Pholiota nameko Polysaccharides Promotes Cell Proliferation and Migration and Reduces ROS Content in H2O2-Induced L929 Cells

Pholiota nameko, a type of edible and medicinal fungus, is currently grown extensively for food and traditional medicine in China and Japan. It possesses various biological activities, such as anti-inflammatory, anti-hyperlipidemia and antitumor activities. However, P. nameko has rarely been discussed in the field of dermatology; identifying its biological activities could be beneficial in development of a new natural ingredient used in wound care. To evaluate its in vitro wound healing activities, the present study assessed the antioxidant and anti-collagenase activities of P. nameko polysaccharides (PNPs) prepared through fractional precipitation (40%, 60% and 80% (v/v)); the assessments were conducted using reducing power, hydroxyl radical scavenging activity, dichloro-dihydro-fluorescein diacetate and collagenase activity assays. The ability of PNPs to facilitate L929 fibroblast cell proliferation and migration was assessed using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and scratch assays. The findings indicated that, among all fractions, PNP-80 showed the best antioxidant and anti-collagenase activity, as measured by their reducing power (IC50 of PNP-80 was 2.43 ± 0.17 mg/mL), the hydroxyl radical scavenging (IC50 of PNP-80 was 2.74 ± 0.11 mg/mL) and collagenase activity assay, and significantly reduced cellular ROS content, compared with that of H2O2-induced L929 cells. Moreover, PNP-80 significantly promoted L929 fibroblast proliferation and migration, compared with the control group. Overall, we suggested that PNP-80 could be a promising candidate for further evaluation of its potential application on wound healing.


Extraction and Fractionation
PNPs were prepared as described in our previous study [16]. PNPs were extracted using ethanol precipitation. Ethanol was added at final concentrations of 40%, 60%, and 80%, and the resulting PNPs were named PNP-40, PNP-60, and PNP-80, respectively. The three different ethanol precipitations of PNP samples were extracted, lyophilized, and refrigerated at 4 • C.

Reducing Power Assay
The reducing power assay was performed as previously described but with modification [24]. Equal volumes (312.5 µL) of PNPs dissolved in ddH 2 O (0.3125-5.000 mg/mL), phosphate buffer (0.2 M, pH 6.6), and 1% potassium ferricyanide were mixed. The mixture was heated to 50 • C for 20 min; subsequently, 312.5 µL of 10% trichloroacetic acid was added to the mixture, followed by 312.5 µL of distilled water and 62.5 µL of 0.1% ferric chloride. Absorbance was immediately detected at 700 nm. Vitamin C (0.0063-0.1014 mg/mL) was used as the positive control. The IC 50 value was equal to the concentration of samples producing 0.5 absorbance at 700 nm.

Hydroxyl Radical Scavenging Activity
Hydroxyl radical scavenging activity was assessed using a previously described Fenton reaction, with modification [25]. Briefly, 50 µL of PNPs dissolved in ddH 2 O (0.3125-5.000 mg/mL) was incubated with 50 µL of sodium salicylate (9 mM), 50 µL of FeSO 4 (9 mM), and 50 µL of H 2 O 2 (0.025%, w/v) at 37 • C for 30 min. Absorbance was then determined at 510 nm. Deionized water was used as the blank control, and vitamin C (0.3125-5.000 mg/mL) was served as the positive control. Hydroxyl radical scavenging activity was calculated using the following equation: Hydroxyl radical scavenging activity (%) = (absorbance of blank control − absorbance of sample/absorbance of blank control) × 100% IC 50 of PNPs was derived from the formula, Y = 100 × A1/(A1 + X), using GraphPad Prism 6.01 and Y denotes the relative content of hydroxyl radical (Y = 100 when X = 0), A1 denotes IC 50 of PNPs and X denotes the concentration of PNPs [26].

