 175.024
10 S-Methyl glutathione C11H19N3O6S 57.1 2.22 6.12E + 04 6.06E + 03 322.077
11 Pyroglutamate C5H7NO3 100 2.4 1.98E + 05 3.12E + 04 130.05
12 Leucine C6H13NO2 98.6 3.07 2.94E + 05 3.08E + 04 132.102
13 Amphetamine C9H13N 50.1 3.17 6.25E + 04 6.71E + 03 136.075
14 Fucose C6H12O5 47.3 3.17 1.13E + 05 1.13E + 04 165.054
15 Glyceraldehyde C3H6O3 93.5 3.35 2.12E + 04 2.05E + 03 121.064
16 Xanthosine C10H12N4O6 53.8 3.42 2.50E + 04 2.85E + 03 285.102
17 Uracil C4H4N2O2 77.9 3.43 1.86E + 04 2.61E + 03 113.034
18 Guanosine C10H13N5O5 100 3.55 2.70E + 05 2.40E + 04 284.1
19 3′-Aenylic acid C10H14N5O7P 100 3.56 5.03E + 04 4.67E + 03 348.071
20 6-Hydroxypurine C5H4N4O 79.8 3.58 1.51E + 05 1.04E + 04 137.046
21 Cyclic AMP C10H12N5O6P 98.8 4.27 2.07E + 05 1.80E + 04 330.06
22 Cyclic GMP C10H12N5O7P 100 5.46 2.17E + 05 4.18E + 04 346.055
23 Adenosine C10H13N5O4 100 5.5 1.41E + 05 4.08E + 04 268.104
24 Guanine C5H5N5O 92.4 5.53 5.25E + 04 8.79E + 03 152.056
25 Rutin C27H30O16 97.2 7.11 2.74E + 05 7.95E + 04 611.16
26 3,4-Dihydroxybenzoate C7H6O4 95.4 8.89 3.26E + 04 9.44E + 03 155.106
27 Pseuoginsenoside F11 C42H72O14 98.2 9.09 2.88E + 05 8.36E + 04 801.498
28 Uvaol C30H50O2 91.3 9.12 3.79E + 05 5.86E + 04 443.388
29 Cortisone 21-acetate C23H30O6 37.5 9.56 2.59E + 04 8.23E + 03 405.351
30 Androsterone C19H30O2 34.1 9.77 3.27E + 04 9.51E + 03 291.195
31 Sclareolide C16H26O2 76.9 9.95 1.70E + 04 4.93E + 03 251.2
32 Methyl linoleate C19H34O2 68.1 10.52 1.49E + 05 1.25E + 04 295.227
33 Methyl dihydrojasmonate C13H22O3 85.4 10.55 6.06E + 04 1.46E + 04 227.164
34 6-Phosphogluconate C6H13O10P 40.5 10.61 6.00E + 04 6.32E + 03 277.216
35 Palmitoleate C16H30O2 44.2 10.65 2.50E + 04 6.40E + 03 255.159
36 Corydaline C22H27NO4 46.8 10.67 1.21E + 04 3.30E + 03 370.241
37 Gamma-linolenate C18H30O2 98.3 10.68 3.66E + 04 3.90E + 03 279.159
38 15-Hydroxyculmorone C15H24O3 40.6 10.87 2.04E + 04 5.64E + 03 253.18
39 Cytidine diphosphate C9H15N3O11P2 60.3 10.92 5.14E + 04 1.41E + 04 404.206
40 Panaxydol C17H24O2 77.7 11.45 2.75E + 04 7.94E + 03 261.184
41 Ursodeoxycholate C24H40O4 100 11.51 1.76E + 06 4.55E + 05 393.285
42 β-Sitosterol C29H50O 96.2 11.73 6.76E + 05 1.74E + 05 432.238
43 Petroselinate C18H34O2 97.8 15.67 4.40E + 05 1.19E + 05 282.278
44 Stearate C18H36O2 84.4 16.67 4.06E + 04 9.57E + 03 285.298
45 Myristic acid (D27) C14H54HO2 38.2 16.88 3.67E + 04 9.08E + 03 256.263
46 Octadecanamide CH3(CH2)16CONH2 64.1 16.95 6.19E + 05 1.39E + 05 284.295
47 Monensin C36H62O11 90.7 17.09 2.31E + 04 5.40E + 03 693.454
48 Buspirone C21H31N5O2 33.9 17.27 7.05E + 04 1.51E + 04 386.342
49 Bisoprolol C18H31NO4 32.6 17.4 4.59E + 04 1.07E + 04 326.342
50 Trimethylamine C3H9N 91.6 17.52 1.14E + 04 1.76E + 03 60.044
51 Phosphoribosyl pyrophosphate C5H13O14P3 98.6 17.58 2.84E + 05 5.84E + 04 391.284
52 Naratriptan C17H25N3O2S 79.8 17.76 3.93E + 04 7.49E + 03 336.326
53 Allyl isothiocyanate C4H5NS 99.5 17.8 4.90E + 04 4.84E + 03 100.075
54 Citramalate C5H8O5 84.9 17.81 2.77E + 04 2.69E + 03 149.023
55 o-Xylene C6H4(CH3)2 97.5 17.92 6.34E + 04 3.15E + 03 107.07
56 7-Ketocholesterol C27H44O2 94.7 17.92 9.92E + 04 2.07E + 04 401.341
57 Danazol C22H27NO2 58.6 18.18 3.34E + 05 7.39E + 04 675.675
58 Leonurine C14H21O5N3 84.1 18.47 8.53E + 04 1.59E + 04 312.326
59 Dibutyl phthalate C16H22O4 99.5 18.51 1.24E + 05 7.23E + 03 279.159
60 Patchouli alcohol C15H24 91.7 18.52 9.20E + 04 5.55E + 03 205.086
61 1-Methyl-2-pyrrolidone C5H9NO 98.1 18.53 8.25E + 04 6.82E + 03 100.075
62 Benzoate C7H6O2 44.5 19.65 2.47E + 05 2.71E + 04 122.964
63 5-Hydroxylysin C6H14N2O3 86.1 19.91 1.84E + 04 5.38E + 03 163.132
64 m-Xylene C6H4(CH3)2 60.6 21.61 6.62E + 03 1.98E + 03 107.07
65 1,2-Dichloroethane C2H4Cl2 97.2 21.68 1.27E + 04 5.46E + 03 100.076
66 β-alanine C3H7NO2 30.1 21.68 3.80E + 03 1.63E + 03 89.939
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Body weight and organ coefficients

