A Study On The Effect Of Calpain And Calpastatin On Incised Skin Wound Healing In Mice | Posted on:2010-01-21 | Degree:Doctor | Type:Dissertation | Country:China | Candidate:W Zhang | Full Text:PDF | GTID:1114360275467482 | Subject:Forensic pathology | Abstract/Summary: | PDF Full Text Request | IntroductionThe initial response to wounding is typically the formation of a blood clot,which together with local damaged tissue- releases proinflammatory signals,which trigger inflammatory cell infiltration in the form of neutrophils and then macrophages into the wound site.These signals and those from the invading inflammatory cells influence both re-epithelialisation and connective tissue contraction of the wound.The most logical method of cellular down-regulation is through apoptosis.Apoptosis allows for the eliminations of entire populations without tissue damage or an inflammatory response.Previous studies during skin wound healing have shown that caspase-3 may play a pivotal role in inducing neutrophil,macrophage and fibroblast apoptosis.Calpains,calpainl and calpain2,are calcium-activated,neutral cysteine proteases proposed to participate in and regulate many important intracellular processes,such as turnover of cytoskeletal proteins,signal transduction cascades,cell cycle control and apoptosis.Calpain1 and calpain2 are ubiquitously distributed in all animal cells examined thus far.Both are heterodimers,consisting of a homologous catalytic 80 kDa subunit and the identical regulatory 30 kDa subunit made up of distinct domains.It is generally agreed that thes enzymes are proenzymes that are regulated by Ca2+binding and autoproteolytic activation.It is still unkown whether calpains are activated during incised skin wound healing.In this study,the expression and activation of calpain1, calpain2 and calpastatin during incised skin wound healing in mice were investigated by immunohistochemical staining,Western blotting and colorimetric measurement of proteolytic activity.In addition,by administering calpain inhibitor MDL28170 we investigated calpains and caspase-3 activities to attempt to intepret the role of calpains during incised skin wond healing in mice.Materials and MethodsA total of 115 Kunming mice(8 week-old,healthy),of either sex,weighting 30±2g were provided by the Animal Breeding Laboratory of China Medical University. The mice divided randomly into three group with five mice in each group,the injury group,the inhibitor group,the control group and the sham-injured group.The injury group was sub-divied into Oh,6h,12h,1d,3d,5d,7d,10d and 14d.The inhibitor group and the control group were sub-divied into 6h,12h,1d,3d,5d and 7d.A 1.5cm-long full-thickness incision was made on the central dorsum of each mouse after the anesthetized by inhaling ether.The first dose,24 mg/kg of MDL 28170,was injected intraperitoneally in the inhibitor group and 12 mg/kg of MDL 28170 was subsequently delivered every 12 h until 7d post-wounding.As control,equal volume of 2%DMSO was delivered in the control group.A 1.5cm×1.0cm specimen was collected from the wounded site after the animal was sacrificed by cervical dislocation at each post-wounding time-point for the following the experimental procedures including H.E staining,immunohistichemical staining,Western blotting,colorimetric measurement of calpain activity.The data were expressed as mean±SE.The data were compared using one-way ANOVA test by SPSS 12.0 for Windows,and were evaluated using t-test.P<0.05 were considered to be statistically significant.ResultsPartâ… Study on the expression of calpain and calpastatin during incised skin wound healing1.The pathomorphological changes during incised skin wound healingPolymorphonuclear leucocytes(PMNs) and mononuclear cells(MNCs) were detected to wound sites during acute exudative phase from 6h to 1d.Then an increase in the cellularity of fibroblasts at the wound margins in 3d post-wounding,and the proliferation is maximal at 5 to 7d after post-wounding.At the same time,granular tissue was formed.In the last phase,scar was constituted until 14d.2.The immunohistochemical study on the expressions of calpain1, calpain2 and calpastatin during incised skin wound healingCytoplasmic immunoreactivity for calpainl,calpain2 and calpastatin was predominantly detected in PMNs,MNCs and FBCs which might be differentiated by their morphological feature.The ratios of calpain1-,calpain2- or calpastatin- positive cells were time-dependent.3.The expressions and cleavages of calpain 1,calpain2 and calpastatin during incised skin wound healing by Western blottingThe expressions of calpain1 and calpain2 were time-dependent through the statistical analysis on the comparative grey intensity.The expressions of calpain1 and calpain2 precursor were maximized at 5d post-wounding and the cleavaged fragment of them significantly increased at 3d and 5d post-wounding respectively.The expression of calpastatin fragment was maximized at 3d post-wounding.4.Measurement of calpain and caspase-3 activity during incised skin wound healingThe relative activity of calpain reached its peak in 1d and 5d post-wounding respectively.The fold increase in caspase-3 activity was noted gradually from 6h to 3d post-injury,reached the maximum in 5d,and then began to decline.Partâ…¡The effect of calpain inhibitor MDL28170 in incised skin wound healing1.The pathomorphological changes affected by