Measurement of Inhibitory Effect on Collagenase
Inhibitory effect on collagenase was performed by modified Wang's method [27]. To measure the collagenase activity, 100 µL of 200 units/mL collagenase and 100 µL PNPs (5000, 2500, 1250 µg/mL) were mixed together and incubated at 37 • C for 15 min, weighed at 1 mg of azo dye-impregnated collagen substrate and mixed with 800 µL 0.1 M Tris-HCl buffer (pH 7.0), then added together at 43 • C for 1 h under shaking conditions. Subsequently, the reaction mixture was centrifuged at 3000 rpm for 10 min, and the absorbance was read at 520 nm using the ELISA reader. Distilled water was used as control, and 100 µL of epigallocatechin gallate (EGCG, 5000, 2500, 1250 µg/mL) was used as positive control.
The inhibitory rate of collagenase (%) = ( where A c represents the absorbance of the control, and A ts represents the absorbance of the test sample.

ROS Generation
The ROS generation assay was performed using a previously described method, with modification [28]. Specifically, the concentration of ROS was evaluated using a DCF-DA probe (Sigma Aldrich). The L929 cells were seeded in 24-well plates at a concentration of 1.5 × 10 4 cells/well and allowed to adhere for 24 h. The cells were pre-incubated with PNPs (500 µg/mL) for 24 h and then exposed to H 2 O 2 (0.75 mmol/L) for an additional 2 h after adhesion. After treatment, the L929 cells were incubated in DMEM, without FBS, containing DCF-DA (10 µM) at 37 • C in the dark for 30 min. After removal of the probe by washing twice in phosphate-buffered saline (PBS), the final results were evaluated using fluorescence microscopy (Olympus IX51, Tokyo, Japan). Images were captured, and the mean density values were analyzed using Image J software.

Cell Proliferation
Cell proliferation was determined through the MTT assay by using a previously described procedure, with modification [29]. Briefly, L926 cells were seeded in three 96-well plates (1800 cells/well). After 24 h (day 0), the PNPs (500 µg/mL) dissolved in DMED medium were added and then incubated for 0 h, 24 h and 48 h at 37 • C with 5% CO 2 in a humidified atmosphere, followed by 100 µL of MTT, and the plate was incubated for 2 h. Absorbance was measured at a test wavelength of 570 nm to evaluate cell proliferation. Absorbance was measured on days 0, 1, and 2.

Scratch Assay
The scratch assay-typically used to evaluate the wound-healing capacity of a substance or molecule-was performed using a previously described method, with modification [30]. The assay was used to study cell migration and proliferation, which are crucial for tissue repair. L929 cells were seeded (10 5 cells/mL) in a 24-well plate and cultured for 24 h. Cell culture monolayers were scratched with a sterile 200-µL pipette tip across the center of the well. After scratching, the wells were gently washed twice with PBS to remove the detached cells. The cells were then treated with 500 µg/mL of the three different PNPs prepared in DMED medium. Control cells were not treated with any PNP. Wound-healing efficiency was monitored at 0 and 24 h. The scratch closure rate is expressed as the percentage of scratch closure on an initial area basis, according to the following Equation: where A t0 is the scratch area at time 0 h and A t is the corresponding scratch area at 24 h. The values shown are the means of three wells from three independent experiments.

Microscopy and Image Analysis
Scratch wound closure was examined using an inverted microscope, and images were captured and analyzed using fluorescence microscopy (Olympus IX51) software. The scratch closure area was monitored at different time intervals (0 and 24 h) to calculate wound closure [31].

Statistical Analysis
All data are expressed as means ± standard deviations. Statistical data processing was implemented through dispersion analysis using SPSS 20 software. Statistical analysis was performed using one-way Antioxidants 2020, 9, 65 5 of 14 ANOVA and Duncan's multiple range tests, and a p value of < 0.05 was considered to indicate statistical significance.

Reducing Power and Hydroxyl Radical Scavenging Activity
It was indicated that the antioxidant activities of natural compounds are associated with their wound-healing properties [9]. Therefore, to evaluate the antioxidant activity of PNPs, we used two different antioxidant assays, the reducing power and hydroxyl radical scavenging activity, to simulate the environmental oxidative stress in non-healing wound. Hydroxyl radicals overproduced via uncontrolled Fenton reaction disturb healing process in delayed wound healing [32]. The reducing power can be used to assess to extent to which a compound reduces Fe 3+ to Fe 2+ , and the higher absorbance value at 700 nm is, the stronger is antioxidant [33]. The reducing power levels derived from PNPs and vitamin C increased in a dose-dependent manner in Figure 1. PNP-80 (IC 50 = 2.43 ± 0.17 mg/mL) had a higher reducing power for ferric ion than did PNP-40 and PNP-60 (p < 0.05). The growth rates of PNP-40 and PNP-60 were slow, with the absorbance values being 0.43 and 0.31 at 5.0 mg/mL, respectively.

