2.1 Drugs and materials

Wuzi Yanzong Pill (WZYZP, a water honey pill) was purchased from Beijing Tong Ren Tang Co., Ltd. (Beijing, China). Cyclophosphamide was purchased from Jiangsu Hengrui Medicine Co., Ltd. (Jiangsu, China). Neomycin, ampicillin, metronidazole, and vancomycin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Hyperoside, verbascoside, kaempferol, and schisandrin were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China).

2.2 Chemical components analysis and quality control of WZYZP

The chemical components of WZYZP were analyzed by UPLC-QTOF-MS. To ensure the quality of WZYZP, high-performance liquid chromatography (HPLC) was used to quantitatively analyze the contents of hyperoside, verbascoside, kaempferol, and schisandrin in WZYZP according to the 2020 edition of Chinese Pharmacopoeia. The detailed methods were provided in the Supplementary methods.

2.3 Animals and WZYZP administration

Specific pathogen-free (SPF)-grade adult male Kunming mice (8 weeks old, 32 − 38 g) were purchased from Chengdu Dossy Experimental Animal Co., Ltd. (Chengdu, China). Animals were housed under the SPF conditions with a temperature of 22 ± 2 °C, a relative humidity of 60 ± 5 %, and a standard 12 h/12 h light/dark cycle. All animal experiments were approved by the Ethics Committee of Chengdu University of Traditional Chinese Medicine (CDUTCM-2022116) and it was carried out following the National Institutes of Health Guidelines for Care and Use of Laboratory Animals.

After 1 week of acclimatization, mice were randomized into five groups (n = 6): the control group, model group, WZYZP-L group, WZYZP-M group, and WZYZP-H group. Except for the control group, the other groups of mice were injected with cyclophosphamide at a dose of 60 mg/kg/d for 5 days to induce spermatogenic dysfunction. In detail, cyclophosphamide was dissolved in 0.9 % NaCl and injected intraperitoneally into the mice (60 mg cyclophosphamide/kg body weight), once a day for 5 days (Chi et al., 2022). The mice in the control group were injected intraperitoneally with 0.9 % NaCl (0.01 ml/g/d) for 5 days in the same manner. Then, in the WZYZP-L, WZYZP-M, and WZYZP-H groups, concerning previous studies, mice were treated with WZYZP at doses of 0.35 g/kg/d, 0.70 g/kg/d, and 1.40 g/kg/d by gavage for 14 days, respectively (Chen et al., 2021). The mice in the control and model groups were given sterile water in the same manner for 14 days. Before the end of the experiment, feces were collected, snap-frozen in liquid nitrogen, transferred, and stored at − 80 °C for the analysis of gut microbiota and fecal metabolomics. At the end of the trial, all mice were fasted overnight (12 h) and anesthetized by intraperitoneal injection of 2 % pentobarbital (50 mg/kg). The plasma was obtained by centrifugation at 3,000 revolutions per minute (rpm) at 4 °C for 15 min and stored at − 80 °C for the determination of testosterone, pyruvate, lactate, and adenosine triphosphate, as well as the analysis of tryptophan metabolism. The testis and epididymis were immediately excised, and the testis was weighed for counting testicular index (i.e., total testicular weight/body weight). The left testis further was photographed. The left epididymis was cut into pieces for sperm count and motility measurement. The right epididymis and left testis used for histopathological analysis were fixed in 4 % paraformaldehyde. The remaining testis were frozen at − 80 °C for further biochemical analysis, including assay kits and Western blotting.

2.4 Sperm count and motility measurement

The cauda epididymis was minced in 500 μL PBS (pH 7.2) and incubated at 37 °C for 5 min to release the sperm. Sperm motility and sperm count were analyzed using a Digital Color Sperm Quality Detection System (WLJY-9000, Beijing, China).

2.5 Histological examination

The testis and epididymis were fixed in 4 % paraformaldehyde for 24 h, dehydrated in graded ethanol, embedded in paraffin, and sectioned at 4 μm in thicknesses. The sections of the testis and epididymis were stained with hematoxylin-eosin (H&E) and observed under a pathological section scanner (NanoZoomer S60, Hamamatsu, Japan).

2.6 Plasma and testicular testosterone assay

The concentrations of testosterone in the plasma and the testis for each mouse were assayed using commercially available enzyme immunoassay kits specific for mice (CSB-E05101m, Cusabio, Wuhan, China).

