Letrozole and high-fat diet induced clinical PCOS-like changes in SD rats
As mentioned above, the pathogenesis of PCOS has been studied from many angles, but no one has investigated the role produced by the NF-κB-TERT pathway in the pathogenesis of PCOS, so we wanted to address this issue in the current study.Hence,a PCOS rat model was established by the treatment of HFD and letrozole. As shown in Fig. 1A and Fig. 1B, the body weight and the ovarian weight of rats in the PCOS group was significantly increased than that of the control group. Compared with the control group, the serum levels of testosterone was significantly higher in PCOS group (Fig. 1C).Estrous cycle disorder is one of the main characteristics of PCOS(38, 39, 40). The normal estrous cycle in rats averages 4–5 days, which is generally divided into four stages—proestrus, estrus, metestrus, and diestrus (34). As shown in Fig. 1D-E, the Ctrl rats had a regular estrous cycle. Briefly, samples dominated by nucleated epithelial cells indicated the proestrus stage, and those with primarily cornified squamous epithelial cells indicated the estrus stage. The metestrus stage was indicated by mixed cells (nucleated, cornified, leucocytes), while the diestrus stage was dominated by leukocytes. Compared with Ctrl rats, the PCOS rats displayed disrupted estrous cycles, which comprised only the metestrus or diestrus phases. Appearance of representative ovaries. Compared with the control group, multiple follicles with cystic expansion presented vacuolated and disorganized structure (Fig. 1F). Assessment of the ovarian morphology showed a significant decrease in the number of follicles and corpus luteum in PCOS rats compared with those in the controls, whereas antral follicles with cystic expansion were increased. The thickness of the granular cell layer was also reduced (Fig. 1G-H).These results proved that we successfully constructed the PCOS rat model.
Figure 1. Effects of Induction of PCOS by Letrozole and High-Fat Diet on Body Weight, Ovarian Weight, Serum Hormone Levels, Estrous Cycle and Ovarian Morphology.
A: body weight of each group; B: Bilateral total ovarian weight in each group;
C: serum testosterone levels in each group;
D: Vaginal smear cytology;
E: Line chart of estrous cycle;
F: ovarian morphology of each group;
G: HE staining scan and magnification of ovarian sections in each group;
H: Counts of cystic follicles and corpus luteum;
CL: corpus luteum; GC: granular cells.**P < 0.01,***P<0.001.
Letrozole and high-fat diet induced PCOS increased the expression of NF-κB-related inflammatory factors and TERT in ovaries
Subsequently, to investigate the association between NF-κB-TERT feedback regulation and ovarian granulosa cell apoptosis in PCOS rats, we initially assessed the levels of NF-κB-related inflammatory factors in ovarian tissues from both groups to elucidate the link between chronic low-grade ovarian inflammation and TERT expression. The qRT-PCR results demonstrated an upregulation of NF-κB, IL-6, TNF-α, and TERT in PCOS rats (Fig. 2A). Western blot analysis further confirmed the elevated levels of NF-κB, IL-6, TNF-α, and TERT in the PCOS group compared to the Ctrl group(Fig. 2B). Immunohistochemistry analysis also supported these findings by revealing increased expression of NF-κB, IL-6, TNF-α ,and TERT in the PCOS group(Fig. 2C). Our findings suggest that induction of PCOS through letrozole administration and a high-fat diet leads to enhanced expression of NF-κB-related inflammatory factors as well as TERT within the ovary. Additionally, telomere length was determined using qPCR analysis on ovarian granulosa cells from PCOS rats. Telomere-specific primers were employed for this purpose. As depicted in Fig. 2D,the telomere length of ovarian granulosa cells exhibited a 1-to2-fold increase compared with that observed in control animals.These outcomes indicate heightened activity of inflammatory markers along with increased telomerase reverse transcriptase (TERT) function within ovarian granulosa cells obtained from PCOS rats.
