HucMSC-Ex Alleviates IBD-associated Intestinal Fibrosis By Inhibiting ERK Phosphorylation In Intestinal Fibroblasts

Objective:The animal model human umbilical cord mesenchymal stem cell-derived exosomes involved in Dextran Sulfate Sodium(DSS)-induced IBD-related fibrosis was established to observe the role of huc MSC-Ex in IBD-related fibrosis.To explore the effect of huc MSC-Ex in fibroblast proliferation,migration and activation.To determine that huc MSC-Ex inhibited IBD-associated fibrosis by reducing ERK phosphorylation in fibroblasts.Methods:(1)Isolate human umbilical cord-derived mesenchymal stem cells from umbilical cords,extract exosomes by ultracentrifugation,observe the morphology under transmission electron microscope,measure the diameter by NTA,extract exosomes protein,detect exosomes protein marker by Western blotting.(2)Select 6-8 weeks old famale C57BL/6 mice and divide them into 3 groups:NC group,DSS group and huc MSC-Ex group,give DSS group and huc MSC-Ex group 2% DSS water for 7 days at the 1st,4th,and 7th week of the experiment and give huc MSC-Ex group huc MSC-Ex every 7 days starting from the 3rd day,record the change of weight and stool;detect the weight length of colon,detect the fibrosis-related proteins(TGF-β,α-SMA,CollagenⅠ,Collagen Ⅲ and Fibronectin)in colon by Western blot,extract RNA to detect expression of fibrosis-related genes(TGF-β,α-SMA,FN1,COL1A1 and COL3A1)in colon by RT-PCR and q RT-PCR,record the results of H-E,Masson and Sirius Red staining by the microscope,observe the expression levels of α-SMA and Fibronectin in colon tissue by immunohistochemistry.(3)Culture colonic fibrobalst CCD-18 Co in vitro,induce cell proliferation and activation by TGF-β,treat cells with huc MSC-Ex,divide cells into 3 groups: NC group,TGF-β group and huc MSC-Ex group,perform CCK8 experiment and cell cloning experiment,extract protein and detect the expression level of the PCNA by Western blot,perform Transwell migration experiment to observe the cell migration under the microscope after 18 hours,extract protein after cluturing for 48 hours and detect the the expression ofα-SMA,Collagen Ⅰ,Collagen Ⅲ and Fibronectin,extract RNA after culturing for 24,48 or 72 hours and perform RT-PCR and q RT-PCR to detect the expression levels of α-SMA,FN1,COL1A1 and COL3A1 in cells.(4)Detect the expression of ERK in DSS-induced mouse IBD-related fibrosis model and in fibroblasts induced by TGF-β,use ERK inhibitor PD98059 to inhibit the phosphorylation of ERK in fibroblasts,extract protein after treated by PD98059 for48 hours and detect the expression of t-ERK and p-ERK in cells by Western blot,detect the expression levels of α-SMA,Collagen I,Collagen III and Fibronectin in cells by Western blot.Results:(1)HucMSC-Ex was isolated and results showed that the diameter was 50-150 nm.The typical shape of exosomes was found.Exosomes expressed CD9,CD81 and HSP70.(2)The DSS-induced mice model was successfully established.The weight of mice showed that weights of NC group continued to increase and weights in DSS group generally showed a downward trend compared with the DAI scores increasing.After treatment with huc MSC-Ex,weights were increasing and DAI scores were decreasing.The length was shortened in DSS group and recovered in huc MSC-Ex group.The colon weight/length ratio increased in the DSS group and recovered after huc MSC-Ex treatment.HucMSC-Ex effectively inhibited the increase of thickness in colon.Masson and Sirius Red staining results showed that collagen deposition increased in DSS group and inhibited after huc MSC-Ex treatment.Western blot results showed that the expressions of TGF-β,α-SMA,Collagen I,Collagen III and Fibronectin in DSS group were increased and were inhibited after treatment with huc MSC-Ex.Results of q RT-PCR showed that the expression of TGF-β,α-SMA,COL1A1,COL3A1 and FN1 in the DSS group were increased,and inhibited after huc MSC-Ex treatment.The results of IHC showed that the expression of Fibronectin was significantly increased in colon of DSS group and inhibited by huc MSC-Ex.(4)The proliferation of human colonic fibroblasts was induced by TGF-β in vitro.The results of CCK8 experiments after treated for 24 or 48 hours showed that intestinal fibroblasts proliferated actively under the stimulation of TGF-β and inhibited after the treatment of huc MSC-Ex.The cell cloning experiments showed that the cell proliferation ability was enhanced after TGF-β treatment for 14 days and weakened after huc MSC-Ex treatment.The result showed the expression of PCNA in the TGF-β group was increased and reduced by huc MSC-Ex.The results of the18-hour Transwell migration experiment showed that TGF-β-induced fibroblast migration ability was enhanced and inhibited by huc MSC-Ex.(5)Fibroblasts were activated by TGF-β.Western blot results showed that the expression of α-SMA was increased and inhibited after treatment with huc MSC-Ex.The expression of Fibronectin and Collagen Ⅰ were increased and reduced by huc MSC-Ex.The expression of Collagen III in TGF-β group increased at 48 hours,and continued to increase after huc MSC-Ex treatment.QRT-PCR results showed that the expression of Collagen Ⅰ in the TGF-β treatment group was increased after cultured for 24 hours and depressed by huc MSC-Ex.The expression of Collagen Ⅲdecreased significantly after the huc MSC-Ex intervention in the cell culture for 72 hours.The results of fluorescence experiments showed that the expressions of α-SMA and Fibronectin decreased after huc MSC-Ex treatment for 48 hours(5)Western blot results of mice colon tissue showed that the ratio of p-ERK/t-ERK was increased in DSS group and decreased after the huc MSC-Ex treatment.Western blot results showed that the ratio of p-ERK/t-ERK in TGF-β group was increased and decreased after huc MSC-Ex treatment.ERK inhibitor PD98059 inhibited ERK Phosphorylation in human intestinal fibroblasts.Results of Western blot showed that the ratio of p-ERK/t-ERK in fibroblasts treated with ERK inhibitor PD98059 was lower than that in TGF-β group;The expression of α-SMA,Fibronectin and Collagen I in fibroblasts treated with PD98059 were decreased.Conclusion:HucMSC-Ex alleviates IBD-associated intestinal fibrosis by inhibiting ERK phosphorylation in intestinal fibroblasts.