Fig 2: KLB expression was elevated in ESCs of patients with IUA. (A) KLB was expressed in both stromal and epithelial cells. Compared with the normal control group, the expression of KLB in ESCs of patients with IUA was increased. Scale bar = 100 μm. (B) The primary ESCs were extracted and identified with Vimentin. Immunofluorescence showed that the positive rate of Vimentin was more than 96%. Scale bar = 50 μm. (C) Through RT-qPCR analysis, it was found that the expression of KLB mRNA in the primary ESCs of the IUA group was higher than in those of the normal control group. (D) The expression of KLB and the fibrosis marker Fibronectin (FN) in primary ESCs of the IUA group and normal control group was detected and analyzed using Western blotting. It was found that the expression of KLB and FN increased in the primary ESCs of IUA patients. (E) Spearman rank correlation analysis showed that the expressions of KLB and FN proteins in ESCs were positively correlated. ** p < 0.01; *** p ≤ 0.001.

Fig 3: TGF-β1 was used to stimulate HESCs to construct an IUA cell model and detect the expression of KLB. (A) Western blot was used to detect the expression of Collagen I and FN in HESCs stimulated with TGF-β1 at the concentration of 10 ng/mL for 0, 12, 24, and 48 h. It was found that the expression of Collagen I and FN increased in a statistically significant manner when treated with 10 ng/mL for 48 h. (B) The expression of Collagen I and FN in HESCs stimulated with TGFβ1 at the concentrations of 0, 2.5, 5, and 10 ng/mL for 48 h was detected by Western Blot. It was found that the expression of Collagen I and FN was significantly increased when the cells were treated with 5 ng/mL and 10 ng/mL. (C) The expression of KLB in HESCs stimulated with TGF-β1 at the concentration of 10 ng/mL was detected by Western Blot, and it was found that the expression of KLB increased after 48 h of TGF-β1 stimulation. (D) The expression of KLB in HESCs stimulated with TGF-β1 at the concentrations of 0, 2.5, 5, and 10 ng/mL for 48 h was detected by Western Blot. It was found that the expression of KLB in HESCs increased when the cells were stimulated with TGF-β1 at the concentration of 10 ng/mL. * p < 0.05; ** p < 0.01; *** p ≤ 0.001.

Fig 4: IHC to detect KLB in rats. The lumen surface of the uterus on the operative side (IUA side) was discontinuous, the uterine lumen was narrowed, and the glands of the damaged uterus were decreased. KLB was expressed in both epithelial and stromal cells of the rats, and KLB staining on the operative side was stronger than on the non-operative side. Statistical analysis of eight pairs of rat endometrial sections (400×) with ESCs showed significant differences (p = 0.0141).

Fig 5: KLB promotes the fibrosis and proliferation of HESCs through the PI3K/AKT signaling pathway. (A) Through Western blot analysis, it was found that under the same stimulation with TGF-β1, the expression of P-AKT in the KLB overexpression group was significantly higher than in the control group transfected with the empty plasmid. (B) Through Western blot analysis, it was found that LY294002, an inhibitor of PI3K, could rescue the protein levels of Collagen I and FN after KLB overexpression. (C) The cell proliferation ability was detected by the CCK8 assay, and it was found that LY294002 could also reduce the cell proliferation promoted by KLB overexpression. (D) The activation the PI3K/AKT signaling pathway by KLB plays a critical role in IUA by regulating ESC proliferation and fibrosis. * p < 0.05; ** p < 0.01.