No differences in body weight were observed among the groups before the experiment. As the experiment progressed, the body weight of each group gradually increased. After the experiment, the body weight of the model group was significantly lower than those of the sham group. With the administration of GLEXG, the weight increased significantly (Figure 2(A)). The epididymal coefficients of the model group were significantly lower than those of the sham group, and those of the GLEXGM and GLEXGH group were significantly higher than those of the model group (Figure 2(B)). The testicular coefficient in each group was not statistically significant (Figure 2(C)).

Figure 2.

Figure 2.

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GLEXG treatment mitigated histopathological lesions of testes and improved semen quality in the epididymis of an OAS rat model induced by GTW. (A–C) Body weights of rats, and organ coefficients of testis and epididymis in each group. (D–F) Effect of GLEXG on sperm concentration, motility, and the number of mobile sperm. (G) Representative sperm images under microscopy in each group. (H) Representative H&E staining of testicular tissue in each group. Red and black arrowheads indicate seminiferous tubule space and spermatogenic cells, respectively. Data are shown as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. the model group, respectively.

GLEXG treatment improved semen quality in epididymis and mitigated histopathological lesions of testes in OAS rat model induced by GTW

GTW administration resulted in a remarkable decrease in sperm concentration, motility, and the number of mobile sperm. However, treatment with GLEXG significantly increased the sperm concentration, motility, and the number of mobile sperm (Figure 2(D–F)). Representative sperm images under microscopy from each group were shown in Figure 2(G). In histological analysis (Figure 2(H)), the sham group exhibited normal arrangement of spermatogenic cells in seminiferous tubules, without histopathologic lesions. In contrast, the model group had obvious damage to testicular tissues, consisting of intertubular edema, exfoliation of the seminiferous tubules, vacuolization in the cytoplasm of Sertoli cells, and unordered arrangement of spermatogenic cells. GLEXG treatment mitigated the histopathologic lesions and demonstrated the greatest improvement in the high dose group.

