Protective Effects of Ginseng Proteins on Photoaging of Mouse Fibroblasts Induced by UVA
Abstract
UVA can penetrate dermis and cause functional damage of dermal fibroblasts leading photoaging. Ginseng is a widely used traditional Chinese medicine for skin aging. However, its effects on skin photoaging induced by UVA are not clear. In this study, we isolated ginseng proteins (GP), with molecular weights of 27 kDa and 13 kDa, and found that they alleviated the inhibitory effects of UVA on cell viability and increased percentage of NIH‐3T3 fibroblasts in the S phase of cells cycle. GP also improved cell contraction ability, increased the expression and secretion of CoL‐I, similar to MAPK phosphorylation inhibitors and reduced expression and secretion of MMP‐1, MMP‐2 and MMP‐9 as well as the enzyme activities of MMP‐2 and MMP‐9. They reduced ROS content, DNA damage and 8‐OHdG content, as well as the protein expression of p53, p21 and p16. The levels of p‐ERK, p‐p38 and p‐JNK, p‐c‐Fos and p‐c‐Jun proteins were decreased by GP. Inactivated GP did not inhibit the cellular activity and expression and secretion of CoL‐I irradiated by UVA. The results showed that GP can improve cell viability and contractile function by inhibiting DNA damage and collagen degradation to inhibit the photoaging effects of skin dermal cells caused by UVA.
Introduction
The ultraviolet component of terrestrial radiation is a major cause of skin aging 1. The wavelength of long‐wave ultraviolet A (UVA) is 320–400 nm, which accounts for about 95% of ultraviolet radiation (UVR) 1. It can penetrate the dermis and cause functional damage to dermal fibroblasts, leading to photoaging 2. The main manifestations of damage are inhibition of the proliferation ability of fibroblasts, which reduces their cell viability and contraction ability, and an increase in collagen degradation 1, 3. Therefore, it is important to find protective agents that are resistant to the photoaging of dermal cells caused by UVA, and as such, can effectively protect skin from photoaging.
UVA causes oxidative stress to promote the excessive production of reactive oxygen species (ROS) in dermal fibroblasts, which leads to oxidative damage in biomacromolecules such as cell membranes, proteins and nucleic acids 4. On one hand, excessive ROS can cause cell DNA damage to produce 8‐hydroxydeoxyguanosine (8‐OHdG), which promotes the expression of p53 protein, thereby increasing the expression of p21 and p16 proteins, causing cell cycle arrest and decreasing the activity of fibroblasts and the contractile capacity of dermal fibroblasts 5, 6. On the other hand, it can activate the mitogen‐activated protein kinase (MAPK) signaling pathway and promote the phosphorylation of AP‐1 protein, causing the activation of matrix metalloproteinases (MMPs) and subsequent degradation of collagen 6-8. Therefore, the decrease of ROS resists the photoaging effects of UVA on dermal cells by inhibiting key regulatory sites in the abovementioned signaling pathways.
Ginseng is the most commonly used traditional Chinese medicine for combating skin aging 9. However, studies on its active substance and mechanism by which it prevents the photoaging caused by UVA have been very limited. It has been reported that ginsenoside Rg3 (Rg3) can reduce dermal thickening and the expression of procollagen‐I, MMP‐1 and interleukin‐6 in a three‐dimensional (3D) skin model irradiated with UVA 10. Recent studies have shown that the protein components of ginseng roots have many important biological functions 11-13. In addition, our previous study showed that ginseng proteins can promote fibroblast proliferation and secretion of type‐I collagen (CoL‐I), through the extracellular signal‐related kinase (ERK) signaling pathway to achieve wound repair 13. Thus, it has been suggested that ginseng proteins may affect UVA‐induced dermal fibroblast aging.
In this study, proteins with protective effects on photoaging in fibroblast cells irradiated with UVA were isolated from ginseng roots and their protective effects on regulatory pathways activated by UVA‐induced fibroblast injury were investigated. The results of this study revealed the effective substances and pathways underlying the protective effects of ginseng on photoaging induced by UVA in fibroblasts.
