5-ALA-PDT Induced Ferroptosis In Keloid Fibroblasts Via ROS And The Mechanisms Involved

Characterized by elevated proliferation of fibroblasts and excessive deposition of extracellular matrix,keloids are fibroproliferative disorders usually caused by abnormal healing of wounds following burns,trauma or infection.Their clinical features include itching,pain and deformed appearance,and in severe cases may be accompanied by bleeding,ulceration and infection,which greatly affect the patient’s quality of life.Keloids display many cancerous properties,including uncontrolled and invasive growth,high rates of recurrence as well as similar bioenergetics.5-aminolevulinic acid-based photodynamic therapy（5-ALA-PDT）is an effective treatment that performs cytotoxic effects by producing reactive oxygen species（ROS）,which is linked to lipid peroxidation and ferroptosis.Ferroptosis,a novel form of cell death characterized by lethal iron-dependent lipid peroxidation,is regulated by multiple cellular metabolic events,including redox homeostasis,iron handling,mitochondrial activity and metabolism of amino acids.This study was conducted with human normal skin tissue and keloid tissue as well as extracted fibroblasts to investigate whether 5-ALA-PDT can induce ferroptosis in keloid fibroblasts and the potential mechanisms that may be involved.This lays the foundation for further understanding of the oxidative stress state and cell death process in keloid fibroblasts under PDT treatment,and also provides new ideas for promoting the therapeutic effect of 5-ALA-PDT on keloids through ferroptosis in clinical practice.Ⅰ.Effect of 5-ALA-PDT on keloid fibroblasts and screening of optimal experimental parameters1.Identification of in vitro tissues and primary cells:HE staining showed that,compared with normal skin,there were more thick collagen fibers in the dermis of keloid,with more complex and compact arrangement and higher cell number.Immunohistochemical staining of vimentin showed that the extracted primary cells were positive for vimentin,and the extracted and cultured primary cells in the experiment were fibroblasts with high purity.2.Cytotoxic effects of 5-ALA-PDT on keloid fibroblasts:The results of CCK-8showed that in the range of 0-8mM（5-ALA concentration）and 0-80 J/cm²（light intensity）,5-ALA-PDT inhibited the cell viability of keloid fibroblasts in a 5-ALA concentration and light intensity dependent manner,and reached the maximum at 8mM and 80 J/cm².3.Conversion of 5-ALA to PpIX in keloid fibroblasts:Observation by fluorescence microscope showed that the fluorescence intensity of PpIX under fluorescence microscope increased with the increase of 5-ALA concentration4.5-ALA-PDT induced ROS production in keloid fibroblasts:The fluorescence intensity of ROS in 4mM group was significantly higher than that in 1mM group under the same light intensity.At the same 5-ALA concentration,ROS fluorescence intensity was light intensity dependent,and reached the highest value at 32J/cm².The trend of ROS induction by 5-ALA-PDT was similar to that of Erastin,a ferroptosis inducer.Ⅱ.5-ALA-PDT induced ferroptosis in keloid fibroblasts1.Differentially expressed genes in keloid fibroblasts treated with 5-ALA-PDT:mRNA sequencing analysis showed that significantly up-regulated genes in keloid fibroblasts treated with 5-ALA-PDT included PTGS2 and ATF3,suggesting that ferroptosis may be involved.2.Western Blot showed that compared with normal skin fibroblasts,CCND1 and ferroptosis-related proteins GPX4 and xCT were highly expressed in keloid fibroblasts,and5-ALA-PDT could inhibit this trend.3.5-ALA-PDT induced ferroptosis in keloid fibroblasts:The results showed that5-ALA-PDT increased the levels of Fe²⁺,lipid peroxide and malondialdehyde in keloid fibroblasts,and the trend was similar to that of Erastin.Pretreatment of Ferrostatin-1,a ferroptosis inhibitor,can reverse the trend of 5-ALA-PDT.Compared with the control group,the mitochondria in the 5-ALA-PDT and Erastin treatment groups showed characteristic changes of ferroptosis in keloid fibroblasts,namely,the mitochondria were compact with smaller size and the membrane density of mitochondria has increased with fewer visible cristae.4.Western Blot showed that the inhibition effect of 5-ALA-PDT on the high expression of CCND1,GPX4 and xCT in keloid fibroblasts was similar to that of Erastin treatment,and Ferrostatin-1 pretreatment could reverse the trend of 5-ALA-PDT.In summary,we identified that 5-ALA-PDT led to elevated levels of ROS and lipid peroxidation in keloid fibroblasts,accompanied by downregulation of xCT and GPX4,which are associated with anti-oxidation effects and ferroptosis inhibition.These results may indicate that 5-ALA-PDT treatment increases ROS while inhibiting xCT and GPX4,thus promoting lipid peroxidation to induce ferroptosis in keloid fibroblasts.