GLEXG improved oxidative stress and iron metabolism of testicular tissue in OAS rat model induced by GTW

Based on previous research (Hou et al. 2016), the MDA and ROS levels can be utilized as a measurement of lipid peroxidation in cells or tissues. GTW administration triggered a remarkable increase in MDA and ROS levels in the testes compared with the sham group, however, the GPX4 and GSH levels significantly decreased, which is a molecular signature of ferroptosis. After GLEXG treatment, the MDA and ROS levels were reduced, while the GPX4 and GSH levels increased (Figure 3(A–E)). In contrast with the sham group, the level of sperm mitochondrial membrane potential significantly decreased in the model group; however, after GLEXG treatment, there was a remarkable increase of the level in sperm mitochondrial membrane potential (Figure 3(F,G)). Additionally, GTW administration also resulted in increases in ferrous and total iron, but not ferric iron (Figure 4(A–C)). We also observed that GTW administration resulted in characteristic morphologic features associated with ferroptosis in the model groups, including shrunken mitochondria, diminished mitochondrial cristae, chromatin condensation, cytoplasmic and organelle swelling and plasma membrane rupture. After GLEXG treatment, sperm mitochondria morphology damage was mitigated (Figure 4(D)).

Figure 3.

Figure 3.

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GLEXG treatment improved oxidative stress of testicular tissues in OAS rat model induced by GTW. (A–B) Representative images of relative fluorescence intensity of testicular tissue ROS and the contents of ROS measured by flow cytometry in each group. (C–E) MDA, GPX4, and GSH levels in testes in each group. (F–G) Representative flow cytometry images of sperm mitochondrial membrane potential, and the levels of sperm mitochondrial membrane potential measured by flow cytometry in each group. Data are shown as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. the model group, respectively.

Figure 4.

Figure 4.

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GLEXG improved iron metabolism of testicular tissue in OAS rat model induced by GTW. (A–C) Levels of total, ferric and ferrous iron in each group. (D) Representative images of transmission electron microscopy of sperm mitochondria morphology in each group. White arrowheads indicate shrunken mitochondria; red arrowheads indicate cytoplasmic and organelle swelling, as well as plasma membrane rupture. Data are shown as the means ± SEM. *p < 0.05, **p < 0.01 vs. the model group, respectively.

Protein and mRNA expression in Keap1/Nrf2/GPX4 signaling pathway associated with ferroptosis

Compared with the sham group, HO1 and nuclear Nrf2 protein expression significantly decreased, while Keap1 and cytoplasmic Nrf2 protein expression increased in the model group. After GLEXG treatment, we observed an increase in HO1 and nuclear Nrf2 protein expression and a decrease in Keap1 and cytoplasmic Nrf2 protein expression. Moreover, Nrf2 and Keap1 mRNA expression significantly increased in the model group, while HO1 mRNA expression decreased. After GLEXG treatment, Nrf2 and Keap1 mRNA expression decreased and that of HO1 increased (Figure 5(A–C)).

Figure 5.

Figure 5.

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GLEXG treatment activated the Keap1/Nrf2 signaling pathway. (A) Representative gel images of Keap1 and HO1 and relative protein levels in each group. (B) Representative gel images of nuclear Nrf2 and cytoplasmic Nrf2, and relative protein levels in each group. (C) Relative mRNA expression levels of Keap1, HO1 and Nrf2 in each group. Data are shown as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. the model group, respectively.

Additionally, we explored the expression of mRNA and proteins implicated in iron metabolism. In contrast with the sham group, GTW administration induced decreased protein and mRNA expression of GPX4 and FPN1. After GLEXG treatment, GPX4 and FPN1 expression significantly increased. (Figures 6(A–B) and 7(A–B)). Furthermore, immunohistochemical analysis revealed that the level of GPX4 was significantly lower in the model groups compared with the sham group. After GLEXG treatment, the GPX4 level significantly increased (Figure 6(C–D)). Immunofluorescence analysis revealed that the level of GPX4 and FPN1 was extensively lower in the model group compared to the sham group. After GLEXG treatment, the GPX4 level significantly increased. (Figures 6(E–F) and 7(C–D)).

Figure 6.

Figure 6.

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GLEXG treatment improved the protein and mRNA expression of GPX4. (A) Representative gel images of GPX4 in each group. (B) Relative protein and mRNA expression levels of GPX4 in each group. (C–D) Immunohistochemical staining and quantification of GPX4 in each group. (E–F) Representative immunofluorescence images and quantification of GPX4 in each group. Data are shown as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. the model group, respectively.

