https://orcid.org/0000-0001-5115-5588
Figures
Abstract
Cordyceps cicadae (Miq.) is an edible fungus with unique and valuable medicinal properties that is commonly used in traditional Chinese medicine, but its anti-aging effects on the skin fibroblast are not well studied. The aim of the present study was to analyze the active components of aqueous C. cicadae extract (CCE), determine the effects of CCE on hyaluronan synthesis in human skin fibroblasts, and explore the underlying mechanisms. The results of this study indicate that CCE was rich in polysaccharides, five alditols (mainly mannitol), eight nucleosides, protein, and polyphenols, which were present at concentrations of 62.7, 110, 8.26, 35.7, and 3.8 mg/g, respectively. The concentration of extract required to inhibit 50% of 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulphonic acid) (ABTS) and 2,2-diphenyl-1-picrylhydrazil (DPPH) radical scavenging capacities were 0.36 ± 0.03 and 4.54 ± 0.10 mg/mL, respectively, indicating that CCE exhibits excellent antioxidant activities. CCE showed no cytotoxicity to skin fibroblasts at concentrations ≤ 100 μg/mL, and promoted HA synthesis in fibroblasts. Treatment of fibroblast cells with 100 μg/mL CCE enhances the HA content to 1293 ± 142 ng/mL, which is significantly more than that in the non-treatment (NT) group (p = 0.0067). Further, RNA sequencing detected 1,192 differentially expressed genes (DEGs) in CCE-treated fibroblasts, among which 417 were upregulated and 775 were downregulated. Kyoto Encyclopedia of Genes (KEGG) and Genomes pathway (GO) analysis based on RNA sequencing revealed that CCE mainly affected cytokine-cytokine receptor interaction regulated by HA synthesis-related genes. CCE upregulated HA synthase 2 (HAS2), epidermal growth factor (EGF)-related genes, heparin-binding EGF-like growth factor, C-C motif chemokine ligand 2, interleukin 1 receptor-associated kinase 2, and other genes related to fibroblast differentiation and proliferation. CCE downregulated the gene of matrix metallopeptidase 12 (MMP12), which leads to cell matrix loss. RT-qPCR further verified CCE significantly upregulated HAS2 expression and significantly downregulated MMP12 expression, thus promoting hyaluronan synthesis. CCE shows potential as a moisturizer and anti-aging agent in functional foods and cosmetics.
Citation: Shao L, Jiang S, Li Y, Yu L, Liu H, Ma L, et al. (2023) Aqueous extract of Cordyceps cicadae (Miq.) promotes hyaluronan synthesis in human skin fibroblasts: A potential moisturizing and anti-aging ingredient. PLoS ONE 18(7): e0274479. https://doi.org/10.1371/journal.pone.0274479
Editor: Manoj B. Menon, Indian Institute of Technology Delhi, INDIA
Received: January 3, 2022; Accepted: August 29, 2022; Published: July 7, 2023
Copyright: © 2023 Shao et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Data Availability: All relevant data are within the paper.
Funding: This work was funded by Shanghai Collaborative Innovation Center of Fragrance, Flavor and Cosmetics (1021ZK202002008). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Abbreviations: ABTS, 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulphonic acid); AGE-RAGE, receptor for advanced glycation end products; BSA, bovine serum albumin; CCE, Cordyceps cicadae (Miq) extract; COG, clusters of orthologous groups; DEGs, differentially expressed genes; DMEM, Dulbecco’s modified eagle medium; DPBS, Dulbecco’s phosphate-buffered saline; DPPH, 2,2-diphenyl-1-picrylhydrazil; ECM, extracellular matrix; ERK/P-ERK, extracellular regulated protein kinases/phosphor- extracellular regulated protein kinases; FRAP, ferric reducing antioxidant power; GO, Gene Ontology; HA, hyaluronan; HAS2, HA synthase 2; HPAEC, high-performance anion-exchange chromatography; HPLC, high-performance liquid chromatography; JNK/PJNK, c-Jun N-terminal kinase/phosphor- c-Jun N-terminal kinase; KEGG, Kyoto Encyclopedia of Genes and Genomes; MMP, matrix metallopeptidase; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide; NR, non-redundant protein sequence database; NT group, non-treatment groupA; Pfam, protein families database; PI3K-Akt, phosphoinositide 3-kinase; RNA-seq, RNA sequencing; RT-qPCR, real-time quantitative polymerase chain reaction; Swiss-prot, Swiss-Prot protein sequence database; TCM, traditional Chinese medicine; TNF, tumor necrosis factor; TPTZ, 2, 4, 6-tripyridyl-s-triazine
1. Introduction
Cordyceps cicadae (Miq.) is a medicinal fungus that parasitizes Hemiptera cicadae larvae [1]. C. cicadae is a valued ingredient in Traditional Chinese Medicine (TCM) that has been used for approximately 1,600 years [2]. Active components, such as polysaccharides, nucleosides, sterols, cyclic dipeptides, sugars, fatty acids, amino acids, aromatic compounds, and other small organic compounds, have been isolated from C. cicadae [3]. Many pharmacological studies have demonstrated that C. cicadae exhibits biological functions such as growth reduction of human hepatocellular carcinoma cells, prevention of cell senescence, bacteriostasis [2, 4, 5] antioxidant activities [2, 4, 5], prevention of liver damage, anti-fatigue and anti-aging [3, 6, 7].