2.7 Determination of pyruvate, lactate, and adenosine triphosphate (ATP)

The levels of testicular pyruvate, lactate, and ATP were detected using the commercially available kit (Nanjing Jiancheng Bioengineering Institute, Nanjing, China) according to the manufacturer’s protocol.

2.8 Gut microbiota analysis

Total bacterial DNA was extracted from feces using an E.Z.N.A.® soil kit (Omega Bio-Tek, Norcross, GA, U.S.) according to the manufacturer’s protocol. DNA was amplified with Gene Amp 9700 PCR system (Applied Biosystems, Singapore) using barcoded universal bacterial primers targeting variable region V3-V4 of 16SrRNA gene with 338F-806R barcoded primers: 338 Forward Primer (5′-ACTCCTACGGGAGGCAGCAG-3′), 806 Reverse Primer (5′-GGACTACHVGGGTWTCTAAT-3′). The amplification system includes 10 ng of template DNA, 4 μL FastPfu buffer, 2 μL dNTPs (2.5 nM), 0.8 μL forward primer (5 μM), and 0.8 μL reverse primer (5 μM). The PCR conditions were as follows: initial denaturation at 95 °C for 3 min, followed by 27 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 30 s, and extension at 72 °C for 30 s, with a final extension at 72 °C for 10 min. Each PCR was performed in triplicate, then combined, and quality checked on an agarose gel. Each PCR amplification was then quantified using QuantiFluor™-ST (Promega, USA), and each sample was pooled into an equal amount of 200 ng to form a library. Sequencing was performed on an Illumina MiSeq PE300 platform (Illumina, San Diego, USA) in Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China).

2.9 Antibiotics (Abx) treatment

After a week of adaptation, 12 mice were randomly divided into the WZYZP-H group (n = 6) and WZYZP-H with antibiotics (WZYZP-H-Abx) group (n = 6). All 12 mice were injected with cyclophosphamide at a dose of 60 mg/kg/d for 5 days to induce spermatogenic dysfunction (Chi et al., 2022). Then, mice in the WZYZP-H and WZYZP-H-Abx groups were treated with WZYZP at doses of 0.70 g/kg/d by gavage for 14 days. Meanwhile, mice in the WZYZP-H group and the WZYZP-H-Abx group were given sterile water and water with Abx for 14 days, respectively. The antibiotics cocktail was composed of 0.5 mg/mL of neomycin, 0.5 mg/mL of ampicillin, 0.5 mg/mL of metronidazole, and 0.125 mg/mL of vancomycin provided in the drinking water changed twice per week (Zhao et al., 2023).

2.10 Untargeted metabolomics analysis of feces by UPLC-QTOF-MS

Metabolomics profiling of fecal samples of mice was performed at Shanghai Majorbio Bio-pharm Technology Co., Ltd. (Shanghai, China). Briefly, 50 mg of feces were homogenized with 100 μL water for 3 min. Then, an aliquot of 400 μL of methanol-containing internals standard was mixed with feces to extract the metabolites. The homogenized sample was centrifuged at 13,000 g and 4 °C for 15 min. The supernatant was transferred to a 200 μL vial for UPLC-Q-TOF/MS analysis. The chromatographic separations were carried on an ACQUITY UPLC® HSS T3 column (1.8 μm, 100 mm × 2.1 mm). The mobile phase was composed of solvent A (acetonitrile/water, 5:95, v/v, containing 0.1 % formic acid) and solvent B (isopropanol/acetonitrile/water, 47.5:47.5:5, v/v/v, containing 0.1 % formic acid). The gradient elution condition was as follows: 100 % A (0 − 0.5 min), 100 %−75 % A (0.5 − 2.5 min), 75 %−0% A (2.5 − 9 min), 0 % A (9 − 13 min), 100 % A (13 − 13.1 min), 100 % A (13.1 − 16 min). The column temperature and flow rate were set at 40 °C and 400 μL/min, respectively. The sample injection volume was 10 μL.

The ESI source conditions were set as follows: the positive and negative ESI spray voltages at 5.0 kV and 4.0 kV, respectively; and ion source temperature at 550 °C. Both the sheath gas and the auxiliary gas were nitrogen. The sheath gas pressure was 30 psi, and the auxiliary gas pressure was 50 psi. The parameters of the full mass scan were as follows: a resolution of 70,000, a maximum isolation time of 50 ms, a normalized collision energy of 30 v, and an m/z range of 50 − 1,000. Data were collected and processed with Progenesis QI (Waters Corp., Milford, USA).

