| Background and ObjectivesWound healing is a complex process including three phases, referring to inflammation,proliferation and tissue reconstruction, which interact with each other. Injury, as an initiatingsignal, involves in the whole process of wound healing. The traditional concept is that thehemostasis is firstly activated after trauma, and followed by successively chemotaxis ofinflammatory cells to the wound site. However, recent studies have revealed that reactive oxygenspecies (ROS) played a critical role in initiating inflammatory response of wound healing. ROSdependent redox system is absolutely indispensable for initiating the cascade reactions of woundhealing. ROS, serving as a second messenger, can regulate numerous signal transductions andgene expressions.5-hydroxytryptamine (5-HT) and prostaglandin E2(PGE2) are two principal inflammatoryfactors in the early phase of wound healing. PGE2is believed to be associated with the signs ofredness, swelling, heat and pain in the wound area, which are major symptoms characteristics ofinflammation. However, the mechanism of rapid increase of PGE2after injury is still unknown.In this study, we investigated the production of PGE2and its upstream and downstreamregulating factors in a human keratinocyte injury model, in order to elucidate the role of ROSsignalings in the early inflammation of wound healing.PART I Mechanical damage induces PGE2release, which depends on ROS in human skinkeratinocytes.Objective To investigate the effect of ROS on inducing PGE2release in human skinkeratinocytes by mechanical damage.Methods1. Human skin keratinocytes were cultured and the cells of passages3-8were subjected tomechanical damage.2. Keratinocytes were divided into three groups as following: a) injury group (treated bymechanical damage); b) Nox inhibitor (DPI) pretreatment plus injury group; c) control group (no treatment).3. Samples were collected at0、1、5、10、15、30and60min after mechanical damage. ROSproduction was detected by measuring the fluorescence of2’,7’-dichlorofluorescein (DCF), aspecific probe for ROS.5. The supernatants were collected at0、1、2、4、6、8、12、24h after mechanical damage.And PGE2secretion was measured by enzyme immunosorbent assay (EIA).Results1. The ROS level increased immediately after mechanical damage, and reached a peak30min after mechanical damage (P<0.05).2. PGE2secretion raised significantly after mechanical damage, and reached a peak6h aftermechanical damage (P<0.05)；PGE2secretion still maintained a significantly high level24h aftermechanical damage (P<0.05).3. PGE2secretion was suppressed obviously by DPI2or4h after mechanical damage（P<0.05）.Conclusions The rise of Nox activity and ROS production led to PGE2secretion immediatelyafter mechanical damage.RART II ERK participates in the process of PGE2production induced by increased ROSafter mechanical damage in human skin keratinocytesObjective To investigate the effect of ERK phosphorylation on PGE2production induced bymechanical damage and ROS increase in human skin keratinocytes.Methods1. Human skin keratinocytes were cultured and the cells of passages3-8cells weresubjected to mechanical damage.2. Keratinocytes were divided into three groups as following: a) injury group (treated bymechanical damage); b) Nox inhibitor (DPI) pretreatment plus injury group; c) ERK inhibitor(PD98059) pretreatment plus injury group; and d) control group (no treatment).3. Samples were collected at0、0.25、0.5、1、2、4、6h after mechanical damage. Westernblot was used to qualify the expression of p-ERK and ERK. 4、DPI was added30min before the mechanical damage. Samples were collected at30minafter mechanical damage. Western blot was used to qualify the expression of p-ERK.5、PD98059was added1h before the mechanical damage. Samples were collected at2h or4h after mechanical damage. PGE2secretion was measured by EIA.Results1、Mechanical damage increased the extent of ERK phosphorylation, which peaked at15minand returned to basal level after60min (p<0.05).2、The NOX inhibitor DPI abrogated the extent of ERK phosphorylation by mechanicaldamage.3、PGE2secretion was reduced obviously by the ERK inhibitor PD98059at2or4h aftermechanical damage (P<0.05).Conclusions Mechanical damage can activate ERK, which participates in the process of PGE2production induced by increased ROS after mechanical damage in human skin keratinocytes..RART III COX-2participates in the process of PGE2production induced by increasedROS production and ERK phosphorylation after mechanical damage in human skinkeratinocytesObjective To elucidate signal pathway and molecular mechanisms in the earlyinflammation post mechanical damage in human skin keratinocytes.Methods1. Human skin keratinocytes were cultured and the cells of passages3-8cells weresubjected to mechanical damage.2. Keratinocytes were divided into five groups as following: a) injury group (treated bymechanical damage); b) Nox inhibitor (DPI) pretreatment plus injury group; c)PD98059pretreatment plus injury group; d)NS398pretreatment plus injury group; and e)control group (notreatment).3. Samples were collected at0、1、2、4、6、8h after mechanical damage. Western blot wasused to qualify the expression of COX-2.4、DPI or PD98059was added30min or1h before the mechanical damage. Samples were collected at2h after mechanical damage. Western blot was used to qualify the expression ofCOX-2.5、NS398was added1h before the mechanical damage. Samples were collected at2h or4h after mechanical damage. PGE2secretion was measured by EIA.Results1、COX-2protein accumulation caused by the mechanical damage was evident at2h, andmaintained until24h.2、The NOX inhibitor DPI abrogated Cox-2protein accumulation induced by mechanical damage.3、The ERK inhibitor PD98059abrogated Cox-2protein accumulation induced by mechanicaldamage.4、PGE2secretion was reduced obviously by COX-2inhibitor NS398at2and4h aftermechanical damage（P<0.05）Conclusions Nox-ROS-ERK-PGE2serves as a critical regulator for early inflammatoryresponse in wound healing. |
PDF Full Text Request