The skin, which is the largest organ in the mammalian ectoderm, ages with time and external factors [8]. Skin aging is characterized by skin water loss, sagging, increased wrinkles, and decreased elasticity. Histologically, the extracellular matrix (ECM) loses flexibility due to the breakdown of skin collagen and elastic fibers and a reduction in hyaluronan (HA) content [4, 9]. HA is a disaccharide unit glycosaminoglycan composed of D-glucuronic acid and N-acetylglucosamine. It is a natural component in the skin that helps absorb moisture from the body and the skin surface and is particularly prominent in rapidly growing and repairing tissues [10]. In addition, HA is involved in wound healing, ECM tissue and joint lubrication, regulation of cell adhesion and movement through receptors that interact with the cytoskeleton, angiogenesis by mediating cell proliferation, cell differentiation, and cell migration and has immunomodulatory, anti-tumor, and anti-proliferative properties [11–13].
Natural products are rich in biological ingredients known for their therapeutic properties. Recent research on the antioxidant, anti-inflammatory, and antibacterial activities of Cordyceps fungus extracts suggests that they have great potential for biomedical and cosmetic applications [14]. C. cicadae belongs to the Cordycipitaceae family, which is known for its therapeutic and health benefits. However, research on the potential skin care applications of C. cicadae is limited. In this study, we obtained C. cicadae extract (CCE) by hot water extraction, analyzed its active components, and evaluated its antioxidant properties and effects on HA synthesis in human skin fibroblasts. Further, we determined the effects of CCE on human fibroblasts using transcriptome analysis and verified the expression of HA-related genes using RT-qPCR. This study will further our knowledge of C. cicadae, its effects on human fibroblasts, and the mechanisms involved in the promotion of HA synthesis.
2. Materials and methods
2.1 Material
C. cicadae samples were purchased from Jingfu Traditional Chinese Medicine Decoction Pieces Co., LTD, Bozhou City, batch number 210901. The product had the characteristics of C. cicadae and met the Shanghai processing specifications of TCM decoction pieces, inspection number JC210901.
2.2 Chemicals
1,1-diphenyl-2-picrylhydrazyl (DPPH) was purchased from Sigma-Aldrich; 2,2-azino-bis (3-ethylbenzothiazo-line-6-sulphonic acid) diammonium salt (ABTS), Trolox, and the ferric reducing antioxidant power (FRAP) assay kit were purchased from the Beyotime Institute of Biotechnology (S0119 and S0116, Nantong, China). Dulbecco’s modified eagle medium (DMEM), amphotericin B, penicillin, bovine serum albumin (BSA), and Dulbecco’s phosphate-buffered saline (DPBS) were purchased from Thermo Fisher Scientific Inc. (China); streptomycin was purchased from Guangzhou Huayueruike Scientific Equipment Co. (China); Hyaluronan Quantikine ELISA Kit was purchased from R&D Systems (DHYAL0). All other reagents and solvents were of analytical grade.
2.3 C. cicadae extraction
C. cicadae fruiting bodies were separated, washed with distilled water, and dried at 60°C until reaching a constant weight. Dried fruiting bodies were ground into powder and passed through an 80 mesh sieve. Ten grams of C. cicadae powder was dissolved in 200 mL water and heated at 85°C for 2.5 h. The extraction solution was separated by centrifugation at 8,500 × g for 15 min and lyophilized to obtain CCE for quantitative assessment and analysis.