2.11 Targeted metabolomics analysis of plasma by UHPLC-QTRAP-MS

The mouse blood samples were centrifuged at 3,000 rpm and 4 °C for 15 min, and the supernatants were collected. Targeted Trp metabolomics in plasma were determined by LC-ESI-MS/MS (UHPLC-QTRAP). Briefly, 80 μL plasma was mixed with 360 μL methanol (containing the internal standards), vortexed, and ultrasound at low temperature for 30 min (5 °C, 40 kHz). After centrifugation at 13,500 rpm and 4 °C for 15 min, the supernatant was freeze-dried in a freeze dryer. Re-dissolved plasma samples (1 % of acetonitrile, containing 0.1 % formic acid) were further vortexed and centrifuged. The supernatant was then injected into the UPLC-MS/MS system. Samples were injected into an ACQUITY UPLC® HSS T3 column (1.8 μm, 150 mm × 2.1 mm) at a flow rate of 0.35 mL/min. The column temperature was set at 40 °C. The sample injection volume was 2 μL. The mobile phase was composed of solvent A (water, containing 0.1 % formic acid) and solvent B (acetonitrile, containing 0.1 % formic acid). The gradient elution condition was as follows: 99 %−89 % A (0 − 2.5 min), 89 % A (2.5 − 5.5 min), 89 %−72 % A (5.5 − 6.5 min), 72 % A (6.5 − 7.5 min), 72 %−50 % A (7.5 − 12.5 min), 50 %−5% A (12.5 − 13.5 min), 5 %A (13.5 − 15.5 min), 5 %−99 % A (15.5 − 15.6 min), 99 % A (15.6 − 18 min). QTRAP 6500 + mass spectrometer was operated in positive and negative polarity mode with a positive ion spray voltage of 5,500 V and a negative ion spray voltage of 4,000 V.

2.12 Quantitative real-time PCR analysis

Total RNA was extracted from the testis using a total RNA isolation kit (RNAqueous®, Invitrogen). Reverse transcription cDNA synthesis and quantitative real-time polymerase chain reaction (qRT-PCR) analysis were performed as described. The relative expression of mRNA was detected by the 2^−ΔΔCt method (Wu et al., 2023). The forward and reverse primers of the detected genes are shown in Table S1.

2.13 Western blotting analysis

The testicular tissues were lysed with RIPA buffer (Servicebio, Wuhan, China) to obtain total protein. Total protein concentration was measured by BCA Protein Assay kit (Servicebio, Wuhan, China), followed by protein denaturation. The equal amounts of denatured total protein from each sample were then loaded onto 8 % or 10 % SDS-PAGE (Servicebio, Wuhan, China) for separation and transferred to PVDF (Merck Millipore, Darmstadt, Germany). Subsequently, the membranes were blocked with 5 % skimmed milk (Servicebio, Wuhan, China) at room temperature for 1 h and incubated overnight at 4 °C with primary antibodies anti-AhR (1:1,000, ABclonal, A1451), anti-AMPKα (1:1,000, ABclonal, A12718), anti-p-AMPKα-Thr172 (1:1,000, ZEN-BIOSCIENCE, R381164), anti-GLUT1 (1:1,000, ABclonal, A11727), anti-LDHA (1:1,000, ZEN-BIOSCIENCE, R24822), anti-MCT4 (1:1,000, ABclonal, A3016), anti-GAPDH (1:1,000, Servicebio, GB15004) and anti-β-Actin (1:1,000, Servicebio, GB15003). Later, the membranes were incubated with secondary antibody anti-rabbit IgG (1:3,000, Servicebio, GB23303) for 1.5 h at room temperature. The protein band in membranes was visualized using an enhanced chemiluminescence reagent (Westernblot, Beijing, China) in the Bio-Rad ChemiDoc MP imaging system (Hercules, CA, USA).

2.14 Statistics

Data were expressed as mean ± standard deviation (SD). Statistical analysis was performed in GraphPad Prism version 8.02. Unpaired two-tailed student’s t-test was used to compare two different groups, and one-way ANOVA with Dunnett’s multiple comparison test was performed to compare more than two groups. Differences with P < 0.05 were considered to be statistically significant.