Materials and Methods
Chemicals and reagents
The NIH‐3T3 mouse fibroblast cell line was purchased from the Cell Resource Center of the Shanghai Institute for Biological Sciences (Shanghai, China). Dulbecco's modified Eagle's medium (DMEM) and fetal bovine serum (FBS) were purchased from Invitrogen (Carlsbad, CA). Penicillin, streptomycin, 3‐(4,5‐dimethylthiazol‐2‐yl)‐2,5‐diphenyltetrazolium bromide (MTT), dimethyl sulfoxide (DMSO) and Triton X‐100 were purchased from Sigma‐Aldrich (St. Louis, MO). Antibodies against CoL‐I, MMP‐1, p53, p21, p16, N‐terminal kinase (JNK)/phosphorylated JNK (p‐JNK), p38/p‐p38, ERK/p‐ERK, p‐c‐Jun, p‐c‐Fos, β‐actin, and the western blotting luminol reagent were purchased from Santa Cruz Biotechnology (Dallas, TX). The TIANamp Genomic DNA Kit was purchased from TIANGEN Biotech Co. Ltd. (Beijing, China). The BCA Protein Assay Kit, propidium iodide (PI), DCFH‐DA fluorescent probe and MAPK phosphatase inhibitors targeting JNK (sp600125), ERK (FR180204) and p38 (SB203580) were purchased from the Beyotime Institute of Biotechnology (Shanghai, China). Mouse CoL‐I, MMP‐1, MMP‐2 and MMP‐9 ELISA Kits were purchased from Boster Biological Technology Co. Ltd. (Wuhan, China). All other reagents were of analytical grade and made in China.
Plant materials
The plant material of 5‐year‐old ginseng roots (Panax ginseng C.A. Meyer) was collected from Fusong, in China's Jilin Province in September 2016, and identified by Jie Wu, a ginseng expert of Jilin Province. The voucher specimen (2145) is deposited at the Herbarium of College of Science, Beihua University, Jilin province, China.
Preparation of Ginseng proteins (GP)
Fresh ginseng roots (1 kg) were cut into small slices and extracted twice with 10 L phosphate‐buffered saline (PBS) at 4°C for 4 h. The supernatant was collected and concentrated with an ultrafiltration membrane (5 kDa, PALL minimate) to remove small molecular compounds. Then the concentrated solution was separated with DEAE Sepharose Fast Flow (1.6 × 20 cm) that had been equilibrated with 25 mm Tris‐HCl buffer at pH 6.0. The columns were eluted with a linear NaCl gradient (0–2.0 m) at a flow rate of 2.0 mL min−1. The adsorbed protein fractions were collected at 280 nm on an UV spectrophotometer 14. The protein fraction was loaded on a Sephadex G‐75 column (1.6 × 60 cm), eluted with water at a flow rate of 1.0 mL min−1, and detected with a UV detector at 280 nm. The yield of each peak was collected by vacuum drying, and the target peak was named GP 15. The protein concentration was determined with the BCA Protein Assay Kit. The molecular weight distribution was detected by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS‐PAGE) and compared with its electrophoretic mobility against molecular mass marker proteins 15.
Identification of GP
The two protein bands were manually excised from the preparative gels, digested with trypsin at 37°C for 20 h and analyzed by using LCMSMS (Q Exactive; Thermo Fisher) as previously described 16. The peptide mass fingerprint was analyzed with MASCOT search engine.
Protein denaturation
To determine the effects of GP on the antiphotoaging of mouse fibroblasts induced by UVA, methods of heat inactivation and protease degradation were used to inactivate the protein. The protein was heated at 100°C for 30 min and centrifuged at 15 294 g for 20 min; this fraction was named GPH. Then the proteins were degraded by protease K (50 μg mL−1) at 54°C for 30 min and incubated at 70°C for 30 min to inactivate protease; this fraction was named GPS.
