Water‐soluble intracellular extract of Desmodesmus sp. YT enhanced the antioxidant capacity of human skin fibroblast to protect the skin from UV damage

The oxidative stress induced by ultraviolet (UV) radiation is a pivotal factor in skin aging and can even contribute to the development of skin cancer.


| INTRODUC TI ON
Excessive free radicals can damage cells, tissues, and organs, and cause oxidative damage, and interfere with cell functions, and severely lead to various diseases, including cardiovascular disease, aging, and cancer. 1 Exposure to UV radiation can lead to increased levels of free radicals, thereby inducing oxidative stress.This oxidative stress is known to be a major factor contributing to skin aging and potentially even the formation of skin cancer. 2,3Human skin fibroblasts (HSF) are the basic cell group of human skin structure and function, so improving the vitality of HSF and protecting HSF from UV damage are of great significance in preventing skin aging. 4tioxidants are important substances for scavenging free radicals.With the deepening of research on microalgae, more natural active substances in microalgae have been identified, such as microalgal polysaccharides, fatty acids, and carotene. 5Meanwhile more related applications have been developed and utilized, such as antioxidants, 6 food additives, 7 and nutritional supplements. 8As a natural antioxidant, microalgae has the advantages of strong antioxidant capacity, high safety, reliable source, and small side effects. 9rthermore, microalgae meets people's requirements for green and health, and has huge market potential in the fields of medicine, health products, and cosmetics. 10Consequently, it has established a solid groundwork for the advancement of highly effective and low-toxic antioxidants.2][13] Desmodesmus sp. is regarded as a promising raw material because it is rich in protein, polysaccharide, lutein, unsaturated fat acid, and other active substances. 14Previous studies have shown that Desmodesmus sp. can be used to remove heavy metals 15 and tetracyclines 16 from wastewater, but there are still few reports on its antioxidant capacity.

•−
) and 2,2′-Azino-bis (3 ethylbenzothiazoline-6sulfonic acid; ABTS) of the water-soluble intracellular extract (WIE) of Desmodesmus sp.YT (YT).In addition, the influences of YT-WIE on the HSF exposed under ultraviolet (UV) radiation were further studied and the mechanism of action was explored using RT-qPCR and western blot.

| Preparation of YT-WIE and determination of its main components
To obtain the water-soluble intracellular extract of microalgae, 50 mg fresh algae powder was dissolved in 1 mL cold phosphate buffered saline (PBS, pH = 7.4).The mixture was then crushed using a cell crusher for 1 min, followed by centrifugation at 10 000 rpm for 15 min at 4°C.The supernatant was freeze-dried to obtain YT-WIE, which is stored at −20°C for future use.Following the instructions of manufacturer, the protein, carbohydrate, and phenolic contents in YT-WIE were evaluated using BCA Protein Assay Kit, Total Carbohydrate Content Assay Kit, and Plant Total (TP) Phenol Content Assay Kit, respectively.These assay kits were all purchased from Solarbio (Beijing, China).Furthermore, compared to the control group, the YT-WIE group exhibited an 89.30% enhancement in HSF viability and a 44.63% increase in survival rate post-UV irradiation.Significant upregulation of antioxidant genes (GCLC, GCLM, TXNRD1, HMOX1, NQO1) was observed with YT-WIE treatment at 400 μg mL −1 , with fold increases ranging from 1.13 to 5.85 times.

Conclusion:
YT-WIE demonstrated considerable potential as an antioxidant, shielding human cells from undue oxidative stress triggered by external stimuli such as UV radiation.This suggests its promising application in cosmetics antioxidants.

| The effect of YT-WIE on the viability of HSF
The cytotoxic effects of YT-WIE and UV irradiation on HSF were evaluated using the Cell Counting Kit- There were three groups replicated for each sample.

| The effect of YT-WIE on HSF under UV damage
To assess the antioxidative potential of YT-WIE on HSF, UV radiation was used to induce oxidative damage.HSF were inoculated into 96well plate (1 × 10 4 /well) 1 day in advance.Subsequently, varying concentrations (100, 200, 400 μg mL −1 ) of both YT-WIE and L-Ascorbic acid (MACKLIN, Shanghai, China) were added to the co-culture and maintained for 24 h.Prior to irradiation, the previous medium was removed, and the cells were rinsed twice with PBS to ensure thorough washing.Subsequently, the plate was placed in a UV Crosslinker and exposed to irradiation for 240 s at a dose of 9999 mJ cm −2 .After irradiation, the cells were cultured with fresh medium for 24 h.Then, 10 μL CCK-8 was added for detection at 450 nm as above.

