Morinda officinalis polysaccharides activate the SIRT1/PGC-1α pathway to reduce oxidative damage in Leydig TM3 cells

Abstract

Oxidative damage can induce serious spermatogenic disorders, cause testicular tissue pathological damage, decrease testicular index and sperm count, and increase sperm deformity rate, thus causing male infertility. The present study aimed to investigate the effects of Morinda officinalis polysaccharide (MOP) upon the function of testicular Leydig cells and the underlying mechanisms, to provide novel insights into clinically treating male infertility. Leydig TM3 cells were treated with H₂O₂ to induce oxidative damage. Cell proliferation was detected using CCK-8 assay and EdU staining. The protein level of testicular interstitial cell biomarkers and mitochondrial signaling pathway-associated proteins was detected using Western blot. SA-β-gal staining was used to detect cell senescence. The levels of intracellular reactive oxygen species (ROS) were detected using flow cytometry. Additionally, SOD, CAT, GSH-Px, MDA, and ATP levels were detected with biochemical methods. Mitochondrial damage was traced using JC-1 and Mito-tracker dyes. For in-vivo validation, the Leydig cell elimination model was established in rats by an intraperitoneal injection of Ethane dimethane sulfonate (EDS). MOP effects on rat reproductive function (such as testicular pathological changes and sperm morphology, activity, and quantity) and Leydig cell function (such as levels of serum testosterone and biomarkers) were evaluated. MOP promoted testicular interstitial cell proliferation and testosterone secretion, attenuated testicular pathological damage, and improved sperm morphology, activity, and quantity. For mechanism, MOP can reduce ROS-induced oxidative damage to cells and mitochondria. Furthermore, MOP can activate the SIRT1/PGC-1α pathway and the level of mitochondrial-related functional biomarkers. SIRT1 inhibitor EX-527 could reverse the protective effect of MOP on Leydig cells. MOP can alleviate ROS damage to testicular Leydig cells, promote testosterone secretion, and improve male rat reproductive function. MOP was a potential drug for treating male infertility in the clinic.

Supplementary Information

The online version contains supplementary material available at 10.1038/s41598-026-46267-6.

Introduction

Male infertility is defined as the inability of a male to make a fertile female pregnant. According to population-based research reports, infertility occurs in about 15% of couples; among these infertile couples, approximately 40% to 50% are caused by male reproductive disorders¹. Male infertility has become a common disease. It has been shown that male semen concentration has dropped by more than 50% in Eastern (including China) and Western countries over the past 50 years². This decrease in male semen concentration may result in pregnancy failure among about 15% of couples.

Under various harmful stimulation inside and outside the body, the oxidative system and antioxidant system in the body is out of balance; in this case, reactive oxygen species (ROS) in mitochondria will accumulate in large quantities, exposing tissue cells to high concentrations of oxygen molecules or oxygen chemical derivatives, thereby leading to oxidative damage³. A certain level of ROS is essential for sperm physiological functions, such as capacitation, hyperactivation, and acrosome reactions. Nevertheless, high ROS levels could result in male infertility not only through lipid peroxidation or DNA damage but also through inactivation of enzymes and oxidation of proteins in sperm⁴. Leydig cells, as the principal cell type in the male testicular stroma, are the only cells that synthesize and secrete testosterone in male animals; testosterone is a primary hormone for spermatogenesis and maintaining male sex characteristics⁵. Mitochondrial dysfunction/damage can cause the production and accumulation of ROS in Leydig cells, thereby inducing the impairment of cell viability and decreased testosterone synthesize/release. Therefore, it has become a hot spot and difficulty to improve Leydig cell functions via regulating oxidative stress for overcoming male infertility.

Morinda officinalis is a perennial vine which belongs to the genius Morinda of the family Rubiaceae; its fleshy roots can be utilized as medicine, with a warm nature, a pungent and sweet taste, showing the effects of kidney tonifying and Yang strengthening, wind-dampness dispelling, and bone and muscle strengthening; clinically, MOP, known as the “essential medicine for tonifying kidney Yang”, is mainly used to treat sexual impotence, frequent micturition, lower abdominal pain, irregular menstruation, and cold-dampness pain^6,7. Carbohydrates are a group of biological macromolecules in living organisms and can provide structural support to many organisms; carbohydrates intrinsically possess various biological activities with almost non-toxic side effects and high safety, rendering their high exploration value. Morinda officinalis contains abundant carbohydrates that show various biological activities including antioxidant, anti-tumor, antidepressant, anti-osteoporosis, hypoglycemic, immune regulation, and promoting angiogenesis activities⁸. Morinda officinalis oligosaccharides have been reported to activate mitophagy to resist mitochondrial damage in astrocytes, thereby alleviating the depressive-like behaviors of hypertensive rats⁹. Recently, Morinda officinalis polysaccharides (MOP) have been evidenced to exert anti-inflammatory and antioxidant effects on various diseases, such as cerebrovascular diseases¹⁰ and osteoporosis¹¹.

