The mechanism of NF-κB-TERT feedback regulation of granulosa cell apoptosis in PCOS rats

Abstract

Patients with Polycystic ovary syndrome (PCOS) have chronic low-grade ovarian inflammation. Inflammation can cause telomere dysfunction, and telomere and telomerase complex are also involved in regulating inflammation. However, the specific mechanisms of inflammatory signaling feedback and telomere-telomerase mutual regulation remain to be discovered. This study elucidates the role of Nuclear factor kappa-B (NF-κB)-Telomerase reverse transcriptase (TERT) feedback in PCOS granulosa cell apoptosis. Using letrozole and a high-fat diet, a PCOS rat model was established, along with a Lipopolysaccharide (LPS) -treated KGN cell inflammation model was established. NF-κB and TERT inhibitors (BAY 11-7082 and BIBR1532) were then administered to LPS-induced KGN cells. PCOS rats displayed disrupted estrous cycles, increased weight, elevated serum testosterone, cystic follicles, granulosa cell layer thinning, and reduced corpora lutea count (P are all less than 0.05). In PCOS rat ovaries, NF-κB, Interleukin-6 (IL-6), Tumor Necrosis Factor α (TNF-α), TERT, Bax, and Caspase-3 exhibited notable upregulation, while Bcl-2 decreased, with telomere elongation (P are all less than 0.05). There were significant correlations among NF-κB-related inflammatory factors, TERT and apoptotic factors, and they were positively correlated with Bax and Caspase-3, and negatively correlated with Bcl-2 (P are all less than 0.05). LPS-treated KGN cells demonstrated increased expression of inflammatory and pro-apoptotic factors, later restored post-treatment with NF-κB and TERT inhibitors (P are all less than 0.05). In conclusion, TERT may induce granulosa cell apoptosis by participating in the regulation of the NF-κB signaling pathway, thereby mediating the chronic inflammatory response of PCOS through downstream inflammatory factors IL-6 and TNF-α.

Fig 1. Effects of letrozole and high-fat diet induction on body weight, ovarian weight, serum hormone levels, estrous cycle, and ovarian morphology.

A: Body weight of each group (n = 6 per group, day 60). B: Bilateral total ovarian weight in each group. C: Serum testosterone levels in each group (n = 6 per group). D: Cytological assessment of vaginal smears (n = 5 per group, day 51–60). E: Line chart of estrous cycle (n = 5 per group, day 51–60). F: Ovarian morphology of each group. G: HE staining scan and magnification of ovarian sections in each group (n = 6 per group, scale bar in upper is 500 μm). The lower panel show the higher magnification of the box area in the upper panel respectively. H: Counts of cystic follicles and corpus luteum (n = 6 per group). CL: corpus luteum; GC: granulosa cells. Significant differences between groups were indicated as **P < 0.01 and ***P < 0.001.

Fig 2. The levels of NF-κB related inflammatory factors, TERT expression and telomerase relative length are significantly increased in the ovarian tissue of PCOS rats.

A: The expression changes of NF-κB, IL-6, TNF-α and TERT at mRNA level were detected by qRT-PCR (n = 3 per group). B-C: 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 (n = 3 per group). D: Immunohistochemistry was used to detect the expression changes of NF-κB, IL-6, TNF-α and TERT at protein level (n = 4 per group), the lowest panel shows the negative control. E: Telomere length quantitative qRT-PCR results showed increased telomere length in the ovarian region of PCOS rats compared to controls (n = 3 per group). T/S ratio (telomere/single gene ratio); Data were presented as mean ± SEM. Significant differences were respectively presented as *P < 0.05, **P < 0.01, ***P < 0.001.

Fig 3. Apoptosis is increased in the ovary of PCOS rats, and there is a correlation between NF-κB-related inflammatory factors, TERT and apoptosis factors.

A: The expression changes of Bax, Bcl-2 and Caspase-3 at the mRNA level were detected by qRT-PCR (n = 3 per group). B-C: 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 (n = 3 per group). D: The expression of Bax, Bcl-2 and caspase-3 at the protein level was detected by immunohistochemistry (n = 4 per group), the lowest panel shows the negative control. E: 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. Data were presented as mean ± SEM. Significant differences were respectively presented as *P < 0.05 and **P < 0.01.

Fig 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 LPS stimulation in KGN cell.

A: The mRNA expression of NF-κB, IL-6, TNF-α, TERT, Bax, Bcl-2 and Caspase-3 in KGN cells in each group was 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. Data were presented as mean ± SEM. Significant differences were respectively presented as *P < 0.05 and **P < 0.01.

Fig 5. Changes in the expression of NF-κB-related inflammatory factors, TERT and telomerase activity in KGN cells in each group.

A: The mRNA expression of NF-κB, IL-6, TNF-α and TERT in KGN cells of each group was detected by qRT-PCR. B-C: Western blotting detected the expression of NF-κB, IL-6 and TNF-α proteins in KGN cells of each group, 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. Data were presented as mean ± SEM. Significant differences were respectively presented as *P< 0.05, **P< 0.01, ***P<0.001.

Fig 6. Changes in the expression of apoptosis-related factors Bax, Bcl-2, and Caspase-3 in KGN cells in each group.

A: The mRNA expression of Bax, Bcl-2 and Caspase-3 in KGN cells of each group was detected by qRT-PCR. B-C: Western blotting detected the expression of Bax, Bcl-2 and Caspase-3 proteins in KGN cells of each group, and ImageJ was used for quantitative analysis. D: The apoptosis of KGN cells in each group was detected by flow cytometry. Data were presented as mean ± SEM. Significant differences were respectively presented as *P< 0.05 and ***P<0.001.

Fig 7. The NF-κB-TERT in PCOS ovarian feedback regulation mechanism of simple model.

TERT may participate in regulating the NF-κB signaling pathway, activating downstream inflammatory factors IL-6 and TNF-α, mediating the chronic inflammatory response of PCOS, and inducing apoptosis of ovarian granulosa cells.