Cell culture and UVA irradiation
The NIH‐3T3 cell line was cultured in DMEM at 37°C with 10% FBS, 100 units mL−1 penicillin and 100 μg mL−1 streptomycin in a humidified 5% CO2 incubator (Thermo Fisher Scientific, Waltham, MA) 13. Before UVA irradiation, cells (70–80% confluent) were washed and coated with 1 mL PBS to prevent UVA absorption by components of the medium. A Philips UVA lamp with an emission spectrum between 320 and 400 nm was used. The dose of UVA irradiation was 10 J cm−2 (16 min of exposure, tested with UV intensity tester) at a distance of 15 cm from the bottom of the opening plate, which was verified with a UV light meter (Sigma, Shanghai, China). After UVA irradiation, cells were incubated in complete medium and maintained with the indicated compounds.
Cell viability assay
A modified version of the MTT assay was used to test the effects of GP, GPH and GPS on NIH‐3T3 cell viability 8, 13. NIH‐3T3 cells (6 × 104 cells/well) were plated in a 96‐well plate for 24 h. The cells were preincubated with various concentrations of GP, GPH and GPS for 1 h, and subsequently irradiated with UVA (10 J cm−2) and incubated for 24 h. Then MTT (10 μL, 5 mg mL−1 stock solution) was added to each well and incubated at 37°C for 4 h. After incubation, 150 μL DMSO was added, and the absorbance was measured at 570 nm in a microplate reader after shaking for 10 min. All tests were performed in triplicate.
Cell cycle assay
Flow cytometry was used to detect effects of GP on the cell cycle induced by UVA 5. Cells treated with the abovementioned method were collected, washed twice with ice‐cold PBS and then incubated with 70% alcohol at 4°C for 4 h. The cell pellets were collected by centrifugation and suspended in PI for 30 min at 37°C. The fluorescence intensity was analyzed by flow cytometry (C6; BD Biosciences, San Jose, CA).
Cell contraction assay
The collagen matrices (150 μL, 1 mg mL−1 collagen, 6 × 104 cells/matrix) were polymerized for 24 h in DMEM, preincubated with various concentrations of GP for 1 h, irradiated by UVA (10 J cm−2) and incubated for 24 h. At the end of the incubation period, the matrices were fixed in 3.7% paraformaldehyde, and the matrix diameter was measured to calculate the extent of contraction 3.
Enzyme‐linked immunoassay
The cells were preincubated with GP for 1 h and subsequently irradiated by UVA (10 J cm−2). After incubation at 37°C for 24 h, the cells were collected and lysed in 1% Triton X‐100 at 4°C for 1 h. The supernatant was collected and measured using an enzyme‐linked immunoassay (ELISA) Kit according to the manufacturer's instructions. The culture medium was directly measured using ELISA Kit.
Gelatin zymography
Gelatin zymography was used to detect the effects of GP on the enzyme activities of MMP‐2 and MMP‐9 induced by UVA 5, 17. The cells irradiated with UVA (10 J cm−2) and incubated for 24 h were collected and washed with PBS, and lysed in RIPA Lysis Buffer at 4°C for 1 h. The supernatant was collected by centrifugation, and the protein concentrations were measured with the BCA Protein Assay Kit. Proteins (30 μg) were electrophoresed on zymography gels (10% polyacrylamide, 0.1% gelatin) at 120 V for 2 h. After electrophoresis, the gel was washed with 2.5% Triton X‐100, incubated with developing buffer (50 mmol L−1 Tris‐HCl, 5 mmol L−1 CaCl2, pH 7.8) overnight at 37°C and stained with staining solution (0.5% w/v Coomassie blue R‐250).
Determination of ROS content
ROS content was monitored using flow cytometry with a DCFH‐DA probe 18. The cells were preincubated with GP for 1 h and subsequently irradiated by UVA (10 J cm−2). After incubated at 37°C for 3 h, the cells were collected, washed with ice‐cold PBS and then suspended in 10 μm DCFH‐DA at 37°C for 20 min. The cells were washed with PBS and the fluorescence intensity was analyzed by flow cytometry (C6; BD Biosciences).
