Effect of 5-luorouracil Carboxymethyl Chitosan Nanoparticles on the Apoptosis Level of Keloid Fibroblasts

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Xiaoguang Su, Bing Han, Xingdong Jia, Yanjun Gao

Abstract

Objective to investigate the effect of 5-fluorouracil (5-FU) loaded carboxymethyl chitosan (CMC) nanoparticles on the apoptosis level of keloid fibroblasts. Methodskeloid tissue (n = 80) was taken from inpatient and outpatient patients who were treated in our hospital from January 2020 to December 2020. The fresh keloid specimen tissues were washed with saline three times. After removing the epithelium, they were cut into 2mm×2mm tissue blocks for subculture. CMC nanoparticles loaded with 5-FU were prepared by ionically crosslinking CMC solution with calcium chloride. The viability of fibroblasts was determined by MTT assay. Transwell was used to assess fibroblast invasion. The wound healing test was used to evaluate the migration of fibroblasts. The apoptotic cells were analyzed by Annexin V-FITC and flow cytometry. The expression of apoptotic protein in fibroblasts was determined by Western blot analysis. The expression of transforming growth factor-β (TGF-β) and Smad2/3 were analyzed by immunohistochemistry. The mRNA expression of ERK1/2, protein kinase B (AKT) and nuclear transcription factor-κB (NF-κB) was analyzed by RT-qPCR. Results compared with the control group, there was no difference in the viability of fibroblasts in the nanoparticle group at 0h (P>0.05), and the viability of fibroblasts at 24h, 48h and 72h decreased by 37.29%, 29.58% and 28.38, respectively (P<0.05). Compared with the control group, the fiber cells composed of nanoparticles were less likely to pass through the pores of the basement membrane and their invasiveness decreased (P<0.05). Compared with the control group, the migration ability of fibroblasts in the nanoparticle group decreased (P<0.05). Compared with the control group, the apoptotic rate of fibers composed of nanoparticles increased (P<0.05). Compared with the control group, the expression levels of Bax, Caspase-3 and Caspase-9 apoptotic proteins in the nanoparticle group increased (P<0.05). Compared with the control group, the expression levels of TGF-β and Smad2/3 in the nanoparticle group decreased(P<0.05). Compared with the control group, the expression levels of ERK1/2, Akt and NF-κB mRNA in the nanoparticle group decreased (P<0.05). Conclusion 5-FU loaded CMC nanoparticles could reduce fibrosis in keloids, lower cell proliferation, migration and invasion, and increase cell apoptosis by inhibiting TGF-β related pathways.

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