2.4 Determination of active components
2.4.1 Polysaccharide extraction and assessment.
Crude polysaccharides were extracted following the standard method with some modifications [2]. Ten grams of CCE powder was dissolved in 20 mL water, and three times the volume of alcohol was added to the extract solution. The mixture was incubated at 4°C for 12 h, and then the precipitate was separated by centrifugation at 8,000 × g for 15 min. The crude polysaccharides precipitate was washed with deionized water and lyophilized for quantitative assessment via the phenol-sulfuric acid method [15]. The linear relationship between the polysaccharide content and the absorbance at 490 nm was determined. Glucose solution was used as the standard.
2.4.2 Monosaccharide composition and molecular weight analysis.
The lyophilized polysaccharide sample was weighed, and 2–5 mg were added to a hydrolysis flask with 3 mL of trifluoroacetic acid (4 mol/L). The mixture was hydrolyzed at 110°C for 3 h. After hydrolysis, 3 mL of methanol was added, and nitrogen was used to blow dry. This was repeated three times, and distilled water was used to dissolve the volume to 50–100 mL. The solution was filtered through a 0.45-μm membrane before determination. The filtrate was assessed using high performance anion exchange chromatography (HPAEC) according to the method described by [16]. The chromatography column was a CarboPac PA20 column (3 mm × 150 mm, Dionex, USA) with pulsed amperometric detection. The mobile phase was ultrapure water (A), 0.25-mol/L NaOH (B), and 1-mol/L NaAc (C), with the following elution gradient: 0–30 min: 99.2% A and 0.8% B; 30–40 min: 79.2% A, 0.8% B, and 20% C; 40–45 min: 20% A and 80% B; 45–60 min: 99.2% A and 0.8% B. The flow rate was 0.5 mL/min and the injection volume was 20 μL. Fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, galacturonic acid, and galacturonic acid were mixed as a standard.
The molecular weight of polysaccharides in CCE was determined according to previously reported methods with modifications [17]. A TSK-GEL G6000 PWXL chromatographic column (7.8 mm × 300 mm, Tosoh Corp., Tokyo, Japan) with an injection volume of 20 μL was used for the analysis. Ultrapure water elution was conducted at a flow rate of 0.6 mL/min at a column temperature of 30°C.
2.4.3 Protein quantification.
Protein content was determined using a Bradford assay [18]. The protein sample (1 mL) was combined with 5 mL of Coomassie Brilliant Blue G-250 solution, mixed, and incubated at room temperature for 20 min. BSA concentrations (0, 0.025, 0.05, 0.1, 0.15, 0.2, and 0.25 μg/mL) were prepared respectively for standards. The absorbance was measured at 595 nm, and the results were reported as milligrams of BSA equivalents per milligram of extract.
2.4.4 Alditols analysis.
Alditols contents were determined by HPAEC. CCE powder (0.025 g) was dissolved in 100 mL distilled water and the solution was filtered through a 0.45-μm microporous membrane before ion chromatography determination using a CarboPac MA1 anion exchange column (4 mm × 250 mm, Dionex, USA). The injection volume was 25 μL, the mobile phase was 48-mmol/L NaOH, the flow rate was 0.4 mL/min, and the column temperature was 30°C.
2.4.5 Nucleoside analysis.
CCE nucleosides were analyzed by high-performance liquid chromatography (HPLC). CCE powder (0.5 g) was dissolved with 50 mL distilled water, and the solution was filtered through a 0.45-μm aqueous phase filter membrane for liquid chromatography analysis. The chromatography column was a Venusil MP C18 column (5 μm, 4.6 mm × 250 mm, Agela Technologies of America). The mobile phase was composed of ultrapure water (A) and methanol (B) with a gradient elution as follows: 0–5 min: 100% A; 5–10 min A: 95 A; 10–30 min: 70% A; 30–40 min: 95% A; 40–45 min: 100% A. The injection volume was 10 μL, the flow rate was 1.0 mL/min, and the column temperature was 40°C. The UV detection wavelength was set at 254 nm.