| Transcriptome sequencing and analysis
The samples were sent to Baimaike (Beijing, China) for transcriptome sequencing.Differentially expressed genes (DEGs) were identified based on fold change (FC) > 1.5 and false discovery rate (FDR) < 0.05.BMKCloud V.2022 software (Biomarker Technologies, Beijing, China) was utilized to perform Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis.

| Real-time quantitative PCR (RT-qPCR) analysis
HSFs were exposed to YT-WIE at a concentration of 400 μg mL −1 for a duration of 24 h.Following the treatment, RNA extraction was carried out using the RNA fast200 Total RNA Rapid Extraction Kit (220010, Fastagen, Shanghai, China).The PrimeScript™ RT reagent Kit (RR047A, Takara, Dalian, China) was employed to synthesize cDNA.For quantitative RT-qPCR analysis, TB Green® Premix Ex Taq™ II (RR820A, Takara, Dalian, China) and ABI QuantStudio 6 Flex (Applied Biosystems, Foster, USA) were utilized.The primer sequences can be found in Table S2.The relative gene expression was calculated using the 2 −△△t method, with GAPDH serving as the reference gene for normalization.

| Western Blot (WB) analysis
WB was conducted following a previously established protocol. 22dio Immunoprecipitation Assay (RIPA) buffer with protease inhibitor was used to lyse the cells on ice for 30 min.After Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE), the protein bands were transferred to a Polyvinylidene Fluoride (PVDF) membrane using a semi-dry transfer method (1.3A, 25 V, 11 min).The PVDF membrane was blocked with 5% skim milk powder at room temperature.After blocking, the membrane was incubated overnight at 4°C with the primary antibody (refer to Table S3 for details).Subsequently, the membrane was incubated with the secondary antibody (as006, Asbio, Guangzhou, China) at room temperature for 1 h.Protein bands were visualized using a chemiluminescence substrate kit (MF074-01, Mei5bio, Beijing, China) and the ChemChemChemeTM+ system (SageCreation, Beijing, China).Quantification was performed using ImageJ software.

| Statistical analysis
The data were expressed as mean ± standard deviation (SD).
Statistical analysis was performed using unpaired t-tests and oneway ANOVA to determine significant differences between groups.
A p-value of less than 0.05 was considered statistically significant.

| Cell identification of Desmodesmus sp.YT and the main components of YT-WIE
The biomass yield of Desmodesmus sp.YT was 0.629 ± 0.037 g L −1 .In addition, the cell morphology was observed with fluorescence microscope.Cell morphology of Desmodesmus sp.YT was an oval monocyte and entered a temporary phase called "palmelloid," which had also been reported in Chlamydomonas 22 (Figure 1A).After preparing YT-WIE, its main component content is measured, and the results showed that the proportions of proteins, carbohydrates, and phenols in YT-WIE were 42.78%, 13.64%, and 2.05%, respectively (Figure 1B).

| The ability of YT-WIE to scavenge free radicals
To examine the antioxidant capacity of YT-WIE, we assessed its ability to scavenge various free radicals, including DPPH, ABTS, •OH, and . The results showed that YT-WIE possessed strong capability for scavenging DPPH radical.The DPPH-scavenging ability of YT-WIE showed a trend of concentration dependence from 0.05 to 0.6 mg mL −1 .

| The effect of YT-WIE on the cell viability and anti-UV capacity of HSF
The results showed YT-WIE significantly increased the viability of HSF at a concentration of 50-1600 μg mL −1 , with a maximum increase of 89.30% (400 μg mL −1 ; Figure 3A).To further explore the antioxidant effect of YT-WIE, the UV radiation (9999 mJ cm −2 , 240 s) was used to cause oxidative stress.We found that YT-WIE could significantly enhance the cell viability, compared with vitamin C, which is a potent antioxidant. 24When the HSF exposed under the UV, the cell viability dropped to 52.32% directly (Damage group).
With 200 μg mL −1 vitamin C co-cultivated, there is no significant difference observed for the cell viability (51.29%), while higher concentration of vitamin C (400 μg mL −1 ) can even cause the death of HSF (cell viability of 4.67%).However, groups treated with YT-WIE showed much higher cell viability compared with other groups.
With 100-400 μg mL −1 of YT-WIE addition, cell viability increased 21.76-44.63%compared with damage group.When the HSF was treated by 400 μg mL −1 of YT-WIE, the cell viability reached up to 96.95%, which improved 92.28% compared with 400 μg mL −1 of vitamin C group (Figure 3B).Above all, the results indicate that YT-WIE can significantly enhance the viability of HSF and exhibit better UV resistance than vitamin C, thereby helping cells resist free radical damage caused by UV radiation.

