A Study On Time-dependent Expressions Of Caspase-9 And Caspase-3 During Skin Incised Wound Healing In Mice

IntroductionIt is well established that skin wound healing can be temporally divided into three phases, the early acute exudative phase, fibro - proliferative phase and tissue remodeling phase. Inflammatory cells including polymorphonuclear cells (PMNs) , mononuclear cells (MNCs) and fibroblastic cells (FBCs) play an important role in remodeling injured skin, removing necrotic skin tissue and invaded pathogens. The number of inflammatory cells increases significantly during early acute exudative phase and fibro - proliferative phase, but decreases significantly during tissue remodeling phase. Recent studies have suggested that apoptosis occurs and plays an important role in achieving a decrease in cellularity during skin wound healing. More and more studies indicated that the family of caspases proteases produce a marked effect during apoptosis. As the important initiate factor and executioner, caspase - 9 and caspase - 3 maybe have some important value. To investigate if caspase - 9 and caspase - 3 play an important role in inducing PMNs, MNCs and FBCs apoptosis during skin wound healing and explore the applicability of caspase - 9 and caspase - 3 to determination of wound age, the expressions of caspase - 9 and caspase - 3 were studied by im-munohistochemical and western blotting techniques on skin incised wounds at different posttraumatic intervals in mice.Materials and Methods1. Establishment of animal modelA total of 33 healthy, adult mice, weighting 35 ~ 40g were emplored in thestudy. A 1.5cm —long incision was made with a scalpel in the skin layer on the central dorsum under sterile technique. After wounding, each mouse was individually housed in a cage and given sterilized chow and redistilled water to prevent bacterial infection. 1. 5 cm x lcm specimens were taken from the wounded sites after the amimals were anaesthetized and sacrificed by cervical dislocation at Oh, 3h, 6h, 12h, Id, 3d, 5d, 7d, lOd, 14d (3 mice in each group) post-wounding. The remaining 3 mice were used as controls.2. Immunohistochemical detection of caspase —9 and caspase —3 Immunohistochemistry SP method was applied to investigate the protein expression of caspase - 9 and caspase - 3. After serum incubated, sections were incubated with polyclonal antibodies against caspase -9 or caspase -3 at 4°t o-vernight at a dilution 1:300. Goat - anti - rabbit IgG - biotiny incubated at 25°C for 20 minutes. The slides were incubated with SP at 251 for 20 minutes. Visualization of antibody binding was the DAB method. Finally, the sections were counterstained with hematoxylin, dehydrated and mounted. H. E staining of the sections was routinely conducted.3. Analysis of caspase - 9 by Western blotWhole cell extract proteins were loaded at 100|xg/lane[w/v] on 12% SDS — PAGE ( SDS — Tris — glycine polyacrylamide gel electrophoresis). Protein was transferred to PVDF membrane, blocked in 5% nonfat dry milk and 0. 1% Tween -20. Membrane was probed with polyclonal antibody against caspase —9 ( Neomarker). Then it was incubated with goat - anti - rabbit secondary antibody. Immunoreactive bands were visualized by ECL for detecting the bands of procaspase -9 and cleaved subunit P20. The procaspase -9 was detected as a 46kD black band and the P20 subunit of caspase - 9 was detected as a 37kD black band. We can learn the intensity of expression from the shade of black bands.Results1. Histopathological changes of the skin incised wound healingIn the wound specimens aged 3h, a few PMNs were detected in injuriedsite, and in the wound specimens aged 12h after wound there were a large number of PMNs. In the specimens aged Id, many PMNs and MNCs infiltrated into wound site. Form 3d to 5d after wound, most of inflammatory infiltrating cells were PMNs and MNCs. The epidermis proliferated remarkably at posttraumatic 7d and covered the wound completely at 14d.2. The expressions of caspase -9 and caspase -3 during skin wound healingIn the control specimens, the caspase - 9 positive cells or caspase - 3 positive cells were detected in the epidermis, hair follicle epithelium and sebaceous gland epithelium. The caspase - 9 positive cells or caspase - 3 positive cells were detected in PMNs in the wound specimens aged 3 hour, and in the wound specimens aged from 6h to 24h after wound caspase - 9 or caspase - 3 was identified in a large number of infiltrating PMNs and part of MNCs. Afterwards, the MNCs and FBCs accounted in the most part of the caspase — 9 or caspase — 3 positive cells.The percentage of caspase -9 and caspase -3 positive cells;The percentage of positive cells of caspase - 9 was low in the wound specimens aged Oh ~ 3h (9. 