2.4.6 Total phenolic content analysis.
The total phenolic content in CCE was determined using a Folin-Ciocalteu assay as described by Bursal et al. [19]. CCE dissolved to 1 mg/mL (1 mL) was added to a test tube with 1 mL of Folin-Ciocalteu reagent and 2 mL of 2% Na2CO3. The reagents were vortexed and incubated at 25°C for 40 min. Absorbance was measured at 765 nm. The results were reported as milligrams of gallic acid equivalents per milligram of extract.
2.5 Antioxidant activity assays
2.5.1 ABTS radical scavenging activity assay.
ABTS radical scavenging activity was assayed according to instructions from the Beyotime Institute of Biotechnology. ABTS solution was prepared by mixing the stock solution and oxidant solution in equal volumes and allowing them to react at room temperature in the dark for 16 h. A 10-μL aliquot of CCE solution was added to 200 μL of fresh ABTS solution. The solution was then diluted with 5-mM phosphate-buffered saline (pH 7.4) to obtain an absorbance of 0.7 ± 0.05 at 734 nm. The absorbance was measured at 734 nm after 6 min at room temperature. Ascorbic acid was used as a reference; the ABTS radical scavenging activity was calculated using Eq 1.
(1)
where At is the absorbance of the sample, B is the absorbance of the sample and distilled water, and A0 is the absorbance of the blank control.
2.5.2 DPPH radical scavenging activity assay.
The free radical scavenging activity of CCE was analyzed by a 2, 2-diphenyl-1-picrylhydrazil (DPPH) assay as described by Sharma et al. [15]. First, the CCE solution (0.5, 1, 2, 3 and 4 mg/mL) was prepared by diluting CCE in distilled water and add 0.4 mL solution in the test tubes respectively. Finally, 3.6 mL of DPPH solution (0.1 mM) was transferred to the test tubes, which were gently stirred and incubated in the dark at 25°C for 30 min. The absorbance was read at 517 nm using a spectrophotometer, and the scavenging activity (%) of each concentration was calculated in triplicate (Eq 2). Distilled water was used as the blank control.
(2)
where A0 is the absorbance of the blank control, As is the absorbance of the sample, and Ac is the absorbance of the sample and ethanol mixture.
2.5.3 FRAP assay.
The ferric reducing ability of CCE was determined by FRAP assay as per instructions from the Beyotime Institute of Biotechnology. Stock solutions were the detective buffer, 2, 4, 6-tripyridyl-s-triazine (TPTZ) solution, TPTZ dilution, 0.5 mL of 10-mM FeSO4 solution, and 0.1 mL of 10-mM Trolox solution. A working solution was prepared by mixing TPTZ dilution, detective buffer, and TPTZ solution in a ratio of 10:1:1 (v/v), respectively. The working solution was warmed to 37°C before use. CCE samples (5 μL) with concentrations ranging from 0.3 to 1.5 mM were mixed with 180 μL of FRAP working solution and incubated at 37°C for 5 min. The absorbance of the reaction mixture was measured at 593 nm. FRAP was expressed as FeSO4 concentrations, which were calculated using standard curves.
2.6 Effects of CCE on fibroblast HA synthesis
2.6.1 Cell culture.
Human skin fibroblasts, which were collected from the facial skin of a 45-year-old woman, were obtained from the Shanghai Engineering Research Center cell bank. The cell culture was prepared in regular DMEM. After resuscitation, fibroblasts were cultured in 175 cm2 flasks for 7 days, and the medium was refreshed every other day. The prepared CCE medium was added to each well containing fibroblasts and cultured at 37°C for 24 h. A 5% BSA blocking buffer, which was devolved in high glucose DMEM and DPBS, was used to block the cells to prevent false positives. Cells treated with regular DMEM served as the not treated (NT) group. For cell viability assays, the cells were cultured in 96-well plates at a concentration of 10,000 cells/well and cultured in 6-well plates at a concentration of 20,000 cells/well for immunofluorescence analysis of HA expression.
2.6.2 Cell viability assay.
Cell viability was determined using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay [20]. Serum-free medium was used to starve the cells for 12 h, and medium solutions with a CCE concentration gradient of 10 to 1,000 μg/mL CCE were added to the wells, followed by incubation at 37°C for 24 h. Three parallel tests were set for each concentration. The medium was removed after 24 h, and 0.5-g/L MTT solution was added to the wells, followed by incubation at 37°C for 3 h. Dimethylsulfoxide was added, and the absorbance was measured at 550 nm.
