| Background and purposeSkin is of great importance in maintaining body’s stablity, preventing it from physical,chemical, mechanical damage and invasion of pathogenic microorganisms. In serious burnpatients, the integrity of skin is severely destroyed, which leads to loss of barrier function,besides, wound infection can provide an important source of systemic infection. Treatmentof burn wounds can significantly affect patient’s condition, that is why we emphasize thepriority for the restoration of an intact skin barrier, also, known as re-epithelialization.During this process, the migration of keratinocyte is not only the initial event, but also therate-limiting step[1], while their proliferation can meet the need of migration. Meanwhile,epithelial-mesenchymal transition of keratinocyte results in the increase of motility, whichis propitious to wound repair.It’s widely known that hypoxia can occur locally and systemically after severe burn.Cardiogenic shock brings sharp cutoff of effective circulating blood volume and tissuehypoperfusion. On the basis of hypoperfusion, local vascular damage, wound infection andrespiratory burst contribute to the hypoxia environment of wound. In clinical application,not only hypoxia environment formed by dressing and vacuum suction can acceleratewound healing[2-4], but also hyperbaric oxygen therapy[5-7], while long-time hypoxia canlead to chronic wound. This phenomenon suggests that oxygen concentration、 timethreshold affect cells in wound repair to a great extent. The mammalian target of rapamycin(mTOR), which plays an important role in cell growth, differentiation, apoptosis, is anindispensible molecule in the body[8-11], its two complexes mTORC1, mTORC2regulatescell growth and movement respectively[12]. Lots of studies reveal that hypoxia inhibits theactivity of mTOR signaling pathway, however, mTOR is activated in various tumor tissuesin spite of their hypoxia environment, furthermore, this signal pathway is closely relatedwith tumors’ invasion and metastasis[13-15]. Take the similarity between wound healing and tumor[16-20]and its universality in body into consideration, we wonder what state it is inhypoxia environment during wound healing and its role in the process of ofre-epithelialization. Considering the importance of this signal molecule, these questions arereally worth studying.In spite of the application of negative pressure device, current researches are focusedon the influence of hypoxia on cell biological behavior in vitro without being applied invivo. Based on this literature[21], two kinds of films, differing in oxygen permeability, areused to cover wound for7days on the purpose of the creation of hypoxia environment.During the process of wound repair, the dynamic change of wound oxygen pressure isrecorded, it turns out that hypoxia environment slower this repair process. Combination ofthe literature with mechanism of hypoxia make us wonder the influence of short timehypoxia. In order to accomplish this goal, we investigated the influence of different hypoxiaduration on cell behavior in vitro firstly, studied hypoxia model in vivo, which is expectedto assist in the development of therapeutic plans to enhance wound healing artificially.Methods1. The hypoxic environment and cell proliferation after woundBiposy Punchs were used to make full-thickness skin wounds on the back of mices,skin samples were taken at day1,2,3,4,7,10,14. HypoxyprobeTM-1Plus Kit was used forimmunofluorescence staining, whose red fluorescence represented hypoxic regions. Theimages were analysed by Image-Pro Plus software to figure out the location、degree、trendof hypoxic regions. Through immunohistochemistry staining of PCNA, we studied cellproliferation after wound.2. Primary skin cells’ isolation and cultureThe neonatal Balb/c mouses’ skin were removed and disposed of DispaseⅡat4°Covernight, then0.25%trypsin at37°C for4-5min. After that, the solution obtained wasfiltered, centrifuged, suspended in order to remove skin debris and obtain skin cells. Thesuspension was divided into several T25flasks after cell density adjustment, those flaskswere put into carbon dioxide incubator at37°C with5%CO2. Attention should be paid tocell morphology, medium color, turbidity etc.3. Effect of short-time hypoxia on the movement of HaCaT cellHaCaT cells were cultured in accordance with the normal protocol and divided intocontrol group, hypoxia1h,3h,6h groups based on the random number table, each group consisted of6samples respectively. Cells’ trajectory paths were recorded by Live CellImaging Workstation, and then pictured by Image J software, their speed were calculated.HaCaT cells were divided into different groups as listed above, each group contained8samples, scratches were made with200μL pipette evenly, after that, these cells receivedtheir respective treatment and then were put into carbon dioxide incubator as long as theirtreatment finished. Pictures were taken at specified time for subsequent analysis.4. Effect of hypoxia on proliferation of HaCaT cell(1) HaCaT cells were routinely cultured and divided into control group, hypoxia1h,3h,6h,9h,12h,24h groups randomly with20samples in each group. Cell Counting Kit-8wasused to detect their OD value so as to estimate their capacity of proliferation.