Comet assay
The effects of GP on the extent of DNA damage were determined by the comet assay 19. The fibroblasts were resuspended in PBS to obtain the irradiated cell suspension (incubated for 24 h, 1 × 105 cells mL−1), and then suspended in 2‐hydroxyethylagarose to 500 μL and spread on a glass microscope slide. The slides were placed at 4°C by the comet assay lysis buffer (2.5 m NaCl, 100 mm EDTA‐Na2, 10 mm Tris, 10% DMSO, 1% Triton X‐100, pH 10.0). After treatment with lysis buffer, the slides were kept in electrophoresis buffer (300 mm NaOH, 1 mm EDTA‐Na2 pH 8.0). Electrophoresis was performed for 30 min (25 V, 300 mA), after which the slides were stained with SYBR Green I solution. The images were taken using Cytation 3 (BioTek, Winooski, VT). A total of 100 cells (50 cells from each of the two replicate slides) were selected and analyzed using Comet Score software.
8‐OHdG contents assay
DNA was extracted from cells treated with the abovementioned method using a TIANamp Genomic DNA kit, and 8‐OHdG was measured using an ELISA kit 20.
Western blot analysis
The cells and culture medium of each group were collected. The protein samples of the irradiated cells (incubated for 24 h or 3 h) were lysed in lysis buffer (20 mm Tris–HCl [pH 7.4], 2 mm EDTA, 0.5 mm PMSF, 20 μm NaCl and 20 μm CHAPS) and then centrifuged at 15 294 g for 20 min. The supernatant was collected, and the protein concentration was determined using the BCA Protein Assay Kit. Proteins (20 μg) of each sample were electrophoresed on a 12% SDS‐PAGE gel and transferred onto a PVDF membrane. The membranes were incubated with monoclonal antibodies (anti‐p53, anti‐p21, anti‐p16, anti‐JNK/p‐JNK, anti‐p38/p‐p38, anti‐ERK/p‐ERK and anti‐β‐actin antibodies) overnight at 4°C. After incubation with secondary antibodies, the proteins were detected using enhanced reagents. The culture medium was directly electrophoresed on a 12% SDS‐PAGE gel. The membranes were incubated with polyclonal antibodies (anti‐CoL‐I and anti‐MMP‐1 antibodies) and detected using enhanced reagents.
Immunofluorescence staining
Immunofluorescence staining was conducted to evaluate the effects of GP on AP‐1 protein phosphorylation levels induced by UVA 21. The cells were treated with the above mentioned in a 96‐well plate and fixed in 2% paraformaldehyde for 20 min at room temperature. After washing with PBS, the plates were incubated with 0.2% Triton X‐100 at 4°C for 10 min. Then, the plates were again washed with PBS and subsequently incubated with 5% BSA at 4°C for 1 h, and with anti‐p‐c‐Jun and anti‐p‐c‐Fos antibodies at 37°C for 1 h. The nuclei were stained with DAPI. Image capture and intensity analysis were analyzed using Cytation 3 (BioTek).
Statistical analysis
Data are expressed as means ± standard deviations (SDs). The Student's t‐test was used for statistical analysis, and P < 0.05 was considered statistically significant.
Results
Purification and characterization of GP
The crude protein extract from ginseng roots was purified by DEAE Sepharose Fast Flow. As shown in Fig. 1A, peak 1 was the fraction of highest protein content purified from the ginseng protein extract. Therefore, further purification of peak 1 was conducted using the Sephadex G‐75 gel filtration column. As shown in Fig. 1B, peak 2 was the target protein for purification. The protein purity of GP was 98.21% ± 1.20%, as determined by a BCA Protein Assay kit. GP was characterized by SDS‐PAGE, which showed two protein bands of molecular mass 27 and 13 kDa (Fig. 1C), indicating that GP was composed of proteins of molecular weights 27 and 13 kDa. We found that 27 kDa protein had the molecular mass of 27.3 kDa (63.03% coverage rate) to Ribonuclease‐like storage protein with 19 peptides matching and 13 kDa protein had the molecular mass of 16.4 kDa (49.35% coverage rate) to Ribonuclease 1 with 6 peptides matching and the molecular mass of 16.5 kDa (40.52% coverage rate) to Ribonuclease 2 with 6 peptides matching.
