Fig 2: IL-11 is a key target in regulating the ovarian matrix stiffness.a, Immunofluorescent staining of COL1A1 and TGFβ1. L and H represent regions with low and high coexpression of COL1A1 and TGFβ1, respectively. b, Measurement of ovarian matrix stiffness in COL1A1loTGFβ1lo or COL1A1hiTGFβ1hi regions. Data are presented as the mean ± s.d. (Welch’s two-sided t-test). n = 20 per group. c, Schematic of RNA-seq of primary human ovarian fibroblasts (pHOFs) after in vitro TGFβ1 treatment (10 ng ml−1, 24 h). d, IL11 RNA expression is upregulated in pHOFs stimulated with TGFβ1. Differential expression was assessed using a two-sided Wald test, with P values adjusted for multiple comparisons through the Benjamini–Hochberg method (n = 3 per group). e, IL-11 protein abundance in human, mouse and rat ovaries across different age groups. f, Representative images of IL-11 immunohistochemical staining in ovaries from individuals of different ages or with ovarian dysfunction due to various diseases. g, Representative images of pHOFs immunostained for COL1A1 and ACTA2 after 24 h of incubation without stimulus (control), TGFβ1 (10 ng ml−1) or IL-11 (10 ng ml−1) or with TGFβ1 (10 ng ml−1) and an anti-IL-11 neutralizing antibody (2 μg ml−1). h, GO and KEGG enrichment analysis of differentially expressed genes in pHOFs incubated without stimulus (control) or IL-11 (10 ng ml−1) for 24 h. Red labels highlight GO terms enriched in pathways related to the ECM and its receptor. The analyses were performed using the hypergeometric test. P values were adjusted for multiple comparisons using the Benjamini–Hochberg FDR method. n = 3 per group. i, COL1A1 mRNA expression in pHOFs treated with IL-11 or IL-11 and NOTCH pathway inhibitor (PF-3084014, IMR-1), PI3K–AKT pathway inhibitor (PI3K/AKT-IN-1, BYL-719), cGMP–PKG pathway inhibitor (KT5823, MBP146-78), p38 MAPK pathway inhibitor (p38 MAPK-IN-1, SB 203580), ERK1/2 pathway inhibitor (ravoxertinib, SCH772984), JNK pathway inhibitor (JNK-IN-7, SP600125) or mTOR pathway inhibitor (rapamycin, everolimus). Red box highlighting indicates the ERK1/2 inhibitor group, which showed the most significant reduction in IL‑11‑induced COL1A1 upregulation. Data are presented as mean ± s.d., n = 3 for each group (unpaired two-tailed t-test or Welch’s two-tailed t-test). j,k, Human phospho-kinase array of pHOFs treated with 10 ng ml−1 IL-11 or vehicle control for 24 h. Numbers 1–8 on the blot (j) and the corresponding bars (k) indicate the following: 1) CREB at serine 133; 2) ERK1/2 at threonine 202 and tyrosine 204; 3) GSK3β at serine 9; 4) WNK1 at threonine 60; 5) β‑Catenin; 6) RSK1/2 at serine 221 and serine 227; 7) RSK1/2/3 at serine 380, serine 386 and serine 377; 8) STAT3 at tyrosine 705. Data are presented as the mean ± s.d., n = 3 for each group (unpaired two-tailed t-test). l, Western blot analysis of COL1A1 and ACTA2 expression in pHOFs treated with IL-11, with or without the ERK1/2 inhibitor SCH772984. m, Representative immunofluorescence images and quantification of COL1A1 and ACTA2 in pHOFs treated with IL-11 or IL-11 + SCH772984. int., integrated intensity. Data are presented as the mean ± s.d., n = 5 per group (one-way ANOVA).Source data

Fig 3: Preparation of Il11 siRNA-loaded liposomes and AAV-sh-Il11ra1 treatment.(A) RT-PCR analyses of the interference efficiency of Il11 siRNAs in primary mice ovary fibroblasts. Data are presented as the mean ± SD, n = 3 per group (unpaired two-tailed t-test). (B) Western blot analyses of the interference efficiency of Il11 siRNAs in primary mice ovary fibroblasts. (C) RT-PCR analyses of the interference efficiency of Il11 siRNA in primary rat ovary fibroblasts. Data are presented as the mean ± SD, n = 3 per group (unpaired two-tailed t-test). (D) Western blot analyses of the interference efficiency of Il11 siRNA in primary rat ovary fibroblasts. (E) Schematic diagram showing the preparation of Il11 siRNA-loaded liposomes. (F) The hydrodynamic diameter, polymer dispersity index (PDI) and zeta potential of the liposomes (blank or siRNA-loaded) were measured by dynamic light scattering (DLS). siRNA entrapment efficiency was measured by RiboGreen assay. (G) Representative transmission electron microscopy (TEM) image of siRNA-loaded liposomes. (H) Hydrodynamic diameter distribution of siRNA-loaded liposomes. (I) Colloid stability of siRNA-loaded liposomes in PBS. (J) Flowchart for the evaluation of matrix stiffness and ovarian function in 48-week-old mice after ovarian micro-injection of AAV-sh-Il11ra1. (n = 25 per group). (K) Assessment of ovarian matrix stiffness using atomic force microscopy. (L) Quantification the collagen content within ovarian tissue by hydroxyproline assay. (M) Representative micrographs of paraffin-embedded ovary stained histochemically for Picrosirius red. (N) Representative images of H&E stained ovarian sections from two groups. (O) Primordial follicle (PMF), primary follicle (PF), secondary follicle (SF), antral follicle (ANF), atretic follicle (ATF), total healthy follicles (THF) and corpus luteum (CL) quantification in ovaries. (P) Average litter size of total mated mice. Data are presented as the mean ± SD, n = 6 per group (unpaired two-tailed t-test). Source data