Figure 2. Effects of letrozole and high-fat diet on PCOS: increase of ovarian NF-κb related inflammatory factors, TERT expression and telomerase relative length
A: The expression changes of NF-κB, IL-6, TNF-α and TERT at mRNA level were detected by qRT-PCR ;
B: Western blotting was used to detect the protein expression of NF-κB, IL-6, TNF-α, and TERT, and ImageJ was used for quantitative analysis;
C: Immunohistochemistry was used to detect the expression changes of NF-κB, IL-6, TNF-α and TERT at protein level.
D: Telomere length quantitative RT-PCR results showed increased telomere length in the ovarian region of PCOS rats compared to controls.
T/S ratio (telomere/single gene ratio);*P < 0.05, **P < 0.01,***P<0.001.
The increase of ovarian cell apoptosis in PCOS rats is positively correlated with NF-κb related inflammatory factors, TERT and apoptosis factors
To further validate our hypothesis, we proceeded to investigate the expression of apoptotic factors at both mRNA and protein levels. In comparison with the Ctrl group, we observed a significant increase in the mRNA levels of Bax and caspase-3, along with a decrease in Bcl-2 mRNA level in GCs from the PCOS group (Fig. 3A). Additionally, compared to the Ctrl group, GCs from the PCOS group exhibited elevated protein levels of Bax and caspase-3, while showing reduced levels of Bcl-2 ,immunohistochemical staining analysis yielded consistent results(Fig. 3B-C). Subsequently, we conducted an extensive analysis to explore any potential relationship between NF-κB-related inflammatory factors, TERT, and apoptotic factors in PCOS; aiming to elucidate whether chronic low-grade ovarian inflammation and up-regulation of TERT are associated with apoptosis. Figure 3D demonstrates a significant correlation among NF-κB-related inflammatory factors, TERT, and apoptotic factors. Specifically, Nf-κb-related inflammatory factors and TERT displayed positive correlations with pro-apoptotic markers such as Bax and Caspase-3; conversely exhibiting negative correlations with anti-apoptotic factor Bcl-2.These data suggest that ovarian granulosa cells undergo apoptosis in PCOS rats, and there is a positive correlation between NF-κB-related inflammatory factors, TERT and apoptosis.
Figure 3. Effects of Induction of PCOS by Letrozole and High-Fat Diet Increases Cell Apoptosis in the Ovary and correlation between NF-κB-related Inflammatory Factors, TERT and Cell Apoptosis Factors in PCOS.
A: The expression changes of Bax, Bcl-2 and Caspase-3 at the mRNA level were detected by qRT-PCR;
B: Western blotting was used to detect the changes in the expression of Bax, Bcl-2 and caspase-3 at the protein level, and ImageJ was used for quantitative analysis;
C: The expression of Bax, Bcl-2 and caspase-3 at the protein level was detected by immunohistochemistry;
D: Correlation analysis of inflammatory factors NF-κB, IL-6, TNF-α, telomerase reverse transcriptase TERT and apoptosis-related factors Bax, Bcl-2, Caspase-3 in ovarian tissue of rats in each group, Correlation between variables was determined by Pearson’s correlation coefficient.P < 0.05 was considered to be statistically significant.
*P < 0.05, **P < 0.01.