GP alleviates the inhibitory effect on the cell viability caused by UVA
We used different concentrations of GP to treat with NIH‐3T3 cells irradiated with UVA and detected the changes in cell viability with the MTT assay. As shown in Fig. 2A, when irradiated by UVA (10 J cm−2), the cell activity significantly decreased to 48.82% ± 0.80% of the control group. However, it significantly increased after pretreatment with GP; a concentration 20 μg mL−1 GP recovered the activity to 86.53% ± 6.60% of the control group, which was 1.77‐fold higher than that in the UVA group. These results showed that GP could reverse the decreased viability of NIH‐3T3 cells caused by UVA. To investigate whether the protein components in GP played a major role in this effect, we used two methods of thermal inactivation and enzymatic hydrolysis to inactivate the proteins of GP and used the MTT assay to determine their effects on the viability of NIH‐3T3 cells. When UVA irradiation caused a decline in cell viability, the cell viability of the heat‐inactivated GPH sample group was lower than that of the control group, but the difference was not statistically significant compared with the UVA group, and the cell activity of the protease‐degraded GPS group did not increase but decreased compared with the UVA group (Fig. 2B). Thus, inactivation of the protein components of GP did not promote the cell viability of UVA‐treated fibroblasts, suggesting that the protein components of GP played a role in alleviating the inhibitory effects on cell viability induced by UVA.
GP inhibits cell cycle arrest induced by UVA
The cell proliferation of fibroblasts can be inhibited via irradiated by UVA. The percentage of cells irradiated by UVA (10J cm−2) in the S stage was increased by 1.30‐fold and decreased by 0.76‐fold and 0.34‐fold in cells pretreated with 10 and 20 μg mL−1 GP, respectively (Fig. 2C). These data showed that GP effectively inhibited the S‐phase arrest of fibroblasts caused by UVA to promote their proliferation.
GP restores the reduced contractile capacity of cells caused by UVA
UVA causes photoaging of fibroblasts to decrease the contraction ability of cells. Therefore, we used a gel to detect the effects of GP on the contraction of cells irradiated with UVA. As shown in Fig. 3A, the gel area of cells irradiated with UVA was increased by 1.50‐fold and decreased by 0.49‐fold in the UVA group pretreated with 20 μg mL−1 GP. Those data demonstrated that GP effectively inhibited the decrease of contractile ability of fibroblasts induced by UVA.
GP inhibits the decreased protein expression of CoL‐I induced by UVA
The decreased contractile capacity of cells irradiated with UVA is mainly due to the degradation of intracellular functional proteins, the most important of which is the degradation of CoL‐I 6. Therefore, we used Western blotting and ELISA to detect the expression and secretion of CoL‐I in cells. The expression of CoL‐I both secreted by fibroblasts and expressed in cells was decreased by UVA irradiation, and increased upon pretreatment with GP (Fig. 3B, C), suggesting that GP can reverse UVA‐mediated inhibition of CoL‐I expression and secretion in fibroblasts. There was no difference in CoL‐I content in the cell extract and medium of GPH and GPS inactivation samples compared with the UVA group (Fig. 3D, E), showing that the primary components of GP increased the expression of CoL‐I is still protein components.