Figure 7.

Figure 7.

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GLEXG treatment improved the protein and mRNA expression of FPN1. (A) Representative gel images of FPN1 in each group. (B) Relative protein and mRNA expression levels of FPN1 in each group. (C–D) Representative immunofluorescence images and quantification of FPN1 in each group. Data are shown as the means ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001 vs. the model group, respectively.

Discussion

Male infertility causes substantial social and psychological distress and imposes a considerable economic burden on patients and health­care systems (Inhorn and Patrizio 2015), which remains poorly understood, and idiopathic OAS accounts for 60–75% of all cases (Barratt et al. 2017). Currently, treatment strategies against OAS remain limited. Thus, combinative therapy and the comprehensive management of OAS are urgent. Investigations involving TCM for OAS treatment have been ongoing for thousands of years. As a significant component of complementary and alternative medicine, TCM plays a crucial role in OAS treatment and is practiced worldwide. Previous animal experiments showed GLEXG to be orally applicable, very safe, and harbor low toxicity (Chou et al. 2018; Wang et al. 2020).

In this study, the chromatographic fingerprint of GLEXG was established and characterized using UPLC-Q/TOF-MS. We identified 122 compounds in positive ion mode and 66 in negative ion mode in GLEXG, indicating that GLEXG is chemically complex with hundreds or thousands of constituents. The exact chemical nature and interaction of these constituents remain still unknown. Among the compounds obtained from GLEXG, it is reported that the β-sitosterol could reduce oxidative stress in sperm, thus improving sperm counts and motility (Zhang et al. 2015). The kaempferol could significantly increase the levels of GPX, SOD in spermatozoa of rats with diabetes and reduce the levels of TNF-α, NF-κB in spermatozoa (Dobrzynska 2004; Jamalan et al. 2016). GLEXG also contains multiple amino acids, which play multiple roles in regulating cell metabolism, proliferation, and differentiation, and are involved in spermatogenesis and sperm maturation (Tosic 1947; Dai et al. 2015).

In this study, we showed that GLEXG (i) significantly improved the semen concentration, motility, and the number of mobile sperm in an OAS rat model induced by GTW; (ii) mitigated histopathological damage in the testicular tissues; (iii) improved the structure and function of sperm mitochondria; (iv) improved the level of oxidative stress and iron metabolism in testicular tissues; (v) activated the Keap1/Nrf2/GPX4 signaling pathway to resist ferroptosis.

Ferroptosis is an atypical form of regulated cell death first proposed by Stockwell et al. (2012) and differs from autophagy, pyroptosis, apoptosis, necrosis, or other kinds of cell death studied in biochemistry, morphology, and genetics. Morphologically, ferroptosis is characterized by ruptured mitochondrial outer membranes, diminished mitochondrial cristae, and shrunken mitochondria (Dixon et al. 2012; Yang et al. 2014). The vital signatures of ferroptosis are iron dependence and aggregation of lipid ROS. Nevertheless, low ROS levels are required for various redox-sensitive physiological processes, such as sperm capacitation, insemination, as well as hyperactivation (Aitken et al. 2012). Emerging evidence shows that ROS-triggered damage to sperm contributes to 30–80% of male infertility (Guérin et al. 2001; Tremellen 2008; Bisht et al. 2017). Oxidative stress constitutes a primary cause of germ cell dysfunction given the impairment of the structural and functional integrity of spermatozoa (Aitken and Baker 2006; Agarwal et al. 2014). Previous research has indicated that excessive ROS could damage sperm membranes, thereby inhibiting sperm motility along with their ability to fuse with oocytes. High levels of ROS could also directly lead to sperm DNA lesions, compromising the paternal genomic contribution to the embryo (Agarwal et al. 2006). In this study, GTW-administered rats manifested typical features of OAS, including decreased semen concentration, motility and mobile sperm count in the epididymis, and histopathological damage in the testis. We also observed that the OAS rat model exhibited characteristic features of ferroptosis, such as increased MDA and ROS levels, decreased GSH and GPX4 levels, iron accumulation, and abnormal sperm mitochondrial morphology. We found that GLEXG treatment markedly improved the sperm quality in the model, mitigated histopathological damage in the testes and lesions in the sperm mitochondria morphology, increased the level of sperm mitochondrial membrane potential, and regulated the level of oxidative stress and iron metabolism in testicular tissue.

