Platelet-derived mitochondria transfer facilitates wound-closure by modulating ROS levels in dermal fibroblasts

Abstract Platelets are known to improve the wound-repair capacity of mesenchymal stem cells (MSCs) by transferring mitochondria intercellularly. This study aimed to investigate whether direct transfer of mitochondria (pl-MT) isolated from platelets could enhance wound healing in vitro using a cell-based model. Wound repairs were assessed by 2D gap closure experiment in wound scratch assay using human dermal fibroblasts (hDFs). Results demonstrated that pl-MT were successfully internalized into hDFs. It increased cell proliferation and promoted the closure of wound gap. Importantly, pl-MT suppressed both intracellular and mitochondrial ROS production induced by hydrogen peroxide, cisplatin, and TGF-β in hDFs. Taken together, these results suggest that pl-MT transfer might be used as a potential therapeutic strategy for wound repair. Plain Language Summary What is the context? During the wound healing process, abnormal regulation of ROS and inflammation delays the healing process, resulting in chronic non-healing wounds. Mitochondria are key organelles responsible for the ROS generation. Mitochondrial dysfunction has been implicated in delayed wound repair. Mitochondria transfer, which utilizes intact mitochondria isolated from healthy cells to recover from disease, has been applied in various clinical studies, but additional evidence is needed to apply it to wound healing. What is new? In this study, we chose platelets as a cell source for mitochondrial transfer. We isolated the functional mitochondria from platelets and applied them to wound healing. What is the impact? This study provides evidence that platelet-derived mitochondria (pl-MT) improve the wound healing progress by increasing the viability of dermal fibroblasts and suppressing intracellular and mitochondrial ROS production. Platelets have also been demonstrated to be a suitable cell source for mitochondrial transfer


Introduction
Wound healing is a complex biological reaction characterized by consecutive and overlapping steps including hemostasis, immune response, inflammation, cell proliferation, and tissue remodeling [1,2].Reactive oxygen species (ROS) play a pivotal role in the progress of wound repair by acting as both antimicrobials and cell survival signaling molecules throughout inflammation and tissue remodeling [3].However, excessive and uncontrolled ROS generation can deregulate inflammation processes and cause cellular damage by creating oxidative stress, resulting in impaired wound healing and chronic non-healing wounds [4,5].
Mitochondria are cell organelles important for cellular functions by providing energy in the form of ATP through oxidative phosphorylation, in which ROS are generated as by-products [6].Mitochondria also play critical roles in apoptosis, inflammation, and calcium homeostasis.Given their important roles in cellular physiology, mitochondrial dysfunction can result in impaired bioenergetic efficiency, which in turn leads to abnormal inflammatory responses and excessive ROS.Recently, application of mitochondria isolated from healthy cells (referred to as mitochondria transfer) has become an attractive therapeutic strategy for treating mitochondrial abnormalities [7][8][9][10].The aim of mitochondrial transfer is to replace intracellular abnormal mitochondria with healthy mitochondria isolated from normal cells, resulting in the recovery of cell function.Therefore, transfer of healthy mitochondria into cells with damaged mitochondria could be beneficial to restore excessive ROS production and mitochondrial dysfunction.
Sources of healthy mitochondria include skeletal muscles, mesenchymal stem cells (MSCs), and platelets.Among them, platelets have attracted attention because they are abundant in blood that they can be simply acquired by venipuncture.In addition, the isolation of mitochondria from platelets is relatively less invasive than that from muscles.Platelets generally contain 5 ~ 8 mitochondria [11] that are metabolically active to provide appropriate ATP for maintaining proper platelet functions such as blood coagulation and calcium homeostasis [12,13].In support of this, transfer of platelet-derived mitochondria (pl-MT) can rescue mitochondrial malfunction and cognitive impairment in diabetic mice [14] and suppress ROS overproduction and mitochondrial apoptotic pathway induced by hypoxia/reoxygenation in neuronal cells [15].Together, these findings imply that pl-MT can be beneficial for restoring mitochondrial dysfunctions.
In the present study, mitochondria were isolated from platelets.Their integrity and activity were assessed by evaluating mitochondrial respiratory chain enzyme activity and citrate synthase activity.We confirmed that pl-MT could be internalized into human dermal fibroblasts (hDFs), resulting in the increase of intracellular ATP and respiratory enzyme activities in hDFs.We next investigated effects of pl-MT on wound closure in hDFs.Our data clearly showed that treatment with pl-MT stimulated cell migration in a scratch-wound assay in vitro.In addition, we found that the administration of pl-MT to hDFs promoted cell proliferation and suppressed both intracellular and mitochondrial ROS production induced by hydrogen peroxide (H 2 O 2 ), cisplatin, and TGF-β.

