| ObjectiveSkin wound healing is a complex process, concluding clotting of blood components, inflammation, granulation tissue formation, re-epithelialization, angiogenesis, fibroplasia and connective tissue retraction. p38MAPK, an important member of MAPK(mitogen-activated protein kinase, MAPK) family, is a serine/threonine kinase that may be phosphorylated and activated by cellular-stresses and inflammatory stimulus. It seems to be involved in the regulation of important cellular response such as apoptosis and inflammatory reactions. In the present study, the level of phospho-p38MAPK in skin incised wound was detected by immunohistochemical technique and western blotting. The aim of this investigation is to expole the possible function of p38MAPK in skin wound healing and its applicability to the wound age estimation.Materials and MethodsA total of 33 mice, each weighting 35～40g, were divided into eleven groups randomly, one control group and ten experiment groups, and each group was divided into 0h, 3h, 6h, 12h, 1d, 3d, 5d, 7d, 10d and 14d intervals three mice in each time point.A 1.5cm-long incision was made with a bistoury in the skin layer on the central dorsum under sterile technique after the mouse was anesthetized by inhalating diethyl ether. Then each mouse was fed with sterilized food and distilled water in individual cage to prevent infection.The 1.5cm×1.0cm specimens were taken from the wounded site after the mice were executed by cervical dislocation at 0h, 3h, 6h, 12h, 1d, 3d, 5d, 7d, 10d and 14d post-wounding to prepare for the subsequent procedures. The level of phospho-p38MAPK in mouse skin incised wound was detected by immunohistochemical and western blotting, and the non-incised mouse skin was used as control. The number and ratio of positive cells were counted and analyzed by microscope and comparative the band integral optical density value by software of Fluochem V2.0.The date were analyzed comparatively by the software of SPSS11.0 for Windows.ResultsA weak immunoreactivity of p-p38MAPK was detected in normal mouse skin. Expression of p-p38MAPK was detectable in polymorphonuclear cells(PMNs) in the wound specimens aged 6h. In the wound specimens aged from 12h to 24h after injury, p-p38MAPK were identified in a large number of infiltrating PMNs and part of mononuclear cells(MNCs). Afterwards from 1d to 3d postwounding, the p-p38MAPK positive cells were mainly found of MNCs and fibroblastic cells(FBCs) and mainly in FBCs from 5d to 14d postwounding. Morphometrically, the ratios of the number of p-p38MAPK stained PMNs, MNCs and FBCs to total number of the cells in the wounds were evaluated and calculated. The ratios of the p-p38MAPK positive cells were low in the wound specimens aged from 0h to 3h, and elevated in the wound specimens aged 12h and keep a highly level from 1d to 5d postwounding, with peak at the 3d. Thereafter, the ratios decreased from 7d to 14d and minimized in the specimens aged 14d. There were significant differences in ratios of p38MAPK positive cells between the neighboring time groups(P<0.05) except that of 5d postinjury. The obvious bands of 43KD were noticed at 12h and 3d and the expression of p38MAPK revealed a time-dependent pattern.ConclusionThe results suggested that p-p38MAPK may play an important role in inducing PMNs, MNCs and FBCs apoptosis during skin wound healing. Furthermore, p-p38MAPK may be used as a marker for the wound age determination. |
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