Berberine exerts a protective effect on rats with polycystic ovary syndrome by inhibiting the inflammatory response and cell apoptosis

All animal experiments were conducted in strict accordance with the animal ethics standards and with ethics approval from the Ethics Committee of Obstetrics and Gynecology Hospital of Fudan University (no. [2015]45).

Female clean Sprague Dawley rats (6 weeks old, total n = 42) were obtained from the Shanghai Laboratory Animal Centre (SLAC, Shanghai, China), and were adaptively fed for 3 days. The PCOS rat models were established following the methods reported by Abramovich et al. [18] with some modifications. The rats were first randomly divided into the following six groups (n = 7 per group): control, control + BBR, PCOS-high fat diet (HFD), PCOS-HFD + BBR, PCOS-normal diet (ND), and PCOS-ND + BBR. The rats in the PCOS-HFD and PCOS-HFD + BBR groups were fed with an HFD and were injected with DHEA (source leaf organisms, S24516; 6 mg/100 g body weight with 0.2 ml of sesame oil) for 21 days to induce PCOS in IR rat models. The rats in the PCOS-ND and PCOS-ND + BBR groups were fed with an ND and injected with DHEA. The other rats were fed with an ND and injected with 0.2 ml of sesame oil (control and control + BBR groups). The main energy materials of an ND and an HFD are shown in Table 1. Methylene blue staining was used to examine the changes of vaginal epithelial cells in the DHEA-treated rats under an optical microscope. The PCOS model was considered successful if the vaginal epithelial cells were continuously keratinized for 10 consecutive days [19]. After 12 h of fasting (20,00 to 8:00), the venous blood was extracted from the tails of the PCOS rats. Fasting plasma glucose (FPG) levels were determined by using the ACCU-CHEK Performa glucose [20] meter (Roche Diabetes Care, USA), and the fasting insulin (FINS) levels were determined using rat insulin ELISA kits (Thermo Scientific, USA). In accordance with the homeostasis model assessment of IR (HOMA-IR) method [21], HOMA-IR = FINS × FPG/22.5. HOMA-IR > 2.8 of the PCOS rats was considered to indicate the presence of IR [11].

Subsequently, the rats in the control + BBR, PCOS-HFD + BBR, and PCOS-ND + BBR groups were gavaged with BBR (150 mg/kg/d, Sigma-Aldrich, Louis, MO, USA). The BBR was dissolved with 0.5% sodium carboxymethyl cellulose [Sangon Biotech (Shanghai) Co., LTD, Shanghai, China]. The rats in the other groups were gavaged daily with an equal volume of 0.5% sodium carboxymethyl cellulose. The treatments were administered for 6 weeks.

At the end of the experiment, the body weights and HOMA-IR of all rats were determined. Further, all rats were euthanized by cervical dislocation, and serum and ovary tissue samples were collected from all rats. Additionally, a part of each ovary tissue sample was fixed in polyformic acid solution. The remaining ovary tissue was stored at − 80 °C for subsequent experiments.

For histological analysis, the fixed ovary tissue was embedded in paraffin, sectioned to a thickness of 5 mm, and stained with hematoxylin and eosin (HE). Slides were scanned using the OPTIKA microscope (M.A.D. Co. LTD, Italy) and the morphological changes of the ovary tissues were observed.

According to the manufacturer’s instructions, the levels of insulin and testosterone in sera were respectively measured using rat insulin and rat testosterone ELISA kits (KGE010, R&D systems, USA).

The cell apoptosis rate was investigated by using the TUNEL assay kit (Roche Applied Science, Penzberg, Germany). Briefly, slides with 5-μm sections of the ovary tissues were dewaxed by xylene and washed with ethyl alcohol. After sealing with H₂O₂ for 30 min, 20 mg/L of protease K was added to the samples. After 20 min, the TUNEL reaction solution was added and incubated for 60 min at 37 °C. Then, anti-luciferin antibodies linked by peroxidase were added and incubated for 30 min at 37 °C. Subsequently, the slides were stained with DAB and restained with hematoxylin. The number of apoptotic cells was observed and calculated under an optical microscope (M.A.D. Co. LTD, Italy). The positive apoptotic cell nuclei were dark brown in color, and the non-apoptotic cell nuclei were blue in color. The Image pro plus 6.0 software was utilized to determine the number of TUNEL-positive cells.

