The following primary antibodies: Anti-Cyp1b1 (Cat#DF6399), Anti-GPR41 (Cat#AF9057) and anti-GAPDH (Cat#AF7021) was purchased from Affinity Biosciences Technology (Jiangsu, China). PrimeScript™ RT reagent Kit with gDNA Eraser (RR047A) and TB Green^® Premix Ex Taq™ II (RR820A) were purchased from TaKaRa(Dalian, China)and PCR primers were purchased from Gencreat (Wuhan, China). RIPA tissue/cell lysate was purchased from Solarbio life sciences (Beijing, China). The ELISA kits [PYY (SYP-R0196), Ghrelin (RX301269R) and 4-OHE2(RXJ303175R)] were purchased from Ruixin Biotechnology Co., Ltd (Quanzhou, China). TG (A110-1-1), TC (A111-1-1), LDL-C (A113-1-1) and HDL-C (A112-1-1) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Lentivirus GV492 and GV492-cyp1b1 were purchased from Shanghai Genechem Co.,Ltd. Rodent Maintenance feed (1025) and Maintenance powder feed (1024) were purchased from Guangdong Vital River Laboratory Animal Technology Co., Ltd.

The lipo-coated NaBu was provided by King Techina Technology Co., Ltd with a purity of 30%. According to previous studies, dietary supplementation with 1% ~5% NaBu has been shown to exert beneficial effects in animal disease models (13, 28, 29). Based on preliminary experimental results, the present study adopted a final NaBu concentration of 3.6% in the diet. Equivalent to adding 12g of lipid-coated NaBu per 1000g of maintenance feed, the effective dose of NaBu is 360mg. Preparation of the treatment feed for PCOS rats: The maintenance powder feed was uniformly blended with lipid-coated NaBu, then process it into feed pellets through granulation for storage.

A 0.5% carboxymethyl cellulose sodium (CMC) solution was prepared by dissolving 0.5 g CMC in 100 mL of boiled double-distilled water with continuous stirring until complete dissolution. After cooling to room temperature, 1 g of letrozole powder was added and thoroughly mixed to prepare a 1 mg/mL working solution. The solution was aliquoted and stored at -20°C.

Eight-week-old female Sprague Dawley rats were purchased from Changsha Tianqin Biotechnology Co., Ltd. [SCXK (Xiang) 2019-0014, No. 430726210100078487] and housed at the Experimental Animal Center of Youjiang Medical University for Nationalities [SYXK (Gui) 2022-0003] at a density of three rats per cage. The rats were maintained in a controlled environment with a temperature of 22 ± 2°C, relative humidity of 55 ± 5%, and a 12/12 hour light/dark cycle. All experimental procedures were approved by the Animal Welfare and Ethics Committee of Youjiang Medical University for Nationalities (No. 2022031005).

After one week of adaptive feeding, the rats were randomly divided into two groups: the normal control group (NC, n=6) and the model group (Model, n=12).The PCOS modeling method refers to the existing literature report (7). In the first stage, the NC group received oral gavage of 0.5% CMC solution (1 mL/kg/day), while the Model group received oral gavage of letrozole solution (1 mg/kg/day) for 28 consecutive days, with weekly weighing. In the second stage, the model group was further divided into the PCOS group and the NaBu group, each consisting of 6 rats. The rats were individually housed in metabolic cages, with the NC and PCOS groups having ad libitum access to maintenance feed, and the NaBu group having feed containing 3.6% NaBu, administered for 21 consecutive days. On day40, the rats were subjected to vaginal smear examination for 10 consecutive days (about 2 estrus cycles). All rats were weighed once a week, and the average food intake was recorded from day29 to day49.

