Effect And Mechanism Of Sestrin2 On Wound Healing Of Deep Second Degree Burns

BACKGROUNDThe treatment of burn wound has always been the focus of clinical and laboratory research.In clinical,the treatment of patient is usually guided according to the degree of burns.The first degree and superficial second degree burns can usually be cured after treatment with wound dressing.The third degree burns usually require surgical operations such as skin grafting or flap transplantation.The schema for deep second degree burns is equivocal between the wound dressing and surgical operation.The wound of deep second degree burns usually takes long time to heal with obvious scar formation.The infection,which is very common during the wound treatment,could easily lead to a deeper wound.Therefore,it is still importantly needed to clarify the underlying mechanisms of wound healing process in deep second degree burns and seek the more efficient therapy to promote the wound healing outcome.Wound healing is a process involving with multiple cells and cytokines,which is usually characterized as three phases: inflammation,formation of new tissues,and remodeling.Previous studies have found that adjust the inflammation and accelerate the epithelialization would promote the wound healing process.Sestrin2(SESN2),a member of the sestrin family,is a stress-inducing protein widely existed in mammalian cells and plays a role in the regulation of metabolism,anti-aging,and inhibiting obesity.Previous studies have showen that the SESN2 expression was dramatically elevated when the cells were stimulated by oxidative stress,DNA damage and hypoxia,and plays a pivotal role in protecting cells by inhibiting inflammation,regulating autophagy,fighting with oxidative stress and maintaining cell homeostasis.Recently,SESN2 was reported to regulate immune responses in many diseases and promote the recovery of epithelial cells during inflammatory injury.Our previous work showed that the expression of SESN2 mRNA and protein were significantly increased in the deep second degree burn wounds,suggesting that SESN2 plays a role in the wound healing.Thus,we hypothesized that the improvement of the SESN2 could promote the wound healing through inhibiting the release of inflammatory cytokines and promoting keratinocytes proliferation and migration.To investigate the role of SESN2 in the wound healing of deep second degree burns,we established a animal model of deep second degree burn wounds.Eupatilin,the agonist of SESN2,was injected in the wound edge to increase SESN2 level,and the si-SESN2 was used to reduce the SESN2 level.The effects of SESN2 on inflammation and tissue regeneration were observed in vivo and in vitro.Finally,the mechanisms of SESN2 in promoting wound healing were also researched.PURPOSETo investigate the related cellular and molecular mechanisms,the role of SESN2 in wound healing of deep second degree burns was studied through the observation and analysis of the expression pattern of SESN2 in deep second degree burn wounds during the inflammatory period and the tissue formation period.Finally,the research provide the effective experimental evidence for effective treatment of deep second degree burns.METHODS1.Build the deep second degree burn model by C57BL/6.C57BL/6 mice were randomly divided into 4 groups including normal control group and scald for 2 s,4 s and 6 s groups.After hair removal,mice were treated with hot steam at 92°C for 2 s,4 s and 6 s to make round wounds with a diameter of 1.5cm on the back.The skin samples were obtained at 12 h,24 h and 48 h after burned.HE staining was performed to observe the depth of skin injuries.2.Detection of the expression of SESN2 in deep second degree burn wounds.Deep second degree burns were made on the back of C57BL/6.Skin samples were collected at 12 h,24 h,2 d,4 d,7 d after injury.We detected SESN2 expression by Real-time PCR,immunohistochemistry and Western blot.The expression of inflammatory cytokines such as TNF-?,IL-1,IL-6 and the cell proliferation marker Ki-67 were also detected.3.The role of SESN2 in the wound healing of deep second degree burns was observed by interfering SESN2 or using eupatilin(the agonist of SESN2).C57BL/6 mice were divided into 4 groups: control group,eupatilin injection group,si-NC injection group and si-SESN2 injection group.The deep second degree burns were made at 24 h after the first time of injection,and the mice were injected around the wound every other day until the wound was healed.Observe and record the wound healing process.The Real-time PCR,immunohistochemistry and Western blot were used to detect the effects of SESN2 on inflammation,cell proliferation and migration in wound healing process,and clarify the role of SESN2 in the deep second degree burns.4.The expression of SESN2 in HaCaT cells with heat treatment and its effects on the cell proliferation and migration.Real-time PCR and Western blot were used to detect the expression of SESN2 in HaCaT