calpain inhibitor MDL28170 during incised skin wound healingThe skin wound healed more rapidly in the inhibitor group than in the control group.2.The immunohistochemical study on the expressions of calpain1, calpain2 and calpastatin after in vivo administration of MDL28170 during incised skin wound healingThe ratios of calpain1- and calpain2- positive cells in the inhibitor group were lower than those in the control group from 6h to 7d post-wounding.In contrast,the ratios of calpastatin-positive cells in the inhibitor group were higher than those in the control group from 12h to 7d post-wounding.3.The expressions and cleavages of calpain1,calpain2 and calpastatin after in vivo administration of MDL28170 during incised skin wound healingThe expression of calpain1 precursor in the inhibitor group was obviously down-regulated as compared with that in the control group with except at 7d post-wounding.In addition,the preducts of calpainl and calpain2 fragment in the inhibitor group were obviously reduced than those in the control group.However,the product of calpastatin fragment was significantly elevated in comparison with that in the control group.4.Measurement of calpain and caspase-3 activity after in vivo administration of MDL28170 during incised skin wound healingThe relative activity of calpain and the fold increase in caspase-3 activity in the inhibitor group werereduced significantly as compared with that in the control group from 12h to 7d post-wounding.DiscussionPresently many studies have focused on the calpain inhibitor,calpastatin,the specific endogenous inhibitor of calpain.It is not a potential reagent for study because of its molecular weight(110KDa).MDL 28170 is a newly-developed potent,and non-peptide,cell-permeable,uncompetitive and selective calpain inhibitor that may rapidly penetrate the blood-brain barrier following systemic administration.Thus,we investigated the role of calpain during skin wound healing by systemic administration of a calpain inhibitor MDL28170.In normal mouse skin,the expression of calpain1,calpain2,and calpastatin were detected in epidermal cell layers,sebaceous glands,hair follicles and subcutaneous muscles.The high expression of calpain2 was detected at 0h in the control group which indicated that calpain2 may participate in kerotinocyte apoptosis in epidermic basal layer and sustain stable homeostasis of the skin,and overactivation of calpainl was an important event in skin wound healing.Using Western blot analysis and kinase activity measurement,the results indicate that calpainl and calpain2 were actived during incised skin wound healing,which were relevant to the mechanism in skin healing.MDL28170 can obviously inhibit calpain and caspase-3 activity from 12h to 7d post-wounding.These results showed that calpain1 and calpain2 were activated and calpain may play a role at upstream of caspase cascade during incised skin wound healing.The skin wound healed more rapidly in the inhibitor group than in the control group following systemic administration of a calpain inhibitor MDL28170.In addition, the cells number of the inhibitor group was apparently more than that of the control group.These findings indicate that MDL28170 can effectively inhibit the activition of calpain and caspase-3 and decrease the number of apoptotic cells during incised skin wound healing.MDL28170,the calpain inhibitor can accelerate skin wound healing in mice.It is found that calpainl activity was low level from 6h to 12h post-wounding and reached its peak at 1d post-wounding.In contrast,the high expression of calpain2 was detected from 6h to 1d post-wounding.The ratios of calpain1- and calpain2- positive cells hayed a drastic increase in parallel and activity of calpain increased obviously at 1d post-wounding The present observations showed that calpain1 and calpain2 activity were a high level from 5d to 14d post-wounding.Furthermore,the expression of calpain1,calpain2 and calpastatin were weak in the metabolic stage fibroblast,and the expression appeared highly in the adult fibroblast.Taken together,these data suggested that calpain and calpastatin may play important role in migration,proliferation and apoptosis in neutrophils,mononuclear cells and fibroblasts.The ratios of positive cells and Western blotting of calpain1,calpain2 and calpastatinin dicated that expression of calpain1,calpain2 and calpastatinin were time dependent.In particular,calpain1 may be used as a marker for the time-estimation of skin wound.Conclusion1.In normal mouse skin,calpain1,calpain2 and calpastatin are distributed in epidermal cell layers,sebaceous glands,hair follicles and subcutaneous muscles.2.Both calpain1 and calpain2 are activated during incised skin wound healing.3.calpain may play a role upstream of caspase-3 during incised skin wound healing.4.Inhibition of calpain activity can accelerate skin wound healing in mice.5.Calpain play an important role in migration and proliferation in monocytes and fibroblasts and it may be related to apoptosis in them.6.Calpain1 may be used as a marker for estimation of skin wound age. | Keywords/Search Tags: | Skin incised wound, apoptosis, Ca2+, calpain, calpastatin, caspase-3, calpain inhibitor, immunohistochemistry, Western blotting, kinase activity, PMNs, MNCs, FBCs, wound age estimation | PDF Full Text Request | Related items |
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