3.1 Chemical component identification and quality control of WZYZP

The chemical profile of WZYZP was analyzed by UPLC-Q/TOF-MS using the positive and negative ion models. In total, 52 compounds were identified, including flavonoids, alkaloids, phenylpropanoids, and organic acids (Table S2 and Fig. 1A–B), such as quercetin, kaempferol, quercetin-3-O-galactoside, isorhamnetin, apigenin, rutin, schizandrin A, verbascoside (Fig. 1C). Many of these compounds are reported to exhibit low oral bioavailability, such as quercetin, kaempferol, and rutin (Aa et al., 2020; Feng et al., 2019), suggesting WZYZP may act indirectly on hosts to treat diseases. Further, the levels of key components were determined by HPLC. Fig. S1A and B displayed the chromatograms of the mixed reference solution and WZYZP, respectively. The levels of major components in the WZYZP (compared to the weight of WZYZP) were hyperoside (0.379 mg/g), verbascoside (0.152 mg/g), kaempferol (0.161 mg/g), and schisandrin (0.136 mg/g). The results met the requirements of Chinese Pharmacopoeia (Committee of National Pharmacopoeia, 2020).

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Fig. 1

Analysis of the chemical components of the WZYZP by UPLC-Q-TOF/MS. (A, B) Total ion chromatography of WZYZP in (A) positive and (B) negative ion modes. (C) The typical structures of the main bioactive compounds.

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3.2 WZYZP improved spermatogenesis and energy metabolism

As shown in Fig. 2E, the testis index was significantly increased in groups of WZYZP-L, WZYZP-M, and WZYZP-H compared with the model group. Similarly, compared with the model group, WZYZP treatment significantly increased the testis size in a dose-dependent manner (Fig. 2A). H&E staining of testis showed that WZYZP significantly increased the number of spermatogenic cells and the thickness of the seminiferous epithelium in a dose-dependent manner (Fig. 2B). Furthermore, epididymal H&E staining showed a small amount of mature sperm in the model group, but the number of mature sperm was notably increased by WZYZP in a dose-dependent manner (Fig. 2C). Similarly, all three WZYZP treatment groups significantly increased sperm concentration, sperm motility, as well as testicular and plasma testosterone concentrations compared with the model group (Fig. 2D–I). Since energy plays an important role in spermatogenesis (Rato et al., 2012), we further examined the contents of pyruvate, lactate, and ATP in testicular tissue. Compared with the model group, WZYZP significantly reversed the decrease of pyruvate, lactate, and ATP in a dose-dependent manner (Fig. 2J–L). Taken together, these results demonstrated that WZYZP significantly improved spermatogenesis and energy metabolism in mice with spermatogenesis dysfunction.

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Fig. 2

The effects of WZYZP on spermatogenesis and energy metabolism. (A) Morphology of the testis. (B) The representative testicular sections with H&E staining (magnification = × 400, scale bar = 50 μm). (C) The representative epididymal sections with H&E staining (magnification = × 200, scale bar = 100 μm). (D) The representative pictures of sperm morphology and quantity. (E) Testis index of mice in each group. (F) Sperm concentration and (G) motility of mice in each group. (H, I) Testosterone levels in testis (H) and plasma (I). (J) Expression of testicular pyruvate. (K) Expression of testicular lactic acid. (L) Expression of testicular ATP. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns represents no significant difference.

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3.3 WZYZP improved spermatogenesis dysfunction in a gut microbiota–dependent manner

The gut microbiota plays a vital role in spermatogenesis (Zhang et al., 2021), therefore, we analyzed the changes of the gut microbiome after the WZYZP-H intervention. As revealed in Fig. 3A–B, the Chao index, Ace index, and Shannon index of gut microbiota were significantly altered, indicating that the richness and diversity of the gut microbiota were reduced in the WZYZP-H group. A distinct clustering of gut microbiota composition in the control group, model group, and WZYZP-H group was observed using principal coordinates analysis (PCoA) (Fig. 3C). Moreover, the clustering of gut microbiota in WZYZP-H group was closer to that of the control group (Fig. 3C). The phylum level of gut microbiota indicated that the relative abundances of Bacteroidota and Proteobacteria were decreased and Desulfobacterota and Patescibacteria were increased in the model group compared with the control group, while WZYZP-H significantly restored these levels (Fig. 3D and Fig. S2A).

