| Background:The skin is the largest and most vulnerable tissue of human body.Skin damge and subsequent scars due to various reasons are among the most concerned issues.In some cases,hypertrophic wound scar not only seriously affects patients’quality of life,but also causes huge physical and psychological pain.Therefore,it is of great significance to explore new treatments on wound healing,because even slight improvement of the scar would be of great significance to the patients.The most prominent histological features of scars are excessive activation of multiple cells and imbalanced destruction/deposition of extracellular matrix,which ultimately lead to abnormal accumulation of collagen and other extracellular matrix components in the wound.As the most important players in scar formation,keratinocytes and fibroblasts communicate closely in the middle and late stages of wound healing,so that the wounds can heal in an orderly manner.However,the two types of cells are not equal,as keratinocytes play a key role in activation of fibroblasts.Therefore,activation of fibroblasts by keratinocytes becomes a popular target to treat hypertrophic scars.A variety of cytokines are known to participate in wound healing.Among them,prostaglandin E2(PGE2),as the most widely distributed and most broadspectrum inflammatory cytokine,plays an essential role in scar formation.PGE2 is synthesized from Arachidonic acid under the catalysis of Cyclooxygenase(COX).During wound repair,PGE2 may promote proliferation and chemotaxis of fibroblasts via direct activation through keratinocytes paracrine pathway or indirectly inducing various downstream,leading to reduced collagen degradation ~9and excessive scar formation.In summary,regulating PGE2 in keratinocytes can largely affect fibroblast activity and scar formation.However,the molecular basis of this seemingly simple process is still poorly understood.This study aims to clarify how PGE production is regulated after keratinocyte activation,and intends to prevent formation of excessive hypertrophy by regulating PGE2 synthesis.Research objectives1.To clarify the specific mechanism of cyclooxygenase(Cox)family in regulating the expression of PGE2 in keratinocytes;2.To clarify the different regulatory effects of Cox-1 and Cox-2 on the expression of PGE2;3.To explore potential drugs that can effectively interfere the PGE2 expression and their pharmacological effects;4.To explore the effect of clinical drugs on wound scar formation in the actual situation;Research methods1.In activated Hu EK-N keratinocytes,the m RNA and protein expressions of Cox-1,Cox-2and PGE2 were analyzed by western blot and polymerase chain reaction(PCR).2.Specific agonists and inhibitors of Cox-1 and Cox-2 were used to determine the roles of the two Cox isoforms in the synthesis of PGE2.3.Test the effect of ibuprofen and Latanoprost on PGE2 expression,4.The mouse wound scar model was used to observe the time and histological differences of wound healing and verify previous cytological experimental results.Research results1.After activated by heparin-binding egf-like growth factor(hb-egf),the Hu EK-N presents the following features:(1)Both Cox-1 and Cox-2 m RNA levels increased,and the degree of elevation was time-dependent.(2)The Cox-2 protein level increased in a time-dependent pattern.However,it change was not observed in the Cox-1 expression(3)Inhibition of Cox-1 did not significantly affect the expression of PGE2,while inhibition of cox-2 significantly did inhibite the expression of PGE2.2.The effects of ibuprofen and latanoprost on the PGE2 expression in Hu EK-N keratinocytes were compared:(1)Ibuprofen inhibited the expression of PGE2 protein in Hu EK-N cells in a concentration-dependent manner(IC50=8.18 ng/ml,95%CI=4.4-17.69,R2=0.929).;(2)Latanoprost promoted the expression of PGE2 protein in a concentration-dependent manner(EC50=133.1 ng/ml,95%ci=76.9–230.1 ng/ml,R2=0.91).3.The effects of ibuprofen and latanoprost on local scar formation on wound surfaces of live mice were compared:(1)Healing time:a.The wound healing time of the control group was 11.1±0.7 days.b.After ibuprofen treatment alone,the wound healing time was significantly longer than that of the control group,which was 14.6±0.9 days(P<0.05).c.After treatment with latanoprost alone,the wound healing time was significantly shorter than that of the control group,which was 10.1±0.4 days(P<0.05).(2)The characteristics of collagen fibers in local scar tissue on the wound surface were observed histologically,and the main evaluation criteria were the difference in collagen arrangement and staining density:In the control group,the collagen arrangement and staining density scores were 4.1±0.3~aand 3.3±0.1~c,respectively.In the Ibuprofen group,collagen fibers in scar tissue were significantly less than those in the control group,and the staining was lighter,but the arrangement was orderly.The scores of collagen arrangement and staining density were 3.5±0.5~b and 2.5±0.5~d,respectively,showing statistically significant differences with the control group(P<0.05).In the Latanoprost group,collagen fibers in scar tissue were arranged in a disordered manner and stained in a dense manner,and the scores of collagen arrangement and staining density were 1.5±0.8~d and 4.8±0.3~a,respectively,which were also significantly different from the control group(P<0.05).Latanoprost can effectively reverse the delay of scar healing caused by ibuprofen,which is concentration-dependent.Research conclusions1.Cox-2 is the main protease promoting the synthesis of PGE2.2.For translational medicine purpose,the ibuprofen and latanoprost can effectively inhibit and activate cox-2,respectively,thereby indirectly regulating the expression of PGE2 protein in keratinocytes.3.Ibuprofen significantly prolonged the wound healing time in vivo by indirectly inhibiting the expression of PGE2 protein,which was confirmed by poor scar formation in histological study.At the same time,Latanoprost can significantly shorten the wound healing time,leading to the excessive healing of the wound surface,leading to the possibility of excessive formation of potential scars.4.Latanoprost can effectively reverse the adverse reactions of Ibuprofen:In this study,we showed that Latanoprost could effectively reverse the delayed wound healing caused by Ibuprofen,providing a theoretical basis for the safe NSAID usage in clinical practice. |
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