Tenascin C activates the toll‑like receptor 4/NF‑κB signaling pathway to promote the development of polycystic ovary syndrome

Abstract

Polycystic ovary syndrome (PCOS) is a globally prevalent gynecological disorder among women of childbearing age. The present study aimed to investigate the role of tenascin C (TNC) in PCOS and its potential mechanisms. Fasting blood glucose and serum insulin, the homeostasis model assessment of insulin resistance and the serum hormone levels were determined in PCOS rats. In addition, H&E staining was used for assessing pathology. In addition, the effects of TNC on oxidative stress and inflammation response in PCOS rat and cell models was assessed. Furthermore, the roles of TNC on KGN cell proliferation and apoptosis were determined employing EdU assay and flow cytometry. TLR4/NF‑κB pathway‑related proteins were measured using western blotting, immunofluorescence and immunohistochemistry. It was found that the mRNA and protein expression was upregulated in PCOS rats and in KGN cells induced by dihydrotestosterone (DHT). Knockdown of TNC relieved the pathological characteristics and the endocrine abnormalities of PCOS rats. Knockdown of TNC inhibited ovarian cell apoptosis, oxidative stress and inflammation in PCOS rats. Knockdown of TNC reversed the DHT‑induced reduction in cell proliferation and increase in apoptosis in KGN cells. Furthermore, knockdown of TNC alleviated oxidative stress and inflammatory responses induced by DHT in KGN cells. Additionally, knockdown of TNC inhibited the toll‑like receptor 4 (TLR4)/NF‑κB signaling pathway in PCOS rats and DHT‑treated KGN cells. In conclusion, knockdown of TNC could ameliorate PCOS in both rats and a cell model by inhibiting cell apoptosis, oxidative stress and inflammation via the suppression of the TLR4/NF‑κB signaling pathway.

Keywords: inflammation; insulin resistance; oxidative stress; polycystic ovary syndrome; tenascin C; toll‑like receptor 4/NF‑κB pathway.

Figure 1.

TNC is highly expressed in PCOS rats. (A) TNC levels in tissues from patients with PCOS and control normal tissues from the Gene Expression Omnibus dataset GSE124226. (B) TNC levels in PCOS rats and normal rats were detected using reverse transcription-quantitative PCR. (C) Immunohistochemistry to assess TNC expression in PCOS rats and normal rats. (D) Western blotting to detect TNC expression in PCOS rats and normal rats. *P<0.05, compared with control group. PCOS, polycystic ovary syndrome; TNC, tenascin C.

Figure 2.

Knockdown of TNC relieves the pathological characteristics and the endocrine abnormalities of PCOS rats. (A) TNC levels in different groups were detected using reverse transcription-quantitative PCR. (B) Fasting blood glucose, (C) fasting serum insulin and (D) HOMA-IR in different groups. ELISAs detecting the serum levels of (E) LH, (F) FSH, (G) testosterone and (H) estradiol. (I) H&E staining in different groups. C, corpora luteum; F, cyst-like follicles. *P<0.05 vs. control group; ^#P<0.05 vs. PCOS + sh-NC group. FSH, follicle-stimulating hormone; HOMA-IR, homeostasis model assessment of insulin resistance; LH, luteinizing hormone; NC, negative control; PCOS, polycystic ovary syndrome; sh, short hairpin RNA; TNC, tenascin C.

Figure 3.

Knockdown of TNC inhibits ovarian cell apoptosis, oxidative stress and inflammation in PCOS rats. (A) Western blot analysis of Bcl-2 and cleaved caspase-3 in rats of different groups. (B) MDA, GSH and SOD levels in rats of different groups. (C) TNF-α, IL-1β, IL-6 and IL-18 levels in rats of different groups. *P<0.05 vs. control group; ^#P<0.05 vs. PCOS + sh-NC group. GSH, glutathione; MDA, malondialdehyde; NC, negative control; PCOS, polycystic ovary syndrome; sh, short hairpin RNA; SOD, superoxide dismutase; TNC, tenascin C.

Figure 4.

Knockdown of TNC inhibits the activation of the TLR4/NF-κB pathway in PCOS rats. (A) KEGG analysis demonstrated that TNC promoted the TLR4/NF-κB signaling pathway. (B) Western blot analysis of TLR4, p-NF-κB and NF-κB in rats of different groups. (C) Immunohistochemistry to assess TLR4 and p-NF-κB levels in rats of different groups. *P<0.05 vs. control group; ^#P<0.05 vs. PCOS + sh-NC group. KEGG, Kyoto Encyclopedia of Genes and Genomes; NC, negative control; p-, phosphorylated; PCOS, polycystic ovary syndrome; sh, short hairpin RNA; TLR4, toll-like receptor 4; TNC, tenascin C.

Figure 5.

Knockdown of TNC reverses the reduced proliferation and elevated apoptosis in KGN cells induced by DHT. (A) TNC levels in different groups were detected using RT-qPCR in KGN cells without DHT treatment but knockdown of TNC. (B) TNC levels in different groups were detected using RT-qPCR in KGN cells with DHT treatment and knockdown of TNC. (C) EdU assay to evaluate proliferation. (D) Flow cytometry was utilized to evaluate apoptosis. (E) Western blot analysis of Bcl-2 and cleaved caspase-3 in KGN cells of different groups. *P<0.05 vs. control group; ^#P<0.05 vs. DHT + si-NC group. DHT, dihydrotestosterone; EdU, 5-ethynyl-2′-deoxyuridine; NC, negative control; RT-qPCR, reverse transcription-quantitative PCR; si, small interfering RNA; TNC, tenascin C.

Figure 6.

Knockdown of TNC alleviates the oxidative stress and inflammatory response in KGN cells induced by DHT. (A) MDA, GSH and SOD levels in KGN cells of different groups. (B) TNF-α, IL-1β, IL-6 and IL-18 levels in KGN cells of different groups. *P<0.05 vs. control group; ^#P<0.05 vs. DHT + si-NC group. DHT, dihydrotestosterone; GSH, glutathione; MDA, malondialdehyde; NC, negative control; si, small interfering RNA; SOD, superoxide dismutase; TNC, tenascin C.

Figure 7.

Knockdown of TNC inhibits the activation of the TLR4/NF-κB signaling pathway in DHT-treated KGN cells. (A) Western blot analysis of TLR4, p-NF-κB and NF-κB in KGN cells of different groups. (B) Immunofluorescence analysis to assess TLR4 expression in KGN cells of different groups (C) Immunofluorescence analysis to assess p-NF-κB expression in KGN cells of different groups. *P<0.05 vs. control group; ^#P<0.05 vs. DHT + si-NC group. DHT, dihydrotestosterone; NC, negative control; p-, phosphorylated; si, small interfering RNA; TLR4, toll-like receptor 4; TNC, tenascin C.

Figure 8.

A schematic diagram showing the findings of the present study. Knockdown of TNC ameliorates PCOS by inhibiting cell apoptosis, oxidative stress and inflammation, and attenuating IR via suppression of the TLR4/NF-κB signaling pathway. IR, insulin resistance; PCOS, polycystic ovary syndrome; TLR4, toll-like receptor 4; TNC, tenascin C