Application of mitogen-activated protein kinase inhibitor SP 600125 for wound healing control

Abstract 
Background: The problem of wound healing remains critical due to lack of methods for direct control of regeneration processes. 
Main purpose of the study: To investigate the ability to

doi: 10.7243/2050-1218-2-9 The discovery of Mitogen-Activated Protein Kinase (MAPK) as universal cascade mechanisms involved in implementation of all stages of wound process enabled to put forward the hypothesis that surgical wound healing process may be controlled by altering the activity of c-Jun N-terminal kinases (JNK) MAPK-one of the key elements in the process of cell differentiation, inflammation and apoptosis [6].
c-Jun N-terminal kinases are activated by cytokines, UV light, heat and osmotic shock [7].JNK group control inhibition of cell growth and inflammatory reactions [8][9][10][11][12].Main purpose of the study: to investigate the ability to control wound healing process by inhibiting JNK mitogenactivated protein kinase.

Materials and methods
Skin-muscle wounds were made to Wistar rats 9 months old, 220-250 g weight.The experiment was carried out under the "The Guide for the Care and Use of Laboratory Animals" as per the record approved by the Local Committee of Biomedical Ethics of Scientific Center of Reconstructive and Restorative Surgery SB RAMS.
The rats were sedated by ketamine 25 mg/kg, droperidol 1.25 mg/kg and atropine 0.2 mg/kg.The wounds were made paravertebral on the chest area 5 cm in length.Then a perforated plate with the tested material with the slow resorption of the active ingredient as have been developed (control group-placebo plate) was inserted into the wound.Five surgical sutures were made at the equal distance.Two groups were examined, each one consisted of 30 animals: control group (inoculation of placebo plate); JNK group (inoculation of the inhibitor of JNK MAPK cascade SP 600125 (Tocris bioscience, cat No 1496 Bath No7 A/B 1710) the dosage per one animal was 2,6 μg/kg).The calculation of the medication dosage was carried out according to IC50 [13][14][15][16].
Solution of active substance was inoculated slowly (over 3 days) by means of resorbable medicated film of our own development [17,18].
The animals were taken out of the experiment in the periods from 2 hours up to 30 days.The segments of the skin-muscle wound were fixed by FineFix (Milestone, Italy), embedded into paraffin for histological, immunohistochemical and immunofluorescent investigations.
To assess the collagen fibers in connective tissue, we measured the ratio of section area occupied by collagen fibers to the total area of tissue on micrographs of tissue sections stained by Van Gieson's, as our own method [19].
The analysis of the contingence tables and Mann-Whitney U test were used during the investigation [20].The critical level of the criteria relevance was equal to 0.05.The analysis of the data was carried out using the statistical package r-project.