Isolation of mitochondria from human platelets
Concentrated platelets were purchased and used with research permission from the Korean Red Cross (permission number #1111).pl-MT were isolated from membrane-disrupted platelets physically followed by differential centrifugation according to procedures described in our previous study [16].The amount of pl-MT was determined by bicinchoninic acid (BCA) assay.

Validation of isolated mitochondria
Isolated pl-MT were stained with 1 μM Mitotracker Red CMXRos (Invitrogen, USA) for 10 min.Samples were loaded into a 96-well black wall/clear bottom plate (SPL, Korea) and visualized using a confocal microscope (Fluoview FV3000, Olympus, Japan).

Measurement of mitochondrial respiratory enzyme activities
Mitochondrial respiratory chain enzymatic activities of isolated mitochondria were assessed following a prior research [17].Complex I+III activities were evaluated with the following procedures.First, 2 μg of pl-MT was added to each well of a 96-wellplate and then adjusted to 166 μl with distilled water (DW).To inhibit complex I+III activity, 6 μl of 1 mg/ml antimycin A (Sigma-Aldrich, USA) as an inhibitor of complex III was added to each well containing pl-MT.In addition, inactivated pl-MT were prepared by boiling them for 10 min.After 2 min, 20 μl of 500 mM potassium phosphate buffer (pH 7.5), 5 μl of 4% BSA (97% purity, Sigma-Aldrich, USA), 6 μl of 10 mM KCN, and 10 μl of 1 mM oxidized cytochrome C in DW were added.After 2 min, 4 μl of 10 mM NADH (Sigma-Aldrich, USA) was added.Oxidized cytochrome C (Sigma-Aldrich, USA) solution was always freshly prepared.Absorbance was measured at 550 nm every 1 min for 20 min to determine its changes.Complex Ⅳ activities were evaluated with the following procedures.First, a solution of 1 mM cytochrome c in 20 mM potassium phosphate buffer (pH 7.0) was prepared and it was then reduced by using a very small amount of sodium hydrosulfite on a tip of a pipette.The cytochrome c solution was vigorously mixed for freshly preparing reduced cytochrome c solution.Reduced cytochrome c solution was freshly prepared before use.Next, 50 μl of 100 mM potassium phosphate buffer (pH 7.0) and 10 μl of reduced cytochrome c were added to each well.To compare with inhibited MT, 6 µl of 10 mM KCN as an inhibitor of complex Ⅳ was added to each well.Inactivated pl-MT were prepared by boiling them for 10 min.After 2 μg of pl-MT or 2 μg of inactivated pl-MT was added into each well, the volume of each well was adjusted to 200 μl with DW.Absorbance was measured at 550 nm every 1 min for 20 min to determine its changes.ATP synthesis activities were evaluated with the following method.5 µg of pl-MT and 10 μl of 500 μM ADP were added to each well of 96-well-white bottom plates.To compare with pl-MT, inactivated pl-MT were prepared by boiling them for 10 min.The volume of each well was adjusted with distilled water (DW) to 100 μl.Then, 100 μl of CellTiter-Glo® reagent (Promega, USA) prepared at room temperature was added into each well.After shaking the plate on a plate reader for 30 s, the luminescent signal was measured every 2.5 s for 20 min.Citrate synthase activities were evaluated with the following procedures.2 μg of pl-MT, 100 μl of Tris (200 mM, pH 8.0) with Triton X-100 (0.2% (vol/vol)), and 6 μl of 10 mM acetyl CoA were added to each well of a 96-well plate.To compare with pl-MT, inactivated pl-MT were prepared by boiling it for 10 min.The volume of each well was adjusted up to 170 μl with DW.After adding 10 μl of 10 mM oxaloacetic acid (Sigma-Aldrich, USA) prepared in DW to start the reaction, 20 μl of 5,5'dithiobis(2-nitrobenzoic acid) (DTNB) (Sigma-Aldrich, USA) in 100 mM Tris (pH 8.0) was then added.DTNB and oxaloacetic solutions were always freshly arranged.Change of absorbance was recorded at 450 nm every 1 min for 20 min.

