Journal: Journal of ovarian research

Article Title: Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

doi: 10.1186/s13048-023-01242-x

Figure Lengend Snippet: Fig. 1 DHEA induces ovarian dysfunction, and IRE1α levels are increased in the ovaries of PCOS rats. Rats received DHEA for induction of polycystic ovarian syndrome. A H&E staining was carried out for showing ovarian and follicular morphology (upper panel). The percentages of each type of the follicles were quantified (lower panel). B The protein levels of AR and cytochrome P450 family enzymes (CYP11α1, CYP19α1) in ovaries were assessed by western blot. C Serum LH and FSH levels were analyzed using ELISA. D-E The expression of NLRP3 inflammasome activation factors (GSDME, GSDMD, GSDMD-C, ASC, IL-1β and IL-18) (D) and fibrosis factors (collagen I, β-catenin, α-SMA, TGF-β) (E) in ovaries was assessed by western blot. F IRE1α expression in ovaries was analyzed by immunohistochemical staining. G Levels of IRE1α were measured using western blot. Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. DHEA, dehydroepiandrosterone; AR, androgen receptor; PAF, preantral and early antral follicle; AF, antral follicle; CF, cystic follicles; CL, corpus luteum; CYP11α1, cytochrome p450 11; CYP19α1, cytochrome p450 19; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; LH, luteinizing hormone; FSH, follicle stimulating hormone; GSDME, gasdermin E; GSDMD, gasdermin D; GSDMD-C, C-terminal fragment of gasdermin D; ASC, apoptosis-associated speck-like protein containing a CARD; IL-1β, interleukin-1β; IL-18, interleukin-18; α-SMA, a-smooth muscle actin; TGF-β, transforming growth factor-beta; IRE1α, inositol-requiring enzyme 1α

Article Snippet: Samples of ovarian tissues were sectioned at a thickness of 4 μm and stained with specific antibodies against IRE1α (1:100, Proteintech, China).

Techniques: Staining, Western Blot, Enzyme-linked Immunosorbent Assay, Expressing, Activation Assay, Immunohistochemical staining

Journal: Journal of ovarian research

Article Title: Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

doi: 10.1186/s13048-023-01242-x

Figure Lengend Snippet: Fig. 2 IRE1α silencing by siRNA rescues DHT-induced dysfunction of GCs. GCs from naïve rats were treated with DHT, or were transfected with IRE1α siRNA followed by treatment with DHT. A The mRNA levels of IRE1α in GCs were analyzed by qRT-PCR. B The protein levels of IRE1α in GCs were analyzed by western blot. C Levels of IRE1α in GCs (Alexa Fluor 488) were analyzed by immunofluorescence staining using an Alexa Fluor 488-conjugated antibody (60 ×). D-F The expression of IRE1α, TXNIP, NLRP3 (D), NLRP3 inflammasome activation factors (GSDMD, ASC) (E) and fibrosis factors (β-catenin, TGF-β) (F) in GCs were assessed by qRT-PCR. G-H The protein levels of ASC (G) and α-SMA (H) in GCs were analyzed by immunofluorescence staining (60 ×). I ROS in GCs were assessed by the DCF-DA probe using confocal microscopy (90 ×). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. IRE1α, inositol-requiring enzyme 1α; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ctrl, control; DHT, dihydrotestosterone; DAPI, 4’,6-diamidino-2-phenylindole; NC, normal contrast; si, siRNA; TXNIP, thioredoxin-interacting protein; NLRP3, NOD-like receptor family pyrin domain containing 3; GSDMD, gasdermin D; ASC, apoptosis-associated speck-like protein containing a CARD; TGF-β, transforming growth factor-beta; DCF-DA, dichlorofluorescein diacetate; ROS, reactive oxygen species

Article Snippet: Samples of ovarian tissues were sectioned at a thickness of 4 μm and stained with specific antibodies against IRE1α (1:100, Proteintech, China).

