CDDO-Im Inhibits The Proliferation Of Keloid Fibroblasts Through Nrf2 Pathway

Objectives: Although keloid is a disease of benign hyperplasia of connective tissue,its itching and pain symptom is obvious.There is treatment resistance and high recurrence after operation,and only surgical resection will show a recurrence rate of 100%.This has a great impact on the patient’s body and mind.However,the exact pathogenesis of keloid is not clear,and it may be related to oxidative stress.Previous studies have shown that Nrf2(Nuclear factor erythroid 2-related factor 2)may be related to the pathogenesis of keloid.Nrf2 is a defense protein with antioxidant stress in cells,which may affect the proliferation of keloid fibroblasts.As an agonist of Nrf2,CDDO(2-cyano-3,12-dioxooleana-1,9(11)-dien-28-oic acid)is widely used in previous studies of other diseases.Therefore,in this study,we selected CDDO as the intervention drug.CDDO-Im(CDDO-Imidazolide)one of the higher effectiveness CDDO derivatives is selected to activate the Nrf2 pathway to explore the role of Nrf2 pathway in the proliferation of keloid fibroblasts and the inhibitory effect of CDDO-Im on keloid formation through this pathway for preventing and treating.Methods:(1)To explore the difference of Nrf2 expression between normal skin and keloid fibroblasts,they were divided into:(1)Normal skin group and(2)Keloid group.At the tissue level,the difference of Nrf2 expression level was determined by immunohistochemical method.At the cell level,the fibroblasts of the two groups were cultured respectively,and the difference of Nrf2 expression level was determined by Western Blot.(2)To explore the effect of Nrf2 induced by CDDO-Im at different concentrations on keloid fibroblasts,they were divided into the following groups:(1)Control/0 group and(2)Experimental groups: CDDO-Im groups with different concentrations(0.5μmol/L、0.7μmol/L、0.9μmol/L).CCK-8assay and scratch experiment were selected to determine the survival rate and invasive ability of fibroblasts respectively.The differences of protein expression level of Nrf2 and selected downstream molecules GCLC(γ-glutamate cysteine ligase catalytic Subunit)and NQO-1(NAD(P)H quinone oxidoreductase-1)in each group were determined by Western Blot.Results: 1.The expression level of Nrf2 in keloid is lower than that in normal skin.(p<0.05).2.After adding different concentrations of CDDO-Im to activate Nrf2 pathway,the results of CCK-8 assay,scratch experiment and Western Blot of keloid fibroblasts were determined.The results of CCK-8 assay showed that the cell survival rate of keloid fibroblasts treated with low concentration of CDDO-Im(0.5μmol/L、0.7μmol/L、0.9μmol/L)was lower than that of the control group(p<0.05),but there was no significant difference among the three groups.The results of scratch experiment showed that the invasive ability of keloid fibroblasts treated with low concentration of CDDO-Im(0.5μmol/L、0.7μmol/L、0.9μmol/L)decreased with the increase of CDDO-Im concentration(p<0.05).The results of Western Blot experiment: compared with the control group,the expression content of Nrf2 in 0.7μmol/L and 0.9μmol/L groups increased(p<0.05).There was no significant difference in Nrf2 expression content between 0.5μmol/L group and control group.There was no significant difference in GCLC and NQO-1 expression content in 0.5μmol/L,0.7μmol/L and 0.9μmol/L groups.The results show that CDDO-Im can inhibit the proliferation of keloid fibroblasts by activating Nrf2 pathway,but CDDO-Im inhibits the proliferation of keloid through other downstream molecules rather than Nrf2-related downstream molecules GCLC and NQO-1.Conclusion: The expression level of Nrf2 in keloid is lower than that in normal skin.CDDO-Im can inhibit the proliferation of keloid fibroblasts by activating Nrf2 pathway.The inhibition of keloid fibroblasts proliferation is through the downstream molecules other than GCLC and NQO-1 related to Nrf2.