Reducing Power and Hydroxyl Radical Scavenging Activity
It was indicated that the antioxidant activities of natural compounds are associated with their wound-healing properties [9]. Therefore, to evaluate the antioxidant activity of PNPs, we used two different antioxidant assays, the reducing power and hydroxyl radical scavenging activity, to simulate the environmental oxidative stress in non-healing wound. Hydroxyl radicals overproduced via uncontrolled Fenton reaction disturb healing process in delayed wound healing [32]. The reducing power can be used to assess to extent to which a compound reduces Fe 3+ to Fe 2+ , and the higher absorbance value at 700 nm is, the stronger is antioxidant [33]. The reducing power levels derived from PNPs and vitamin C increased in a dose-dependent manner in Figure 1. PNP-80 (IC50 = 2.43 ± 0.17 mg/mL) had a higher reducing power for ferric ion than did PNP-40 and PNP-60 (p < 0.05). The growth rates of PNP-40 and PNP-60 were slow, with the absorbance values being 0.43 and 0.31 at 5.0 mg/mL, respectively. Hydroxyl radicals are among the most reactive and hazardous free radicals. Overproduction hydroxyl radical overwhelms oxidation-reduction system and causes damage in cellular protein, DNA, lipid and wound-healing related cells, fibroblast, arising delayed healing of wounds [9]. Therefore, to evaluate the hydroxyl radical scavenging activity of the PNPs in vitro, we used the Fenton reaction system as a model. The hydroxyl radical scavenging activities of the PNPs and vitamin C are illustrated in Figure 2. All samples exhibited hydroxyl radical scavenging activity in a dose-dependent manner. The IC50 value of PNP-80 and PNP-60 was 2.74 ± 0.11 mg/mL (95% confidence intervals = 2.520 to 2.965 mg/mL and R 2 = 0.9406) and 4.25 ± 0.09 mg/mL (95% confidence intervals = 4.069 to 4.436 mg/mL and R 2 = 0.9816), respectively; additionally, at 5 mg/mL, the scavenging abilities of PNP-80, PNP-60, PNP-40, and vitamin C were 60.17%, 51.44%, 47.25%, and 94.29%, respectively. These results indicate that PNP-80 exhibited greater potency to donate hydrogen to hydroxyl radicals than did the other fractions (p < 0.05) [34].
Low-molecular-weight polysaccharides have a less compact structure than do high-molecularweight polysaccharides. This signifies that low-molecular-weight polysaccharides have more free functional groups, such as carboxyl, amino, and hydroxyl groups which could react with free radicals and then stabilize them, than do high-molecular-weight polysaccharides [35]. The antioxidant properties of chitosan weighing 2.2-300.0 kDa are inversely related to their molecular weights [36], explaining why PNP-80 (4.40 kDa) exhibited a more pronounced scavenging activity (measured through its reducing power and hydroxyl radical scavenging activity) than did PNP-60 (21.57 kDa) and PNP-40 (333.49 kDa) at a concentration of 5 mg/mL. In terms of reducing power, the absorbance Hydroxyl radicals are among the most reactive and hazardous free radicals. Overproduction hydroxyl radical overwhelms oxidation-reduction system and causes damage in cellular protein, DNA, lipid and wound-healing related cells, fibroblast, arising delayed healing of wounds [9]. Therefore, to evaluate the hydroxyl radical scavenging activity of the PNPs in vitro, we used the Fenton reaction system as a model. The hydroxyl radical scavenging activities of the PNPs and vitamin C are illustrated in Figure 2. All samples exhibited hydroxyl radical scavenging activity in a dose-dependent manner. The IC 50 value of PNP-80 and PNP-60 was 2.74 ± 0.11 mg/mL (95% confidence intervals = 2.520 to 2.965 mg/mL and R 2 = 0.9406) and 4.25 ± 0.09 mg/mL (95% confidence intervals = 4.069 to 4.436 mg/mL and R 2 = 0.9816), respectively; additionally, at 5 mg/mL, the scavenging abilities of PNP-80, PNP-60, PNP-40, and vitamin C were 60.17%, 51.44%, 47.25%, and 94.29%, respectively. These results indicate that PNP-80 exhibited greater potency to donate hydrogen to hydroxyl radicals than did the other fractions (p < 0.05) [34]. convert them into more stable products. In terms of hydroxyl radical scavenging activity, the IC50 value of Auricularia auricular polysaccharides that was reported to be greater than 5.0 mg/mL is similar to that of PNPs [38]. Our results show that among the three fractions, PNP-80 had the greatest antioxidant activity. Moreover, although PNPs did not exhibit antioxidant activity as good as vitamin C did, PNPs possess a variety of wound healing-related functions, such as moisturizing [16] which vitamin did not hold.