Techniques: Transfection, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, Expressing, Activation Assay, Confocal Microscopy, Control

Journal: Journal of ovarian research

Article Title: Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

doi: 10.1186/s13048-023-01242-x

Figure Lengend Snippet: Fig. 3 Exercise suppresses the IRE1α-TXNIP/ROS-NLRP3 pathway and improves the phenotype of PCOS rats. Rats received DHEA to induce polycystic ovary syndrome followed by exercise treatment. A Tissues were stained with H&E for revealing ovarian and follicular morphology. B Abdominal adipose tissue were stained with H&E. C Serum LH and FSH levels were analyzed using ELISA. D The expression of AR and CYP11α1 in ovaries was assessed by western blot. E–F The expression of NLRP3 inflammasome activation factors (GSDMD, ASC, IL-1β and IL-18) (E) and fibrosis factors (TGF-β, α-SMA, β-catenin and fibronectin) (F) in ovaries was assessed by qRT-PCR. (G-H) The expression of NLRP3 inflammasome activation factors (GSDMD, GSDMD-C, ASC, IL-1β and IL-18) (G) and fibrosis factors (collagen I, β-catenin, P-SMAD3, α-SMA, TGF-β) (H) in ovaries was assessed by western blot. I Collagen in ovarian slices was revealed by Masson staining. J-K Serum MDA levels (J) and SOD activity (K) were analyzed using an enzymatic colorimetric method. L The expression levels of IRE1α, TXNIP and NLRP3 in ovaries were analyzed by western blot assay. Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. DHEA, dehydroepiandrosterone; D + E, DHEA + Exercise; LH, luteinizing hormone; FSH, follicle stimulating hormone; AR, androgen receptor; CYP11α1, cytochrome p450 11; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; GSDMD, gasdermin D; ASC, apoptosis-associated speck-like protein containing a CARD; IL-1β, interleukin-1β; IL-18, interleukin-18; TGF-β, transforming growth factor-beta; α-SMA, a-smooth muscle actin; GSDMD-C, C-terminal fragment of gasdermin D; MDA, malondialdehyde; SOD, superoxide dismutase; IRE1α, inositol-requiring enzyme 1α; TXNIP, thioredoxin-interacting protein; NLRP3, NOD-like receptor family pyrin domain containing 3

Article Snippet: Samples of ovarian tissues were sectioned at a thickness of 4 μm and stained with specific antibodies against IRE1α (1:100, Proteintech, China).

Techniques: Staining, Enzyme-linked Immunosorbent Assay, Expressing, Western Blot, Activation Assay, Quantitative RT-PCR, Activity Assay

Journal: Journal of ovarian research

Article Title: Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

doi: 10.1186/s13048-023-01242-x

Figure Lengend Snippet: Fig. 5 Irisin mediates skeletal muscle-ovary crosstalk and inhibits the IRE1α-TXNIP/ROS-NLRP3 signaling pathway in GCs. GCs were treated with DHT, in the presence or absence of various concentrations of irisin. A-B Cell viability was measured by CCK8. (C-D) The mRNA (C) and protein (D) levels of IRE1α, TXNIP and NLRP3 in GCs were analyzed by qRT-PCR and western blot. (E) The expression of IRE1α in GCs was analyzed by immunofluorescence staining (60 ×). F-G GC MDA levels (F) and SOD activity (G) were analyzed using an enzymatic colorimetric method. H ROS in GCs was assessed by the DCF-DA probe using confocal microscopy (90 ×). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. DHT, dihydrotestosterone; IRE1α, inositol-requiring enzyme 1α; TXNIP, thioredoxin-interacting protein; NLRP3, NOD-like receptor family pyrin domain containing 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ctrl, control; DAPI, 4’,6-diamidino-2-phenylindole; MDA, malondialdehyde; SOD, superoxide dismutase; DCF-DA, dichlorofluorescein diacetate; CCK8, Cell Counting Kit-8

Article Snippet: Samples of ovarian tissues were sectioned at a thickness of 4 μm and stained with specific antibodies against IRE1α (1:100, Proteintech, China).