Western blot analysis
Mitochondria fraction was separated by centrifugation at 12000 x g for 15 min at 4°C from cytosol fraction.Each fraction was boiled in an equal volume of SDS-sample buffer and loaded onto 12% SDS-polyacrylamide gels.Samples were transferred to Amersham Protran polyvinylidene fluoride membranes (GE Healthcare, USA).Membranes were blocked with Tris-buffered saline with 0.1% Tween 20 (TBST) containing 5% (w/v) skim milk.After blocking, membranes were incubated with primary antibodies diluted in PBS containing 0.1% BSA.The following primary antibodies were used: mouse monoclonal anti COX Ⅳ (Abcam; 1:2000) and mouse monoclonal anti β-actin (Sigma-Aldrich; 1:5000).Membranes were incubated with a goat anti-mouse HRP-conjugated secondary antibody (1:5000, Abcam, UK).Protein signals were measured using a Western Bright ECL assay kit (Advansta, USA).Results were obtained with an Azure biosystems c280 quantitative western blot imaging system (Azure biosystems, USA).

Transfer of pl-MT into hDFs
6 × 10 4 of hDFs were seeded into a 24 well plate and stained with 1 μM CellTrace TM CFSE cell proliferation kit (Invitrogen, USA) for 20 min at 37°C.Next, hDFs were washed twice with DPBS (Sigma-Aldrich, USA).6μg of pl-MT stained with 1 μM Mitotracker Red CMXRos (Invitrogen, USA) were transferred to hDFs for 24 h.The cells were harvested and washed with DPBS.The fluorescent intensity of hDFs was measured using CytoFLEX (Beckman Coulter, USA).To image the fluorescence, 4 × 10 3 of hDFs were stained with 1 μM Mitotracker Green (Invitrogen, USA) and pl-MT were stained with 1 μM Mitotracker Red CMXRos (Invitrogen, USA), respectively.The hDFs were incubated with pl-MT and washed twice by DPBS.The nuclei were stained with 1 μM of Hoechst33342 (Invitrogen, USA) for 10 min.The images were obtained using a confocal microscope (Fluoview FV3000, Olympus, Japan).

Measurement of intracellular ATP
5 × 103 of hDFs were seeded into a 96 well white tissue plate and incubated with pl-MT for 24 h.After washing with DPBS, 100 μl of CellTiter-Glo® reagent (Promega, USA) was added each well.hDFs were incubated for 10 min at RT on orbital shaker to induce cell lysis.The luminescence signal was measured using a SYNERGY HTX multi-mode reader (BioTek, USA).