Herein, the effects of MOP upon rat reproductive function and mitochondrial damage in Leydig cells were investigated using the in-vivo and in-vitro models. MOP has been shown to inhibit ROS production and alleviate rat reproductive function damage and mitochondrial damage in Leydig cells, as well as promote testosterone secretion by Leydig cells and cell viability and proliferation. We also demonstrated that MOP may exert protective effects on Leydig cells via activating the sirtuin (SIRT1)/peroxisome proliferator-activated receptor-γ coactivator α (PGC-1α) pathway and upregulating mitochondrial-related functional biomarkers. MOP was a promising drug for the clinical treatment of male infertility.

Materials and methods

Establishment of rat Leydig cell elimination model

Healthy male SPF grade 8-week SD rats (weighing 300–320 g) were purchased from the SLAC Experimental Animal Co., Ltd. All experiments involving animals were performed under the approval of the Third People’s Hospital of Haikou Animal Ethics Committee (No. SC20210045). All animal experiments were conducted in accordance with the relevant designated guidelines and regulations and in compliance with the ARRIVE Guidelines. Before the experiment, all rats were fed adaptively for one week. The rats were allocated into 3 groups (six rats for each group): Control, EDS, EDS + MOP. Next, rats in the ethane dimethane sulfonate (EDS) group were intraperitoneally injected with 75 mg/kg EDS alone (single dose) to remove Leydig cells¹². After 14-day EDS administration, EDS + MOP rats were subjected to 28-day treatment with MOP (100 mg/kg/day; Shanghai Winherb Co. Ltd, Shanghai, China) by gastric lavage^13,14. The control rats were administrated with an equal amount of saline. Finally, rats were euthanized (an intraperitoneal injection of 200 mg/kg sodium pentobarbital, and death was confirmed by a secondary physical method cervical dislocation), and their peripheral blood, testicles, and epididymis tissues were collected for subsequent analyses.

Cell culture and treatment

Leydig cell line TM3 cells (procured from ATCC, Manassas, Virginia, USA) were cultured (37 °C, 5% CO₂) in a complete culture medium (a mixture of Ham’s F12 media and DMEM in 1:1 ratio with 2.5 mM L-glutamine, 0.5 mM sodium pyruvate, 1.2 g/L sodium bicarbonate, 15 mM HEPES, 5% horse serum, 2.5% fetal bovine serum, 1 × 10⁵ U/L penicillin, and 100 mg/L streptomycin) with saturation humidity. Two or three days later, the media was refreshed, and cells were passaged at a 1:2 ratio. For oxidative stress, TM3 cells were subjected to 48-h treatment with 100 µM H₂O₂^15,16. For MOP treatment, TM3 cells were subjected to 48-h treatment with MOP of various concentrations (50 mg/L, 100 mg/L, and 250 mg/L^17,18. To verify the involvement of the SIRT1 pathway, cells were co-treated with H₂O₂ (100 µM), MOP (100 mg/L), and the SIRT1 specific inhibitor EX-527 (10 µM, MedChemExpress)¹⁹ for 48 h.

Cell counting kit-8 (CCK-8) assay

After being prepared into a single-cell suspension and counting, TM3 cells were seeded (5000 cells/well) onto 96-well plates for an overnight culture, followed by 48-h incubation with various concentrations of MOP. CCK-8 (Beyotime Biotechnology Co. Ltd., Shanghai, China) was employed as per the protocol of the manufacturer to measure cell viability. In short, 10 µL CCK-8 solution was supplemented to each well, prior to 2-h further incubation in the incubator. Finally, a microplate reader (Bio-Rad, Inc., Hercules, CA, USA) was employed to measure the optical density (OD) value at 450 nm.