Anti-Collagenase Activity of PNPs
Collagenase is one of matrix metalloproteinases (MMPs) capable of breaking down the extracellular matrix (ECM), the major components of connective tissue, which can support the skin structure, maintain skin elasticity and play an important role in wound healing [39]. However, it has been shown that overexpression of collagenase was associated with chronic wound and treatment chronic wound with metalloproteinase inhibitor could improve delayed healing wound [22,23]. To evaluate if PNPs have the inhibitory ability against collagenase, we used in vitro collagenase activity assay as an evaluation model. Epigallocatechin gallate (EGCG), the predominant catechin in tea, possesses the inhibition of collagenolytic activity by collagenase and is usually used as positive control in collagenase activity assay [40][41][42]. The collagenase inhibitory activity of PNPs and epigallocatechin gallate (EGCG) was shown in Figure 3. All of samples inhibited collagenase in a dose-dependent manner at a range of concentration from 125 to 500 µg/mL. The inhibitory activity of PNP-40, PNP-60 and PNP-80 ranged from 25% to 33%, from 26% to 39% and from 32% to 61% at 125-500 µg/mL, respectively. The inhibitory activity of PNP-80 at a concentration of 500 µg/mL was significantly higher than that of PNP-60, PNP-40 and EGCG. Although the collagenase inhibitory activity of all samples did not show significant differences at a concentration of 125 µg/mL and 250 µg/mL, the inhibitory activity of PNP-80 was slightly higher than that of PNP-40, PNP-60, and EGCG. The result indicated that PNPs might improve delayed healing wound by reducing the collagenase activity Collagenases are a group of zinc-containing proteinases, which contains Zn ion at its active site that facilitates interaction with an inhibitor [43]. Previous studies showed that PNPs have the ability to chelate metal [16], which might suggest that the inhibitory mechanism of PNPs against collagenase is due to their ability to chelate Zn ion at collagenase's active site and hamper the interaction between the substrate and its active site. Low-molecular-weight polysaccharides have a less compact structure than do high-molecular-weight polysaccharides. This signifies that low-molecular-weight polysaccharides have more free functional groups, such as carboxyl, amino, and hydroxyl groups which could react with free radicals and then stabilize them, than do high-molecular-weight polysaccharides [35]. The antioxidant properties of chitosan weighing 2.2-300.0 kDa are inversely related to their molecular weights [36], explaining why PNP-80 (4.40 kDa) exhibited a more pronounced scavenging activity (measured through its reducing power and hydroxyl radical scavenging activity) than did PNP-60 (21.57 kDa) and PNP-40 (333.49 kDa) at a concentration of 5 mg/mL. In terms of reducing power, the absorbance value at 700 nm of G. lucidum polysaccharides-80 was 0.138 at a concentration of 2.0 mg/mL [37], suggesting that PNP-80 could be more effective electron donors that can react with free radicals and convert them into more stable products. In terms of hydroxyl radical scavenging activity, the IC 50 value of Auricularia auricular polysaccharides that was reported to be greater than 5.0 mg/mL is similar to that of PNPs [38]. Our results show that among the three fractions, PNP-80 had the greatest antioxidant activity. Moreover, although PNPs did not exhibit antioxidant activity as good as vitamin C did, PNPs possess a variety of wound healing-related functions, such as moisturizing [16] which vitamin did not hold.