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Fig. 3

The effects of WZYZP-H on the gut microbiota. (A) The richness and (B) diversity of gut microbiota. (C) Principal coordinate analysis (PCoA) plot of gut microbiota based on the operational taxonomic units (OTUs) abundance. (D) Bacterial taxonomic profiling at the phylum level of gut microbiota. (E) Heatmap of differential gut microbiota abundance among control, model, and WZYZP-H groups. The color of the spots in the left panel represents the relative abundance of the genus level in each group. (F) Spearman’s correlation analysis of the gut microbiota of genus level with sperm concentration and motility. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, and ***P < 0.001.

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We further analyzed the change of gut microbiota at the genus level, in which fifteen bacteria were altered in the control, model, and WZYZP-H groups (Fig. 3E). Most of these changed bacteria belonged to phyla Firmicutes and Bacteroidota. Among these genus-level bacteria, the relative abundances of Lactobacillus and Bacteroides were significantly decreased in the model group compared with the control group, while WZYZP-H significantly increased these levels (Fig. S2B). Moreover, the relative abundances of Lachnospiraceae_NK4A136_group, Colidextribacter, and Candidatus_Saccharimonas were significantly decreased by WZYZP-H treatment (Fig. S2B). The Spearman’s correlation showed that genera Lactobacillus and Bacteroides were positively while genera Lachnospiraceae_NK4A136_group, Colidextribacter, and Candidatus_Saccharimonas were negatively correlated with the sperm concentration and motility (Fig. 3F).

To investigate whether the ameliorating effect of WZYZP on impaired spermatogenesis depends on the presence of gut microbiota, we treated mice in the WZYZP-H group with a cocktail of antibiotics (Abx) containing metronidazole, neomycin, vancomycin, and ampicillin to deplete gut microbiota (Zhao et al., 2023). After Abx treatment, the OTUs of gut microbiota in the WZYZP-H-Abx group (48) were significantly lower than those in the WZYZP-H group (2183), which is equivalent to 97.9 % reduction in OTUs (Fig. S3A). Moreover, the alpha diversity of gut microbiota and the vast majority of the gut microbiota at genus levels were significantly reduced by Abx treatment (Fig. S3B–D). These results suggested that the Abx protocol efficiently depleted the majority of gut microbiota in our study. Importantly, the depletion of gut microbiota suppressed the ability of WZYZP-H to improve spermatogenesis dysfunction (Fig. 4A–K). These findings indicated that the ameliorating effect of WZYZP on impaired spermatogenesis acted in a gut microbiota-dependent manner.

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Fig. 4

Effect of antibiotic-induced microbiota depletion on WZYZP-H in improving spermatogenesis. (A) Morphology of the testis. (B) The representative H&E staining of testicular sections (magnification = × 400, scale bar = 50 μm) and epididymal sections (magnification = × 200, scale bar = 100 μm). (C) The representative pictures of sperm morphology and quantity. (D) Testis index of mice. (E) Sperm concentration and (F) motility of mice. (G, H) Testosterone levels in testis (G) and plasma (H). (I) Levels of testicular pyruvate in mice. (J) Levels of testicular lactic acid in mice. (K) Levels of testicular ATP in mice. Data were shown as mean ± SD. *P < 0.05.

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3.4 WZYZP modulated fecal microbial metabolites

Since the fecal metabolome, a functional readout of the gut microbiota, is considered an intermediate phenotype mediating interactions between host and microbes (Zierer et al., 2018), we investigated the changes of fecal metabolites in mice after treatment with WZYZP-H by untargeted metabolomics. The partial least squares discriminant analysis (PLS-DA) score plot showed a distinct clustering of fecal metabolites between control and model groups, as well as the model and WZYZP-H groups in positive ion mode (Fig. S4A–D). Moreover, PLS-DA showed that WZYZP-H treatment showed a trend to shift toward the control group (Fig. S4E–F). Compared with the control group, 133 metabolites were significantly altered in the model group, of which 83 metabolites were significantly reduced and 50 metabolites were significantly increased (Fig. 5A). Compared with the model group, 101 metabolites were significantly changed in the WZYZP-H group, of which 42 metabolites were significantly decreased and 59 metabolites were significantly increased (Fig. 5B). However, only 22 metabolites changed significantly between the control and the model and could be significantly reversed by WZYZP-H (Fig. 5C, Table S3). To investigate the functional information of the 22 differential metabolites in the feces regulated by WZYZP-H, we performed functional annotation of KEGG pathway. KEGG pathway functional annotation showed that the differential metabolites have the highest distribution ratio in the amino acid metabolism (Fig. 5D). The KEGG pathway enrichment analysis displayed that tryptophan metabolism was the most important metabolic pathway (impact value: 0.148) (Fig. 5E). These results suggested that WZYZP-induced tryptophan metabolism may play an important role in improving spermatogenesis.