Quantitation of ROS
ROS levels were assessed using CM-H2DCFDA (Invitrogen, USA), a chloromethyl derivative of H 2 DCFDA (Invitrogen, USA), and MitoSOX TM red (Invitrogen, USA), a mitochondrial superoxide indicator.After 8 � 10 3 cells were seeded into a 96-well black plate with a clear flat bottom (Corning, USA), the plate was incubated for 18 h in serum-free DMEM, and treated with 50 μM of hydrogen peroxide (H 2 O 2 , DAEJUNG, Korea), 20 μM of cis-diammineplatinum (II) dichloride (cisplatin) (Sigma-Aldrich, USA), or 5 ng/ml of recombinant human TGF-β (Peprotech, USA) in the presence or absence of pl-MT for 24 h.Cells from each well were washed with DPBS buffer and incubated with 10 μM of CM-H2DCFDA for intracellular ROS and 5 μM of MitoSOX for mitochondrial superoxide.Cells were washed twice with DPBS and measured using a SYNERGY HTX multi-mode reader (BioTek, USA).ROS levels were normalized against Hoechst33342 (Invitrogen, USA).To visualize ROS fluorescence, cells were treated with cis-diammineplatinum(II) dichloride and TGF-β1.After that, pl-MT were stained with 1 μM of MitoTracker TM Red FM (Invitrogen, USA) for 10 min and then added to each well.Before measurement, nuclei were stained with 1 μM of Hoechst33342 for 10 min.Fluorescence images were obtained using a confocal microscope (Fluoview FV3000, Olympus, Japan).

Proliferation assay
Cell proliferation was evaluated with Cell Proliferation Reagent WST-1 (Roche, Switzerland) known to label replicating DNA.After 6 � 10 3 cells were seeded into a 96 well plate, the plate was incubated at 37°C with 5% CO 2 overnight.Then, 0.6 μg of pl-MT was added.Each well was then washed twice with DPBS buffer.After adding 100 μl of medium containing 10 μl of Reagent WST-1 solution to each well, the plate was incubated at 37°C with 5% CO 2 for 1 h in an incubator.The absorbance was measured using a SYNERGY HTX multi-mode reader at wavelengths of 450 nm and 650 nm for background subtraction.

Wound scratch assay
Cell migration was evaluated by performing a scratch assay.Briefly, 8 � 10 3 of hDFs were seeded into a 96 well plate and incubated at 37°C with 5% CO 2 overnight to allow cells to adhere onto the plate.Scratching was done with a P-1000 pipette tip to central cells to make a straight line.hDFs were washed twice with DPBS to remove detached cells and debris.After 0.8 μg of pl-MT was added to each well in culture medium containing 10% FBS and 1% antibiotics, cells were incubated for 48 h.Each well was examined by microscopy.

Experimental design and statistical analysis
All statistical analyses were performed using GraphPad Prism 5.03 (GraphPad Software, Inc.).One-way ANOVA and two-way ANOVA were used for multiple comparisons.Statistical significance criteria were set as *p < .05,**p < .01,and ***p < .001.

Validation of isolated pl-MT
Mitochondria were first isolated from human platelets and stained with MitoTracker Red CMXRos to label mitochondria in a membrane potential-dependent manner.As shown in Figure 1a, pl-MT isolated from human platelets were clearly labeled with MitoTracker Red CMXRos, suggesting that pl-MT were functional.A flow cytometric assay was then carried out to investigate the quality of isolated pl-MT.The purity of pl-MT was approximately 97.4% in the tested sample (Figure 1b).Next, we performed western blot assay to check the purity of pl-MT using anti-COX4 (subunit Ⅳ of cytochrome c oxidase) as a mitochondrial marker and anti-β-actin as a cytosol marker.Immunoreactive band (Figure 1c) of western blotting indicated that the expression of COX-4, but not that of β-actin was observed in the fraction of isolated pl-MT.Next, we tested the activity of pl-MT by evaluating activities of mitochondrial respiratory chain enzymes involved in the synthesis of ATP in OXPHOS [17].Complex I+III activity of pl-MT was significantly higher (0.012 ΔO.D./min) than that of pl-MT+AA (0.0038 ΔO.D./min), which were treated with an inhibitor of complex III of the electron transport chain (antimycin A) and that of heat-inactivated pl-MT (pl-MT+heat; 0.0005 ΔO.D./min) (Figure 1d).Similarly, in complex IV activity, pl-MT showed 4.3 times higher activity (0.0026 ΔO.D./min) than pl-MT+heat (0.0006 ΔO.D./min) and KCN-treated pl-MT (0.0006 ΔO.D./min) (Figure 1e).KCN is an inhibitor of complex IV.ATP synthase activity of pl-MT was higher than that of pl-MT+heat (0.35 pmol/μg protein/ min and 0.008 pmol/μg protein/min, respectively) (Figure 1f).Citrate synthase activity of pl-MT was 241.6 pmol/μg protein/min and that of pl-MT+heat was 10.6 pmol/μg protein/min (Figure 1g).Together, these results indicate that pl-MT have mitochondrial activity preserved compared to pl-MT treated with inhibitors of the mitochondrial respiratory chain and heat.Platelet-derived mitochondria transfer 3