5-ethynyl-2’-deoxyuridine (EDU) labeling assay

Cell DNA synthesis was detected using the EdU Apollo567 in Vitro Kit (CA1170, Solarbio). The solution was diluted with the culture media (dilution ratio: 1:1000), which was then added to 24-well plates, followed by cell incubation for 2 h. Cells were labeled with EdU, and then the culture media was discarded. Cells were added and fixed (room temperature, 15 min) in 1 mL of 4% paraformaldehyde. After 3 washes with 1 mL phosphate-buffered saline (PBS) (each time for 3–5 min), 50 µL 2 mg/mL glycine solution was added to each well for 5-min incubation. Next, cells were incubated for 15 min at room temperature (RT) with 0.3% Triton X-100. Subsequently, 100 µL 1×Apollo staining reaction solution was added, followed by 30-min incubation at RT away from light. The cell nucleus was stained with 1000 × Hoechst 33,342 (diluted with PBS at 1:1000 ratio; #62249, Thermo Fisher Scientific Inc., Waltham, MA, USA). In short, 0.5 mL staining solution was added to each well, followed by 10-min incubation at RT away from light. Cells were rinsed thrice in the washing solution (each time for 3–5 min), and then a fluorescence microscope (Olympus, CKX53, Japan) was employed to observe cells; the proliferating cells showed red fluorescence and the nuclei showed blue fluorescence.

Senescence-associated-β-galactosidase (SA-β-gal) staining

The treated TM3 cells were stained with a β-gal staining kit (K146501, ThermoFisher). Firstly, after 1 washing in PBS, an appropriate amount of staining fixative was added to cells in the well for fixing (room temperature, 10 min). Following 3 washes with PBS, 3 min for each, cells were added with an appropriate amount of staining solution for staining (room temperature, 0.5–2 h). Finally, the staining solution was removed and an inverted microscope (Nexcope, NIB610) was applied to observe and photograph cells.

Biochemical detection

The levels of superoxide dismutase (SOD; S0101S, Beyotime), catalase (CAT; S0051, Beyotime), glutathione peroxidase (GSH-Px; S0057S, Beyotime), malondialdehyde (MDA; S0131S, Beyotime), and adenosine triphosphate (ATP; A095-1-1, NanJing JianCheng Bioengineering Institute, Jiangsu, China) in various groups of TM3 cells, as well as the levels of testosterone (CSB-E05100r, Wuhan CUSABIO Biotech Co., Ltd, Wuhan, Hubei, China), luteinizing hormone (LH; H206-1-1, JianCheng), and follicle-stimulating hormone (FSH; CSB-E06869r, CUSABIO) in rat serum were detected using the corresponding kits. The operation was performed strictly following the corresponding instructions.

Flow cytometry detecting intracellular ROS

ROS level in TM3 cells was detected using the ROS detection kit (S0033S, Beyotime) strictly as per the instructions. Dichlorofluorescein diacetate (DCFH-DA) was diluted in a serum-free medium (1:1000) to a final concentration of 10 µM. TM3 cells (1 × 10⁷ cells/mL) were harvested and suspended within the diluted DCFH-DA, followed by incubation (37 °C, 20 min) in an incubator, shaking and mixing every 3–5 min for thorough contact with the probe. After that, cells were rinsed thrice in serum-free media to completely eliminate free DCFH-DA. After staining for 20–30 min, flow cytometry was used for detection.

Western blot

The total protein of TM3 cells and rat testicular tissues were extracted with RIPA lysis buffer (Beyotime). A bicinchoninic acid (BCA) reagent kit (Beyotime) was applied to determine the protein content. Next, the corresponding protein volume was supplemented to the loading buffer (Beyotime) and mixed, and then the protein denaturation was carried out by 5-min heating in a boiling water bath. Following electrophoresis by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), the separated proteins (50 µg) were electroblotted from the gel onto polyvinylidene fluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). Subsequently, after 1–2-min washing with the washing solution, membranes were blocked for 60 min at RT in a 5% skim milk-contained sealing solution, followed by an overnight incubation at 4 °C on a shaking table using primary antibodies: Lhcgr (19968-1-AP, 1:1000, Proteintech, Wuhan, China), Insl3 ( PA5-76807, 1:1000, ThermoFisher), Cyp11a1 (ab272494, 1:1000, Abcam), Hsd3b1 (PA5-96905, 1:1000, ThermoFisher), Hsd11b1 (10928-1-AP, 1:1000, Proteintech), PGC-1α (ab188102, 1:1000, Abcam), SIRT1 [#8469, 1:1000, Cell Signaling Technology (CST), Beverly, MA, USA], optic atrophy (OPA1; ab157457, 1:2000, Abcam), mitochondrial transcriptional factor A (TFAM; ab252432, 1:1000, Abcam), B-cell lymphoma 2 (BCL2; ab182858, 1:2000, Abcam), steroidogenic acute regulatory protein (StAR; ab203193, 1:1000, Abcam) and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 5174 S, 1:1000, CST). After 3 washes in washing solution the next day, 10 min for each, the membranes underwent incubation (room temperature,1 h) with horseradish peroxidase (HRP)-conjugated secondary antibody (goat anti-rabbit or anti-mouse IgG (1:2000, Proteintech ). Finally, the membranes were developed and visualized using an enhanced chemiluminescence (ECL) reagent (Beyotime) and detected with a chemiluminescence imaging system (Bio-Rad).