The vaginal smear of a rat was stained with HE and observed under an optical microscope(Olympus, Japan). The estrus cycle in rat averages 4~5 days and is generally divided into four stages: proestrus, estrus, metestrus, and diestrus. The stages of estrus are distinguished by identifying different cell types, following methods from our previous research (7). At the end of the experiment, all rats were fasted for 12 hours and anesthetized with isoflurane. Blood samples were collected from the tail artery to measure fasting blood glucose levels. Subsequently, the abdominal cavity was opened, and blood was collected from the abdominal aorta. Serum, ovaries, colon, and fecal samples were collected for further experimental analysis.

Blood samples collected from rats were allowed to stand at 4°C overnight and then centrifuged at 4000 rpm for 10 minutes. The supernatant was collected and stored at -80°C for further use. The radioactive immunoassay for serum T, INS, E₂, P₄, FSH, and LH levels was conducted by Beijing North Institute of Biological Technology by using their assay kit (220220). The serum levels of Ghrelin, PYY, TC, TG, HDL-C and LDL-C were determined following the instructions provided in the ELISA assay kit.

SCFAs analysis of rat feces and iTRAQ proteomics analysis of rat ovaries were both conducted by Nanning Current Science Biotechnology Co., Ltd (Guangxi, China).

The ovarian and colon tissue samples were fixed in 4% paraformaldehyde solution at 4°C over 24h, then embedded in paraffin and cut into 3~4μm thickness for hematoxylin-eosin staining. Analysis was performed using an optical microscope (Olympus, Japan). Follicles with a thinned granulosa cell layer and a thickened theca cell layer were recognized as cystic follicles. The numbers of cystic follicles and corpora lutea were counted, and the results including colon morphology analysis were confirmed by a pathologist.

The rat colon and ovarian tissues were fixed in 4% paraformaldehyde at 4°C for 48 hours, followed by dehydration, embedding in paraffin wax, and sectioning. After dewaxing and hydration, sections were subjected to endogenous peroxidase blocking at room temperature for 10 minutes. Subsequently, they were rinsed with PBS buffer (3 min×3 times) and incubated with a primary antibody (1:200) at 37°C overnight. On the following day, sections were rinsed again with PBS buffer (3 min×3 times), treated with goat anti-rabbit IgG polymer for 20 minutes at 37°C, and washed with PBS buffer (3 min×3 times). DAB was used for color development, followed by incubation with hematoxylin staining solution for 20 seconds. After dehydration with alcohol, sealing with xylene transparent and neutral gum, the staining results were observed under an optical microscope.

RNA was extracted from ovarian tissue by Trizol method, cDNA was synthesized by reverse transcription, and the target gene was amplified by quantitative real-time PCR. The primer sequences are shown in Supplementary Table S1 . The mRNA expression levels of the target genes were normalized by 2^−ΔΔCT with β-actin as the internal reference.

Initially, 40 mg of ovarian tissue was homogenized in RIPA tissue lysate, and the resulting supernatant was collected after centrifugation. Protein concentration was determined using the BCA method. The protein sample was then mixed with sample buffer, denatured at 100°C for 5 minutes, and the supernatant was obtained after centrifugation. Next, an 8% SDS-PAGE gel was prepared, and proteins were separated by electrophoresis at 100 V for 100 minutes. The proteins were transferred from the gel to a PVDF membrane at 250 mA for 80 minutes, followed by blocking with 5% BSA for 1 hour. After washing with TBST, a primary antibody (1:1000) was added and incubated overnight at 4°C. The following day, TBST washes were performed, and a secondary antibody (1:5000~10000) was added for 1 hour. After additional TBST washes, ECL developer was applied for visualization.