Anti-Collagenase Activity of PNPs
Collagenase is one of matrix metalloproteinases (MMPs) capable of breaking down the extracellular matrix (ECM), the major components of connective tissue, which can support the skin structure, maintain skin elasticity and play an important role in wound healing [39]. However, it has been shown that overexpression of collagenase was associated with chronic wound and treatment chronic wound with metalloproteinase inhibitor could improve delayed healing wound [22,23]. To evaluate if PNPs have the inhibitory ability against collagenase, we used in vitro collagenase activity assay as an evaluation model. Epigallocatechin gallate (EGCG), the predominant catechin in tea, possesses the inhibition of collagenolytic activity by collagenase and is usually used as positive control in collagenase activity assay [40][41][42]. The collagenase inhibitory activity of PNPs and epigallocatechin gallate (EGCG) was shown in Figure 3. All of samples inhibited collagenase in a dose-dependent manner at a range of concentration from 125 to 500 µg/mL. The inhibitory activity of PNP-40, PNP-60 and PNP-80 ranged from 25% to 33%, from 26% to 39% and from 32% to 61% at 125-500 µg/mL, respectively. The inhibitory activity of PNP-80 at a concentration of 500 µg/mL was significantly higher than that of PNP-60, PNP-40 and EGCG. Although the collagenase inhibitory activity of all samples did not show significant differences at a concentration of 125 µg/mL and 250 µg/mL, the inhibitory activity of PNP-80 was slightly higher than that of PNP-40, PNP-60, and EGCG. The result indicated that PNPs might improve delayed healing wound by reducing the collagenase activity. The experiments were conducted in triplicate independently (n = 3), and the data are expressed as the means ± standard error (SE). a-c Means within the same concentration followed by the same letter were not significantly different (p > 0.05).

Effect of Intracellular ROS Generation
Wound healing is a dynamic and precisely controlled process and can be divided into three phases: inflammatory, proliferative and remodeling phase. During the inflammatory phase, neutrophils infiltrate to the wound area in order to combat microbes and clear cell debris via secreting H2O2 and proteases. Even though these substances have a beneficial effect on the wound healing process, overproduction H2O2 gives rise to the non-healing wound via weakening wound-healing related cell, fibroblast [17,44]. In our previous study, we proved that pre-treated with PNPs reduced the H2O2-induced damage to L929 cell using MTT assay, suggesting PNPs might protect L929 cell from programmed cell death by ameliorating oxidative stress in L929 cell [16]. To assess if PNPs possess the ability against H2O2-induced oxidative stress, we utilized ROS generation assay as a model. We had already proved that PNPs did not exhibit cytotoxicity toward L929 cells under a concentration of 500 µg/mL [16]. The levels of ROS, determined through DCFH-DA staining, produced in control cells, H2O2-induced cells without PNPs pretreatment and with PNPs pretreatment (500 µg/mL) are shown in Figure 4a; in this figure, green fluorescence indicates ROS production. This study observed a notable increase in ROS production in the H2O2-induced cells without PNPs, however, the ROS levels were lower in the PNPs-pretreated groups. ROS production was also quantified using image J software (Figure 4b). Compared with the H2O2-induced L929 cells without PNPs pre-treatment, the H2O2-induced L929 cells pre-treated with PNPs had significantly reduced ROS levels. The relative fluorescence intensity of H2O2-induced L929 cells without PNPs was 0.15, H2O2-induced L929 cells pre-treated with PNP-80, PNP-60 and PNP-40 which were reduced to 0.06, 0.10, and 0.09, respectively. Overall, PNP-80 showed the greatest free-radical scavenging activity and reduced the level of ROS in the H2O2-treated cells by 53.33%. Moreover, it was indicated P. nameko polysaccharides have the potential to increase the activity of cellular antioxidant enzymes, such as superoxide dismutase (SOD), thereby reducing cellular ROS contents [45], suggesting PNP-40, PNP-60 and PNP-80 might also ameliorate cellular oxidative stress via up-regulating SOD expression. According to these results, we infer that PNPs could attenuate H2O2-induced damage via improving cellular oxidative stress. These results are similar to that of sulfated polysaccharides isolated from the edible marine algae Padina tetrastromatica which weaken H2O2-induced cellular damage via the reduction of intracellular reactive oxygen species level [46]. Collagenases are a group of zinc-containing proteinases, which contains Zn ion at its active site that facilitates interaction with an inhibitor [43]. Previous studies showed that PNPs have the ability to chelate metal [16], which might suggest that the inhibitory mechanism of PNPs against collagenase is due to their ability to chelate Zn ion at collagenase's active site and hamper the interaction between the substrate and its active site.

