| BackgroundAcute and chronic wound injuries are very common in clinics and the aim of their treatments is to promote the healing of damaged tissues. Wound healing is an intricate process in which the skin repairs itself in response to the injury. This process can be divided into hemostasis, inflammation, proliferation and remodeling phases, which sequentially occur accompanied with a closely orchestrated biochemical events/cascade to repair the damaged tissues. The speed of wound healing can be impacted by many local and systemic intrinsic factors, of which the inflammation status and the levels of growth factors are the critical determents. Therefore, it is important to well control the inflammatory reaction and to regulate the expression of wound repair-beneficial genes.It has long been recognized that medicinal maggot Lucilia sericata can be used to remove wound debridement by addressing them on the wound area. This therapy is especially helpful in managing the chronic wounds and ulcers with inflammation and necrosis [4]. Although the maggots are able to get rid of the devitalized or contaminated tissue, not all patients with wounds accept this treatment manner. Further studies reveal that maggots promote wound healing not only by ―eating‖ but also through excreting and secreting certain bioactive elements. Moreover, the injuries on maggot body show very efficient healing rates, indicating the presence of unique wound repair promoting factors in maggot cells and the possibility to use maggot extracts instead of the living maggots in wound treatments. In this context, it would be necessary to identify the more valuable part(s)/extract(s) of maggots in the management of skin wounds and to investigate the molecular mechanisms underlying their wound repair promoting effects.A body of evidence reveals that several signaling pathways are involved in wound healing via triggering their target gene expression. On the other hand, multiple rather than single factor in the maggot extracts are supposed to be involved in wound healing processes. However, no datum has been available concerning the influences of maggot extracts in wound healing-related signaling transduction. The current study aims to address the above issues by treating rat skin wounds and human keratinocytes and fibroblasts with the maggot excretion/secretion mixture and the extracts prepared from the maggots before and after extensive excretion/secretion.Materials and methods1. Maggots and maggot extract preparationL cuprina blowflies were maintained under standard condition and their larvae were reared to later second or early third Maggots. By putting maggot container in the darkness and facing its opened door to the sunlight, the maggots actively moved toward the light direction and were harvested easily without any hurt. The maggots collected(about 120g) were rinsed with autoclaved pure water for three times, stood in 3.5% formaldehyde normal saline solution for 5 minutes, in 2% H2O2 for 3 minutes and, finally, in 1% hydrochloride acid for 5 minutes, followed by 3 washes with autoclaved pure water. Part of the maggots(60g) was directly snap frozen in liquid nitrogen and stored at-80 o C until extract preparation. The remaining part(60g) were put separately(20g/time) in the same phosphate buffer saline/PBS solution(15ml) for 10 minutes to collect the concentrated mixture of excretion and secretion; afterword, the maggots were snap frozen and stored at-80 o C. The extracts of maggots without and with excretion/secretion were prepared by series sectioning the frozen maggot tissues into pieces(5mm in thickness) and then putting the frozen sections into the test tubes containing p H7.5 PBS with 0.02% sodium azide supplementation. By this way, the high quality maggot extracts were prepared in high yields.2. Rat wound healing model and treatmentsThe design of the current study had restrictively followed the guidelines of the Association for Assessment and Accreditation of Laboratory Animal Care, International. After getting the permission to conduct the animal experiment from Institutional Ethics Committee and the Committee on Research Animal Care of Dalian Medical University, thirty-two 10-week old male Sprague Dawley rats were provided by the Experimental Animal Center of Dalian Medical University and reared under specific pathogen-free/SPF condition. The rats were anaesthetized with 12mg/kg xylazine via intraperitoneal injection and f1.5 cm2 round open wounds down to the muscle fascia were made on the left flank by removing the skin layer(epidermis and dermis). The animals were divided into 4 experimental groups in 8 animals/group as Group 1(G1): dressed with vaseline only as untreated control; G2: treated with a mixture of vaseline and 150mg/ml extract from the maggots without excretion/secretion; G3: treated with a mixture of vaseline and 150mg/ml extract from the maggots with excretion/secretion; G4: treated with a mixture of vaseline and 15mg/ml maggot excretion/secretion. The treatments lasted for 16 days by daily