The Keap1/Nrf2 pathway is among the most remarkable defense mechanisms against oxidative stress and can regulate the redox process, maintaining cellular homeostasis (Lu et al. 2016). Keap1, a negative repressor of Nrf2, could target Nrf2 for ubiquitin-dependent proteasomal degradation (Yamamoto et al. 2018). Nrf2 has been shown to play a crucial role in ferroptosis regulation, and the expression of Nrf2 along with its target gene, GPX4 can inhibit ferroptosis (Song and Long 2020; Takahashi et al. 2020; Ge et al. 2021). GPX4 is considered to be an indispensable modulator of ferroptosis. Repressing the activity of GPX4 or depleting GSH, the substrate of GPX4, induced ferroptosis (Yang et al. 2014). Male sperm quality and level of seminal plasma GPX4 are positively correlated (Ou et al. 2020), and low expression of GPX4 in rat testes could further influence semen concentration and motility, which could cause sperm deformity (Zhou et al. 2017; Wang et al. 2021). Nevertheless, the mechanism of GPX4-triggered male infertility remains poorly understood. Additionally, clinical investigations have demonstrated that Nrf2 mRNA levels were drastically diminished in the spermatozoa of individuals with OAS (Chen et al. 2012; Yu et al. 2013). Moreover, silencing of the Nrf2 gene in mice diminished sperm quality in an age-dependent manner, illustrating that Nrf2 has an indispensable role in spermatogenesis and maturation (Nakamura et al. 2010). Increasing evidence demonstrated that Nrf2 positively inhibits ferroptosis (Sun et al. 2016; Shin et al. 2018; Zhao et al. 2020). In this study, we demonstrated that Nrf2 and GPX4 expression significantly decreased, while Keap1 expression increased in the testes of rats with OAS induced by GTW. After GLEXG treatment, Nrf2 and GPX4 levels increased remarkably, while Keap1 expression decreased. Because ferroptosis is a newly discovered iron-dependent form of cell death, we also examined protein expression involved in iron metabolism. Specifically, FPN1 is the only iron exporter (Chen et al. 2020), and deletion of FPN1 in mice increases cellular iron burden (Zhang et al. 2011, 2012; Li et al. 2019). We established that FPN1 expression was lower in the testes of the OAS rats compared with normal rats. After GLEXG treatment, the expression of FPN1 increased significantly. Schematic representation of GLEXG resisting ferroptosis and improving semen quality via the Keap1/Nrf2/GPX4 signaling pathway is presented in Figure 8.

Figure 8.

Figure 8.

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Schematic representation of GLEXG resisting ferroptosis and improving semen quality via the Keap1/Nrf2/GPX4 signaling pathway.

Conclusions

In this study, GLEXG improved the semen quality of an OAS rat model partially through ferroptosis resistance via the Keap1/Nrf2/GPX4 signaling pathway. These findings suggest that targeting ferroptosis could be a potential strategy for OAS therapy. In further studies, we intend to validate the underlying mechanism of ferroptosis regulated by GLEXG on mouse spermatogenic cell lines (GC-1 spg, GC-2 spd) in vitro.

Acknowledgements

The authors thank all members of the andrology laboratory of the Hunan University of Chinese Medicine for helpful discussions and comments on the manuscript.

Funding Statement

This study was supported by the National Natural Science Foundation of China [Grant Number: 81774324], the Open Fund Project of Integrated Traditional Chinese and Western Medicine of Hunan University of Chinese Medicine [Grant Number: 2020ZXYJH29], the Fundamental Research Project of Medical and Health in Shenzhen Bao’an District [Grant Number: 2020JD505], the Key Discipline Projects of Hunan University of Chinese Medicine [Grant Number: 2021ZXYJH03], and the Dongjian Postgraduate Innovation Project of Hunan University of Chinese Medicine [Grant Number: 2021DJ03].

Author contributions

Qinghu He and Jin Ding designed and conceived the study. Jin Ding, Lumei Liu, Zixuan Zhong and Bonan Li conceived the study and drafted the manuscript. Jin Ding, Wen Sheng, Baowei Lu, and Neng Wang retrieved and analyzed the data. Qinghu He, Jin Ding, and Wen Sheng revised the manuscript. All authors have read and approved the final manuscript.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Data availability statement

All datasets generated for this study are included in the article.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

All datasets generated for this study are included in the article.

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