GP reduces the UVA‐induced increase in MMP expression and MMP‐2 and MMP‐9 activities
The degradation of collagen caused by UVA is mainly due to its ability to induce the secretion of MMPs from fibroblasts. The secretion of MMP‐1, MMP‐2 and MMP‐9 from NIH‐3T3 cells was increased by 1.30‐fold, 1.61‐fold and 1.27‐fold with UVA, and was decreased by 0.61‐fold, 0.65‐fold and 0.80‐fold with treatment of 20 μg mL−1 GP (Figs. 3B and 4B). The expression of MMP‐1, MMP‐2 and MMP‐9 in NIH‐3T3 cells was increased by 2.69‐fold, 1.37‐fold and 1.54‐fold by UVA, and was decreased by 0.42‐fold, 0.77‐fold and 0.63‐fold by 20 μg mL−1 GP (Figs. 3C and 4C). These data indicated that GP reduced the UVA‐induced increase of MMP‐1, MMP‐2 and MMP‐9 expression and secretion from cells. Next, we used gelatin zymography to analyze the effects of GP on the enzyme activities of MMP‐2 and MMP‐9. The enzyme activities of MMP‐2 and MMP‐9 were significantly increased after UVA induction, and GP treatment inhibited this effect (Fig. 4A). These results showed that GP effectively reduced the increased expression and enzyme activity of MMPs induced by UVA to inhibit CoL‐I degradation.
GP effectively reduces the UVA‐induced ROS increase in cells
ROS is an important second messenger of UVA‐induced photoaging injury of fibroblasts, which can increase the expression of MMP family members, promote collagen degradation, inhibit cell proliferation and decrease contractile ability 22. Therefore, we used flow cytometry to detect the content changes. After 3 h of UVA treatment, the intracellular ROS content increased by 0.91‐fold compared with the control group, and the content of ROS was reduced by 0.43‐fold in the UVA group treated with 20 μg mL−1 GP (Fig. 5A). These data indicated that GP effectively inhibited the increase of ROS content in NIH‐3T3 cells induced by UVA.
GP effectively reduces DNA damage in cells caused by UVA
ROS induced by UVA can cause DNA damage in NIH‐3T3 cells, so we used the comet assay to detect the repair effects of GP on DNA damage caused by UVA. The percentage of DNA in the tail is proportional to the frequency of DNA strand‐breaks, and the results can be expressed by dividing cells into different damage categories according to the amount of DNA in the tail 23. The cells irradiated by UVA had a significantly higher percentage tail DNA content compared with the control group, whereas 20 μg mL−1 GP reduced the percentage of DNA in the tail (Fig. 5B). These data indicated that GP effectively reduced the DNA damage in NIH‐3T3 cells induced by UVA.
GP effectively reduces the UVA‐induced increase of 8‐OHdG content
8‐OHdG is mainly caused by the formation of purines in DNA oxidized by ROS, which marks the oxidative DNA damage of cells. UVA irradiation increased the content of 8‐OHdG, whereas adding 20 μg mL−1 GP reduced the content of 8‐OHdG by 0.61‐fold (Fig. 5C). These data indicated that GP effectively inhibited the increase of 8‐OHdG content in NIH‐3T3 cells induced by UVA.
GP improves the expression of cell cycle regulatory proteins in NIH‐3T3 cells treated with UVA
DNA damage caused by UVA can increase the expression of p53 protein and its downstream effectors p21 and p16, which plays a role in regulating the cell cycle 4. The expression of p53, p21 and p16 protein was increased upon irradiation with UVA and was reduced by treatment with 20 μg mL−1 GP by 0.86‐, 0.64‐ and 0.33‐fold that of the UVA group (Fig. 5D). These results showed that GP effectively reduced expression of cell cycle regulatory proteins induced by UVA in NIH‐3T3 cells.
GP inhibits the phosphorylation levels of ERK, p38 and JNK caused by UVA irradiation
The MAPK family is an important downstream signaling pathway of ROS that mainly regulates downstream signaling factors through the phosphorylation of ERK, p38 and JNK signaling factors 7, 8. As shown in Fig. 6A, the expression levels of p‐ERK, p‐p38 and p‐JNK proteins were increased by UVA in NIH‐3T3 cells, whereas GP inhibited these levels in a dose‐dependent manner. When the phosphatase inhibitors of ERK, p38 and JNK were separately added, both the expression and secretion of CoL‐I increased compared with the UVA group, whose effects were similar to those GP (Fig. 6B). These data showed that similar to the phosphatase inhibitors of MAPK proteins, GP may play a role in increasing the synthesis of CoL‐I by inhibiting the phosphorylation of proteins in the MAPK signaling pathway.
