Hydrogen-rich water treatment targets RT1-Db1 and RT1-Bb to alleviate premature ovarian failure in rats

Experimental Sprague Dawley (SD) rats

All adult, virgin, female Sprague Dawley (SD) rats (8–10 weeks old) were purchased from the Beijing HFK Bioscience Co., Ltd. (Beijing, China), and they were housed in the animal center of Tianjin Medical University (Tianjin, China). The animal experiments as well as humane care were approved by the Institutional Animal Care and Use Committee of Tianjin Medical University, in accordance with the National Institute of Health Guide for Care and Use of Laboratory Animals (protocol code: IRB2021-DW-16). All SD rats were housed under standard cycles (12 h light; 12 h dark) and specific pathogen-free conditions, with chow and water ad libitum.

Preparation of hydrogen-rich water

The HRW was freshly prepared before the administration weekly, mainly according to the descriptions in previous report (Meng et al., 2015). Briefly, hydrogen was generated using a hydrogen generator (Beijing Zhongke Huiheng Technology, Beijing, China), and it was dissolved in physiological saline under 0.4 MPa pressure for 4 h. Then, the concentration of HRW was confirmed to be >0.6 mmol/L, employing a needle-type hydrogen sensor (Unisense A/S, Aarhus, Denmark). The HRW was stored in an aluminum bag with no dead volume at 4 °C, to ensure the eligible concentration of HRW.

POF model construction and group settings

There were nine total SD rats in this work, and all rats were randomly divided into three groups, including the control group (con, n = 3), cyclophosphamide (CTX) induced POF group (POF, n = 3), and HRW treated POF groups (POF+HRW, n = 3). For the POF group and HRW treated POF group, all rats were given 50 mg/kg CTX (200–400 µL, NO.6055-19-2, Shanghai Yuanye Bio-Technology, Shanghai, China) by a single intraperitoneal injection to generate the POF rat model. Regarding control group, the rats received the equal volume of saline by intraperitoneal injection. In HRW treated POF group, the rats received intraperitoneal injection with HRW (10 ml/kg body weight) once a day, between the 1st and the 4th week. Meanwhile, the rats in control group and POF group were intraperitoneally injected with saline (10 ml/kg body weight).

Before sample collection, all rats were anesthetized by intraperitoneal injection of 3–4 µL/g 10% chloral hydrate. After sample collection, the rats were euthanized with carbon dioxide.

Serum 17-β-estradiol (E2), follicle-stimulating hormone (FSH), and anti-mullerian hormone (AMH) levels determined by enzyme-linked immunosorbent assay (ELISA)

The blood samples in three groups were all withdrawn from the retro-orbital plexus of unconscious rats. The serum samples were obtained after centrifuging blood samples at 1800 r/min for 15 min. The E2 (NO. E-EL-0152c), FSH (NO. E-EL-R0391c), and AMH (NO. E-EL-R3022) serum levels were measured using corresponding ELISA Kits (Elabscience Biotechnology, Wuhan, China), and the protocols were strictly in accordance with manufacturer’s instructions.

Ovarian histomorphological analysis

Before the paraffin embedding, three groups of ovarian tissues were firstly maintained in 10% buffered formalin for 48 h, which were then sliced (5 µm). Briefly, the tissue sections were then air-dried, dewaxed, then stained with haematoxylin and eosin (H&E). The slides were observed under Olympus BX40 light microscope (Olympus, Tokyo, Japan), and analyzed in ImageJ software. The numbers of primordial, primary, secondary, antral and atretic follicles were counted as previously described (Kamal et al., 2022).

TUNEL assay

After the isolation of ovarian, the adipose tissues were eliminated from the ovarian. The ovarian tissues were then embedded in paraffin and sliced. The TUNEL assay was conducted using TUNEL Apoptosis Detection Kit (NO.11684817910; Roche, Basel, Switzerland), following the manufacturer’s instructions. Fluorescence microscope (Olympus, Tokyo, Japan) was used to observe the apoptotic cells. The proportions of apoptotic cells were calculated according to apoptotic cell counts/ total cell counts (five random fields per section and five sections per tissue from a rat).

Ovarian tissue protein extraction and tandem mass tag (TMT)-based quantitative proteomic analysis

Briefly, the flowchart of the TMT-based quantitative proteomic analysis mainly involved protein extraction, trypsin digestion, TMT labeling, and liquid chromatography-tandem mass spectrometry (LC-MS/MS) data acquisition. Proteins of ovarian tissues were quantified using bicinchoninic acid (BCA) assay, and filter aided proteome preparation (FASP) was utilized for the trypsin digestion (Wisniewski et al., 2009). Subsequently, 100 µg peptides were taken from each sample (three biological repeats for each sample), which was subjected to the TMT labeling using TMT labeling kit as per the instructions of manufacture (Thermo Fisher Scientific, Waltham, MA, USA). Finally, the samples were processed using high pH reversed-phase high-performance LC (HPLC), and the raw data were assigned to the peptide sequences basing on the database comparison.

Differentially expressed protein analysis

The differentially expressed proteins (DEPs) between various groups were analyzed using limma function of R (version 4.1.0, the same below). The DEPs with fold change <0.9 or >1.1, and p < 0.05 were considered statistically significant.

Functional enrichment analysis

All significant candidate DEPs were then subjected to the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) functional enrichment analysis, in “clusterProfiler” of R (Yu et al., 2012). Significant GO and KEGG terms were screened with p < 0.05.