Effect of Intracellular ROS Generation
Wound healing is a dynamic and precisely controlled process and can be divided into three phases: inflammatory, proliferative and remodeling phase. During the inflammatory phase, neutrophils infiltrate to the wound area in order to combat microbes and clear cell debris via secreting H 2 O 2 and proteases. Even though these substances have a beneficial effect on the wound healing process, overproduction H 2 O 2 gives rise to the non-healing wound via weakening wound-healing related cell, fibroblast [17,44]. In our previous study, we proved that pre-treated with PNPs reduced the H 2 O 2 -induced damage to L929 cell using MTT assay, suggesting PNPs might protect L929 cell from programmed cell death by ameliorating oxidative stress in L929 cell [16]. To assess if PNPs possess the ability against H 2 O 2-induced oxidative stress, we utilized ROS generation assay as a model. We had already proved that PNPs did not exhibit cytotoxicity toward L929 cells under a concentration of 500 µg/mL [16]. The levels of ROS, determined through DCFH-DA staining, produced in control cells, H 2 O 2 -induced cells without PNPs pretreatment and with PNPs pretreatment (500 µg/mL) are shown in Figure 4a; in this figure, green fluorescence indicates ROS production. This study observed a notable increase in ROS production in the H 2 O 2 -induced cells without PNPs, however, the ROS levels were lower in the PNPs-pretreated groups. ROS production was also quantified using image J software (Figure 4b). Compared with the H 2 O 2 -induced L929 cells without PNPs pre-treatment, the H 2 O 2 -induced L929 cells pre-treated with PNPs had significantly reduced ROS levels. The relative fluorescence intensity of H 2 O 2 -induced L929 cells without PNPs was 0.15, H 2 O 2 -induced L929 cells pre-treated with PNP-80, PNP-60 and PNP-40 which were reduced to 0.06, 0.10, and 0.09, respectively. Overall, PNP-80 showed the greatest free-radical scavenging activity and reduced the level of ROS in the H 2 O 2 -treated cells by 53.33%. Moreover, it was indicated P. nameko polysaccharides have the potential to increase the activity of cellular antioxidant enzymes, such as superoxide dismutase (SOD), thereby reducing cellular ROS contents [45], suggesting PNP-40, PNP-60 and PNP-80 might also ameliorate cellular oxidative stress via up-regulating SOD expression. According to these results, Antioxidants 2020, 9, 65 8 of 14 we infer that PNPs could attenuate H 2 O 2 -induced damage via improving cellular oxidative stress. These results are similar to that of sulfated polysaccharides isolated from the edible marine algae Padina tetrastromatica which weaken H 2 O 2 -induced cellular damage via the reduction of intracellular reactive oxygen species level [46].

Cell Proliferation and Migration
Proliferation and recruitment of fibroblasts in the wound area are particularly important to wound healing process because fibroblasts are directly liable for depositing ECM, forming granulation tissues and contracting wound lesions. Consequently, substances that could enhance the proliferation and migration activity of fibroblast implies they have the potential to accelerate the wound healing process [47].
In wound healing, a major concern is the positive response of fibroblasts toward the materials of interest; L929 is widely used for testing if the samples possess the stimulation activity by studying its cell proliferation activity [48,49]. The MTT assay was used to determine the proliferation of L929 fibroblast cells. Proliferative activity can be determined by measuring the reduction of yellow tetrazolium salt to purple formazan crystals, which indicates cells' metabolic state [50]. The relative fold changes in cell proliferation are shown in Figure 5. The proliferative activity of L929 cells treated with PNP-80 increased significantly compared with that of the control cells on day 1. However, on day 2, all three PNPs significantly promoted the proliferative activity of L929 cells compared with that of the control cells (p < 0.05). The proliferative activity of L929 cells treated with PNP-80, PNP-60, and PNP-40 exhibited 5.80-, 5.29-, and 5.16-fold changes on day 2 compared with the changes