2.6.3 Determination of HA content.
CCE solutions were prepared to two specific concentrations (50, 100 μg/mL), with regular DMEM (high glucose DMEM with 10% bovine calf serum, penicillin, streptomycin, and amphotericin b) and passed through a 0.22-μm filter. Fibroblasts were starved in a 24-well plate using serum-free medium for 16 h. Subsequently, cells were further treated with CCE (50, 100 μg/mL) at 37°C for 48 h. Cell supernatants were collected and assayed for HA content according to the guidelines of the ELISA kit (R&D systems, Cat. Number: DHYAL0) [21].
2.6.4 Immunofluorescence analysis of HA expression.
Sheep anti-HA antibody was diluted with BSA (1:500) to prepare the antibody-BSA working solution. One milliliter of the antibody-BSA working solution was added to each well and incubated at 4°C for 24 h. The antibody-BSA working solution was removed from each well, and the wells were rinsed with 3 mL of DPBS twice. Goat anti-sheep antibody 488 and 4′,6-diamidino-2-phenylindole fluorescent stain were diluted in 5% BSA blocking buffer (1:500 and 1:1,000, respectively) to prepare the anti-sheep 488 working solution. One milliliter of the working solution was added to each well and incubated at 4°C for 1 h. Next, the antibody-BSA working solution was removed from each well, and the wells were rinsed with 3 mL DPBS twice. Three pictures of each well were taken at random using an A1 fluorescence microscope (Zeiss Group, Germany).
2.7 Transcriptome sequencing
Total RNA was extracted from fibroblasts using the RNeasy mini kit (Qiagen, Hilden, Germany). The RNA sequencing (RNA-seq) samples were collected from the cell experiments described above. β-actin was used as the reference gene, and no template was used as the negative control. Sequencing files were constructed by Majorbio, sequenced using Illumina HiSeq2000, and annotated against NR, Swiss-Prot, Pfam, COG, GO, and KEGG functional databases.
2.8 Real-time quantitative PCR
Total RNA was extracted from the same batch of fibroblasts using TRIzol reagent (Gibco-BRL, Paisley, UK), and RNA concentration and purity were measured using a NanoDrop 2000 (Thermo Scientific of America). For the RT-qPCR analysis, 1 μg total RNA was extracted from cell samples and transformed into cDNA (HiScript Q RT SuperMix for RT-PCR [+gDNA wiper], Vazyme). Five genes were selected based on the RNA-seq results; the target gene and primer sequences for amplification and fluorescence detection are shown in Table 1. Β-actin was used as a control gene for all samples. Reaction cycles consisted of denaturation at 95°C for 5 min, followed by 40 cycles of 95°C for 5 s, 58°C for 30 s, and 72°C for 40 s. mRNA levels were quantified using Applied Biosystems sequence detection software and an ABI7500 fluorescence quantitative PCR instrument (LongGene Instruments, Hangzhou China).
2.9 Statistical analysis
All data are represented as mean ± standard deviation (X ± SD) unless otherwise indicated. Astra software (Version 7.1.2) was used to analyze the polysaccharide molecular weight data. The quantitative analysis of immunofluorescence-stained images was performed using Imagej1.53C (Wayne Rasband, USA) image processing software. RNA-seq data were analyzed using the Majorbio Cloud Platform (www.majorbio.com), and relative changes in target gene expression from PCR were analyzed by the −ΔΔCT method [22].
3. Results and discussion
3.1 CCE extraction
CCE was obtained from C. cicadae by hot water extraction, centrifugation, and lyophilization, and the extraction rate was 41.17%. The main active components of CCE were mannitol (10.70%), crude polysaccharides (6.27%), protein (3.57%), and polyphenols (0.38%). Nucleosides and alditols were also detected (data shown in 3.1.3 and 3.1.4), which were indicators of actives that are often of interest to researchers in C. cicadae studies [7, 23].
3.1.1 CCE polysaccharide composition and average molecular weight.
The average molecular weight and monosaccharide composition of CCE polysaccharides were analyzed. Chromatograms of the mixed standard and CCE are shown in Fig 1A and 1B, respectively. The monosaccharide components of the crude polysaccharides in CCE are galactose, glucose, and mannose (Fig 1B). Glucose was the predominant monosaccharide, and the molar ratio of galactose, glucose, and mannose was 6:22:13, which is consistent with that reported in relevant studies [15, 17, 24]. Previous research has demonstrated that polysaccharides are among the most abundant and bioactive components extracted from C. cicadae fruiting bodies, cultured mycelium, and fermentation broth [25]. The crude polysaccharides in CCE were composed of three fractions (Table 2), peak 1 (20.27%), peak 2 (18.64%), and peak 3 (61.09%), with average molecular weights of 2.217 × 107, 3.752 × 104 and 2.316 × 103 Da, respectively.