Detection of mitochondrial membrane potential

A JC-1 detection kit (M34152, ThermoFisher) was employed to detect mitochondrial membrane potential. TM3 cells entering the logarithmic growth phase were planted onto six-well plates at a density of 5 × 10⁵ cells per well. Upon 80% confluence or more, cells were subjected to treatment with H₂O₂ or MOP. Next, after one washing in pre-cooled PBS, cells were added with 1 mL complete culture media and 1 mL JC-1 (10 µM) solution for 20-min staining at 37 °C in the dark. After staining, cells were rinsed twice in pre-cooled 1×JC-1 buffer to remove the staining solution that failed to aggregate in the matrix of mitochondria. Next, cells were added with 2 mL PBS, and then flow cytometry was used for mitochondrial membrane potential detection.

Mito-tracker probes for tracing mitochondria in living cells

Firstly, the Mito-Tracker Red CMXRos (C1035, Beyotime) stock solution was diluted in DMEM medium at a 1:1000 ratio to prepare a 200 nM working solution. Next, cells in each well were added and incubated (37 °C, 30 min) with 1 mL working solution, which was subsequently replaced with 1 mL fresh culture media. The nucleus was stained with DAPI. Lastly, a fluorescence microscope was applied to observe and photograph cells. The mitochondria showed red fluorescence.

Rat sperm count and motility detection

Sperm collected from the right cauda epididymis were minced in 2 mL of pre-heated PBS (32 °C, pH 7.2) to mimic physiological scrotal temperature and preserve motility^13,20. The resulting suspension was filtered through an 80 μm nylon mesh. An aliquot (0.05 mL) of the filtrate was diluted 1:40 with PBS and loaded into a hemocytometer. The sperm count was determined by assessing 8 squares (1 mm² each) and calculating the total number of sperm per epididymis. Sperm motility was recorded and analyzed immediately using a microscope video system and the percentage of progressively motile sperm was calculated by ImageJ CASA plugin (NIH, USA). All assessments were conducted independently by two observers.

Hematoxylin and eosin (HE) staining

Rat testicular tissue samples were fixed in 4% paraformaldehyde, paraffin-embedded, and then sectioned (4 μm). After that, the sections were cleared in xylene, rehydrated using ethanol of gradient concentration, and washed with PBS, followed by 10-min staining with hematoxylin. Slices were washed with running tap water, and then subjected to 5-s immersion in acid alcohol (hydrochloric acid in alcohol). Following tap water washing, the sections were immersed in dilute ammonia water for 5 min. After tap water washing, slices were subjected to 10-min staining with eosin, dehydrated using gradient ethanol, cleaned with xylene, and then sealed using neutral resin. Finally, a microscope (Olympus) was employed to observe slices.

Immunohistochemical (IHC) staining

The sections of rat testicular tissues were dewaxed using xylene and rehydrated using gradient alcohol. After that, the sections were subjected to antigen retrieval and blocking of endogenous peroxidase activity and non-specific binding sites, followed by an overnight incubation with the primary antibodies Cyp11a1 (ab272494, 1:5000, Abcam) and Hsd11b1 (10928-1-AP, 1:200, Proteintech). Next, the IHC universal type 2-step method detection kit (PV-9000, ZSGB-Bio Co., Ltd., Beijing, China) and DAB chromogenic solution were used for the subsequent steps. Hematoxylin served as a counterstain. Slices were observed under a light microscope (Olympus, Tokyo, Japan) and images were captured. The Leydig cell number/field was recorded.

Determination of the Johnsen score

To evaluate the quality of spermatogenesis, the Johnsen score was calculated for each animal. At least 50 seminiferous tubules per rat were randomly examined under a light microscope. Each tubule was scored on a scale from 1 to 10 based on the organization of the germinal epithelium and the presence of germ cells, where a score of 10 indicates complete spermatogenesis with many spermatozoa and a score of 1 indicates no cells within the seminiferous tubules²¹. The mean score was calculated for each group.