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Fig. 5

The effects of WZYZP-H on the fecal metabolites. (A) Volcano plot of differential metabolites between control and model groups. (B) Volcano plot of differential metabolites between model and WZYZP-H groups. (C) Heatmap showing the relative abundances of differential metabolites in fecal samples. (D) The functional annotation of differential metabolites in fecal samples. (E) Key altered pathway of differential metabolites with metabolomics pathway analysis (MetPA).

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3.5 WZYZP modulated microbial tryptophan metabolism

Given that microbial metabolites entering the host blood circulation is an important way for gut microbiota to have a functional impact on the host pathophysiology (Koh and Bäckhed, 2020), we further performed targeted detection of the Trp metabolite levels in the plasma of mice. We observed a decreased level of Trp in the model group compared to the control group, while WZYZP-H significantly increased the concentration of Trp compared to the model group (Fig. 6A). As for indole and its derivatives, the concentrations of indole-3-lactic acid (ILA, Fig. 6B), indole-3-acrylic acid (IA, Fig. 6C), indole-3-propionic acid (IPA, Fig. 6D), indole (IND, Fig. 6E), indole-3-carboxylic acid (I3CA, Fig. 6F), indole-3-acetamide (IAM, Fig. 6G), indole-3-acetic acid (IAA, Fig. 6H), indole-3-carboxaldehyde (IAId, Fig. 6I) were lower in the model group than that of the control group. After WZYZP-H treatment, the concentrations of indole and its derivatives in model mice were increased (Fig. 6B–I). Among these derivatives, IA, IAA, and IAId are ligands of aryl hydrocarbon receptor (AhR) and they originate from the metabolism of Trp by microbe (Agus et al., 2018). Notably, there were significant reductions in ligands of AhR in the model group, compared with the control group, while WZYZP-H treatment significantly attenuated these reductions (Fig. 6J). The 5-hydroxytryptophan (5-HTP) production pathway and kynurenine (Kyn) pathway are the other two metabolism pathways of Trp. Metabolites corresponding to the Kyn pathway were decreased in the model group and were significantly increased by WZYZP-H, while 5-HTP pathway-related metabolites including 5-hydroxyindole-3-acetic acid (5-HIAA), and 3-hydroxyanthranilic acid (3-HAA) were not significantly altered (Fig. 6K–N). Taken together, these findings demonstrated that WZYZP treatment facilitated the conversion of Trp towards AhR ligands in mice with spermatogenesis dysfunction.

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Fig. 6

The effects of WZYZP-H on tryptophan metabolites. Quantification of tryptophan metabolites, (A) Trp, (B) ILA, (C) IA, (D) IPA, (E) IND, (F) I3CA, (G) IAM, (H) IAA, (I) IAId, (J) AhR ligands, (K) 5-HTP, (L) 5-HIAA, (M) Kyn, and (N) 3-HAA in plasma from mice. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns represents no significant difference.

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3.6 WZYZP modulated the AhR/AMPK pathway to improve spermatogenesis