(A) Monosaccharide composition of standard sample; peaks 1–10: fucose, rhamnose, arabinose, galactose, glucose, xylose, mannose, fructose, glucuronic acid, galacturonic acid. (B) Monosaccharide composition of C. cicadae extract crude polysaccharides; peaks 1–3: galactose, glucose, mannose.
3.1.2 CCE alditols.
CCE alditols contents were determined by HPAEC. As shown in Fig 2, five alditols components were detected: 65.25 mg/g trehalose, 107.00 mg/g mannitol, 29.09 mg/g glucose, 13.87 mg/g galactose, and 24.09 mg/g fructose. The mannitol content of CCE is significantly higher than that of Paecilomyces cicadae (53.6 mg/g) [26]. Mannitol content is used as an indicator of C. sinensis quality, and demand for this polyol group is increasing in food and medical fields due to favorable characteristics, such as low metabolism, no glycemic index, non-hygroscopic, anti-oxidative, and natural sweetening properties [27–29]. These attributes reduce consumers’ risk of developing diseases such as diabetes and obesity and can reduce elevated intracranial pressure. Moreover, mannitol in combination with HA can significantly reduce HA degradation [29]. These properties could make CCE an ideal skin care ingredient.
3.1.3 CCE nucleosides.
Nucleosides are one of the main active components of Cordyceps fungi [30] and are often used as markers to distinguish Cordyceps fungi from knockoffs. Many studies have shown that nucleosides can regulate physiological processes via purine and pyrimidine receptors. Exogenous nucleosides regulate immune function, participate in metabolism, have antibacterial and antiviral effects, and exert therapeutic effects on liver, cardiovascular, and nervous system diseases [31]. Zeng et al. [32] determined the nucleosides of C. cicadae from different regions, which included uracil, uridine, 2′-deoxyuridine, inosine, guanosine, adenine, thymidine, adenosine, 2’-deoxyadenosine, and cordycepin, with total nucleoside contents ranging from 3.69439 to 8.39772 mg/g [31]. HPLC chromatograms of CCE and mixed standards are shown in Fig 3. Eight nucleosides were detected in CCE, namely uridine, guanosine, adenosine, cytidine, hypoxanthine, inosine, uracil, and adenine, with respective contents of 1.8497, 1.2787, 1.1352, 0.2925, 0.2871, 0.1503, 0.1419, and 0.1237 mg/g. These results were consistent with other reports that uridine content was the highest among nucleosides in Cordyceps [32, 33].
3.2 CCE antioxidant activities
The antioxidant activities of CCE were estimated using ABTS (Fig 4A), DPPH (Fig 4B), and FRAP assays (Fig 4C). Using ascorbic acid as a control, the concentration of CCE required to inhibit 50% (IC50) of DPPH and ABTS free radical was 4.54 ± 0.10 and 0.36 ± 0.03 mg/mL, respectively. CCE exhibited concentration-dependent ABTS and DPPH scavenging activity; the antioxidant capacity increased with increasing CCE concentrations. Among the antioxidant results of CCE, the IC50 value of DPPH was significantly higher than that of ABTS, and CCE at this concentration is already cytotoxic, which may because the ethanol dissolution system of DPPH radical scavenging experiment, which affected the antioxidant effect of aqueous extract CCE. Similarly, the FRAP results showed that the ferric reducing capacity increased in a dose-dependent manner. These results indicate CCE has a strong free radical scavenging capacity.
(A) ABTS [2,2-azino-bis (3-ethylbenzothiazo-line-6-sulphonic acid)] radical scavenging; (B) DPPH (2,2-diphenyl-1-picrylhydrazil) radical scavenging assay; (C) ferric reducing antioxidant power assay.