Statistical analysis

All data were represented in terms of mean ± SD. GraphPad Prism 8.0 software was applied to perform one-way analysis of variance (ANOVA)followed by a Tukey’s multiple comparisons. The threshold for statistical significance was p < 0.05.

Results

MOP promoted Leydig TM3 cell proliferation

Firstly, the MOP effect on the proliferation and function of Leydig TM3 cells was detected at the cellular level. As indicated by the results, compared with the control group, MOP of low concentrations (50 mg/L) showed no significant influence on TM3 cell proliferation, the level of interstitial cell markers (Lhcgr, Insl3, Star, Cyp11a1, Hsd3b1, and Hsd11b1), and testosterone production (Fig. 1A–D). However, MOP of higher concentrations (100 and 250 mg/L) remarkably promoted Leydig TM3 cell proliferation (Fig. 1A, B), and upregulated the level of interstitial cell markers (Fig. 1C). MOP (100 and 250 mg/L) also notably promoted testosterone generation and release (Fig. 1D). Considering the similar effects of 100 mg/L MOP and 250 mg/L MOP on TM3 cells, MOP of 100 mg/L concentration was hence selected for subsequent experiments.

Fig. 1.

MOP promoted Leydig TM3 cell proliferation. TM3 cells were treated with various concentrations of MOP (50, 100, and 250 mg/L) for 48 h. (A) Cell viability was assessed using the CCK-8 assay. (B) Cell proliferation was evaluated using EdU staining (scale bar = 20 μm). (C) The protein levels of Leydig cell markers (Lhcgr, Insl3, Star, Cyp11a1, Hsd3b1, and Hsd11b1) were determined by Western blot. (D) Testosterone concentration in the culture medium was measured by ELISA. Data are presented as mean ± SD. ** p < 0.05, ** p < 0.01, vs. Control group.

MOP inhibited oxidative damage in Leydig TM3 cells

Next, TM3 cells were induced with H₂O₂ for oxidative damage. The effect of MOP on the oxidative damage of Leydig TM3 cells was evaluated. According to the results, after H₂O₂ (100 µM) treatment, compared to the control group, Leydig TM3 cell senescence was significantly promoted (Fig. 2A); and the intracellular ROS level was notably upregulated (Fig. 2B); meanwhile, the intracellular SOD and CAT activities (Fig. 2C, D) and GSH-Px level were downregulated (Fig. 2E) and the intracellular MDA level was elevated (Fig. 2F). However, MOP treatment can effectively reverse the aforementioned results induced by H₂O₂ damage on cells. These findings indicated that MOP can inhibit oxidative damage in Leydig TM3 cells.

Fig. 2.

MOP inhibited H₂O₂-caused oxidative damage within Leydig TM3 cells. After Leydig TM3 cells were subjected to 48-h co-treatment with 100 µM H₂O₂ and MOP, (A) Leydig TM3 cell senescence was detected using SA-β-gal staining; (B) The ROS levels in Leydig TM3 cells were detected using flow cytometry; (C-F) SOD, CAT, GSH-Px, and MDA levels within Leydig TM3 cells were detected using kits. **p < 0.01, vs. Normal control; ##p < 0.01, vs. the H₂O₂ group.

MOP inhibited mitochondrial damage in Leydig TM3 cells

Mitochondria have been revealed to play a critical mediating role in cellular metabolism, which also act as ROS producers and targets²². Hence, the effects of MOP upon H₂O₂-triggered mitochondrial damage in Leydig TM3 cells were further investigated. JC-1 dye was employed to detect the mitochondrial membrane potential of Leydig TM3 cells. As revealed by the results, H₂O₂ treatment remarkably increased mitochondrial membrane permeability, thereby downregulating mitochondrial membrane potential (Fig. 3A). Moreover, after H₂O₂ treatment, Leydig TM3 cells showed significantly decreased mitochondrial ATP production (Fig. 3B). Additionally, the active mitochondria in Leydig TM3 cells were traced using Mito-tracker probes; it was found that H₂O₂ treatment noticeably decreased the number of mitochondria in the cells (Fig. 3C). Nevertheless, MOP treatment effectively attenuated H₂O₂-induced mitochondrial damage (Fig. 3A–C).

Fig. 3.