Studies have shown that AhR activation by AhR ligands can activate the AMP-activated protein kinase (AMPK) (Muku et al., 2019). Moreover, AMPK regulates adenosine triphosphate (ATP) through the glycolytic pathway, which is critical for the energy requirements of spermatogenesis (Xie et al., 2022). Thus, we detected AhR activation, protein expression related to the AhR/AMPK pathway, and expression level of spermatogenesis-related genes in testicular tissues. It was shown that Ahr gene expression was suppressed in the model group, and was significantly increased by WZYZP-H (Fig. 7A). Furthermore, we observed an increase in the expressions of the Ahr transcriptional targets Cyp1a1 and Cyp1b1 in the WZYZP-H group compared to that of the model group (Fig. 7B, C). WZYZP-H significantly reversed the decrease of AhR protein expression (Fig. 7D, E). Meanwhile, the protein expression level of p-AMPKα was significantly increased in the WZYZP-H group compared to that of the model group (Fig. 7D, F). Similarly, GLUT1, LDHA, and MTC4 expression levels were significantly elevated after WZYZP-H treatment in comparison to the model group (Fig. 7G, H). In addition, similar outcomes were obtained in the expression level of spermatogenesis-related genes in testicular tissues. As shown in Fig. 7I, the expression level of Plzf, which is essential for spermatogonia self-renewal (Hobbs et al., 2010), was significantly up-regulated in the WZYZP-H group compared with the model group. Other genes, including Dmc1, Sycp3, and Stra8, well-known markers of spermatocyte meiosis (Liu et al., 2022), were significantly increased by WZYZP-H treatment (Fig. 7I). Together, these results suggested that WZYZP-H improved spermatogenesis dysfunction possibly via regulation of the AhR/AMPK pathway.

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Fig. 7

WZYZP modulated AhR/AMPK pathway to improve spermatogenesis. Relative mRNA expressions of (A) Ahr, (B) Cyp1a1, and (C) Cyp1b1 in the testis. (D) Representative Western blotting bands and quantitation of (E) AhR, (F) p-AMPKα protein expression in the testis. (G) Representative Western blotting bands and quantitation of (H) GLUT1, LDHA, MCT4 protein expression in the testis. (I) Relative mRNA expressions of spermatogenesis-related genes, including Plzf, Dmc1, Sycp3, and Stra8 in the testis. Data were shown as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001, and ns represents no significant difference.

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3.7 Integrated mechanism of WZYZP on spermatogenesis dysfunction

To further explore the relationship between the WZYZP-H-induced changes in gut microbiota composition and gut microbial Trp metabolites, we performed Spearman rank correlation. As shown in Fig. 8A, four bacterial strains (phyla Bacteroidota and Proteobacteria, genera Lactobacillus and Bacteroides) were positively while five bacterial strains (phylum Desulfobacterota Patescibacteria, genera Lachnospiraceae_NK4A136_group, Colidextribacter, and Candidatus_Saccharimonas) were negatively correlated with ILA, IAId, AhR ligands, IAA, IND, and IA. Moreover, most of the correlations between the gut microbiota and AhR ligands (including IA, IAA, and IAId) were significant (Fig. 8A). We further performed the Mantel test to explore the relationship among the WZYZP-H-induced changes in Trp metabolites, AhR-related targets, and functional genes related to spermatogenesis. The results showed a significant correlation between Trp metabolites (including AhR ligands, IAA, IA, and Trp) and the gene expression levels of Ahr, Cyp1a1, and Cyp1b1 (Mantel’s r > 0.25, Mantel’s P < 0.05, Fig. 8B), indicating AhR activation is potentially correlated with Trp metabolites. Moreover, AhR activation (Ahr, Cyp1a1, and Cyp1b1) was significantly positively correlated with energy metabolism (pyruvate, lactic acid, and ATP) (Fig. 8B). The energy metabolism (pyruvate, lactic acid, and ATP) was significantly positively correlated with spermatogenesis-related genes (Plzf, Dmc1, and Sycp3) (Fig. 8B). Additionally, spermatogenesis-related genes (Plzf, Dmc1, and Sycp3) were positively correlated with sperm concentration (Fig. 8B). Taken together, these results showed the intrinsic relationship that WZYZP improved spermatogenesis impairment through gut microbial Trp metabolites.

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Fig. 8

Association map of the three-tiered analyses integrating the gut microbiota, Trp metabolites, and phenotypes as well as their associated genes. (A) Heat maps of the Spearman rank correlation coefficient and significant tests between the differential bacteria and Trp metabolites. (B) Pairwise comparisons of phenotypes and their associated genes, with a color gradient denoting Spearman’s correlation coefficient. Trp metabolites were related to phenotypes and their associated genes by partial (geographic distance–corrected) Mantel tests. Edge width corresponds to the Mantel’s R statistic for the corresponding distance correlations, and edge color denotes the statistical significance based on 9,999 permutations.

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