3.3 Cell viability
The cytotoxicity of CCE was assessed using an MTT assay of cultured human skin fibroblasts treated with various concentrations of CCE. As shown in Fig 5, CCE enhanced cellular viability at concentrations of 5 (p = 6.04×10−5), 10 (p = 6.37×10−5), 50 (p = 1.34×10−7) and 100 μg/mL (p = 4.43×10−6), and cell viability values remained 94% at concentrations up to 500 μg/mL (p = 0.01) and possessed cytotoxicity at 1000 μg/mL (p = 2.45×10−7). CCE did not reduce fibroblast cell viability at concentrations ranging from 5 to 100 μg/mL and was slightly toxic to fibroblasts at concentrations above 500 μg/mL.
3.4 CCE promotes HA synthesis in human fibroblasts
Skin aging is characterized by uneven pigmentation, impaired skin elasticity and strength, and rough texture. Skin integrity is maintained via the ECM, in which HA plays an important role. Many studies have demonstrated the effects of natural extracts on skin [34]. The hyaluronic acid content in fibroblasts cells was determined using ELISA kits. As shown in Fig 6, treatment of fibroblast cells with 50 μg/mL and 100 μg/mL CCE enhances the HA content to 950 ± 52 and 1293 ± 142 ng/mL respectively, which is significantly more than that in the non-treatment (NT) group (p = 0.0067). This result demonstrated CCE could promote HA synthesis in skin fibroblasts. And the promotion of HA synthesis induced by CCE might be related to its flavonoid content [35].
* P < 0.05, ** P < 0.01 compared with the control group.
3.5 Immunofluorescence staining analysis of HA expression
Using immunofluorescence staining of fibroblasts treated with CCE to further reveal the effects on HA expression. Under the fluorescence microscope, the nucleus was blue, and HA was green. Compared to the NT group (Fig 7A), 50 μg/mL CCE (Fig 7B) and 100 μg/mL CCE (Fig 7C) increased HA expression, and the negative control (Fig 7D) showed that there were no false positives.
Fibroblasts were non-treated (A), treated with C. cicadae extract at concentrations of 50 μg/mL (B) and 100 μg/mL (C), treated without the antibody 488 as negative control (D).
3.6 Transcriptional analysis
To determine the effects of CCE on HA synthesis in fibroblasts, we quantified HA-related gene expression in cell cultures treated with 50 μg/mL CCE.
3.6.1 Transcriptional response.
In the CCE group, 1.48 × 108 reads were obtained by sequencing, and 1.47 × 108 clean reads were obtained by filtration, accounting for 99.18% of the total reads. In the NT group, 1.57 × 108 reads were obtained by sequencing, and 1.56 × 108 clean reads were obtained by filtration, accounting for 99.21% of the total reads. The percentage of clean reads obtained by each sample was greater than 99%, and the sequencing quality was high, meeting the information analysis requirements.
3.6.2 Differentially expressed gene (DEG) analysis.
The transcriptional regulation of CCE-treated (50 μg/mL) and NT fibroblasts was analyzed by RNA-seq. A total of 1192 significant DEGs (up or downregulated > 1.2) were detected between CCE-treated and NT fibroblasts, of which 417 genes were upregulated, and 775 genes were downregulated. DEGs were selected based on P-values generated by the Benjamini-Hochberg correction using a uniform threshold of P < 0.05.
The GO analysis detected 20 significantly enriched terms (Fig 8). GO terms are divided into biological processes, cell components, and molecular function categories. The most significantly enriched GO terms were involved in biological processes, namely, cellular processes, biological regulation, and metabolic processes.
According to the KEGG metabolic pathway analysis (Fig 9), DEGs were mostly enriched in pathways related to immune diseases and signal transduction. Total KEGG enrichment showed that viral protein interaction with cytokine and cytokine receptor, cytokine-cytokine receptor interaction, AGE-RAGE signaling pathway in diabetic complications, protein digestion and absorption, TNF signaling pathway, ECM-receptor interaction, and PI3K-Akt signaling pathway had a high significance and number distribution.
HA is an intercellular matrix component. Of the genes upregulated in the CCE-treated group compared with the NT group, nine of them were associated with HA metabolism, intercellular matrix metabolism, and fibroblast proliferation and differentiation (Table 3). The effects of CCE on skin fibroblasts may be attributed to the regulation of these genes.