Improved mitochondrial function of hDFs by the transfer of pl-MT
To assess the internalization of pl-MT into cells, hDFs and isolated pl-MT were stained with MitoTracker Green and MitoTracker Red CMXRos, respectively, and then hDFs were incubated with pl-MT.We confirmed the presence of pl-MT stained with MitoTracker Red CMXRos within hDFs whose endogenous mitochondria were stained with MitoTracker Green by confocal microscopy (Figure 2a,b).Confocal microscopy images clearly demonstrated that transferred pl-MT co-localized with endogenous mitochondria from hDFs, as evidenced by the Figure 1.Mitochondria isolated from human platelets (pl-MT).Mitochondria were isolated from human platelets.pl-MT stained with 1μm MitoTracker red CMXRos were confirmed by confocal imaging (a, scale bar, 10 μm.), flow cytometry analysis (B), and western blot analysis (c).pl-MT were evaluated by measuring OXPHOS enzyme activity.To induce mitochondrial damage, pl-MT were incubated with 54.6 μM antimycin a or 300 μM KCN (referred to as an pl-MT+AA and pl-MT+KCN, respectively).To inactivate mitochondria, pl-MT were heated at 90°C for 10 min and referred to as a pl-MT+Heat.complex I+III activity (d), complex IV activity (e), ATP synthase activity (f), and citrate synthase activity (g) were measured as described in materials and methods.Each group was repeated four times.Results are indicated as means � SD and examined by one-way analysis of variance.*p < .05compared with pl-MT, **p < .01compared with pl-MT, ***p < .001compared with pl-MT.merged yellow staining, suggesting that pl-MT can be transferred into hDFs by simple coincubation.In contrast, heat-inactivated pl-MT, which were pre-labeled with MitoTracker Red CMXRos, could not be internalized into hDFs, indicating that active pl-MT could be only taken up by hDFs.In order to investigate whether the transfer of pl-MT has any impact on the mitochondrial functions of hDFs, we assessed intracellular changes of ATP content, complex I+III, complex IV, and ATP synthase activities.The intracellular ATP content, complex I+III activity, complex IV activity, ATP synthase activity, and citrate synthase activity after transfer of pl-MT were significantly increased by 20%, 38%, 27%, 32%, and 13%, respectively, compared with before pl-MT transfer (Figure 2c-g).The transfer of heat-inactivated pl-MT could not show any effect on the intracellular changes of ATP content, complex I+III, complex IV, and ATP synthase activities at all.Together, these results show that pl-MT can be internalized into hDFs by the simple incubation and increase the ATP content and metabolic activities.

Stimulation of scratch wound closure by pl-MT
To investigate the effect of pl-MT on dermal wound repair, we performed 2D gap closure experiment in wound scratch assay using hDFs.Results demonstrated that treatment with pl-MT was able to close the wound gap over time more effectively compared with the control (Figure 3a,b).
Wound closure depends on migration and proliferation of cells.Thus, the effect of pl-MT on proliferation of hDFs was further evaluated using WST-1 assay.As shown in Figure 3c, the proliferation rate was higher for cells treated with pl-MT than that of control cells.Taken together, these results indicate that direct transfer of pl-MT can improve wound closure by facilitating the proliferation and migration of hDFs.