Six 30-day-old SPF female SD rats were subcutaneously injected with 50 IU of pregnant mare serum gonadotropin (PMSG) behind the neck to stimulate follicular development. After 48 hours, the rats were euthanized, and soaked in 75% alcohol for disinfection for 30 minutes. The ovaries were aseptically removed and washed twice in PBS containing 2% antibiotics (penicillin 100 IU/mL, streptomycin 100 μg/mL). The surrounding fat tissue was carefully removed, and the ovaries were placed in DMEM/F12 medium containing 2% antibiotics. Follicles were punctured using a 1 mL sterile syringe to collect granulosa cells. The cell suspension was filtered through a 200-mesh stainless steel cell strainer, centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cell pellet was resuspended in red blood cell lysis buffer and incubated at 37°C for 10 minutes. The mixture was then centrifuged at 1000 rpm for 5 minutes, and the supernatant was discarded. The cells were resuspended in PBS and centrifuged at 1000 rpm for 3 minutes. The cell pellet was collected and resuspended in complete medium containing 10% fetal bovine serum, and cultured at 37°Cwith 5% CO2 for 48 hours before changing the medium. When the cells reached about 70% confluence, they were identified. Granulosa cells were then infected with either the empty lentiviral vector GV492 or the lentiviral vector GV492-cyp1b1 to determine the optimal infection conditions. Finally, the effects of the lentiviral vectors under optimal infection conditions on granulosa cell function were analyzed.

The adaptive feeding, grouping and feeding methods of rats are the same as 2.3. Male rats with normal reproductive function were put into cages for 21 days at the second stage in a ratio of 1:3. In the third stage, male rats were taken out at day49, and female rats were kept for 21 days, during which they were fed maintenance diets.

Data are expressed as mean ± standard deviation (SD). SPSS 20.0 and GraphPad Prism 8.0 were used for data analysis and mapping, respectively. Image Pro Plus 6.0 and Image J software were used to measure the optical density and protein band gray value of the immunohistochemical results, respectively. For the data conforming to the homogeneity test of variance, LSD(L) in one-way analysis of variance (ANOVAs) was used for statistical analysis. For the data that did not meet the homogeneity test of variance, Kruskal Wallis rank sum test in the non-parametric test was used for statistical analysis, p < 0.05 was considered statistically significant.

According to the letrozole-induced PCOS rat model and treatment process ( Figure 1A ), although initial body weights did not differ between the NC and model groups ( Figure 1B ), the PCOS and NaBu groups exhibited significantly higher body weights than the NC group after 28 days of letrozole administration. After 21 days of NaBu treatment (day29 to day 49), rats in the NaBu group exhibited significantly lower body weight and reduced average food intake compared to the PCOS group ( Figures 1C, D ). Notably, NaBu treatment showed no significant effect on fasting blood glucose (FBG) levels ( Figure 1E ). Biochemical analysis demonstrated that PCOS rats had significantly elevated triglycerides (TG) and low-density lipoprotein cholesterol (LDL-C) levels relative to NC controls, while total cholesterol (TC) and high-density lipoprotein cholesterol (HDL-C) levels remained comparable between these groups. Importantly, NaBu treatment effectively ameliorated the dyslipidemia in PCOS rats, as evidenced by significant lower in both TG and LDL-C levels ( Figures 1F–I ).

Vaginal smears were performed on the rats for 10 consecutive days from day40 to day49, and the morphology of vaginal exfoliated epithelial cells of rats was observed to distinguish different stages of estrus in order to evaluate the effect of NaBu treatment ( Figure 2A ). The results showed that the rats in the NC group still maintained regular estrus cycles ( Figure 2B ), the rats in the PCOS group were always in the diestrus stage ( Figure 2C ), and the rats in the NaBu group began to show estrus on the day45 ( Figure 2D ). A normal estrus cycle should have four consecutive estrus stages; thus, we counted the estrus stages for each rat. A complete estrus cycle observed is defined as estrus regular and conversely as irregular. According to statistics, all rats in the NC group had regular estrus cycles, while those in the PCOS group were always irregular; 4 out of 6 rats in the NaBu group were observed to have regular estrus cycles, with a recovery rate of 66.7% ( Figure 2E ).

To evaluate the effects of NaBu on endocrine hormones in rats, serum was collected for hormonal assays. Compared with the NC group, the PCOS group showed significantly higher levels of T, LH, and INS ( Figures 3A–C ) but lower levels of E2 and P4 ( Figures 3E, F ), while FSH was not significantly different ( Figure 3D ). After NaBu treatment, T, LH and INS were significantly lower while E₂ and P₄ were significantly higher, suggesting that NaBu treatment could reverse endocrine disorders in PCOS rats.

