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

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

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. 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

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. 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. 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. 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. 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. 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. 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. 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.

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.

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.

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.

Additional information

Funding

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].