Subcellular localization prediction

The amino acid sequences can be converted into numerical localization features in WoLF PSORT database (https://wolfpsort.hgc.jp/) (Horton et al., 2007). The subcellular localization of candidate target proteins was predicted using a k-nearest neighbor classifier.

Protein-protein-interaction (PPI) analysis

The STRING database (https://string-db.org/) integrated numerous known and predicted associations between proteins. Herein, we used STRING database (version 11.0) (Szklarczyk et al., 2019) to analyze functional associations and interactions between proteins.

Additionally, the candidate target proteins were analyzed using GeneMANIA database (http://genemania.org). GeneMANIA was a flexible database that predicted the function of genes/ proteins and prioritized genes for functional assays. For the given gene list, functionally similar genes would be identified via searching vast amounts of genomics and proteomics data. Then, GeneMANIA could weight each functional dataset according to the predicted value of the given gene list.

Hydrogen-rich water alleviated ovarian injury of POF rat models

Firstly, we have constructed the POF rat models via CTX inducing. Compared to rats in control group, rats in POF group had markedly reduced serum E2 (Fig. 1A) and AMH (Fig. 1C) levels, while increased serum FSH (Fig. 1B) levels throughout the four weeks. After the determination of crucial hormone levels in serum, we confirmed that the POF rat models have been successfully built.

Next, we evaluated the effects of HRW treatment on POF rats. The rats in HRW treatment group had significantly increased serum E2 (Fig. 1A) and AMH (Fig. 1C) levels and significantly decreased serum FSH (Fig. 1B) level compared to POF group without HRW treatment. In addition, the primordial, primary, secondary, antral and atretic follicles in control group, POF group and POF+HRW group were counted after H&E staining, respectively (Fig. 1D). The representative HE images were displayed in Fig. 1E. Follicle counting results indicated that HRW treatment significantly affected the number of primordial, secondary, antral and atretic follicles in POF rats (Figs. 1D and 1E). Counting of primordial follicles showed that HRW treatment POF group significantly increased primordial follicles in POF+HRW group compared to POF group (Figs. 1D and 1E). Significantly more follicles were counted at different developmental maturation stages in POF+HRW group than in POF group (Figs. 1D and 1E). In the TUNEL assay, the numbers of apoptotic cells in ovarian tissues were assessed in all three groups, more TUNEL-positive cells were detected in POF group than in POF+HRW group and control group (Fig. 1F). Therefore, our results indicated that the HRW treatment alleviated ovarian injury of POF rat models.

Candidate treatment targets of HRW in POF rat models

Then, TMT-based quantitative proteomic analysis was performed on the ovarian tissue samples from three groups, in order to obtain the potential targets of HRW treatment in POF (raw data was provided in Table S1). Compared to control group, there were totally 106 DEPs in POF group, comprising 48 upregulated proteins and 58 downregulated proteins (Fig. 2A). In total, 27 proteins were upregulated and 58 were downregulated in POF+HRW group, compared with POF group (Fig. 2B). The DEPs between POF vs. control, POF+HRW vs. POF exhibited significantly differential expression, separately (Figs. 2C–2D). A total of 16 DEPs were then identified after the cross analysis of DEPs from POF vs. Control and POF+HRW vs. POF (Fig. S1, Table S1), which were important candidate targets of HRW treatment in POF.

Functional enrichment results of candidate targets of HRW

The GO and KEGG functional enrichment analysis was performed on the above 16 candidate targets to reveal the potential functions affected by HRW treatment. Our results suggested that these 16 proteins were significantly enriched in totally 296 GO terms (p < 0.05, Table S2), comprising 232 biological process (BP) terms, 17 cellular component(CC) terms, and 47 molecular function (MF) terms (top 10 sub terms were shown in Fig. 3A). The 36 significant KEGG pathways involved in immune disease (seven terms), immune system (six terms), carbohydrate metabolism (five terms), cell growth and death (one term), endocrine and metabolic disease (one term), and so on (Fig. 3B, Table S3).

Additionally, the subcellular localization of the 16 candidate targets was predicted using the WoLF PSORT database, indicating that these proteins were mainly localized to extracellular matrix (31%), nucleus(25%), cytosol (13%), mitochondrion (13%), and plasma membrane (12%) (Fig. 3C).

RT1-Db1 and RT1-Bb were crucial targets of HRW treatment in POF rat models

We subsequently conducted the PPI analysis on these 16 candidate proteins, using STRING online tool. Among these proteins, there were 15 nodes and two edges (average degree = 0.267) (Fig. 4A), and the interactions between RT1-Db1 and RT1-Bb were notable. To further screen more reliable crucial targets of HRW treatment more reliably, the GeneMANIA network was also analyzed basing on the candidate targets. As shown in Fig. 4B, the GeneMANIA interaction network involved in co-expression, shared protein domains, co-localization, and the potential functions of the proteins. We found that significant co-expression was observed between RT1-Db1 and RT1-Bb, and they showed significant co-expression with RT-Ba and RT1-Da. The functions of RT1-Db1 and RT1-Bb were significantly correlated with the antigen processing. Based on all 16 candidate targets and their related interaction proteins in Fig. 4B, GO and KEGG enrichment results were in line with our former functional results (Figs. 4C–4D, Tables S4 and S5). The corresponding human orthologous genes of RT1-Db1 and RT1-Bb were HLA-DRB5 and HLA-DQB1, respectively.