Humanm ubilical cord mesenchymal stem cell exosomes regulate the proliferation and apoptosis of keloid fibroblasts by up-regulating pregnancy-specific beta-1 glycoprotein and cytokeratin7
Objective To explore the molecular mechanism by which human umbilical cord mes-enchymal stem cell exosomes(HUCMSCs-Exos)regulate the proliferation and apoptosis of keloid fibroblasts(KFbs).Methods KFb cell samples(from January to December 2021 in the Second Affiliated Hospital of Soochow University)before and after co-culture with HUCMSCs-Exos(umbilical cords from 3 fetuses after delivery in the Department of Obstetrics and Gynecology of the Second Affiliated Hospital of Soochow Uni-versity from January to December 2021)Eight patients with KD from the plastic surgery department of the hospital performed transcriptome sequencing.Based on the bioinformatics analysis results,the molecules with the most significant changes in gene expression,pregnancy-specific β1 glycoprotein(PSG1)and kera-tin 7(KRT7),were selected as targets for subsequent experiments.Genes were selected to enrich the most relevant NF-α/nuclear factor-κB(NF-κB)pathway for subsequent experimental target pathway research.KFbs were isolated and cultured,and then transfected with pregnancy-specific β1 glycoprotein(PSG1)overexpression plasmid(pcDNA-PSG1 group),transfected with keratin 7(KRT7)overexpression plasmid(pcDNA-KRT7 group),and transfected with PSG1 small interfering RNA.(siPSG1 group),transfected with KRT7 small interfering RNA(siKRT7 group),transfected with PSG1+KRT7 small interfering RNA(siPSG1+KRT7 group),and added tumor necrosis factor-α(TNF-α)/NF-κB signaling pathway inhibitor(QNZ)(QNZ group)After co-culture of HUCMSCs-Exos at different concentrations,the protein level expression of target genes and downstream proteins Col Ⅰ,Col Ⅲ,α-smooth muscle actin(α-SMA),TNF-α,and NF-κB was detected by Western blotting;the protein level expression was detected by cell counting kit(CCK-8)to detect cell proliferation ability;detect cell apoptosis levels through flow cytometry.comparisons between groups were performed using independent sample t test.Results The expression lev-els of Col Ⅰ,Col Ⅲ,α-SMA,TNF-α,and NF-κB proteins in the pcDNA-PSG1 group were lower than those in the control group(Col Ⅰ:0.64±0.09 vs.1.00±0.07,t=5.47,P＜0.05;Col Ⅲ:0.71±0.08 vs.1.00±0.03,t=5.88,P＜0.05;α-SMA:0.62±0.05 vs.1.00±0.08,t=6.98,P＜0.05;TNF-α:0.74±0.07 vs.1.00±0.05,t=5.24,P＜0.05;NF-κB:0.69±0.09 vs.1.00±0.05,t=5.22,P＜0.05);while siPSGl group Col Ⅰ,Col Ⅲ,α-SMA,TNF-α,NF-κB proteins The expression was higher than that of the control group(Col Ⅰ:1.64±0.12 vs.1.00±0.07,t=7.98,P＜0.05;Col Ⅲ:1.53±0.11 vs.1.00±0.03,t=8.05,P＜0.05;α-SMA:1.40±0.09vs.1.00±0.08,t=5.75,P＜0.05;TNF-α:1.63±0.14vs.1.00±0.05,t=6.73,P＜0.05;NF-κB:1.47±0.12vs.1.00±0.05,t=6.26,P＜0.05).The expression levels of Col Ⅰ,Col Ⅲ,α-SMA,TNF-α,and NF-κB proteins in the pcDNA-KRT7 group were lower than those in the control group(Col Ⅰ:0.63±0.08 vs.1.00±0.04,t=7.17,P＜0.05;Col Ⅲ:0.75±0.09 vs.1.00±0.05,t=4.21,P＜0.05;α-SMA:0.67±0.05 vs.1.00±0.08,t=6.06,P＜0.05;TNF-α:0.72±0.06 vs.1.00±0.06,t=5.72,P＜0.05;NF-κB:0.66±0.07 vs.1.00±0.05,t=6.85,P＜0.05);while siKRT7 group Col Ⅰ,Col Ⅲ,α-SMA,TNF-α,NF-κB proteins The expression was higher than that of the control group(Col Ⅰ:1.59±0.10 vs.1.00±0.07,t=9.49,P＜0.05;Col Ⅲ:1.50±0.12vs.1.00±0.03,t=6.67,P＜0.05;α-SMA:1.48±0.08 vs.1.00±0.08,t=7.35,P＜0.05;TNF-α:1.73±0.15 vs.1.00±0.05,t=7.83,P＜0.05;NF-κB:1.51±0.12 vs.1.00±0.05,t=6.80,P＜0.05).The protein levels of Col Ⅰ,Col Ⅲ,α-SMA,TNF-α,and NF-κB in the QNZ group were lower than those in the control group(Col Ⅰ:0.57±0.07 vs.1.00±0.04,t=7.24,P＜0.05;Col Ⅲ:0.64±0.03 vs.1.00±0.05,t=10.69,P＜0.05;α-SMA:0.62±0.06vs.1.00±0.08,t=6.58,P＜0.05;TNF-α:0.59±0.07vs.1.00±0.06,t=7.70,P＜0.05;NF-κB:0.67±0.08 vs.1.00±0.05,t=6.06,P＜0.05);while the siPSG1+KRT7 group had higher protein levels of Col Ⅰ,Col Ⅲ,α-SMA,TNF-α,and NF-κB In the siPSG1 group(Col Ⅰ:1.99±0.15 vs.1.63±0.11,t=3.35,P＜0.05;Col Ⅲ:1.75±0.12 vs.1.52±0.10,t=2.99,P＜0.05;α-SMA:1.77±0.14vs.1.39±0.10,t=3.83,P＜0.05;TNF-α:1.99±0.10vs.1.61±0.15,t=3.65,P＜0.05;NF-κB:1.96±0.17vs.1.49±0.11,t=4.02,P＜0.05)and siKRT7 group(Col Ⅰ:1.99±0.15 vs.1.58±0.11,t=3.82,P＜0.05;Col Ⅲ:1.75±0.12 vs.1.49±0.10,t=2.88,P＜0.05;α-SMA:1.77±0.14 vs.1.49±0.07,t=3.10,P＜0.05;TNF-α:1.99±0.10 vs.1.75±0.10,t=2.94,P＜0.05;NF-κB:1.96±0.17 vs.1.50±0.11,t=3.94,P＜0.05).Conclusion HUCMSCs-Exos inhibits the biological activity of KD fibroblasts by upreg-ulating the expression of PSG1 and KRT7 genes in cells and reducing the protein expression of TNF-α and NF-κB in KFbs.
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