GP inhibits the phosphorylation of AP‐1 (c‐Fos and c‐Jun) in cells exposed to UVA
AP‐1 is an important downstream transcription regulator of MAPK, and the phosphorylation of MAPK leads to the increased activity of AP‐1. AP‐1 is composed of c‐Fos and c‐Jun. When c‐Fos and c‐Jun are phosphorylated, AP‐1 is activated to induce transcription of important photoaging effect factors such as MMPs 7, 8. In this study, after UVA induction, the expression levels of p‐c‐Fos and p‐c‐Jun in cells were significantly increased, whereas GP significantly inhibited their phosphorylation (Fig. 6C). Thus, GP effectively reduced the UVA‐mediated increase in AP‐1 phosphorylation level.
Discussion
The purpose of this study was to isolate GP with protective effects on UVA‐induced photoaging of NIH‐3T3 fibroblasts. In this study, we successively used DEAE Sepharose Fast Flow and Sephadex G‐75 to purify proteins from ginseng roots with resistance to UVA‐induced photoaging, with molecular weights of 27 and 13 kDa. We found that 27 kDa protein was Ribonuclease‐like storage protein (63.03% coverage rate) with 19 peptides matching. It is a storage protein providing a nitrogen source in ginseng and has no RNase activity 24. And 13 kDa protein was Ribonuclease 1 (49.35% coverage rate) with 6 peptides matching or Ribonuclease 2 (40.52% coverage rate) with 6 peptides matching. The two protein sequences have the high homology, and the amino acid sequences have the homology of 26% 25. A number of Ribonucleases (RNases) have been extracted from ginseng with antihuman immune‐deficiency virus, antifungal, ribonuclease activities 26-28. Here, we found that GP, contained Ribonuclease‐like storage protein and Ribonuclease 1/2, promoted the cell cycle to increase cell vitality and cell gel contraction ability, reduced the expression, secretion and enzyme activities of MMPs and increased the CoL‐I expression and secretion, leading to antiphotoaging effects induced by UVA in NIH‐3T3 cells. The inactivated samples of GP did not increase the decreased cell viability or CoL‐I expression and secretion from NIH‐3T3 cells irradiated with UVA. This is the first study to extract protein components from ginseng roots with antiphotoaging effects.
UVA together with the short‐wave ultraviolet ray UVB (290–320 nm) constitutes UVR 29. Compared with UVA, although UVB only accounts for about 5% of UVR, its intensity is high, so it is thought to be the main cause of UV damage 2. Studies on the antiphotoaging effects of ginseng have mainly focused on the protective effects of ginsenoside against UVB‐induced skin photoaging. It has been shown that extracts of red ginseng 30, total saponins of red ginseng 31 and a variety of ginsenosides such as Rb1 9, Rb2 32, Rg1 9, 20(S)‐Rg3 33, Rb0 34, CK 35 and F2 36 have good protective effects on the photoaging of fibroblasts induced by UVB. In recent years, an increasing number of studies have shown that although UVA has low energy, its penetration is strong, 20–30% of which can penetrate the dermis and cause functional damage to dermis fibroblasts that leads to photoaging 22. Rg3 (S) was found to reduce dermal thickening in a 3D skin model irradiated with UVA 10. In this study, we found that GP had protective effects on the UVA‐induced photoaging of NIH‐3T3 fibroblasts. This is of great significance for elucidating the mechanisms underlying the basis and efficacy of ginseng on antiaging induced by UVA.
