Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The effects of macrophage-derived CCL24 on MMT in PMCs. A-D. A PD-related peritoneal fibrosis rat model was constructed with intraperitoneal injection of 4.25% glucose PD solution and LPS. Then the peritoneal tissues were collected ( n = 6/group). ( A ) HE staining was conducted to measure the thickness of the peritoneal membrane in control rats and PD rats (quantified in the right). Scale bars: 100 μm. ( B ) Masson’s trichrome staining of peritoneal tissues from rats of different groups (quantified in the right). Scale bars: 100 μm. ( C ) An ELISA kit was used to detect the abundance of CCL24 in peritoneal tissues. ( D ) Macrophages were obtained from the peritoneal tissues, and then the CCL24 mRNA level in macrophages and other cells in the peritoneal tissues from rats with different treatments was measured by qRT-PCR. ( E ) After differentiation into M0 macrophages with stimulation of phorbol 12-myristate 13-acetate (PMA) for 48 h, THP1 cells were transfected with si-NC or si-CCL24. After being treated with recombinant protein IL-4 (20 ng/mL) and IL-13 (20 ng/mL) for another 48 h, CCL24 protein expression in macrophages was detected with western blotting (quantified in the right). ( F ) ELISA kit was adopted to detect the content of CCL24 in medium or the supernatant of THP-1-derived macrophages exposed to si-NC (Mφ si−NC -S) or si-CCL24 (Mφ si−CCL24 -S). ( G ) Western blotting was used to measure the protein expression of E-cadherin, α-SMA, FN, and collagen I in HPMCs exposed to medium or the supernatant from THP1-derived macrophages with different treatments (quantified in the right). (H) Rat primary peritoneal macrophages (PMφ) were transfected with si-NC or si-CCL24 for 48 h. Then CCL24 protein expression in macrophages was detected with western blotting (quantified in the right). ( I ) ELISA kit was adopted to detect the content of CCL24 in medium or the supernatant of rat primary peritoneal macrophages exposed to si-NC (PMφ si−NC -S) or si-CCL24 (PMφ si−CCL24 -S). ( J ) Western blotting was used to measure the protein expression of E-cadherin, α-SMA, FN, and collagen I in RPMCs exposed to medium or the supernatant of rat primary peritoneal macrophages with different treatments (quantified in the right). Values are the mean ± SD; Student’s t-test (panel: A-E and H) or One-way ANOVA with Tukey’s post hoc test (panel: F-G and I-J); * p < 0.05, ** p < 0.01, ns p > 0.05. PD: peritoneal dialysis; Mφ: THP1-derived macrophages; PMφ: rat primary peritoneal macrophages; MMT: Mesothelial-to-mesenchymal transition; HPMCs: human peritoneal mesothelial cells; FN: fibronectin; RPMCs: rat peritoneal mesothelial cells.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Derivative Assay, Construct, Injection, Staining, Membrane, Control, Enzyme-linked Immunosorbent Assay, Quantitative RT-PCR, Transfection, Recombinant, Expressing, Western Blot

Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The effects of CCL24 and HpMSCs-CM on macrophage-driven MMT in HPMCs. A-C. THP1-derived macrophages were treated with medium or HpMSCs-derived conditional medium (HpMSCs-CM) together with recombinant protein IL-4 (20 ng/mL) and IL-13 (20 ng/mL) for 24–48 h. ( A ) The mRNA level of CCL24 in THP1-derived macrophages with different treatments was measured by qRT-PCR assay. ( B ) Western blotting was adopted to detect the protein expression of CCL24 in THP1-derived macrophages from different groups (quantified in the right). ( C ) An ELISA kit was used to detect the content of CCL24 in the supernatant of THP1-derived macrophages. ( D ) ELISA kit was used to measure the content of CCL24 in medium or the supernatant of THP1-derived macrophages exposed to medium (Mφ Con -S) or HpMSCs-CM (Mφ CM -S), together with or without recombinant protein CCL24 (100 ng/mL). ( E ) Western blotting was adopted to measure the protein expression of E-cadherin, α-SMA, FN, and collagen I in HPMCs stimulated with medium or the supernatant of THP1-derived macrophages with different treatments (quantified in the right). Values are the mean ± SD; Two-way ANOVA (Panel: A-C) or One-way ANOVA (Panel: D-E) with Tukey’s post hoc test; * p < 0.05, ** p < 0.01. MMT: Mesothelial-to-mesenchymal transition; Mφ: THP1-derived macrophages; HPMC: human peritoneal mesothelial cells; HpMSCs: human peritoneal mesenchymal stem cells; FN: fibronectin.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Derivative Assay, Recombinant, Quantitative RT-PCR, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The role of CCR3 and the MAPK pathway in the inhibitory effects of HpMSCs-CM on macrophage-driven MMT in HPMCs. ( A - B ) After pre-treatment of CCR3 inhibitor ALK4290 (5 µM) for 1 h, HPMCs were exposed to medium or the supernatant from THP1-derived macrophages treated with medium (Mφ Con -S) or HpMSCs-conditional medium (Mφ CM -S). Then, western blotting was used to detect the protein expression of E-cadherin, α-SMA, FN, and collagen I (A, quantified in the right) and PI3K/AKT and MAPK pathways (B, quantified in the right) in HPMCs. ( C ). After pre-treated with P38 inhibitor SB203580 (10 µM) for 1 h, HPMCs were incubated with medium or macrophage-derived supernatant. Then the phosphorylation level of P38 and the protein level of E-cadherin, α-SMA, FN, and collagen I in HPMCs were measured by western blotting (quantified in the right). Values are the mean ± SD; One-way ANOVA with Tukey’s post hoc test; * p < 0.05, ** p < 0.01, ns p > 0.05. MMT: Mesothelial-to-mesenchymal transition; Mφ: THP1-derived macrophages; HPMC: human peritoneal mesothelial cells; HpMSCs: human peritoneal mesenchymal stem cells; FN: fibronectin.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Derivative Assay, Western Blot, Expressing, Incubation, Phospho-proteomics

Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The effects of HpMSCs-Exo on CCL24 synthesis and secretion in macrophages. ( A - B ). The exosomes in the supernatant of HpMSCs (HpMSCs-Exo) were collected. ( A ) Western blotting was adopted to measure the protein expression of CD9, CD63, CD81, and Calnexin in exosomes and cell lysis (quantified in the right). ( B ) The morphology of HpMSCs-Exo was observed under a transmission electron microscope. Scale bars: 200 nm. C-E. THP1-derived macrophages were incubated with HpMSCs-conditional medium (HpMSCs-CM) or exosome-free HpMSCs-CM (HpMSCs-CM Exo−free ) or different concentrations of HpMSCs-Exo (12.5, 25, 50 µg/mL). qRT-PCR ( C ) and western blotting ( D ) were used to measure the mRNA level and protein expression of CCL24 in macrophages, and an ELISA kit was adopted to detect the protein content of CCL24 in the supernatant from macrophages ( E ). Values are the mean ± SD; Student’s t-test (Panel: A) or One-way ANOVA with Tukey’s post hoc test (Panel: C-E); * p < 0.05, ** p < 0.01, ns p > 0.05. Mφ: THP1-derived macrophages; HpMSCs: human peritoneal mesenchymal stem cells.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Western Blot, Expressing, Lysis, Transmission Assay, Microscopy, Derivative Assay, Incubation, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The effects of HpMSCs-Exo and miR-320d on CCL24 expression in macrophages. ( A ) The miRNA level in THP1-derived macrophages treated with or without HpMSCs-derived exosomes (HpMSCs-Exo) was measured with qRT-PCR assay. ( B ) After incubating with HpMSCs-Exo with or without FITC-labeled miR-320d, FITC in macrophages was observed under a fluorescence microscope. Scale bars: 20 μm. C-D. THP1-derived macrophages were treated with or without HpMSCs-Exo together with miRNA inhibitor NC (inhibitor NC) or miR-320d inhibitor (miR-320d in). ( C ) qRT-PCR assay was conducted to detect the level of miR-320d in macrophages. ( D ) The protein levels of p-STAT3, STAT3, and CCL24 in macrophages with different treatments were measured with western blotting (quantified in the right). Values are the mean ± SD; Student’s t-test (Panel: A) or One-way ANOVA with Tukey’s post hoc test (Panel: C-D); * p < 0.05, ** p < 0.01, ns p > 0.05. Mφ: THP1-derived macrophages; HpMSCs: human peritoneal mesenchymal stem cells.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Expressing, Derivative Assay, Quantitative RT-PCR, Labeling, Fluorescence, Microscopy, Western Blot

Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The effects of KLF7 on HpMSCs-Exo or miR-320d-mediated STAT3/CCL24 pathway inhibition in macrophages. ( A ). After transfection with luciferase reporter plasmids containing wild or mutated KLF7 mRNA 3’ UTR sequence (displayed in the left), which binds to miR-320d in TargetScan, THP1-derived macrophages were treated with miRNA mimics NC (mimics NC) or miR-320d mimics (miR-320d mi). Then the luciferase activity in macrophages was measured. ( B ). Western blotting was conducted to measure the protein expression of KLF7 in macrophages treated with or without HpMSCs-derived exosomes (HpMSCs-Exo) in the presence of miR-320d mimics or miR-320d inhibitor (quantified in the right). ( C - D ). THP1-derived macrophages were transfected with miRNA mimics NC or miR-320d mimics together with vector or KLF7 overexpression plasmids. Western blotting was adopted to detect the protein expression of KLF7 and the phosphorylation level of STAT3 ( C ) and the protein level of CCL24 ( D ) in macrophages. E-F. After being transfected with vectors or KLF7 overexpression plasmids, THP1-derived macrophages were incubated with or without HpMSCs-Exo. Western blotting was used to measure the protein expression of KLF7 and the phosphorylation level of STAT3 ( E ) and the protein level of CCL24 ( F ) in macrophages. Values are the mean ± SD; Student’s t-test (Panel: A) or One-way ANOVA with Tukey’s post hoc test (Panel: B-F); * p < 0.05, ** p < 0.01, ns p > 0.05. Mφ: THP1-derived macrophages; HpMSCs: human peritoneal mesenchymal stem cells.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Inhibition, Transfection, Luciferase, Sequencing, Derivative Assay, Activity Assay, Western Blot, Expressing, Plasmid Preparation, Over Expression, Phospho-proteomics, Incubation