The ovaries of NC group rats displayed normal histological architecture, featuring multiple well-developed corpora lutea and follicles at various developmental stages. In stark contrast, the PCOS model group exhibited characteristic pathological changes, including a marked increase in cystic follicles, significant reduction in corpora lutea count, and notable thinning of the granulosa cell layer. Notably, NaBu treatment substantially attenuated these polycystic ovarian alterations, as evidenced by the restoration of corpora lutea numbers and the reappearance of a well-organized, thickened granulosa cell layer ( Figures 3G–I ).

SCFAs profiles ( Figure 4A ) revealed that compared with NC group, the levels of propionic acid and butyric acid in NaBu group were significantly higher, while isovaleric acid was significantly lower; the levels of pentanoic acid and isovaleric acid in PCOS group lower significantly. Compared with PCOS group, the levels of propionic acid, butyric acid and isovaleric acid in NaBu group were significantly higher. There were no significant differences in acetic acid levels between the groups.

Histopathological examination of rat colonic tissues revealed that NaBu treatment did not significantly alter colonic morphology. The mucosal architecture remained intact in all experimental groups, demonstrating that NaBu treatment induces no adverse effects on intestinal histology ( Figure 4B ). Immunohistochemical analysis showed that the expression level of colon GPR41 protein was significantly up-regulated in the NaBu group ( Figures 4C, D ). Serum PYY levels in PCOS rats were significantly higher after NaBu intervention, but Ghrelin levels were not affected ( Figures 4E, F ). Our findings indicate that NaBu treatment upregulates GPR41 expression in the colon and increases serum PYY levels, suggesting a potential role of GPR41 in mediating PYY secretion.

A total of 1384875 spectrums were processed using Proteome Discoverer software. On matching 230372 spectrums to Oreochromisniloticus Uniprot database 30736 peptides (28353 unique peptides) were obtained. A total of 4709 proteins were identified and 3345 of them were quantifiable ( Figure 5A ). The Venn diagram showed that there were 100 up-regulated and 84 down-regulated differentially expressed proteins in PCOS vs. NC group while 48 upregulated and 78 downregulated differentially expressed proteins were in the NaBu group vs. PCOS group. There were 83 overlapping differentially expressed proteins (DEPs), and 30 were upregulated and 53 were downregulated after NaBu treatment ( Figure 5B ). KEGG enrichment analysis was performed on these 83 DEPs using STRING database, and the most significant first 11 gene subsets were obtained, including metabolism of xenobiotics by cytochrome P450, PPAR signaling pathway, cholesterol metabolism, fatty acid degradation, tryptophan metabolism, steroid hormone biosynthesis, complement and coagulation cascades, peroxisome, ferroptosis, lysosome, pentose and glucuronate interconversions. Among them, we focused on partially differentially expressed proteins in the metabolism of xenobiotics by cytochrome P450 and steroid hormone biosynthesis pathways ( Figure 5C ). DEPs are listed in Supplementary Table S2 . Next, heatmaps were constructed to show the expression patterns of the overlapping DEPs, and found they shared the gene Cyp1b1. Cyp1b1 and Ephx1, which are involved in metabolism of xenobiotics by cytochrome P450, were upregulated in the PCOS rats but recovered in the NaBu rats, whereas Aldh3b1 and Idh1 were downregulated in the PCOS rats and recovered in the NaBu rats. The expression levels of Cytochrome P450 family 11 subfamily a member 1 (Cyp11a1), 3β-hydroxysteroid dehydrogenase (Hsd3b) and Steroidogenic acute regulatory protein (StAR) proteins related to steroid hormone biosynthesis signaling pathway were decreased in the PCOS group, and levels were increased in the NaBu group except Hsd3b; whereas Cytochrome P450 family 17 subfamily a member 1(Cyp17a1) was upregulated in the PCOS rats and reduced in the NaBu rats. We then performed qPCR to validate the DEPs presenting similar expression patterns for the genes selected from proteomic sequencing ( Figures 5D, E ).