MOP inhibited mitochondrial damage in Leydig TM3 cells. (A) Alterations in mitochondrial membrane potential of Leydig TM3 cells were detected using JC-1 dye and flow cytometry; (B) ATP detection kit was used to determine the ATP content in Leydig TM3 cells; (C) Mito-tracker probes (red) were used to stain active mitochondria in Leydig TM3 cells, and a fluorescence inverted microscope was applied to observe the fluorescence intensity; the nucleus was labeled with DAPI (blue). **p < 0.01, vs. Normal control; #p < 0.05, ##p < 0.01, vs. the H₂O₂ group.

MOP activated the SIRT1/PGC-1α pathway and upregulated mitochondrial-related functional biomarkers

A previous study has evidenced that the SIRT1/PGC-1α signaling exerts a crucial effect on mitochondrial protection²³. Therefore, the role of MOP in the SIRT1/PGC-1α pathway was detected. Firstly, Leydig TM3 cells were treated with 100 mg/L MOP and then detected for the SIRT1/PGC-1α pathway-related protein levels using Western blot. The result showed that MOP notably upregulate SIRT1 and PGC-1α levels (Fig. 4A). Moreover, it was worth noting that MOP can also significantly activate the SIRT1/PGC-1α signaling within H₂O₂-treated cells, as evidenced by the elevated protein contents of SIRT1 and PGC-1α (Fig. 4B). Meanwhile, MOP effects on the level of mitochondrial inner membrane-associated proteins (OPA1 and TFAM) were also evaluated. The results revealed that MOP could upregulate OPA1 and TFAM protein contents within testicular stromal cells in the presence or absence of H₂O₂ treatment, (Fig. 4A, B). Additionally, MOP also dramatically upregulated the protein level of the pro-survival factor BCL2 (Fig. 4A-B). From all the above results, MOP may exert protective effects on mitochondria via activating the SIRT1/PGC-1α signaling and upregulating the mitochondrial-related functional biomarkers.

Fig. 4.

MOP activated the SIRT1/PGC-1α signaling and upregulated mitochondrial-related functional biomarkers. (A-B) Leydig TM3 cells (with or without H₂O₂ treatment for 48 h) were treated with 100 mg/L MOP and then detected for the protein levels of SIRT1, PGC-1α, OPA1, TFAM, and BCL2 using Western blot. *p < 0.05, **p < 0.01, vs. Normal control; ##p < 0.01, vs. the H₂O₂ group.

Inhibition of SIRT1 partially abolished the protective effects of MOP on Leydig TM3 cells

To confirm that the protective effects of MOP against oxidative damage are mediated by the SIRT1 signaling pathway, we utilized the specific SIRT1 inhibitor EX-527 (10 µM). As shown in Fig. 5, compared to the H₂O₂+MOP group, the addition of EX-527 significantly reversed the beneficial effects of MOP. Specifically, EX-527 treatment increased the cellular senescence rate (Fig. 5A) and intracellular ROS levels (Fig. 5B) that had been reduced by MOP. Furthermore, the improvements in antioxidant enzyme activities (SOD, CAT, and GSH-Px) induced by MOP were notably inhibited by EX-527, while the MDA content was re-elevated (Fig. 5C–F). Regarding mitochondrial function, EX-527 abolished the MOP-induced restoration of mitochondrial membrane potential (Fig. 5G) and ATP production (Fig. 5H). Similarly, the fluorescence intensity of active mitochondria traced by Mito-Tracker Red was significantly decreased in the EX-527 co-treatment group compared to the MOP group (Fig. 5I). These results strongly demonstrate that SIRT1 activation is required for MOP to exert its antioxidant and mitochondrial protective effects in Leydig TM3 cells.

Fig. 5.

Inhibition of SIRT1 reversed the protective effects of MOP against oxidative stress. Leydig TM3 cells were treated with H₂O₂ (100 µM) alone, or co-treated with MOP (100 mg/L) and the SIRT1 inhibitor EX-527 (10 µM) for 48 h. (A) Cell senescence was evaluated by SA-β-gal staining. (B) Intracellular ROS levels were measured by flow cytometry. (C-F) The levels of SOD, CAT, GSH-Px, and MDA were detected using biochemical kits. (G) Mitochondrial membrane potential was analyzed using JC-1 staining and flow cytometry. (H) ATP content was measured using a biochemical kit. (I) Active mitochondria were visualized using Mito-Tracker Red staining. Data are presented as mean ± SD. ** p < 0.01 vs. H2O2 group; ## p < 0.01 vs. H2O2 + MOP group.