Suppression of ROS production by pl-MT
The redox environment of the wound site plays a key role in healing outcomes [18,19].Thus, we examined whether pl-MT could control ROS levels induced by H 2 O 2 .hDFs were treated with H 2 O 2 followed by pl-MT treatment.Intracellular ROS was evaluated by DCFH-DA and determined using confocal imaging analysis (Figure 4a).When hDFs were administrated with H 2 O 2 , they showed higher fluorescent intensity than control hDFs (no treatment).As shown in Figure 4b, the intracellular ROS level was increased by 1.2fold after H 2 O 2 treatment.However, pl-MT treatment restored intracellular ROS level almost to the control level.(Figure 4a,b).
Additionally, we investigated intracellular ROS and mitochondrial superoxide levels induced by cisplatin and TGF-β both of which could increase the production of ROS by impairing mitochondrial function [20,21].Similarly, hDFs treated with cisplatin or TGF-β showed stronger fluorescent intensity than control.The intracellular ROS level was increased by 1.3-fold after cisplatin treatment (Figure 4c,d) and by 2.2-fold after TGF-β treatment (Figure 4c-e).However, pl-MT treatment suppressed intracellular ROS level increase practically to the control level.Similar results were also observed for mitochondrial superoxide levels induced by cisplatin or TGF-β (Figure 4f), suggesting the role of pl-MT transfer in regulating ROS levels.Taken together, these results suggest that pl-MT can benefit wound repair by suppressing the overproduction of ROS at wound sites.

Discussion
The present study demonstrated the efficacy of pl-MT transfer using an in vitro wound healing model.Intact and functional mitochondria were isolated from platelets.The administration of pl-MT to hDFs facilitated the closure of wound gap by stimulating cell proliferation and attenuating intracellular ROS.Results of this study suggest that pl-MT transfer might be used as a potential therapeutic strategy for wound repair.
Platelets are anucleate blood cells derived from megakaryocytes located in the bone marrow.They play an important role in physiological processes such as hemostasis, inflammation, and wound healing [22].Although having no nucleus, platelets contain functional mitochondria that are responsible for ATP generation, regulation of ROS, calcium homeostasis, and apoptosis.In support of this, delivery of pl-MT into the brain of diabetic animal improved mitochondrial function in the hippocampus and enhanced the cognitive function [14].In addition, transfer of pl-MT restored mitochondrial dysfunction and attenuated the apoptosis in an in vitro neuronal ischemia/ reperfusion injury model [15].Recently, it has been demonstrated that platelets contribute to the wound repair process by transferring pl-MT to mesenchymal stem cells (MSCs) via dynamin-dependent clathrin-mediated endocytosis, thus improving the pro-angiogenic activity of MSCs through metabolic reprogramming [23].Taken together, these reports suggest that pl-MT are sufficiently functional and that direct transfer of pl-MT to injured tissues might be applicable for improving wound repair.
Practically, we could obtain approximately 50 µg mitochondria from 1 ml PRP (platelet-rich plasma) where ~1×10 9 platelets exist.Thus, preparation of mitochondria from platelets has advantages much more than that from other cell sources because platelets are easily acquired from the peripheral blood.We investigated whether direct delivery of pl-MT could promote wound repair using hDFs.As shown in Figure 3, pl-MT isolated from platelets were successfully internalized into hDFs.They promoted cell proliferation and facilitated wound closure in a wound scratch assay, suggesting that direct treatment with pl-MT might be a potential strategy for wound repair.
Although ROS play a pivotal role in the wound healing process [4], excessive and uncontrolled ROS can delay the healing process of a wound lesion by dysregulating inflammatory responses necessary for the onset of wound healing [3,24,25].Therefore, controlling the deregulation of ROS and inflammation might be a promising therapeutic path for wound healing.In line with this, SkQ1, a mitochondria-targeted antioxidant, can stimulate wound healing progresses in aging mice and type II diabetic mice model by controlling excessive inflammatory responses and improving tissue remodeling [26,27].Our previous report has demonstrated that mitochondria isolated from platelets and human umbilical cord mesenchymal stem cells (UC-MSCs) could regulate LPS-induced inflammation by blocking the NFκB signaling pathway both in vitro and in vivo [16].In addition, the present study clearly shows that pl-MT could rescue the increase of intracellular ROS and mitochondrial ROS induced by TGF-β and cisplatin in hDFs (Figure 4).Therefore, it is reasonable to presume that beneficial effects of pl-MT on wound repair are attributed to the anti-inflammatory activity and the role of ROS scavenger.
Platelets are abundant in the peripheral blood.More importantly, active and structurally intact mitochondria can be easily acquired from patient blood.This strategy can be developed as a new alternative for autologous treatment in the field of mitochondrial transfer.The use of autologous mitochondria will expedite its use as a clinical therapeutic agent in various diseases for which the therapeutic effect of mitochondrial transfer has been validated.
Collectively, our study demonstrates that pl-MT can regulate intracellular and mitochondrial ROS and accelerate cell proliferation, thereby improving the wound healing process.This finding may provide the possibility of pl-MT as a therapeutic agent for acute and chronic wounds.

