Protective effect of hUC-MSCs regulating peritoneal mesothelial cell EMT through cGAS-STING signaling pathway in high glucose-induced peritoneal fibrosis

Figure 1 Activation of the cyclic GMP-AMP synthase-stimulator of interferon genes signaling pathway in in vitro and in vivo models of peritoneal fibrosis. A: 5-ethynyl-2’-deoxyuridine assay was performed to assess the proliferation of HMrSV5 cells under different treatments; B: Quantitative analysis of 5-ethynyl-2’-deoxyuridine assay results; C: Western blot analysis of cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) protein expression in HMrSV5 cells; D and E: Quantitative analysis of protein expression in HMrSV5 cells: CGAS (D); STING (E); F: Western blot analysis of cGAS and STING protein expression in peritoneal tissues from different mouse groups; G and H: Quantitative analysis of protein expression in mouse peritoneal tissues: CGAS (G); STING (H). Data are presented as mean ± SD. ^aP < 0.05 vs control. cGAS: Cyclic GMP-AMP synthase; STING: Stimulator of interferon genes; EdU: 5-ethynyl-2’-deoxyuridine.

Figure 2 Effects of human umbilical cord-derived mesenchymal stem cells on migration and invasion of high glucose-treated HMrSV5 cells. A: Identification of human umbilical cord-derived mesenchymal stem cell surface markers via flow symmetry (negative markers: CD34, CD45; positive markers: CD29, CD44); B and C: Migration ability of HMrSV5 cells assessed via wound healing assay and corresponding quantitative analysis; D and E: Invasion ability of different HMrSV5 cells assessed via Transwell assay and corresponding quantitative analysis. Data are presented as mean ± SD. ^aP < 0.05 vs control; ^bP < 0.05 vs high glucose. hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; HG: High glucose; CD34: Cluster of differentiation 34; CD45: Leukocyte common antigen; CD29: Integrin beta 1; CD44: CD44 antigen.

Figure 3 Effects of human umbilical cord-derived mesenchymal stem cells on epithelial-mesenchymal transition-related gene and protein expression in high glucose-treated HMrSV5 cells. A: Epithelial cadherin mRNA; B: Vimentin mRNA; C: Alpha-smooth muscle actin mRNA; D: Transforming growth factor-beta1 mRNA levels detected via quantitative real-time polymerase chain reaction; E: Representative western blot bands; F-I: Quantitative analysis of protein expression: Epithelial cadherin (F); vimentin (G); alpha-smooth muscle actin (H); transforming growth factor-beta1 (I). Data are presented as mean ± SD. ^aP < 0.05 vs control; ^bP < 0.05 vs high glucose. hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; HG: High glucose; E-cadherin: Epithelial cadherin; α-SMA: Alpha-smooth muscle actin; TGF-β1: Transforming growth factor-beta1.

Figure 4 Effects of human umbilical cord-derived mesenchymal stem cells on activation of the cyclic GMP-AMP synthase-stimulator of interferon genes signaling pathway in various HMrSV5 cells. A: Cyclic GMP-AMP synthase mRNA; B: Stimulator of interferon genes mRNA levels detected via quantitative real-time polymerase chain reaction; C: Representative western blot bands; D and E: Quantitative analysis of protein expression: Cyclic GMP-AMP synthase (D); stimulator of interferon genes (E). Data are presented as mean ± SD. ^aP < 0.05 vs control; ^bP < 0.05 vs high glucose. hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; cGAS: Cyclic GMP-AMP synthase; STING: Stimulator of interferon genes; HG: High glucose.

Figure 5 Human umbilical cord-derived mesenchymal stem cells suppress high-glucose-induced epithelial-mesenchymal transition in HMrSV5 cells by inhibiting the cyclic GMP-AMP synthase-stimulator of interferon genes signaling pathway. A: Representative immunofluorescence images of fibronectin expression in different HMrSV5 groups; B: Quantitative analysis of fibronectin fluorescence intensity; C: Representative images of the wound healing assay; D: Quantitative analysis of cell migration; E-H: TANK-binding kinase 1 mRNA (E); interferon regulatory factor 3 mRNA (F); alpha-smooth muscle actin mRNA (G); transforming growth factor-beta1 mRNA (H) levels detected via quantitative real-time polymerase chain reaction; I: Representative western blot bands; J-M: Quantitative analysis of protein expression: TANK-binding kinase 1 (J); interferon regulatory factor 3 (K); alpha-smooth muscle actin (L); transforming growth factor-beta1 (M). Data are presented as mean ± SD. ^aP < 0.05 vs high glucose; ^bP < 0.05 vs DMXAA. hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; HG: High glucose; EMT: Epithelial-mesenchymal transition; cGAS: Cyclic GMP-AMP synthase; STING: Stimulator of interferon genes; TBK1: TANK-binding kinase 1; IRF3: Interferon regulatory factor 3; α-SMA: Alpha-smooth muscle actin; TGF-β1: Transforming growth factor-beta1.

Figure 6 Effects of human umbilical cord-derived mesenchymal stem cells on peritoneal fibrosis and inflammatory cytokines in peritoneal fibrosis mice. A-C: Tracking the distribution of human umbilical cord-derived mesenchymal stem cells (hUC-MSCs) injected in mice. In vivo imaging of whole-body fluorescence (A). Fluorescent imaging of isolated organs (B). Fluorescence imaging of peritoneal tissues following hUC-MSC injection (C); D: Representative hematoxylin and eosin staining images; E: Representative Masson staining images; F and G: Tumor necrosis factor-alpha (F) and interleukin-6 (G) levels in peritoneal tissues were detected via enzyme-linked immunosorbent assay. Data are presented as mean ± SD. ^aP < 0.05 vs control; ^bP < 0.05 vs peritoneal fibrosis. hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; PF: Peritoneal fibrosis; HE: Hematoxylin and eosin; TNF-α: Tumor necrosis factor-alpha; IL-6: Interleukin-6.

Figure 7 Effect of human umbilical cord-derived mesenchymal stem cells on fibrosis-related protein expression in mouse peritoneal tissues. A-F: Alpha-smooth muscle actin (A and B); vimentin (C and D); and epithelial cadherin (E and F) expression detected by immunohistochemistry; G: Representative western blot bands; H-J: Quantitative analysis of protein expression: Alpha-smooth muscle actin (H); vimentin (I); epithelial cadherin (J). Data are presented as mean ± SD. ^aP < 0.05 vs control; ^bP < 0.05 vs peritoneal fibrosis. hUC-MSCs: Human umbilical cord-derived mesenchymal stem cells; α-SMA: Alpha-smooth muscle actin; E-cadherin: Epithelial cadherin; PF: Peritoneal fibrosis.