dressing the reagents. The margins of individual wounds were outlined in regular changed red and black colors at Day-1,-3,-5,-7,-10,-12,-14 and Day-16 by directly placing a transparency model sheet on the wounds.3. Digital-transparency wound area measurementFor high throughput and more accurate wound area measurement, a transparency model sheet with the traced wound margins of the four experimental groups at different observation times was scanned by HP Laser Jet M1005 MFP and the image was directly saved in JPG form and opened at Adobe Photoshop CS4 site; the pixel values of individual wounds were calculated and all of the calculation data were recorded in an excel form by the method described elsewhere. A standard area unit was chosen according to the overall sizes of the wounds and its pixel value was cited as numerator. The areas of all wounds were calculated simultaneously by dividing each of the wound pixel values(Y) in the excel form with the pixel value of standard area unit(X) and then times the real area of the standard unit(Z). Calculation was conducted by three independent researchers and the data they obtained were analyzed together.4. Cell culture and treatmentsKeratinocytes and fibroblasts as the major components of the skin play pivotal roles in wound healing processes and can be used as in vitro wound healing models. Therefore, a human immortalized keratinocyte line Ha Cat purchased from American Type Cell Culture/ATCC was cultured in RPMI1640 medium supplemented with 10% fatal bovine serum/FBS(Gibco Life Science, Grand Island, NY, USA). After getting donor’s consent, the culture of normal fibroblasts was conducted in DMEM medium containing 10% FBS using a piece of normal skin specimen removed for medical cosmetic purpose. For comprehensive in situ analyses, the novel coverslip preparation dishes/NEST-Dish(China Patent for Invention: ZL200610047607.0) were used, by which 46 cell-bearing coverslips/per dish could be prepared under the same culture condition. All together, 138 coverslips collected from 3 NEST-Dishes, which were randomly separated to the following experimental groups as Group 1: cultured in the medium containing 0.01% Na O3; Group 2-A,-B and-C: cultured in the media containing high(200mg/ml), middle(150mg/ml) and low(100mg/ml) maggot extract without excretion/secretion(Extract+S/E); Group 3-A,-B and-C: cultured in the media containing high(200mg/ml), middle(150mg/ml) and low(100mg/ml) maggot extract after excretion/secretion(Extract-S/E). The treatments lasted for 72 hours and the cells were observed in 12 hour intervals. The cell-bearing coverslips in each of the experimental groups were used for immunocytochemical staining and modified MTT cell proliferation assay. Each of the experiments was repeated for at least three times to establish confidential conclusion.5. 4,5-dimethyl-2-thiazolyl-2,5-diphenyl-2-H-tetrazolium bromide/MTT cell proliferation assayTo estimate the proliferation activities of Ha Cat cells and fibroblasts under different experimental conditions, MTT assay was performed on the freshly collected cell-bearing coverslips from the three experimental groups. Briefly, the coverslips were put in f 0.5 container(1 piece/container) filled with 120ml medium containing 0.5mg/ml MTT, incubated at 37 o C for 4 hours. After that, the medium containing MTT was aspirated off and 100μl DMSO was added to dissolve the formazan crystal formed in viable cells. After the reaction, 100ml supernatants were removed orderly from the containers to a panel of wells in a 96-well plate and their OD values were determined at 492 nm on a micro ELISA plate spectrophotometer(Thermo Fisher Scientific, Finland). The remaining coverslips were used for immunocytochemical evaluation of the statuses of wound healing related signaling pathways and their downstream gene expression.6. Wound tissue biopsyFor protein preparation, the tissues in the size of 0.3cm X 0.3cm X 0.2cm were biopsied from the wound margins of the four experimental groups at the post-wounding times of Day-3,-7 and-12 and snap frozen in liquid nitrogen. The frozen tissues were vertically sectioned in 5mm thickness and 80-100 pieces of the slices were immediately put into 40ml cell lysate buffer. The remaining parts of the sample tissues were sectioned for immunohistochemical staining.7. Immunohistochemical and immunocytochemical stainingThe signaling pathways mediated by Wnt2, Notch 1 and Notch 2, NF-kB, STAT3 and TGFb1/Smad3 are known to influence the wound healing processes through regulating the expression of some growth promoting genes such as c-Myc, cyclin D1 and VEGF. Therefore, the statuses of their expression and intracellular distribution patterns in the tissue and cell samples treated differently were analyzed immunohistochemically and immunocytochemically by the method described elsewhere. The antibodies against those target proteins were purchased from Santa Cruz Biotechnology, Inc, CA except HES-1 as a kind gift of Dr. Tetsuo Sudo, Toray Industries, Tokyo, Japan. Color reaction was developed