Figure 2 .
Figure 2. Intracellular mitochondrial function of hDfs after the transfer of pl-MT.(a) confocal images of hDfs in the absence or presence of pl-MT.Nuclei and mitochondria of hDfs were stained with hoechst (blue) and MitoTracker green (green), respectively.pl-MT were prestained with MitoTrackerred CMXRos.Scale bar, 10μm.(b) Transfer of pl-MT (red fluorescence) into hDfs were shown by fluorometry analysis.(c) intracellular ATP content, (d) complex I+III activity (e) complex IV activity, and (f) ATP synthase activity in hDfs were compared in the presence of pl-MT and inactivated pl-MT.*p < .05,**p < .01,***p < .001compared with NC. #p < .05,##p < .01,###p < 0.001 compared with pl-MT.

Figure 3 .
Figure 3. Scratch assay and cell proliferation assay of hDfs after stimulation with pl-MT.(a) Representative images of scratch wound closure in hDfs in presence of pl-MT and heat-inactivated pl-MT.The black dot-line indicates the primary injured area.pl-MT, but not heat-inactivated pl-MT, promoted wound closure of injured hDfs.Scale bar, 100 μm.(b) quantification of wound closure area after stimulation with pl-MT.Bar represents standard error of the mean of three independent experiments.The scratched range at time 0 hour was set as 100%.Results are indicated as means � SD and examined by one-way analysis of variance (n = 3, *p < .05 and ***p < .001compared with NC at the same time).(c) cell proliferation of hDfs was measured at 24 and 48 hrs in the absence or presence of pl-MT using WST-1 assays.Results are indicated as means � SD and examined by one-way analysis of variance (n = 4, ***p < .001compared with control).

Figure 4 .
Figure 4. Suppression of ROS by pl-MT.pl-MT were used to treat hDfs with accelerated ROS-production induced by 50 μM H 2 O 2 (a and b), 20 μM cisplatin or 5ng/ml TGF-β (c, d, e and f) after starvation.(A) Intracellular ROS level was evaluated by 10 μM DCF-DA and confocal imaging.the image shows that intracellular ROS (green color) is elevated in the cytosol of hDfs stimulated with 50 μM H 2 O 2 compare to that in unstimulated cells.Transferring pl-MT, but not heat-inactivated pl-MT, effectively reduced intracellular ROS in hDfs.pl-MT in red and DAPI nuclear staining in blue.Scale bar, 10μm.(b) quantification of intracellular ROS levels.(c) intracellular ROS (green color) is elevated in the cytosol of hDfs stimulated with cisplatin or TGF-β compare to that in unstimulated cells.Transferring pl-MT effectively reduced intracellular ROS.pl-MT in red and DAPI nuclear staining in blue.The image shown is a representative of at least three independent experiments.(d and e) quantification of intracellular ROS levels.(f) mitochondrial superoxide level was determined by MitoSOX.These results are indicated as means � SD and examined by one-way analysis of variance.*p < .05 and ***p < .001compared with NC. #p < .05,##p < .01,###p < .001compared with H 2 O 2 , cisplatin or TGF-β only treatment.