MOP improved the sperm quality of male rats and their potential reproductive ability

Furthermore, the effect of MOP upon the potential reproductive ability of rats was investigated using a rat Leydig cell elimination model. As shown in Fig. 6A-C, compared to the control group, EDS-injected rats exhibited significantly reduced testis weight, sperm count, and sperm motility. EDS injection also significantly decreased serum testosterone levels (Fig. 6D). However, MOP treatment markedly reversed these EDS-induced impairments (Fig. 6A–D). Moreover, MOP did not exert significant effects on serum LH and FSH levels (Fig. 6D).

Fig. 6.

MOP improved the sperm quality of male rats and their potential reproductive ability. Male SD rats were intraperitoneally injected with EDS (75 mg/kg) and kept for 14 days, and then treated with MOP (100 mg/kg/day) via gastric gavage for another 28 days. (A) The testis weight of rats was recorded; (B-C) the sperm count and sperm activity of rats were observed under a microscope; (D) the level of testosterone, LH, and FSH within rat serum was evaluated using kits. **p < 0.01, vs. Normal control; #p < 0.05, ##p < 0.01, vs. the EDS rats.

MOP improved Leydig cell activity of male rats

Subsequently, the pathological changes in rat testicular tissues and the level of interstitial cell markers were further investigated. As evidenced by HE staining results, in the testicular tissues of control rats, seminiferous tubules were abundant and tightly arranged; the epithelium of the tubules is lined with a layer of seminiferous epithelium composed of spermatogenic cells and supporting cells, with normal and tightly arranged cell morphology; there were long spindle-shaped sperm in the lumen and numerous Leydig cells within the seminiferous tubules, with normal numbers and morphology; no significant abnormality was found. However, after EDS treatment, rat testicular tissues showed expanded seminiferous tubules, enlarged lumen, thinning tube walls, and loose arrangement; some epithelial cells were detached in lumen; some seminiferous tubules atrophied; the seminiferous cells swelled, with a loose cytoplasm; some epithelial cells necrotized and shed, and the nucleus was condensed, deeply stained or fractured (Fig. 7A). To quantify these histological changes, the Johnsen score and Leydig cell numbers were assessed. The results showed that EDS treatment significantly decreased both the Johnsen score and the number of Leydig cells, whereas MOP treatment remarkably restored these parameters (Fig. 7B). Moreover, the protein contents of Leydig cell markers (Lhcgr, Insl3, Star, Cyp11a1, Hsd3b1, and Hsd11b1) in rat testicular tissues were detected using IHC and Western blot. It was found that EDS treatment significantly downregulated the protein level of these markers (Fig. 7C–E). However, MOP treatment effectively improved the pathological damage in testicular tissues and upregulated the protein levels of Leydig cell markers (Fig. 7A-E). Taken together, MOP could improve male rats’ reproductive capacity.

Fig. 7.

MOP improved Leydig cell activity of male rats. (A) HE staining was used to detect the pathological damage in rat testicular tissues; (B) The Johnsen score and Leydig cell number per field were quantified to evaluate spermatogenic function and interstitial cell recovery; (C) Cyp11a1 and Hsd11b1 protein levels in rat testicular tissues were detected using IHC staining; (D) The relative optical intensity of the IHC staining was quantified; (E) Lhcgr, Insl3, Star, Cyp11a1, Hsd3b1, and Hsd11b1 protein contents in rat testicular tissues were detected using Western blot. **p < 0.01, vs. Normal control; ##p < 0.01, vs. the EDS rats.

Discussion

The reduction in Leydig cell number and function is responsible for the pathogenesis of hypogonadism and subsequent diseases²⁴. Adult Leydig cells are essential for spermatogenesis initiation and maintenance, and for promoting male secondary characteristics. Leydig cells migrate into the testes to secrete testosterone. However, Leydig cells cannot proliferate in the body, and can only derive from testicular mesenchymal stem cells by rapid expansion through mitosis and subsequent differentiation²⁵. Therefore, it was of great significance to improve the activity and survival ability of Leydig cells for treating hypogonadism and subsequent diseases in the clinic. Herein, compared to MOP of a low concentration (50 mg/L), MOP of a high concentration (100 mg/L) has been found to noticeably promote Leydig cell functions both in vitro and in vivo. In short, MOP (100 mg/L) notably promoted Leydig cell proliferation and testosterone secretion, as well as improved sperm morphology, count, and activity. These results indicated the enormous potential of MOP in improving hypogonadism.