Fig 4: IL-11 induces increased ovarian matrix stiffness in vivo.a, Schematic of the rmIL-11 intervention in young female mice. b, Changes in body weight of mice during IL-11 intervention. Data are presented as mean ± s.d., n = 13 for control group and 16 for rmIL-11 group (unpaired two-tailed t-test). n.s., not significant. c, Representative macroscopic images of organs from control and rmIL-11-treated mice. d, Assessment of ovarian matrix stiffness in mice using AFM. Data are presented as mean ± s.d., n = 6 per group (Welch’s two-tailed t-test). e, Collagen content in mouse ovaries, assessed by hydroxyproline assay. Data are presented as mean ± s.d., n = 6 per group (unpaired two-tailed t-test). f, Representative images of picrosirius red staining of mice ovaries. Data are presented as mean ± s.d., n = 8 per group (unpaired two-tailed t-test). g, Representative immunohistochemical staining of COL1A1 in ovaries from control and rmIL-11-treated mice. Data are presented as mean ± s.d., n = 6 per group (unpaired two-tailed t-test). IOD, integrated optical density. h, Representative images of ACTA2 immunohistochemistry in mouse ovary sections. Quantification is shown on the right. Data are presented as the mean ± s.d., n = 5 per group (unpaired two-tailed t-test). i, Western blots of IL-11, ACTA2, ERK1/2 and p-ERK1/2 protein expression in control ovaries and mouse ovaries treated with rmIL-11. j, Quantification of IL-11, ACTA2 and p-ERK1/2 and ERK1/2 protein expression in the two groups. p‑ERK1/2/ERK1/2 indicates the ratio of phosphorylated ERK1/2 to total ERK1/2. Data are presented as mean ± s.d., n = 6 for each group (unpaired two-tailed t-test). k, The proportion of regular or irregular estrous cycles in mice. n = 16 per group (Fisher Freeman Halton, two-sided). l, Statistical analysis of the number of follicles at each level in H&E-stained ovarian sections. PMF, primordial follicle; PF, primary follicles; SF, secondary follicle; ANF, antral follicle; THF, total healthy follicle; ATF, atretic follicle. Data are presented as the mean ± s.d., n = 6 per group (unpaired two-tailed t-test). m, AMH, E2 and FSH serum levels in the two groups of mice. Data are presented as mean ± s.d., n = 8 per group for AMH, 7 per group for E2, 5 per group for FSH (unpaired two-tailed t-test). n, The litter size of mice treated with rmIL-11 or control after mating with young male mice. Data are presented as mean ± s.d., n = 6 per group (unpaired two-tailed t-test).Source data

Fig 5: IL-11 is a key target in regulating the ovarian matrix stiffness.(A) Heatmap of differentially expressed genes in primary human ovary fibroblasts (pHOF) after in vitro TGFβ1 treatment. (B) GO and KEGG analysis of differentially expressed genes between the control and TGFβ1-treated pHOF. Red labels highlight ECM secretion as the key biological process. (C) Western blots of COL1A1, COL1A2, and ACTA2 protein expression in pHOF in response to TGFβ1. (D) IL-11 expression in pHOF following stimulation with various fibrotic factors. (E) Detection of IL-11 concentration in the culture medium of pHOF following TGFβ1 treatment using ELISA. Data are presented as the mean ± SD, n = 5 per group (unpaired two-tailed t-test). (F) Human ovarian IL11 mRNA levels in young (18–28 years), middle-aged (36–39 years), and old (47–49 years) groups. Murine ovarian Il11 mRNA levels in young (3 months), middle-aged (9 months), and old (12 months) mice. Rat ovarian Il11 mRNA levels in young (3 months), middle-aged (9 months), and old (12 months) rats. Data are presented as the mean ± SD, n = 6 per group (one-way ANOVA). (G and H) Analysis of age-related IL-11 protein abundance in human, mouse, and rat ovaries. Data are presented as the mean ± SD, n = 6 per group (unpaired two-tailed t-test). (I) Immunohistochemical detection of IL-11 in human ovarian tissue across different ages and pathophysiological states. (J) Correlation of ovarian IL-11 expression with age and AMH levels. n = 40 (Pearson correlation analysis, two-sided). (K) Immunofluorescence staining for IL-11RA in pHOF. (n = 3 experiments). (L) Quantification of pHOF immunostained for COL1A1 and ACTA2 after 24 h incubation without stimulus (Control), TGFβ1 (10 ng/ml) or IL-11 (10 ng/ml) or with TGFβ1 (10 ng/ml) and an Anti-IL-11 neutralising antibody (2 μg/ml). Data are presented as the mean ± SD, n = 5 per group (one-way ANOVA). (M and N) Representative images and quantification of wound-healing assay in pHOF treated with TGFβ1 or IL-11 or with TGFβ1 and an Anti-IL-11 neutralising antibody. Data are presented as the mean ± SD, n = 5 per group (one-way ANOVA). (O) Representative images and quantification of stained pHOF migrating from the upper chamber to the lower chamber after treatment of TGFβ1 or IL-11 or with TGFβ1 and an Anti-IL-11 neutralising antibody. Data are presented as the mean ± SD, n = 5 per group (one-way ANOVA). Source data