Journal: Scientific Reports

Article Title: Peritoneal MSCs-derived exosomes suppress CCL24 synthesis through miR-320d delivery contributing to the improvement of peritoneal dialysis-associated fibrosis

doi: 10.1038/s41598-026-42489-w

Figure Lengend Snippet: The effects of modified HpMSCs-Exo loaded with miR-320d on KLF7/STAT3/CCL24 pathway, MMT, and PD-related fibrosis. ( A ). Exosomes were separated from the supernatant of HpMSCs transfected with miRNA mimics NC or miR-320d mimics, and then the abundance of miR-320d in native exosomes (Exo-C) and modified exosomes (Exo-M) was measured with qRT-PCR assay. B-C. THP1-derived macrophages were incubated with Exo-C or Exo-M. And then qRT-PCR ( B ) and western blotting ( C ) were adopted to measure the level of miR-320d and the protein expression of KLF7 and CCL24, and the phosphorylation level of STAT3 (quantified in the right). ( D ). Western blotting was used to measure the phosphorylation level of p38 and the protein content of E-cadherin, α-SMA, FN, and collagen I in HPMCs treated with the supernatant of THP1-derived macrophages stimulated with medium (Mφ-S) or Exo-C (Mφ Exo−C -S), or Exo-M (Mφ Exo−M -S) (quantified in the right). ( E - F ). Rat primary peritoneal macrophages were incubated with Exo-C or Exo-M. And then qRT-PCR ( E ) and western blotting ( F ) were adopted to measure the level of miR-320d and the protein expression of KLF7 and CCL24, and the phosphorylation level of STAT3 (quantified in the right). ( G ). Western blotting was used to measure the phosphorylation level of p38 and the protein content of E-cadherin, α-SMA, FN, and collagen I in RPMCs treated with the supernatant of rat primary peritoneal macrophages stimulated with medium (PMφ-S) or Exo-C (PMφ Exo−C -S), or Exo-M (PMφ Exo−M -S) (quantified in the right). ( H - L ). A PD-associated peritoneal injury rat model was established with intraperitoneal injection of 4.25% glucose PD solution and LPS. And then PD rats were divided into three groups and subjected to different treatments, including tail vein injection of PBS or Exo-C or Exo-M ( n = 6/groups). Meanwhile, 6 control rats were only exposed to intraperitoneal and tail vein injection of PBS. Then the peritoneal tissues were collected. ( H ). HE staining was conducted to measure the thickness of the peritoneal membrane in control rats and PD rats with different treatments (quantified in the right). Scale bars: 100 μm. ( I ). Masson staining of peritoneal tissues from rats of different groups. Scale bars: 100 μm. ( J ). The level of miR-320d in the peritoneal tissues from different rats was detected by qRT-PCR. ( K ). ELISA kit was used to measure the content of CCL24 in peritoneal tissues. ( L ). Western blotting was conducted to detect the phosphorylation level of p38 and the protein content of E-cadherin, α-SMA, FN, and collagen I in the peritoneal tissues (quantified in the right). Values are the mean ± SD; Student’s t-test (Panel: A) or One-way ANOVA with Tukey’s post hoc test (Panel: B-L); * p < 0.05, ** p < 0.01, ns p > 0.05. Mφ: THP1-derived macrophages; PMφ: Rat primary peritoneal macrophages; MMT: Mesothelial-to-mesenchymal transition; HpMSCs: human peritoneal mesenchymal stem cells; HPMC: human peritoneal mesothelial cells; FN: fibronectin; PD: peritoneal dialysis.

Article Snippet: To induce polarization into M0-type macrophages, THP-1 cells in the logarithmic growth phase with optimal viability were seeded into 6-well plates at a density of 1 × 10 6 cells per well, and then treated with 100 ng/ml phorbol 12-myristate 13-acetate (PMA; ABclonal, RPM0015, Wuhan, CN) for 48 h. Subsequently, the PMA-containing medium was replaced with serum-free medium, and cells were rested for an additional 24 h. To obtain M2 macrophages, THP1-derived M0 macrophages were then treated with medium containing IL-4 (ABclonal, RP00995, 20 ng/ml), IL-13 (ABclonal, RP01320, 20 ng/ml) , and exosome-free FBS for 48 h. Primary rat peritoneal macrophages were isolated from unstimulated rats via peritoneal lavage .

Techniques: Modification, Transfection, Quantitative RT-PCR, Derivative Assay, Incubation, Western Blot, Expressing, Phospho-proteomics, Injection, Control, Staining, Membrane, Enzyme-linked Immunosorbent Assay