Total RNA in the ovary tissues was isolated using the TRIzol reagent (TIANGEN, Shanghai, China) according to the manufacturer’s instructions. The RNA quantity and quality were evaluated with a microplate reader (Tecan Group LTD, Männedorf, Switzerland). Afterward, cDNA was synthesized using a cDNA Reverse Transcription Kit (TaKaRa Bio Inc., Shiga, Japan) following the manufacturer’s instructions. RT-qPCR was performed using a fluorescence ration PCR instrument (Applied Biosystems Inc., Foster City, CA, USA) with SYBR Green Ι (Thermo, Waltham, MA, USA). The sequences of all primers are shown in Table 2. The PCR conditions were as follows: 3 min at 50 °C, 3 min at 95 °C, 40 cycles of 10 s at 95 °C, 30 s at 60 °C, and 15 s at 72 °C. GAPDH was used as the reference gene.

The protein levels of toll-like receptor 4 (TLR4), phosphatidylinositol 3-kinase (PI3K), AKT serine (AKT), tumor necrosis factor-α (TNF-α), LYN proto-oncogene, Src family tyrosine kinase (LYN), and caspase-3 were determined by western blotting. The total proteins of ovary tissues were extracted with a protein extraction kit (Beijing Baiolaibo Technology Co. LTD, Beijing, China) and a BCA protein assay kit (Boster Biological Technology Co. LTD, Wuhan, China) was used to determine the concentration of total protein. Furthermore, the proteins were separated by 12% SDS-PAGE, transferred onto a PVDF membrane, and blocked with 5% BSA. The membranes were then incubated with anti-TLR4 (1:2000, Abcam), anti-T-PI3K (1:5000, Abcam), anti-p-PI3K (1:5000, Abcam), anti-T-AKT (1:5000, Abcam), anti-p-Akt (1:5000, Abcam), anti-TNF-α (1:2000, Abcam), anti-IL-1 (1:2000, Abcam), anti-IL-6 (1:2000, Abcam), anti-caspase-3 (1:5000, Abcam), anti-cleaved caspase-3 (1:5000, Abcam), anti-LYN (1:5000, Abcam), and anti-GAPDH (1:5000, Abcam) antibodies, respectively, at room temperature overnight. Thereafter, IgG-HRP was added to the membranes and incubated at room temperature for 2 h. After washing five times, the protein bands were visualized with ECL.

Statistical analyses were performed using GraphPad InStant 3 (San Diego, CA, USA). The data are reported as mean ± standard deviation (SD). One-way analysis of variance was used for comparison among more than two groups, and the Tukey’s test was subsequently performed to assess group differences. For comparisons of two groups, the Student’s t-test was applied. A p-value of less than 0.05 was considered to indicate a significant difference.

We compared the rat ovarian morphology in the control, PCOS-ND, and PCOS-HFD groups to identify any changes. HE staining and electron microscopy results showed that the rats in the control group had normal ovarian histology with multiple lutea and eight to nine layers of granulosa cells with the dominant follicle (Fig. 1a, Supplementary Fig. 1). However, in PCOS-ND and PCOS-HFD groups, the ovarian tissues exhibited follicular cysts, a reduction of the granulosa cell layer, and hyperplasia (Fig. 1b, c, Supplementary Fig. 1). These results indicated that the PCOS model was successfully established.

To evaluate the effects of BBR on PCOS rats, the body weight and HOMA-IR were evaluated. There were no significant differences in the body weight and fasting blood glucose levels among the control, control + BBR, PCOS-ND, and PCOS-ND + BBR groups (P > 0.05, Fig. 2a, b). However, the body weights of rats in the PCOS-HFD and PCOS-HFD + BBR groups were significantly higher than those of other groups (P < 0.05, Fig. 2a), which indicated that an HFD caused significant weight gain in PCOS rats. Additionally, the fasting blood glucose levels in the PCOS-HFD and PCOS-HFD + BBR groups were 9.57 ± 0.59 and 7.63 ± 0.50 mM, respectively, indicating a significant decrease after BBR treatment in PCOS-HFD rats (P < 0.05, Fig. 2b). For fasting insulin, there was no significant difference in its levels between the control and control + BBR groups (P > 0.05), while its levels were higher in other groups (P < 0.05, Fig. 2c). The intra-assay coefficient of insulin measurements by ELISA was 8.6%. Moreover, compared to the groups not treated with BBR, the HOMA-IR value was significantly decreased after BBR administration (P < 0.05, Fig. 2d), indicating that BBR might have a protective effect on IR in PCOS rats.