cells after heat treatment.Then the HaCaT cells were divided into control group,eupatilin group,si-NC group and si-SESN2 group.The Real-time PCR was used to clarify the effect of SESN2 on the expression of inflammatory cytokines.The EDU assay and CCK-8 assay were performed to detect the effects of SESN2 on the proliferation of keratinocytes.The scratch assay and Transwell assay were used to determine the effects of SESN2 on the migration of keratinocytes.5.Role of SESN2 in promoting the wound healing of deep second degree burns.Through the regulation of SESN2 expression in HaCaT cells,we observed the phosphorylation status of AMPK,NF-?B,and ERK signaling pathways,and explored the possible molecular mechanism of SESN2 in promoting wound healing of deep second degree burns.RESULTS1.HE staining showed that the skin epidermis and deep dermis were damaged but hair follicles and sebaceous glands remaining in the 4 s scald mice.The degree of wounds was stable at 24 hours after injury,which was consistent with the pathological diagnostic criteria for deep second degree burns.2.Immunohistochemical staining analysis showed that SESN2 staining was most obvious at the wound edge of C57BL/6 mice at 24 h after injury.Real-time PCR and Western blot results showed that SESN2 mRNA and protein expression level increased transiently after burn injury,peaked at 24 h(P < 0.05),and then gradually decreased.There was no difference of SESN2 expression compared with control at 7 days after injury(P > 0.05).The expression of TNF-?,IL-1? and IL-6 were significantly upregulated at 24 h after injury(P < 0.01).HE staining showed a dramtic increase of inflammasome in the dermis.Ki-67 level was significantly higher than the control group at 4 days after injury(P < 0.05),and decreased to the normal level at 7 days after injury.3.Calculate and analysis the wound healing rate between each groups by comparing the percentage of remaining area of the wound with the initial burn area.The wound healing rate of the si-SESN2 group was significantly reduced at 7 days and the 21 days compared with the control group(P < 0.05).There was no significant difference between the eupatilin group and the control group within 7 days after injury,but the healing rate of the eupatilin group was much higher than that of the control group at the day 14 and day 21(P < 0.01).The results suggested that SESN2 plays an important role in promoting wound healing in deep second degree burns.4.SESN2 silencing in C57BL/6 mice could dramatically upregulate the expression of cytokines such as TNF-?,IL-1?,and IL-6 but inhibit the expression of Ki67 compared with the control group by Real-time PCR analysis.The results suggested that low expression of SESN2 leads to excessive inflammatory reaction and reduced cell proliferation in deep second degree burns.5.There was a transient increase of the mRNA expression of SESN2 at 1h after heat treatment of HaCaT cells,and the expression of SESN2 has no difference with that of the control group after 4 h.Western blot results also showed a transient increase in SESN2 protein level,with the peak appearing at 2h after heat treatment(P < 0.01).Real-time PCR detection of TNF-?,IL-1?,and IL-6 showed that the levels of inflammatory cytokines in the eupatilin treatment group were significantly lower than those in the control group(P < 0.01),while those in the si-SESN2 treatment group were significantly higher(P < 0.01).It was confirmed by scratch assay and Transwell assay that the increased SESN2 expression could enhance the migration ability of HaCaT cells,CCK8 and EDU assay confirmed that the interference of SESN2 inhibits the proliferation of keratinocytes.6.Results of studies on SESN2/AMPK/NF-?B pathway showed that up-regulating SESN2 expression could increase the phosphorylation level of AMPK and inhibit the phosphorylation level of NF-?B.Although the phosphorylation of NF-?B was increased by the AMPK inhibitor,it could not be inhibited by up-regulated SESN2 expression.Results of studies on SESN2/ERK pathway showed that the phosphorylation of ERK could be increased by up-regulated SESN2 expression,and it could be down-regulated by si-SESN2.It suggests that SESN2 inhibits inflammatory response through SESN2/AMPK/NF-?B signaling pathway,and promotes proliferation and migration of keratinocytes through SESN2/ERK signaling pathway.CONCLUSIONS1.A stable deep second degree burns mouse model is established with the C57BL/6 mice continuously burned at the back skin for 4 s by 92°C hot steam.2.Improving the expression of SESN2 in the wound tissue of deep second degree burns can promote the wound healing process in C57BL/6 mice.3.SESN2 promotes the wound healing of deep second degree burns by inhibiting inflammation and promoting the migration and proliferation of keratinocytes.4.SESN2 regulates the inflammatory action through the AMPK/NF-?B signaling pathway,and promotes the proliferation and migration of keratinocytes through the ERK signaling pathway.