using 3, 3’-diaminobenzidine tetrahydrochloride(DAB).8. Western blot analysesTo validate the immunocytochemical results, total cellular proteins were prepared from the tissues of wound margins without and with different treatments and the cells under different culture conditions [15, 20]. The sample proteins(50μg/lane) were separated in 10% sodium dodecylsulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membrane(Amersham, Buckinghamshire, UK). The membrane was blocked with 5% skimmed milk in TBS-T(10m M Tris-HCl, p H8.0, 150 m M Na Cl and 0.5% Tween 20) at 4 o C, rinsed 10 minutes for three times with TBS-T, followed by 3 hour incubation at room temperature with the first antibody and then 1 hour incubation with HRP-conjugated anti-mouse or anti-rabbit Ig G(Zymed Lab, Inc). The bound antibody was detected using the enhanced chemiluminescence system(Roche Gmb H, Mannheim, Germany). After removing the labeling signal by incubation with stripping buffer, the membrane was reprobed with other antibodies one by one until all of the parameters were examined. The parameters checked are in parallel with that of immunohistochemical staining.9. RNA isolation and reverse-transcription polymerase chain reactionTo further validate the results of immunocytochemical staining and Western blotting, total cellular RNA samples were isolated from normally cultured human Ha Cat keratinocytes and fibroblasts and their 150mg/ml extract+S/E treated counterparts using Trizol solution(Life Technologies, Grand Island, NY). By the method described elsewhere, reverse transcription(RT) was performed on RNA samples, followed by polymerase chain reaction(PCR) with the primers for STAT3, NF-kB/p65, Notch1, Wnt2, TGF-b, Smad3, c-Myc, cyclin D1, COX-2 and VEGF(Table 1). The PCR products were resolved on 1% agarose gel containing ethidium bromide(0.5μg/ml), visualized and photographed using UVP Biospectrum Imaging System(UVP, Inc, Upland, CA). The β-actin PCR products generated from the same RT solution were cited as quantitative controls.10. Statistical analysesThe wound healing statuses of individual experimental groups at different tracing times and MTT data were evaluated with the independent-samples t-test and one-way ANOVA. Statistical significance was defined as P<0.05.Results1. High yield isolation of high quality maggot extracts20g of frozen maggot blocks were sectioned by microtome and the tissue slides in 5mm thickness were immediately put into 15 ml PBS containing 0.02% Na O3(Sigma Inc., USA). Protein concentrations of maggot extracts and the secretion/excretion mixture were calculated, which revealed that 15 ml of maggot secretion/excretion mixture and the extracts prepared from the maggots without and with excretion/secretion contained 9mg(0.6mg/ml), 89.85mg(5.99mg/ml) and 88.05 mg proteins(5.87mg/ml), respectively. 20mg/40ml of the three samples were subjected to SDS-PAGE, which showed that the proteins in the range of 7k Da to 210 k Da were found in the two types of maggot extracts, while only a band less than 7k Da was visualized from the secretion/excretion mixture.2. Best wound healing effects of the extract without secretion/excretionAll together, 243 pieces of tracing message were collected from 32 open skin wounds at 8 time points in the experiment and documented totally in one transparency model sheet. The repairing rates of the skin wounds without and with maggot extract treatments were sequentially evaluated by transparency tracing-digital calculation method.The average repairing rates of the wounds treated with the maggot extracts were higher than that of the wounds addressed only with vaseline(P<0.05). Of the three maggot samples, the extracts prepared from the maggots without excretion/secretion(Extract+S/E) in the final concentration of 150mg/ml in vaseline showed the best promoting effects on the wound healing because the wounds treated by it repaired in the quickest rates throughout the processes and completely closed at Day 14 or Day 16. The average closure rates of the wounds treated by 150mg/ml of the extract from the maggots after excretion/secretion(Extract-S/E), by 15mg/ml maggot excretion/secretion mixture(S/E mixture) and by vaseline were 99.3%, 99.7% and 96.3% by the end of the observation(Day 16). Ki-67 proteins are produced in G1 and S phase cells and therefore reflect the cell proliferation activity. Immunohistochemical staining performed on the wound tissues of the four experimental groups demonstrate more frequent labeling of Ki-67-positive cells in the wound tissues treated with Extract+S/E and Extract-S/E. For instance, Ki-67 index at post-wound Day-3 was 19%(+) in the untreated tissues, which increased to 37%(++) in S/E mixture, 57%(++) in the extract+S/E and 46%(++) in the extract-S/E treated tissues.3. Maggot extracts promoted in vitro cell proliferationHuman Ha Cat immortalized keratinocytes and the fibroblasts at early passages(Passage 8-10) on the coverslips were treated by Extract+S/E and Extract-S/E in the final concentrations of 200mg/ml, 150mg/ml