Cell Proliferation and Migration
Proliferation and recruitment of fibroblasts in the wound area are particularly important to wound healing process because fibroblasts are directly liable for depositing ECM, forming granulation tissues and contracting wound lesions. Consequently, substances that could enhance the proliferation and migration activity of fibroblast implies they have the potential to accelerate the wound healing process [47].
In wound healing, a major concern is the positive response of fibroblasts toward the materials of interest; L929 is widely used for testing if the samples possess the stimulation activity by studying its cell proliferation activity [48,49]. The MTT assay was used to determine the proliferation of L929 fibroblast cells. Proliferative activity can be determined by measuring the reduction of yellow tetrazolium salt to purple formazan crystals, which indicates cells' metabolic state [50]. The relative fold changes in cell proliferation are shown in Figure 5. The proliferative activity of L929 cells treated with PNP-80 increased significantly compared with that of the control cells on day 1. However, on day 2, all three PNPs significantly promoted the proliferative activity of L929 cells compared with that of the control cells (p < 0.05). The proliferative activity of L929 cells treated with PNP-80, PNP-60, and PNP-40 exhibited 5.80-, 5.29-, and 5.16-fold changes on day 2 compared with the changes observed on day 0, respectively, which were significantly higher than those in the control cells. These results indicate that in L929 cells, PNPs greatly promote proliferation, which is crucial in skin wound healing.
Antioxidants 2020, 9, 65 9 of 14 results indicate that in L929 cells, PNPs greatly promote proliferation, which is crucial in skin wound healing. We next determined whether PNPs affect L929 cell migration. Cellular migration is an essential process in wound and cutaneous repair, and fibroblasts can traverse tissue environments to degrade, repair, and remodel the ECM [51]. We used the in vitro scratch assay to measure the migration of L929 cells into a cell-free gap in the tissue culture dish. As presented in Figure 6a,b, during the time from 0 to 24 h, PNPs significantly increased the closure speed in the cells relative to the control cells (11.83%). L929 cells that were treated with PNPs showed a significant increase in closure rate, with the rates being 35.82% (PNP-40), 34.81% (PNP-60), and 54.75% (PNP-80). The closure rates of L929 cells treated with PNPs at 24 h were higher than those of the control cells by 194.25-362.80%. The increased migratory activity of L929 cells treated with PNPs suggests that PNPs have the potential to enhance cutaneous repair [47,52].
In human skin fibroblasts, 24-h exposure to ammonium-chitosan conjugates was reported to result in a significantly increased closure rate (approximately 70%) compared with the rate observed in control cells (approximately 45%) [53]. Sargassum ilicifolium aqueous extracts were also reported to engender an enhanced wound closure rate (97.83%) in L929 cells at 24 h compared with the rate observed in control cells (46.11%); hence, the wound closure rate of the treated cells was higher than that of the control cells by 112.17% [31], indicating PNPs demonstrated better enhancement in fibroblast migration. We next determined whether PNPs affect L929 cell migration. Cellular migration is an essential process in wound and cutaneous repair, and fibroblasts can traverse tissue environments to degrade, repair, and remodel the ECM [51]. We used the in vitro scratch assay to measure the migration of L929 cells into a cell-free gap in the tissue culture dish. As presented in Figure 6a,b, during the time from 0 to 24 h, PNPs significantly increased the closure speed in the cells relative to the control cells (11.83%). L929 cells that were treated with PNPs showed a significant increase in closure rate, with the rates being 35.82% (PNP-40), 34.81% (PNP-60), and 54.75% (PNP-80). The closure rates of L929 cells treated with PNPs at 24 h were higher than those of the control cells by 194.25-362.80%. The increased migratory activity of L929 cells treated with PNPs suggests that PNPs have the potential to enhance cutaneous repair [47,52].
