Preliminary Study Of Ferroptosis In Primary Cutaneous Amyloidosis

Primary cutaneous amyloidosis(PCA)is a skin metabolic disorder caused by the deposition of amyloid in the skin.At present,the etiology of the disease has not been completely clarified and there are hypotheses that amyloids originate from the epidermal basal keratinocytes and are released after death.A large number of previous studies have shown that OSMR gene mutations are involved in the pathogenesis of PCA.But how the basal keratinocyte dead and whether the OSMR control it still undefined.Ferroptosis is a process involving the accumulation of reactive oxygen species caused by the fail or deficiency in GPX4,the overexpression of ACSL4 and the iron ion paticipation,which ultimately leads to cell death.Integrinα6β4,which is responsible for anchoring basal keratinocytes on the basement membrane,can inhibit the occurrence of ferroptosis.By qPCR,Western Blot and immunofluorescence,we detected the expression of ferroptosis markers GPX4,ACSL4,OSMR,integrin α6β4 and 4-HNE.Consider that ferroptosis may not occurred in basal keratinocyte in PCA and ruducion with OSMR expression may increase the sensitivity of cells to ferroptosis.Objective:1.Explore wthethe ferroptosis occurred in PCA keratinocyte;2.Explore whether OSMR is involved in regulating ferroptosis of PCA keratinocyte.Methods:1.8 skin lesion tissues and 8 healthy skin tissues were collected from PCA patients and health volunteers who were divided into 2 groups:PC A group and healthy control group;2.Western Blot was used to detect the expression of apoptotic execution protein cleaved caspase-3,pyropotosic execution protein GSDMD,and ferroptosis protein GPX4 in two groups’tissues;3.QPCR and Western Blot were used to detect the RNA and protein expression of GPX4,ACSL4,OSMR,integrin a6 and integrin β4 in PCA tissues;4.Immunofluorescence detection of 4-HNE expression in tissues;5.HaCaT cells were cultured in vitro and siRNA was used to interfere with OSMR expression,and cultured at 20μmol/L erastin for 48h.Flow cytometric were used to detect lipid peroxidation.QPCR and Western Blot were used to detect RNA and protein expression of GPX4,ACSL4,OSMR,integrin α6 and integrin β4.Results:1.The expression of GPX4 and GSDMD in the PCA tissue was lower than healthy tissue.Cleaved caspase-3 was not significantly different between the PCA group and the healthy control group.2.Compared with the healthy control group,the RNA and protein expression of GPX4 and OSMR in PCA skin lesions decreased.The RNA and protein expression of integrin α6,integrin β4,and ACSL4 were no significant difference.3.No obvious expression of 4-HNE in PCA group;4.In the case of unsuccessful induction of ferroptosis in HaCaT cell,compared with the control group,the RNA and protein expression of OSMR in the siRNA treatment group was significantly decreased.The RNA expression of GPX4,ACSL4 and integrin β4 reduced but the protein expression was not significantly different from the control group.5.After 48 hours of stimulation with erastin and siRNA,OSMR reduced and ACSL4 increased.GPX4,integrin α6 and integrin β4 decreased in mRNA while were not significantly changed in protein.Conclusion:Reducion of OSMR may increase the ferroptosis sensitivity in HaCaT cell,but ferroptosis may not occurred in keratinocytes in PCA.