and 100mg/ml, respectively. Phase contrast microscopic observation and hematoxylin-eosin/HE staining revealed that Ha Cat cells and the fibroblasts grew well in the media containing 100mg/ml or 150mg/ml maggot extracts; however, the cells treated by 200mg/ml extract showed distinct cell death(data not shown). MTT assay performed at 24 h and 48 h experimental points showed that the low(100mg/ml) dose of the two extracts exerted growth promoting effects on both types of cells; the middle(150mg/ml) dose was suitable only for fibroblasts and the extracts in high concentration(200mg/ml) were cytotoxic to the treated cells.4. Maggot extract enhanced TGF-b/Smad3 and STAT3 activitiesTissue microarrays were constructed using the samples regularly collected from the wound margins with different treatments by the method described elsewhere. Immunohistochemical staining was then performed on the tissue microarray sections. Nuclear translocation of NF-kB subunit p65 was not detectable in all tissue spots, indicating the inactivated status of this signaling. Wnt2 was expressed in the regenerating tissues but not activated because b-catenin was distributed on the cell surface without nuclear translocation irrespective to maggot extract treatments. Similarly, the expression levels of Notch1 were low and nuclear translocations of Hes1 were rare in either the control wounds or the wounds treated by the maggot extracts. In the case of STAT3, the enhanced expression and remarkable nuclear translocation were evidenced in maggot extract-treated wounds. TGF-b/Smad3 co-expression could be observed in the untreated wound tissues throughout the experimental time in steady and relatively low levels(+). In contrast, increased levels(++ to +++) of these pair of proteins were found in the epidermal cells and fibroblasts in the wound tissues treated by the maggot extracts, especially by the one without excretion/secretion. The wound healing promoting effects of maggot S/E mixture were relatively weaker than that of the extracts(data not shown). The above immunohistochemical findings were further confirmed the results of Western blotting in terms of increased levels of TGF-b, Smad3 and p-STAT3 but not Notch1, Wnt2 and NF-kB/p65 proteins.5. Maggot extract up-regulated wound healing related genesc-Myc, cyclin D1 and VEGF play active roles in cell proliferation and tissue regeneration. IHC and Western blotting revealed that these three genes were expressed earlier in the treated wound tissues and up-regulated in the ones treated by the maggot extracts. Hes1 were known as the downstream genes of Notch and COX-2 as NF-kB or Wnt signaling. Hes1 was expressed in the regenerating tissues and were not influenced by the treatments. COX-2 was expressed in low and stable levels in the treated and the untreated samples. CD68-positive cells could be observed in low incidences(+; 5-8 cells/view area; X20) in the untreated tissues at Day-3 and Day-7 time points but were not observed(-) or rarely found(+; < 2 cells/view point) in the three treated groups, indicating the minimal inflammatory reaction in the untreated and especially the treated wound regions.6 Maggot extracts increased TGF-b/Smad3 and STAT3 expression in Ha Cat and fibroblastsImmunocytochemical staining, RT-PCR and Western blotting were performed on the cell-bearing coverslips and the sample RNA and proteins prepared from Ha Cat cells and fibroblasts under normal culture or treated by maggot extracts. It was found that STAT3, Wnt2, Notch1 and TGF-b/Smad3 could be detected in both fibroblasts and Ha Cat cells. After maggot extract treatment, STAT3, TGF-b and Smad3 were up-regulated in the two types of cells; Notch1 and Wnt2 expression patterns were not distinctly changed and nuclear translocation of Hes1 and b-catenin was rarely seen. Upon maggot extract treatment, the levels of c-Myc, cyclin D1 and VEGF were remarkably up-regulated; Hes1 was more or less increased in the two types of cells but still remained in the cytosolic space; COX-2 was undetectable in Ha Cat cells and weakly positive in fibroblasts regardless of maggot extract treatment.ConclusionThe current study has addressed which parts of maggots play better growth promoting roles in the healing processes and what molecular effects they exert on the wound tissues by the following evidence:1. The impacts of the three maggot reagents in the progression of rat skin wound healing were evaluated by transparency tracing-digital calculation method. The results revealed that the average repairing rates of the wounds treated with the three maggot samples increased apparently in comparison with the ones in the untreated group. Moreover, the extracts prepared from the maggots without excretion/secretion(Extract+S/E) showed the best promoting effects than the extract of maggots with S/E(Extract-S/E) and the S/E mixture in terms of shorter wound closure times and the higher Ki-67 labeling incidences in the wound regions. Lack of infiltration of CD68-positive cells reflects the minimized inflammatory reaction and