The aging of fibroblasts caused by UVA irradiation is mainly dependent on excessive ROS production, which results in DNA damage in fibroblasts 1. DNA damage caused by UVA mainly includes the formation of pyrimidine dimers, single chain and double chain breakage. ROS can also oxidize purines in DNA to form 8‐OHdG, a marker of oxidative DNA damage 37. DNA damage can activate the expression of p53 protein and increase expression of the downstream effector p21 protein, a cyclin‐dependent protein kinase inhibitor, to inhibit cell proliferation and activity by cell cycle stasis 5. In this study, we found that GP significantly reduced the increase of ROS content in NIH‐3T3 cells irradiated with UVA, reduced cell DNA damage and the production of 8‐OHdG, and reduced the expression of p53 and its downstream p21 protein to reduce the effects of S‐phase arrest, promote cell proliferation and enhance cell vitality. When the fibroblast was damaged by some factors, such as UVR, the change in cell shape occurred during collagen contraction 38. Nakyai et al. 3 reported that fibroblasts irradiated with 5 J cm−2 UVA demonstrated greater contractility and repeated UVA (5 J cm−2 × 3 times) exposure caused an increase in the gel area. Our results showed that 10 J cm−2 UVA can cause an increase in gel area as when the single irradiation intensity increased to 10 J cm−2, the contractile capacity of fibroblasts in the 3D collagen matrix was decreased; treatment with GP reversed this effect. Thus, the results of this study provide insights into the mechanisms by which GP enhances the viability of fibroblasts treated with UVA.
UVA irradiation can induce the excessive production of ROS in fibroblasts, which activates the MMP family, and degrades the collagen of dermal cells and other photoaging phenomena 7. In the human body, the skin collagen type is mainly CoL‐I. MMP‐1 is the main degradation product of CoL‐I collagenase secreted by fibroblasts, and the two gelatinases MMP‐2 and MMP‐9 can further degrade the fragments hydrolyzed by MMP‐1 to cause collagen damage 8. Based on the different modes of action of the two enzyme types, the expression of MMP‐1 protein and activities of MMP‐2 and MMP‐9 were used to evaluated 39, 40. Excessive ROS production induced by UVA activates the phosphorylation of ERK, p38 and JNK of the MAPK protein family, and those proteins further activated the downstream transcription regulator AP‐1 and phosphorylated its c‐Fos and c‐Jun subunits to cause the protein expression of the MMP family 41. In this study, we found that GP reduced ROS content in NIH‐3T3 cells treated by UVA; and reduced the phosphorylation of ERK, p38 and JNK protein and phosphorylation of c‐Fos and c‐Jun protein. It also reduced MMP‐1, MMP‐2 and MMP‐9 expression and the secretion and gelatin enzyme activity of MMP‐2 and MMP‐9 to improve the expression and secretion of CoL‐I. The study of the functional mechanism of GP conducive to elucidating the regulatory mechanisms underlying GP‐enhanced collagen expression and secretion in fibroblasts treated with UVA.
In conclusion, our study showed that GP can prevent UVA‐induced antifibroblast photoaging and may improve fibroblast proliferation, contraction and other functions by inhibiting DNA damage and collagen degradation caused by ROS.
Acknowledgements
This work was supported by the National Key Research and Development Program of China (No. 2017YFC1702100), Science and Technology Development Plan of Jilin Province, China (Nos. 20180201075YY and 20180101128JC), Science and Technology Project of Jilin Province, China (No. JJKH20180378KJ), Science and Technology Development Plan of Jilin City, China (No. 201731200), and Science and Technology Development Plan of Changchun City (No. 18YJ013). We would like to thank LetPub (www.letpub.com) for providing linguistic assistance during the preparation of this manuscript.
References
Citing Literature
Number of times cited according to CrossRef: 2
- Zhidong Zhang, Zhongyao Wang, Cuizhu Wang, Analysis of Proteins in Ginseng, Ginseng Nutritional Components and Functional Factors, 10.1007/978-981-15-4688-4, (29-35), (2020).
- Yueh-Hsiung Kuo, Hung-Lung Chiang, Po-Yuan Wu, Yin Chu, Qiao-Xin Chang, Kuo-Ching Wen, Chien-Yih Lin, Hsiu-Mei Chiang, Protection against Ultraviolet A-Induced Skin Apoptosis and Carcinogenesis through the Oxidative Stress Reduction Effects of N-(4-bromophenethyl) Caffeamide, A Propolis Derivative, Antioxidants, 10.3390/antiox9040335, 9, 4, (335), (2020).