Accumulating studies have provided evidence that mammalian seminal plasma contains various enzymatic antioxidants such as SOD, GSH-Px, glutathione reductase (GSR), and CAT and non-enzymatic antioxidants (such as uric acid, pyruvate, and ascorbic acid); these antioxidants are crucial for improving male reproductive function^26–28. In pathological conditions, excessive ROS accumulation within cells can damage the mitochondrial membrane, causing the abnormal opening of the mitochondrial permeability transition pore (MPTP) and release of apoptosis regulatory factors on the membrane, thereby inducing cell apoptosis^29,30. A previous study has evidenced that the extract of Morinda officinalis exhibits cytoprotective effects upon H₂O₂-caused oxidative damage within mouse testicular Leydig TM3 cells³¹. Similarly, herein, MOP has been proven to markedly decrease intracellular ROS levels and alleviate H₂O₂-induced damage to mitochondria; meanwhile, MOP significantly increased the levels of antioxidants (such as SOD, GSH-Px, and CAT) in Leydig TM3 cells. Taken together, MOP can inhibit ROS production to protect mitochondria.

Interestingly, our results showed that while serum testosterone levels were significantly decreased in the EDS group, the levels of upstream gonadotropins (LH and FSH) remained comparable to the control group. Classically, reduced testosterone triggers a rise in LH and FSH via negative feedback³². The observed profile in this study is likely attributable to the specific timing of our assessment (42 days post-EDS) and the regenerative nature of the model. While EDS selectively eliminates adult Leydig cells, stem Leydig cells remain intact and initiate regeneration³³. Consequently, the testosterone levels observed at day 42 reflect the partial functional recovery mediated by these newly regenerated cells, a finding consistent with previous reports³⁴. Furthermore, this regeneration appears sufficient to reset the hypothalamic-pituitary feedback loop. Although LH surges acutely immediately after Leydig cell destruction³⁵, it tends to normalize as steroidogenesis resumes. Thus, at day 42, the axis has stabilized even if total serum testosterone has not fully recovered to control baselines. Additionally, the unchanged FSH levels suggest that Sertoli cell function was relatively preserved or recovered, likely maintaining normal secretion of Inhibin B, which selectively inhibits FSH release^36,37. The underlying molecular mechanism was further investigated. SIRT1 is a nicotinamide adenosine dinucleotide (NAD)+-dependent histone deacetylase, which can deacetylate and upregulate PGC-1α that can activate mitochondrial biosynthesis³⁸. OPA1 and TFAM are critical in promoting mitochondrial fusion and maintaining mitochondrial cristae^39,40. Moreover, SIRT1 plays an important role in the development of germ cells during spermatogenesis. Reduced levels of SIRT1 are associated with high levels of DNA fragmentation in sperms⁴¹. In this study, we found that MOP notably upregulated the protein level of SIRT1, PGC-1α, OPA1, and TFAM with or without H₂O₂ treatment; these further confirmed the protective effect of MOP on mitochondria. SIRT1 inhibitor effectively reduced the protective effect of MOP on H₂O₂-treated Leydig TM3 cells. This study for the first time elucidated the molecular mechanism of MOP in protecting Leydig TM3 cells against ROS damage.

In summary, this study demonstrated that MOP protected mitochondria via upregulating mitochondrial-related proteins (SIRT1, PGC-1α, OPA1, and TFAM), thereby improving the reproductive function of male rats. This study provides a theoretical reference for MOP treating male infertility in the clinic.

Supplementary Information

Below is the link to the electronic supplementary material.

Author contributions

Minhao Fu, Shengyun Wu, Zhenting Wang conception and designed the experiments. Shengyun Wu drafted the article. Minhao Fu revised the article critically for important intellectual content. Zhenting Wang provided the fund and revised the article critically for important intellectual content. Xianlai Yin, Peng Yang and Binghao Gong contributed to experiments, also the analysis and manuscript preparation. All the authors read and approved the manuscript. Yes, all the authors read and approved the manuscript.

Funding

This work has been supported by Key Science and Technology Program Project of Haikou City (2022-031 and 2024-035).

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Data availability

The datasets used and/or analysed during the current study available from the corresponding author on reasonable request.

Competing interests

The authors declare no competing interests.

Ethics approval

All experiments involving animals were performed under the approval of the Third People’s Hospital of Haikou Animal Ethics Committee (No. SC20210045). All animal experiments were conducted in accordance with the relevant designated guidelines and regulations and in compliance with the ARRIVE Guidelines.