In human skin fibroblasts, 24-h exposure to ammonium-chitosan conjugates was reported to result in a significantly increased closure rate (approximately 70%) compared with the rate observed in control cells (approximately 45%) [53]. Sargassum ilicifolium aqueous extracts were also reported to engender an enhanced wound closure rate (97.83%) in L929 cells at 24 h compared with the rate observed in control cells (46.11%); hence, the wound closure rate of the treated cells was higher than that of the control cells by 112.17% [31], indicating PNPs demonstrated better enhancement in fibroblast migration. It was indicated that β-glucans are a multi-functional modulator of wound healing [54], for instance, (1→3)-(1→6)-β-D-glucan from Aureobasidium pullulans stimulates dermal fibroblast proliferation and migration [55]. Moreover, our previous study showed that the β-D-glucan contents in PNP-40, PNP-60 and PNP-80 were 20.20%, 12.20% and 10.15%, respectively [16], and other report was indicated as a β-D-glucan-(1→3)-linked, substituted at O-6 by β-D-Glcp or (1→6)-linked β-D-Glcp side chains using NMR and methylation analyses [56], suggesting that β-d-glucan might be one of active substances in PNPs elevating cell migration and proliferation.
A moist wound environment has a variety of beneficial effects on wound healing process, such as prevention of tissue dehydration and cell death, accelerated angiogenesis, increased breakdown of dead tissue and fibrin, compared with a dry wound environment [57]. Our previous study showed the moisture-retention rate of PNP-80 was 64.17% after 96 h exposed to 10% relative humidity (RH) and it was higher than that of PNP-40 (63.42%) and PNP-60 (63.21%) and far higher than that of glycerol (49.53%) [16]. In this study, we found that PNP-80 exhibited the best reducing power and It was indicated that β-glucans are a multi-functional modulator of wound healing [54], for instance, (1→3)-(1→6)-β-d-glucan from Aureobasidium pullulans stimulates dermal fibroblast proliferation and migration [55]. Moreover, our previous study showed that the β-d-glucan contents in PNP-40, PNP-60 and PNP-80 were 20.20%, 12.20% and 10.15%, respectively [16], and other report was indicated as a β-d-glucan-(1→3)-linked, substituted at O-6 by β-d-Glcp or (1→6)-linked β-d-Glcp side chains using NMR and methylation analyses [56], suggesting that β-d-glucan might be one of active substances in PNPs elevating cell migration and proliferation.
A moist wound environment has a variety of beneficial effects on wound healing process, such as prevention of tissue dehydration and cell death, accelerated angiogenesis, increased breakdown of dead tissue and fibrin, compared with a dry wound environment [57]. Our previous study showed the moisture-retention rate of PNP-80 was 64.17% after 96 h exposed to 10% relative humidity (RH) and it was higher than that of PNP-40 (63.42%) and PNP-60 (63.21%) and far higher than that of glycerol (49.53%) [16]. In this study, we found that PNP-80 exhibited the best reducing power and hydroxyl radical scavenging activity of all fractions, greatly reduced ROS content in H 2 O 2 -induced L929 cells and significantly enhanced the proliferation and migration rate of L929 cells, compared with control group, suggesting that PNP-80 might be a promising candidate for further evaluation of its potential application on wound healing. In addition, we did further assess if PNPs have the antibacterial activity against Escherichia coli and Staphylococcus aureus (the data was not shown here). Although the result indicated that PNPs did not exhibit antibacterial activity against E. coli and S. aureus in agar well diffusion assay at a concentration of 10 mg/mL, it does not mean PNPs are not appropriate to be used as functional ingredients in wound healing related agents. That is because the flaw might be made up by adding additional antibacterial agents.
Furthermore, identifying and refining the active compounds in PNPs using gel filtration chromatography and ion-exchange liquid chromatography is important and necessary because PNPs did not show great potency in in vitro wound healing assay.

Conclusions
In our study, PNPs extracted using fractional precipitation show great antioxidant activity in vitro; additionally, they significantly reduce ROS production, promote proliferation, and increase the wound closure rate at the cellular level. These attributes provide information for the first time on the effectiveness of PNPs in enhancing in vitro wound healing. The findings indicate that PNP-80 shows the greatest antioxidant, anti-collagenase, proliferative, and migratory activities among the fractions. PNP-80 was the most promising candidate of all fractions for further evaluation of its potential application on wound healing. Further investigations into the effects of PNPs on wound healing must be conducted on an animal model and their stability; moreover, the structure and mechanisms underlying the wound-healing effects of PNPs warrant investigation.