The genes of interest were searched in GO and KEGG, and some of these genes (e.g., IL6 and CCL2) involved in cell proliferation and intercellular matrix assembly were related to immune regulation. The results of GO enrichment and KEGG enrichment analyses (Figs 8 and 9) also showed that most terms were related to inflammatory response and cytokine activity. Thus, CCE may play a role in immune regulation, which can be further explored in the future; however, the DEGs also have other known functions. The TNFAIP6, IL6, CCL2, HBEGF, IRAK2, and AREG genes are associated with the mitogen-activated protein kinase (MAPK) pathway, which transmits regulatory signals to the nucleus and is related to fibroblast proliferation and division. The MAPK pathway includes ERK/P-ERK, P38/P-P38, and JNK/P-JNK [36]. The extracellular-regulated protein kinase (ERK) pathway is mainly involved in cell proliferation, differentiation, migration, survival, and apoptosis [37]. The ERK pathway promotes HA synthesis when activated by appropriate external stimuli such as photodynamic therapy [36]. According to the GO analysis, the upregulation of CCL2 positively regulates the ERK1 and ERK2 cascade.
The upregulated gene hyaluronan synthase 2 (HAS2) encodes one of the isoenzymes that catalyze the addition of N-acetyl-glucosamine and glucuronic acid monosaccharides to the newly formed HA polymer [38]. HAS2 is the major HA synthase in dermal fibroblasts [39], and it specializes in the synthesis of high polymer mass HA. Matrix metalloproteinases (MMPs) are a family of zinc-dependent endopeptidases involved in physiological processes, such as cell proliferation and apoptosis, tissue remodeling, hematopoietic regulation, wound repair, and angiogenesis [40]. In terms of the effect of CCE on the ECM, MMP12 was downregulated in CCE-treated fibroblasts. Previous studies have shown that the ECM is continuously lost during externally induced skin aging, which is often associated with MMP upregulation [8]. Thus, CCE inhibited the gene associated with ECM catabolic processes.
Some studies have shown that polysaccharides derived from Lactobacillus plantarum downregulate MMPs to prevent skin aging and ECM loss, the molecular weights of those polysaccharides ranged from 221 to 79,967 Da [41]. The polysaccharides extracted from CCE were 20.27% macromolecular polysaccharides and 79.73% small molecular weight polysaccharides (2,316 to 37,520 Da), which is on the order of magnitude indicated in Lee’s study. Lee et al. [41] also found that the anti-aging polysaccharide was composed of ribose, glucose, and mannose in a ratio of 4.0:1.5:1.0 [8]. CCE contains glucose and mannose, which is similar to Lee’s study. The effect of CCE on HA synthesis in fibroblasts may involve other regulatory pathways, and future studies on HA synthesis are warranted.
3.7 RT-qPCR verification
To further verify HA-related gene expression, five selected genes (CCL2, MMP12, IL-6, HAS2, and HBEGF) were amplified and quantified using RT-qPCR. Compared with the NT group, CCL2 (p = 0.0017), IL-6 (p = 3.02×10−5), HAS2 (p = 0.0002), and HBEGF (p = 0.006) gene expression was higher and MMP12 (p = 0.0002) expression was lower in the CCE-treated group (Fig 10). CCE-treated fibroblasts exhibited upregulated expression of HAS2, which is involved in synthesizing large HA molecules. Genes such as epidermal growth factor-related gene HBEGF, CCL2, and other genes related to fibroblast differentiation and proliferation were also upregulated, whereas MMP12 was significantly downregulated in fibroblasts treated with CCE. The results were consistent with the transcriptome data, confirming the reliability of the RNA-seq.
4. Conclusion
CCE promoted HA synthesis in human skin fibroblasts. Furthermore, RNA-seq analysis and RT-qPCR verification demonstrated that CCE promoted the expression of HAS2, epidermal growth factor-related genes, HBEGF, CCL2, IRAK2, and genes related to fibroblast differentiation and proliferation, and significantly downregulated the expression of MMP12. The results of this study indicate that CCE has strong antioxidant activity, which may be related to its composition of eight nucleosides, six alditols, active polysaccharides, protein, and polyphenols. Based on the traditional role of C. cicadae as antioxidant and anti-aging medicine, we studied the antioxidant activity and HA synthesis-promoting effects. Thus, CCE can be used as a potential moisturizer and anti-aging agent in functional foods and cosmetics in the future.
Acknowledgments
The authors sincerely acknowledge Pref. Qingjiu Tang for her help in the detection of nucleosides and alditols. We also appreciate Yue Wang for his guidance on cell experiments.
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