uninfected state in the wound regions after dressing the maggot samples. These findings thus suggest that either the body extract or the secreted and excreted materials of the maggots are bacteria-free and contain the elements that are able to promote wound healing and have anti-inflammatory activities.2. To further confirm our in vivo results, human immortalized keratinocyte line Ha Cat and the cultured human normal fibroblasts were employed respectively as a combined in vitro wound healing model for evaluating the cellular and molecular effects of maggot samples on them. The results demonstrated that fibroblasts and especially keratinocytes showed increased proliferation rates after being treated by low(100mg/ml) concentration of the extracts of maggots with and without excretion/secretion. It was also noticed that the cells exposed to a higher concentration(200mg/ml) of the extracts showed distinct growth arrest and cell death. These phenomena suggest that the maggot extract as a kind of bioactive agent may exert adverse effects when it is used in overdose. It is therefore necessary to ascertain the safety dose or addressing concentration of maggot extract before its practical application.3. Although the promoting effects of maggots and their extracts on wound healing have long been recognized, their underlying molecular mechanism(s) remains largely obscure. It has been found that VEGF, c-Myc and cyclin D1 are overexpressed in wound healing region, which can be regulated by multiple signaling pathways. Therefore, the statuses of Wnt2-, NF-kB-, Notch1-, STAT3- and TGF-b/Smad3-mediated signaling pathways in the wound tissues of the four experimental groups were evaluated. It was found that NF-kB, Wnt and Notch signaling seem in less activated states irrespective to maggot extract treatments because of the rarities of p65, b-catenin and Hes1 nuclear translocations.4. STAT3 expression with p-STAT3 nuclear translocation can be found in the wound tissues, especially in the epidermal cells, which become increased and intensified after maggot extract treatments, suggesting the potential active role(s) of this signaling in maggot extract promoted wound healing. Similarly, co-expression of TGF-b and Smad3 can be observed in the untreated wound tissues, which are up-regulated in both epidermal cells and fibroblasts in the wound tissues treated by the extracts of maggots with and particularly without excretion/secretion.5. The levels of c-Myc, VEGF and cyclin D1 expression are increased in the treated tissues. In accordance, similar molecular changes can be found in Ha Cat keratinocytes and fibroblasts upon maggot extract treatments. Consequently, maggot extract promoted wound healing in rat in vivo and human in vitro experimental models is closely correlated with the more activated TGF-b/Smad3 and STAT3 signaling pathways.6. The pivotal roles of TGF-b signaling in cutaneous wound healing have been well documented, which are accomplished, at least in large, under the cooperation of Smad3 proteins. On the other hand, some growth-promoting genes over-expressed in the maggot extract treated wound tissues are not regulated and, in the case of c-Myc, can even be suppressed by TGF-b/Smad3 signaling. These phenomena indicate that the enhanced activation of STAT3 signaling in maggot extract-treated wound tissues may be responsible for maintenance and up-regulation of those gene expression, because 1) c-Myc, VEGF and cyclin D1 are the well known STAT3 downstream genes and 2) the statuses of NF-kB, Wnt2 and Notch1 signaling pathways that also target those genes remain largely unchanged in the control and maggot extract treated wounds. Given the evidence of co-activated TGF-b/Smad3 and STAT3 signaling in the wound tissues and their remarkable enhancements by maggot extract, it is reasonable to consider that these two events would be the major molecular effects of maggot extracts on the wound tissues.Taken together, the wound healing and growth promoting efficacies of three kinds of maggot samples are elucidated using the rat skin wound model and cultured human keratinocytes and fibroblasts. The results reveal that the extract prepared from the maggots without secretion and excretion shows the best wound healing promoting effects in vivo and in vitro when being used in suitable amount, suggesting that both maggot body and the secretion/excretion materials contain favorable elements for wound healing. Of the 5 pathways involved in wound healing, the ones mediated by TGF-?/Smad3 and STAT3 are activated in the untreated wounds and further enhanced by the maggot extracts, accompanied with c-Myc, VEGF and cyclin D1 up-regulation. We therefore propose that the enhanced activities of TGF-?/Smad3 and STAT3 signaling pathways may be the major molecular effects of maggot extracts, which can accelerate wound healing processes by up-regulating the expression of a series of genes involved in cell proliferation, fibrogenesis, re-epithelialization and remodeling. |
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