(3) HaCaTcells were divided into control, hypoxia1h,3h,6h,24h groups with5samples respectively.Western Blot was accomplished to study the expression of proliferating cell nuclearantigen (PCNA).5. Effect of hypoxia on epithelial-mesenchymal transition (EMT) of primary cellAfter isolation and culture, primary epidermal cells were randomly divided intocontrol group, hypoxia0.5h,1h,3h,6h,24h groups with6samples in each of them.Western Blot was used to detect the expression of vimentin. With immunofluorescencestaining performed on the control and hypoxia24h groups, we observed the location andexpression of vimentin directly, distribution and state of F-actin showed byimmunofluorescence staining helped us to understand the EMT better.6. Effect of hypoxia on the expression of Akt, P70S6K, mTORPrimary epidermal cells were randomly divided into control group, hypoxia10min,0.5h,1h,3h,6h,12h,24h groups, all of them were consisted of6samples individually.Western Blot was used to detect the expression of phosphorylated Akt, P70S6K, mTOR,total P70S6K, total mTOR.7. Effect of short-time hypoxia on wound healing in vivoThe body hair of mices was depilated with hair removal cream, two symmetricalfull-thickness skin wounds were made on the two sides of each one’s back with BiopsyPunches(diameter=5mm). These mices were randomly divided into hypoxia1h,3h,6hgroups with5mices individually. Oxygen impermeable (polyvinylidene chloride)membrane was applied to wound on the left side, while oxygen permeable(polymethylpentene) membrane was applied to symmetrical wound on the right side. Once the treatment completed, the membranes were removed and the repair process was recordeduntil the accomplishment of re-epithlialization. Image-Pro Plus was used to analysed thesepictures. Wound on both sides were harvested and analysed by HE staining, Massonstaining and immunohistochemical staining of PCNA to study the difference between them.Results1.The location, degree, trend of hypoxic regionsThe hypoxia at the leading zone of the wound appeared at day2and reached its peakat day3. As time went on, the red fluorescence intensity decreased gradually. The positiveexpression could be seen at the leading zone and the migrating keratinocyte, which stoodfor proliferating cells activity.2. Primary skin cells’ isolation and cultureThe primary cell in good condition became adherent to culture-flask in24hours. Theirsize were mostly large with big nucleus, most of them were round or oval in a paving-stonestate with clear outline and good refraction. Its good condition laid a solid foundation forthe experiment next.3. Effect of short-time hypoxia on the movement of HaCaT cell3.1Movement region: The trajectories of control group were more concentrated,movement range of cell in hypoxia1h,3h,6h groups increased gradually. The movementdistance of cells recorded by Live Cell Imaging Workstation in control group, hypoxia1h,3h,6h groups were (100.320±43.102)μm,(129.144±48.139)μm,(134.828±43.005)μm,(169.183±54.588)μm.3.2Movement speed: In the first hour, the trajectory velocity of cells in hypoxia1h,3h,6h group were respectively (43.315±18.429),(43.965±16.305),(55.005±19.471)μm/h, which were higher than control group (33.832±13.450)μm/h(t=2.840~7.468，P<0.05or0.01). Hypoxia6h was significantly higher than hypoxia1h,3h(t=3.678,3.924，P<0.05or0.01). In two hours, the trajectory velocity of each group were(43.625±17.380),(43.509±13.732),(54.465±17.201)μm/h, which were higher thancontrol group (33.011±11.667) μm/h (with t values from3.936to8.465, P<0.05or0.01).Hypoxia6h was significantly higher than hypoxia1h,3h (t=3.474,4.043, P<0.05or0.01).In three hours, the trajectory velocity of hypoxia groups were (43.048±16.046),(44.943±14.335),(56.394±18.196)μm/h, which were higher than control group (33.440±10.23)μm/h (with t values from2.840to7.468，P<0.05or0.01). Hypoxia6h was obviously higher than hypoxia1h,3h (t=4.545,4.475, P<0.01). Trajectory velocity of control,hypoxia1h,3h,6h groups had no difference at different time point(with t values from-0.523to0.596，P>0.05).In the first hour, the displacement speed of cells in hypoxia1h,3h,6h group wererespectively (22.295±11.374),(19.445±13.732),(31.555±19.12)μm/h, which werehigher than control group (15.219±9.985)μm/h (with t values from1.990to6.976，P<0.05or0.01). Hypoxia6h was significantly higher than hypoxia1h,3h (t=3.394,7.468，P<0.05or0.01). In two hours, the displacement speed of hypoxia3h,6h were (12.156±7.687),(20.533±12.747)μm/h, which were higher than control group (8.801±6.654) μm/h(t=2.386,8.000, P<0.05or0.01). Hypoxia6h was significantly higher than hypoxia1h,3h(t=5.955,5.732, P<0.01). In three hours, displacement speed of hypoxia3h,6h groups were(9.884±6.411),(17.051±11.743)μm/h, which were obviously higher than control group(6.427±5.295)μm/h (t=2.808,8.231，P<0.01). Hypoxia6h was significantly higher thanhypoxia1h,3h (t=6.008,5.558, P<0.01). Displacement speed of control, hypoxia1h,3h,6hgroups had obvious difference at different