Immunohistochemical and Western blot analyses of rat ovaries revealed that Cyp1b1 expression was lower in both the NC group and the NaBu group, whereas it was significantly upregulated in the PCOS group and predominantly localized in granulosa cells ( Figures 6A, B ). These findings indicate that Cyp1b1 expression is increased in the ovaries of PCOS rats, and its expression is downregulated following NaBu treatment. The serum 4-hydroxy-estradiol (4-OHE₂) level of rats was detected by ELISA, and the results showed that there was no significant difference in the serum 4-OHE₂ level of rats in the three groups. However, E₂ was significantly higher in the NaBu group than in the PCOS group, and therefore the ratio of 4-OHE₂/E₂ was significantly higher in the PCOS group ( Figure 6C ).

To investigate the effect of Cyp1b1 overexpression on the mechanism of action in rat ovarian granulosa cells, we conducted primary cell culture. Granulosa cells exhibited excellent growth after being cultured in vitro for 96 hours. Immunofluorescence staining revealed that the cell nuclei were stained blue by DAPI, while the FSHR on the cell membrane showed red fluorescence, confirming that the cultured cells were highly pure granulosa cells ( Figure 7A ). Furthermore, we introduced a lentiviral vector into the granulosa cells to achieve overexpression of Cyp1b1. Immunofluorescence results showed that the GV492-cyp1b1 group exhibited stronger fluorescence signals ( Figure 7B ), indicating successful overexpression of Cyp1b1. RT-qPCR analysis revealed that the mRNA levels of Cyp1b1 were significantly higher compared to the GV492 group. Meanwhile, we observed a significant decrease in the mRNA expression levels of Cyp11a1, StAR, and Hsd3b ( Figure 7C ), suggesting that overexpression of Cyp1b1 suppressed the expression of these key steroidogenic enzyme genes. Western blot analysis at the protein level also confirmed this phenomenon: the expression of Cyp1b1 protein was significantly higher, while the protein levels of Cyp11a1, StAR, and Hsd3b were correspondingly lower ( Figure 7D ). To assess the specific impact of these gene expression changes on steroid hormone synthesis, we measured the concentrations of E₂, P₄, and 4-OHE₂ in the granulosa cell culture medium. The results showed that overexpression of Cyp1b1 significantly reduced the secretion of E₂ and P₄, while the levels of 4-OHE₂ and the ratio of 4-OHE₂ to E₂ were significantly higher ( Figure 7E ). These data suggest that overexpression of Cyp1b1 not only alters the expression patterns of specific genes in granulosa cells but also affects the biosynthesis pathway of steroid hormones.

As previously mentioned, dietary addition of NaBu can improve the morphology of PCOS rats’ polycystic ovaries. Here, the beneficial effects of NaBu on ovaries can be further verified by testing the reproductive performance of the rats. Based on the breeding experiment procedure( Figure 8A ), the rats in the NC group began to give birth on the 20th day after cage confinement, and all 6 rats gave birth on the 33rd day after cage confinement. The rats in NaBu group began to give birth on the 31st day after the cage was closed, and there was still one unpregnant rat within the set time. In the PCOS group, one rat gave birth on day 35 and 39 respectively, and the other four rats were not pregnant ( Figure 8B ). The gestation period of rats generally lasted from 19 to 21 days. In the set time (42d), all the rats in the NC group gave birth, the average litter size was 12, and the reproductive rate was 100%. In the NaBu group, 5 rats gave birth, the reproductive rate was 83.3%, and the average litter size was 10.2. In the PCOS group, only 2 litter were born, the reproduction rate was only 33.3%, and the average litter size was 9 ( Table 1 ). These results suggest that NaBu treatment can improve the reproductive performance of PCOS rats.