38 ± 0. 28% ) , and maximized in the specimens aged 3d (55. 53 ± 1. 72%) , thereafter, the percentage decreased and minimized in the specimens aged 14d( 13.20 ±0. 85% ). The percentage of positive cells of caspase - 3 was low in the wound specimens aged Oh ~3h(4. 53 ±6. 53% ) , and maximized in the skin wound aged 3d (62. 66 ±4. 84% ), thereafter, the percentage minimized in the specimens aged 14d(21.56 ±4. 54% ). By statistical analysis, except Oh group, there is a significant difference in the comparison of the caspase -9 or caspase -3 positive cell ratio between the neighboring groups (P <0. 01).3. Analysis of caspase - 9 by Western blotThe procaspase - 9 with moleculer weight of 46 kD, was detected at sameintensity in control group and experimental groups. The large subunit of caspase-9 named P20, with moleculer weight of 37kD, processed weakly in controlgroup and Oh group. The expression of P20 strengthened slightly from 3h to 6hbut remarkably increased from 12h. At 3d it reached the maximum. After that,the intensity of P20 was decreased slowly from 3d to 14d and it was still detectable in 14d.DiscussionRecently, more and more studies have confimed that mechanical injury, post ischemic reperfusion injury, radiotherapy, chemotherapy and virus infection are associated with cell apoptosis induced by caspase - 9 and caspase - 3, among which studies on the effects of caspase - 3 during apoptosis are the most extensive. However studies on the effects of caspase - 9, comparing with those of caspase - 3 , are relative less. Furthermore there is no related report on the effect of caspase - 9 in the skin incised wound healing process home and abroad. Recent studies have i-dentified that both caspase - 9 and caspase - 3 are detectable when brain and optical nerve are injured. Meanwhile, it is well known that caspase -9 is one of the upsteam initiator to activate caspase - 3. Therefore it is most likely that both of them may be expressed during skin incised wound healing.In the present experiment, the immunohistochemical and western blotting techniques were applied to detect caspase - 9 and caspase - 3 during healing process of skin incised wound. We found that PMNs, MNCs and FBCs expressed caspase - 9 and caspase - 3 in the skin wounded regions. It was confirmed that because of the specificities of the substrates on which the caspases effected, multi - caspase could appear in these three cell types at the same time. Furthermore, by calculating the caspase - 9 and caspase - 3 positive cell ratios at each posttraumatic time interval, we noted that both caspase - 9 and caspase - 3 positive cell ratios changed regularly with the extension of posttraumatic interval. The positive cell ratios were the lowest in the 3h after injury, and began to increase from 6h to 12h, then significantly increased at Id, and reached themaximum at 3d. After that, the positive cell ratios gradualy decreased and reached the minimum at 14d. The result detected by Western blot suggested that the procaspase - 9 was expressed at same intensity in control group and experimental groups. The P20 subunit of caspase - 9 was processed weakly in control group and Oh group. The expression of P20 strengthened from 3h to 3d and maximized at 3d. After that, the intensity of P20 was decreased slowly from 3d to 14d and it was still detectable at 14d.The results suggest that caspase -9 and caspase -3 may play an important role in inducing PMNs, FBCs and MNCs apoptosis during skin incised wound healing. Futhermore, caspase - 9 and caspase - 3 may be used as markers for the wound age determination, since PMNs, FBCs and MNCs express caspase - 9 and caspase - 3 time - dependency during skin wound healing.Conclusion1. The present study demonstrates on the first time that the proeaspase - 9 and P20 subunit of caspase - 9 is expressed in the epidermis, hair follicle epithelium and sebaceous gland. It notes that caspase -9 maybe induce the apoptosis of epidennis, hair follicle epithelium and sebaceous gland cells and take part in the homeostasis of normal skin;2. The study certifies firstly that PMNs, FBCs and MNCs in skin express caspase - 9 during skin incised wound healing;3. PMNs, FBCs and MNCs express caspase - 9 and caspase - 3 regularly during skin incised wound healing. It suggests that caspase - 9 and caspase - 3 maybe play an important role in inducing the apoptosis of PMNs, FBCs and MNCs;4. The results suggest that caspase - 9 and caspase - 3 can be used as markers for the skin wound age determination.