After LPS stimulation, NF-κb related inflammatory factors, hTERT expression and apoptosis of KGN cells increased, and there was a correlation among three
The results of animal experiments demonstrated a significant correlation between NF-κB, its downstream inflammatory factors, and TERT with apoptosis-related factors in PCOS rats. To further investigate the relationship between NF-κB-TERT regulation and PCOS granulosa cell apoptosis, KGN cells were treated with LPS at a concentration of 1µg/ml to establish an inflammatory cell model. This model was divided into two groups: Ctrl group and LPS group. qPCR assay was employed to assess changes in mRNA expression levels of NF-κB, its downstream inflammatory factors, hTERT, and apoptosis-related factors in both groups. In the LPS group, there was a significant upregulation of mRNA expression for NF-κB p65 and its downstream inflammatory factors IL-6, TNF-α, hTERT as well as pro-apoptotic factors Bax and Caspase-3 (Fig. 4A). Conversely, the expression of anti-apoptotic factor Bcl-2 was significantly downregulated. Based on our previous animal experiments which revealed a significant correlation between NF-κB, its downstream inflammatory factors, TERT with apoptosis-related factors in PCOS rat ovaries; we found that NF-κB along with its downstream inflammatory factors were positively correlated with pro-apoptotic markers while negatively correlated with anti-apoptotic markers. To validate these findings from animal experiments further analysis was conducted to explore the association between NF-kappa B along with its downstream inflammatory mediators (IL-6,TNF-α), hTERT and apoptotic markers within KGN cells. As depicted in Fig. 4B it is evident that there exists a significant correlation among NF-kappa B along with its downstream inflammatory mediators (IL6,TNF-α) as well as hTERT concerning apoptotic markers. Specifically speaking,NF-κB p65, IL-6 ,TNF-α,and hTERT are positively associatedwith proapoptic proteins such as Bax and Caspase-3 whereas they exhibit negative associationwith antiapoptic protein Bcl-2.These results indicated that LPS-induced KGN cells exhibited increased expression of inflammatory factors and apoptosis, and NF-κB-related inflammatory factors and TERT were positively correlated with apoptosis.
Figure 4. LPS treatment Increases the Expression of NF-κB-related Inflammatory Factors ,hTERT and Cell Apoptosis in KGN Cell and Correlation between NF-κB-related Inflammatory Factors, TERT and Cell Apoptosis Factors after Lipopolysaccharide stimulation in KGN Cell.
A: The expression changes of NF-κB, IL-6, TNF-α、TERT、Bax, Bcl-2 and Caspase-3 at the mRNA level were detected by qRT-PCR;
B: The correlation between inflammatory factors (NF-κB, IL-6, TNF-α, TERT) and apoptosis-related factors (Bax, Bcl-2, Caspase-3) in KGN cells after LPS stimulation was analyzed. Pearson correlation coefficient was used to determine the correlation between variables. P < 0.05 was considered statistically significant.
*P < 0.05, **P < 0.01.
Inhibition of NF-KB and TERT reduced the expression of NF-κB-related inflammatory factors and TERT in KGN cells treated with LPS
To further elucidate the mechanism underlying NF-κB-TERT feedback regulation on apoptosis of ovarian granulosa cells in PCOS rats, we treated LPS-induced KGN cells with 10µg/ml BAY 11-7082 and 40 µg/ml BIBR1532, respectively. The experimental groups were divided into Ctrl group (control), LPS group (LPS induction only), LPS + NF-κB inhibitor group (LPS induction + NF-κB inhibitor treatment), and LPS + TERT inhibitor group (LPS induction + TERT inhibitor treatment). qPCR and Western Blot analyses were employed to assess the expression levels of NF-κB, its downstream inflammatory factors, and hTERT in these four groups. Our results demonstrated a significant upregulation of NF-κB p65 along with its downstream inflammatory factors IL-6, TNF-α, and hTERT in the LPS group compared to the Ctrl group. However, both the groups treated with NF-κB inhibitor BAY 11-7082 and TERT inhibitor BIBR1532 exhibited a significant downregulation of NF-κB p65 as well as its downstream inflammatory factors IL-6, TNF-α, and hTERT when compared to the LPS group (Fig. 5A-B). To further investigate changes induced by LPS in KGN cells regarding inflammatory factors secretion, we quantified TNF-α and IL-6 levels in cell culture medium using ELISA assay (Fig. 5C). Our findings revealed significantly increased levels of these inflammatory factors in the LPS group compared to both control and inhibitor groups.
Figure 5. Changes in the expression of NF-κB and its downstream inflammatory factors, TERT and telomerase activity in KGN cells in each group.
A: The expression changes of NF-κB, IL-6, TNF-α and TERT at mRNA level were detected by qRT-PCR;
B-C: western blotting was used to detect the protein expression of NF-κB, IL-6, and TNF-α, and ImageJ was used for quantitative analysis;
D: ELISA was used to detect the content of inflammatory factors (TNF-ɑ, IL-6) in the cell culture medium;
*P < 0.05, **P < 0.01,***P<0.001.