time point. Speed in two hours fell sharply incomparison with data in one hour (with t values from-7.036to-4.232，P<0.01). Speed inthree hours also decreased in comparison with data in one hour (with t values from-8.208to-5.540, P<0.01).3.3Scarification assays: Scarification assays showed that hypoxia treatment for1h,3h,6h could promote lateral migration of cells, this trend became much more evident ashypoxia time extended.4. Effect of hypoxia on proliferation of HaCaT cell4.1CCK8assays: The OD value of control, hypoxia1h,3h,6h,9h,12h,24h groupwere1.110±0.069,1.358±0.099,1.387±0.047,1.379±0.049,1.104±0.14,1.063±0.089,0.992±0.064(F=39.19，P<0.01). Hypoxia1h,3h,6h groups showed increasedproliferation when they were compared with control group (with t values from6.639to7.403, P<0.01), while hypoxia24h suppressed proliferation in comparion with controlgroup(t=-3.135, P<0.05). Hypoxia24h,12h,9h lowered proliferation compared withhypoxia1h,3h,6h(with t values from-10.538to-6.775, P<0.05or0.01).4.2Western Blot assays: The PCNA protein levels of control, hypoxia1h,3h,6h,24hgroups were respectively0.926±0.121,0.972±0.142,1.621±0.18,0.945±0.09,0.661±0.21(F=20.11, P<0.01). The expression of hypoxia1h,3h,6h groups were significantly higher than control group(with t values from2.235to5.783, P<0.05or0.01). Hypoxia24hlowered PCNA expression compared with control group(t=-1.998, P<0.05).5. Effect of hypoxia on epithelial-mesenchymal transition (EMT) of primary cellNot only the numbers of cells that expressed vimentin increased, but also the proteinlevel expressed by each cell after hypoxia treatment. The data got from Western Blotshowed that the results of control, hypoxia0.5h,1h,3h,24h group were0.319±0.09,0.983±0.22,1.03±0.11,1.392±0.43,1.329±0.13,1.317±0.14. Meanwhile,immunofluorescence staining showed that F-actin was maily distributed at cells’ edge withthe formation of stress fibers and lamellipodia, which would be benifitcial to cells’movement.6. Effect of hypoxia on Akt, P70S6K, mTOR6.1Phosphorylated Akt: The expression of phosphorylated Akt of control, hypoxia10min,0.5h,1h,3h,6h,12h,24h groups were respectively0.666±0.035,1.845±0.111,2.132±0.452,2.963±0.161,2.263±0.494,0.886±0.171,0.377±0.084,0.142±0.093.6.2P70S6K: The expression of phosphorylated P70S6K of control, hypoxia10min,0.5h,1h,3h,6h,12h,24h groups were respectively0.744±0.156,1.771±0.312,1.639±0.374,2.013±0.227,1.791±0.484,1.679±0.231,0.549±0.197,0.268±0.066, and the totalP70S6K were1.05±0.369,1.435±0.283,1.151±0.214,1.549±0.472,1.578±0.17,1.348±0.724,0.603±0.226,0.657±0.3616.3mTOR: The expression of phosphorylated mTOR(Ser2481) of control, hypoxia10min,0.5h,1h,3h,6h,12h,24h groups were respectively0.62±0.190,1.133±0.286,1.5±0.298,1.967±0.473,1.931±0.449,1.876±0.324,0.878±0.342,0.22±0.082, and thetotal mTOR were0.865±0.05,1.003±0.122,0.961±0.226,1.410±0.242,1.517±0.289,1.468±0.185,0.9±0.298,0.92±0.42.7. Effect of short-time hypoxia on wound healing in vivoDuring the process of wound repair, the wound size of hypoxia groups were smallerthan control group. Taken hypoxia6h for an example, HE staining found that the thicknessof hypoxia wound increased significantly when compared to normoxia;immunohistochemical staining of PCNA showed the number of proliferating cells was morethan those in control group. Masson staining of collagen fibers found no obvious differencebetween two groups. In addition, HE and Masson staining both revealed that there weremore vessels in the hypoxia group. Conlusions1. The hypoxia at the leading zone of the wound appeared at day2and reached itspeak at day3, that was consistent with where proliferating cells located.2. Short-time hypoxia groups gained increased motility. Live Cell ImagingWorkstation showed short-time hypoxia could promote cells’ motility, this trend was mostobvious in hypoxia6h group. Scratch assays showed hypoxia treatment could promotelateral migration.3. Short-time hypoxia could promote cell proliferation and EMT, long-time hypoxiarestrained proliferation. CCK8found that short time hypoxia (1h,3h,6h) promotedproliferation, when the treatment time prolonged to9h,12h,24h, hypoxia slowed down thespeed of cell proliferation or even suppressed it. Hypoxia treatment could not only increasethe expression of vimentin, but also redistributed F-actin and influenced its pattern, whichindicated that hypoxia promoted EMT.4. Short-time hypoxia activated mTOR. Western Blot showed that mTORC1,mTORC2signals were activated by short time hypoxia (30min,1h,3h,6h), but inhibited ashypoxia treatment continued.5. Short-time hypoxia could promote the process of wound healing in vivo. HEstaining, immunohistochemical staining of PCNA found that the number of proliferatingcells was more than that in control group. Masson staining of collagen fibers showed noobvious difference between control and hypoxia group. In addition, HE and Massonstaining both revealed that there were more vessels in the hypoxia group.6. Results listed above provided new sights for mechanism in wound repair andtherapeutic plans to enhance wound healing artificially. |
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