The intra-assay coefficient of testosterone determined by ELISA was 7.3%. There was no significant difference in testosterone levels between the control and control + BBR groups (P > 0.05, Fig. 3). Additionally, compared with the control group, the testosterone levels in the PCOS rats were significantly greater (P < 0.05, Fig. 3). However, after BBR administration, the levels of testosterone decreased (P < 0.05); in the PCOS-ND + BBR group, the levels returned to a level which was similar to that of the control group (P > 0.05).

To investigate the effect of BBR on cell apoptosis in PCOS rats, a TUNEL assay kit was used. The values of apoptosis of interest (AOI) in the control and control + BBR groups were similar (P > 0.05), while those in the PCOS-ND and PCOS-HFD groups were significantly higher (P < 0.05, Fig. 4). After BBR treatment, the AOI value decreased (P < 0.05, Fig. 4), indicating that BBR inhibited cell apoptosis caused by PCOS.

RT-qPCR was performed to evaluate the mRNA expression of TLR4, LYN, PI3K, Akt, NF-kB, TNF-α, IL-1, IL-6, caspase-3, CD14, COL15A1, LY96, CXCL6, CXCL16 and SHC4 among the different groups. The expression levels of TLR4, LYN, PI3K, Akt, NF-kB, TNF-α, IL-1, IL-6, and caspase-3 in the control and control + BBR groups were similar (P > 0.05, Fig. 5), suggesting that BBR exerted minor side effects on PCOS. However, in PCOS rats (rats in PCOS-ND and PCOS-HFD groups), these levels were significantly higher (P < 0.05, Fig. 5); compared with the PCOS-ND group, the expression levels of TLR4, LYN, PI3K, NF-kB, TNF-α, IL-1, and IL-6 in the PCOS-HFD group were significantly higher (P < 0.05, Fig. 5). Further, after BBR administration, the expression levels significantly decreased (P < 0.05, Fig. 5), and this decrease was greater in the PCOS-HFD group compared to that in the PCOS-ND group, indicating that BBR demonstrated a better therapeutic effect on PCOS-HFD rats. For CD14, COL15A1, LY96, CXCL6, CXCL16 and SHC4, there were no significant differences in their expression among these different groups (P > 0.05, Supplementary Fig. 2).

Western blotting results showed that there were no significant differences in the protein levels of related genes in ovarian tissues (Fig. 6). The protein levels of TLR4 in PCOS rats were higher than those in control rats. Meanwhile, these levels in the PCOS-ND + BBR group were similar to those of the PCOS-ND group (P > 0.05). Finally, the protein levels in the PCOS-HFD + BBR group were lower compared to those in the PCOS-HFD group (P < 0.05, Fig. 6a, b). Furthermore, the protein expressions of LYN, TNF-α, IL-1, and IL-6 in PCOS rats were significantly upregulated compared to those of the control rats (P < 0.05). Additionally, after BBR treatment, the expression levels were significantly downregulated (P < 0.05, Fig. 6a, c, d, i, j). The levels of p-PI3K/T-PI3K and p-Akt/T-Akt in the PCOS rats were significantly higher than those in the control group (P < 0.05), whereas these levels were evidently decreased after BBR treatment (P < 0.05, Fig. 6a, e, f). Additionally, there were no significant differences in caspase-3 protein levels among the control, PCOS-ND, and PCOS-ND + BBR groups (P > 0.05). However, compared with the control group, the expression of caspase-3 in the PCOS-HFD group was significantly upregulated (P < 0.05), while after BBR administration, its expression was downregulated in the PCOS-HFD + BBR group (P < 0.05, Fig. 6a, g). For cleaved caspase-3, its protein levels significantly increased in the PCOS rats compared to those of the control rats. Meanwhile, after BBR treatment, the protein levels were markedly decreased (P < 0.05, Fig. 6a, h).