Results
The effect of JNK MAPK SP 600125 inhibitor onto healing process of a surgical wound was investigated.The study determined that in control group of animals the process of connective tissue formation in place of skin-muscle wound corresponded to classic development pattern of inflammatory process as a response to damage in aseptic conditions.Apparent edema of dermis and subcutaneous tissue as well as initial neutrophilic infiltration were observed already after 2 hours after modeling of the wound.At 12 hours after the wounding, both edema and infiltration increased and reached the peak of the entire observation period.After 1 day since the start of experiment, intensity of edema and infiltration reduced slightly.Formation of granulation tissue predictably began since the 3 rd day of experiment.Enlargement zone of immature connective tissue and density of fibroblastic cell elements were increasing toward the 7 th day of experiment.On the 14 th doi: 10.7243/2050-1218-2-9 day, complete epithelialization of the wound was observed, and maturation of newly generated connective tissue began.Density of fibroblasts in the wound area was on the decline on the 30 th day of experiment.A wide and pronounced scar was forming.
A different development pattern was observed in animals inoculated with JNK MAPK SP 600125 inhibitor.Start of neutrophilic reaction was observed in 2 hours after the start of experiment.After 12 hours elapsed, expression of neutrophilic infiltration was growing, although edema was virtually absent.Neutrophilic reaction in the damaged zone reached its peak expression after 1 day since infliction of the wound.Apparent enlargement of immature connective tissue with expressed extracellular collagen deposits in the damaged zone was observed in animals of this group already on the 3 rd day of the experiment.A clear and Immunofluorescence, primary antibodies-CD34 (Santa cruz), 1:300, secondary antibodies-Alexa Fluor 488 (green), primary antibodies-CD45 (Santa cruz), 1:300, secondary antibodies -Alexa Fluor 568 (red), nucleuses were stained with DAPI (blue).Scale bars=10μm.D. JNK group, 3 days.Co-expression of CD34+ CD45+ markers by cells in the zone of surgical wound.Immunofluorescence, primary antibodies-CD34 (Santa cruz), 1:300, secondary antibodies-Alexa Fluor 488 (green), primary antibodies-CD45 (Santa cruz), 1:300, secondary antibodies-Alexa Fluor 568 (red), nucleuses were stained with DAPI (blue).Scale bars=10μm.
apparent scar was formed in 7 days.Further maturation of scar occurred over the remainder of time.
It was determined in the course of investigation that relative volume of collagen in the post-operative scar formation zone in control group of animals was increasing since the 3 rd till the 30 th day of the experiment, predictably reaching its peak on the 30 th day of observation (median value of 73.54%, 25-75% quartiles 66.87% -78.01%).Concentration of collagen in the zone of intact dermis has not demonstrated meaningful changes over the period of observation.Credible increase of collagen concentration by the 14 th day (p=0.029)was observed in derma only in proximity of the wound.
Intensity of collagen generation in the area of surgical wound increased considerably after application of with JNK MAPK SP 600125 inhibitor.In particular, already on the 3 rd day of the wound process, the amount of collagen surpassed the respective value of control group by 4 times (median value 44.44 [23.26-70.30]and 11.57[8.53-27.85]accordingly, z=4.00, p=0.00063).Eventually, at the end of observation period (30 th day of experiment), density of collagen in the experimental group was higher than in control group (median value 78.14 [72.77-81.14]and 73.54 [66.87-78.01]accordingly, z=2.29, p=0.0219).Alongside with that, application of SP 600125 has not produced any considerable effect onto concentration of collagen neither in intact dermis, nor the zone in wound proximity.
In order to assess the effect of JNK MAPK inhibitors onto differentiation of fibroblasts in the focus of connective tissue formation, we investigated expression of marroworiginated cell markers (CD34), white blood cell markers (CD45), procollagen of type I (ColIAI), MMP9, as well as actin in fibroblasts at the wound healing zone.
It has been determined that CD34+ was expressed in the area of scar formation in control group of animals on the 3 rd day of the wound process (Figure 1A), while faint staining of only a few cells in the wound zone was observed on the 7 th day.No specific staining was observed at other terms.
At the same time, application of JNK inhibitor caused a rapid increase of CD34+ cells in the area of the wound.In particular, individual CD34+ cells were observed already after 12 hours of the wound process modeling start.The number of these cells increased rapidly up to the 3 rd day (Figure 1B), whereas not only fibroblasts were stained, but also endotheliocytes of new vessels, which is an indirect indication of angiogenesis activation.Numerous fibroblasts bearing CD34+ markers remained in scar forming area for the period of up to 7 days.
Co-expression of CD34+ CD45+, which is indicative of presence of marrow-derived fibroblast progenitors in doi: 10.7243/2050-1218-2-9 the focus, was only registered in control group on the 7 th day (Figure 1C).
When JNK inhibitor was applied, early appearance of progenitor cells was observed in regeneration zone-on the 3 rd day after modeling of skin-muscle wound (Figure 1D).No progenitor cells were discovered in the regeneration zone at later observation points.
In order to evaluate presence of myofibroblasts in the area of connective tissue formation, the method of actin staining was used, which allows to differentiate these cells [21].We determined that moderate number of stained cells was observed in control group on the 3 rd and 7 th day  of wound process (Figure 2A).
At the same time, when JNK inhibitor was used, staining of individual cells for actin was observed starting with 12 hours of the wound process.Staining remained for up to 7 days, whereas an enormous number of stained cells was observed on the 3 rd and 7 th days (Figure 2B), which indicates at numerous presence of myofibroblasts in the wound and potentially high capacity for collagen production in the wound healing zone).Lack of myofibroblasts in the wound at later periods of the wound process indicates at completion of restorative process and lack of potential for formation of keloid scar [22].
When stained for Col IAI, which enables to identify procollagen of I type in fibroblasts, only individual positively stained cells were identified in scar formation zone on the 3 rd and 7 th days in control group.In the group where JNK inhibitor was used, staining for procollagen was recorded since the 3 rd till the 14 th day, which confirms elevated synthesis activity among fibroblasts in this group of animals.
Presence of fibroblasts with collagen resorption activity in the wound, which is determined as high intensity staining on MMP9+ fibroblasts [23], was observed in the control group only on the 7 th day (Figure 3A).In JNK group, fibroblasts positive for MMP9+ were recorded as solitary cells over an extensive period -since the 3 rd till the 30 th day (Figure 3B).

Discussion
It is generally known that fibroblasts may originate from marrow precursor cells circulating in peripheral blood [24].These fibroblast progenitors constitute approximately 0.05% of peripheral blood cells [25][26].Their amount may increase in response to effect of certain cytokines and chemokines, as well as during inflammation or fibrosis development [27].Fibroblast progenitors emerge from blood stream into damaged areas, and are required for formation of granulomas, scars, as well as tissue remodeling [28].
The study revealed that application of JNK MAPK inhibitor affects the wound healing process considerably, stimulates early connective tissue formation, increases collagen production rate and accelerates maturation of connective tissue in the wound area.
Early completion of scar formation and lack of cells in this area that capable of prolonged increased secretion of collagen allows suggesting that high risk of keloid scar formation does not exist.The accomplished results enable doi: 10.7243/2050-1218-2-9 to consider local inhibition of JNK MAPK cascade associated with a wound process to be a prospective approach to acceleration of surgical wound healing.
For instance, application of JNK inhibitor as component of surgical suture may discover totally new prospects for creation of a new generation of surgical suture and mesh.It is also apparent that increase of scar strength is capable of promoting faster rehabilitation, shorter hospitalization time and temporary incapacity to work.

Conclusions
It has thus been established that application of MAPK inhibitor considerably affects marker expression of various fibroblasts, which increases expression of marrow precursors in the focus, being indicative of a more active attraction of progenitor cells into connective tissue formation zone.Application of this inhibitor increases the number of myofibroblasts in the wound at early periods and elevates expression of procollagen in fibroblasts, which indicates at increased functional activity of these cells in the scar formation zone.
The developed original method of local application of JNK MPAK inhibitor SP 600125 to surgical wound, which enables to modify the surgical wound healing process with a single application, is recommended for pre-clinical and clinical trials for further wide clinical application.
Application of JNK MAPK SP 600125 may be prospective in surgical suture, drainage and other implantable medical goods.