Techniques: Quantitative RT-PCR, Western Blot, Expressing, Immunofluorescence, Staining, Activity Assay, Confocal Microscopy, Control, Cell Counting

Journal: Journal of ovarian research

Article Title: Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

doi: 10.1186/s13048-023-01242-x

Figure Lengend Snippet: Fig. 6 Irisin mediates skeletal muscle-ovary crosstalk and inhibits the IRE1α-TXNIP/ROS-NLRP3 signaling pathway in TCs. TCs were treated with DHT, in the presence or absence various concentrations of irisin. A Cell viability was measured by CCK8. B and D The protein (B) and mRNA (D) levels of IRE1α, TXNIP and NLRP3 in TCs were analyzed by western blot and qRT-PCR. (C) The expression of NLRP3 in TCs was analyzed by immunofluorescence staining (60 ×). E–F TC MDA levels (E) and SOD activity (F) were analyzed using an enzymatic colorimetric method. G ROS in TCs was assessed by the DCF-DA probe using confocal microscopy (90 ×). Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. DHT, dihydrotestosterone; IRE1α, inositol-requiring enzyme 1α; TXNIP, thioredoxin-interacting protein; NLRP3, NOD-like receptor family pyrin domain containing 3; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Ctrl, control; DAPI, 4’,6-diamidino-2-phenylindole; MDA, malondialdehyde; SOD, superoxide dismutase; DCF-DA, dichlorofluorescein diacetate; CCK8, Cell Counting Kit-8

Article Snippet: Samples of ovarian tissues were sectioned at a thickness of 4 μm and stained with specific antibodies against IRE1α (1:100, Proteintech, China).

Techniques: Western Blot, Quantitative RT-PCR, Expressing, Immunofluorescence, Staining, Activity Assay, Confocal Microscopy, Control, Cell Counting

Journal: Journal of ovarian research

Article Title: Exercise-induced irisin improves follicular dysfunction by inhibiting IRE1α-TXNIP/ROS-NLRP3 pathway in PCOS.

doi: 10.1186/s13048-023-01242-x

Figure Lengend Snippet: Fig. 8 Irisin inhibits TCs inflammasome activation and fibrosis. TCs were treated with DHT, in the presence or absence of irisin. A The expression of ASC and GSDMD in TCs was assessed by qRT-PCR. B The expression of NLRP3 inflammasome activation factors (GSDMD, GSDMD-C, ASC, IL-1β and IL-18) in TCs was analyzed by western blot assay. C The expression of fibronectin and β-catenin in TCs was assessed by qRT-PCR. D The expression of fibrosis factors (collagen I, P-SMAD3, α-SMA, TGF-β) in TCs was analyzed by western blot assay. E Exercise-induced irisin improves follicular dysfunction by inhibiting the IRE1α-TXNIP/ROS-NLRP3 pathway in the PCOS signaling pathway. Data are shown as the mean ± SD. *p ≤ 0.05, **p ≤ 0.01. Ctrl, control; DHT, dihydrotestosterone; ASC, apoptosis-associated speck-like protein containing a CARD; GSDMD, gasdermin D; GSDMD-C, C-terminal fragment of gasdermin D; IL-1β, interleukin-1β; IL-18, interleukin-18; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; α-SMA, a-smooth muscle actin; TGF-β, transforming growth factor-beta; ERS, endoplasmic reticulum stress; ROS, reactive oxygen species; IRE1α, inositol-requiring enzyme 1α; TXNIP, thioredoxin-interacting protein; NLRP3, NOD-like receptor family pyrin domain containing 3

Article Snippet: Samples of ovarian tissues were sectioned at a thickness of 4 μm and stained with specific antibodies against IRE1α (1:100, Proteintech, China).

Techniques: Activation Assay, Expressing, Quantitative RT-PCR, Western Blot, Control