| The influence of YT-WIE on the level of ROS in HSF after UV radiation
Fluorescence microscopy as an efficient, visualized, and costeffective method was also widely used for the ROS measurement. 19,21The stronger the green fluorescence intensity means the higher the ROS level (Figure 3C).The results demonstrated that the ROS level in the Damage group was 4.24 times higher compared to the control group, indicating that UV irradiation significantly elevated the ROS level in HSF cells.Additionally, the ROS level in the Vitamin C (VC) group showed a decrease of 24.29% compared to the Damage group.Surprisingly, the fluorescence intensity of YT-WIE at 200 μg mL −1 was reduced 89.15% compared with the Damage group, and reduced 85.67% compared with the VC group.Furthermore, it was even 2.20 times weaker than the control group (Figure 3D).Therefore, the aforementioned results suggest that YT-WIE was beneficial for reducing the ROS level caused by UV radiation, with stronger ROS scavenging ability than vitamin C at the same concentration.

| Analysis of transcriptome data quality
The preceding findings revealed that 400 μg mL −1 YT-WIE had the most significant effect on HSF, not only on cell viability but also on UV resistance.Therefore, a group of HSF that had been treated with YT-WIE (400 μg mL −1 ) for 24 h (Y1, Y2, Y3) and a group of HSF that had not been treated with YT-WIE (B1, B2, B3) were sent for RNA-sequencing.The sequencing results showed that the GCcontent was 48.77-50.33%,and the Q30 of all samples was more than 94.97%, which indicated that the sequencing quality was high and could be used for subsequent analysis (Table S1).The cluster heatmap and volcano plot of DEGs clearly indicated that YT-WIE can significantly affect some genes expression (Figure 4A,B).A total of 1306 DEGs (FDR < 0.05, FC > 1.5) were identified.Among these DEGs, 668 were upregulated, while 638 were downregulated, as shown in Figure 4C. .The data were expressed as mean ± SD.

| GO and KEGG analysis
To gain a deeper understanding of the impact of YT-WIE on gene function and metabolic pathways, GO and KEGG analysis were conducted on DEGs.GO enrichment analysis includes three parts, namely biological processes, cellular components, and molecular functions.In the GO enrichment analysis, a total of 1120 differentially expressed genes (DEGs) were annotated.Among these DEGs, rhythmic process (17 DEGs), nucleoid (1 DEG), and antioxidant activity (6 DEGs) exhibited the most significant differences in terms of biological processes, cellular components, and molecular functions, respectively (Figure 5A).The gene function with a significant proportion difference indicates that the enrichment trend of DEGs is different from that of all genes.It can be analyzed

| The effect of YT-WIE on the expression level of antioxidant genes
To verify the reliability of transcriptome data and explore the antioxidant mechanism of YT-WIE, RT-qPCR was used to detect the expression of some key antioxidant genes in DEGs.The results revealed that treatment with YT-WIE (400 μg mL −1 ) increased the mRNA expression of antioxidant genes in HSF cells.Specifically, the mRNA levels of glutamate cysteine ligase catalytic subunit (GCLC), glutamate cysteine ligase modified subunit (GCSM), thioredoxin reductase 1 (TXNRD1), NAD (P) hydroquinone oxidoreductase-1 (NQO1), and heme oxygenase-1 (HMOX1) were 1.13, 1.69, 1.65, 1.54, and 5.85 times higher than those in the control group,

| DISCUSS ION
UV radiation is known to increase the generation of ROS within cells.The accumulation of ROS-induced oxidative stress can subsequently lead to damage in lipids, proteins, nucleic acids, and organelles.This process is considered one of the key mechanisms involved in skin aging. 25The use of antioxidants is an effective way to prevent symptoms related to photoinduced skin aging. 26In this study, our result showed that YT-WIE had a significant scavenging efficiency of 21.63-54.95%for free radicals at a concentration ranging from 0.6 to 1.0 mg mL −1 .It was reported that DPPH scavenging activity of Chlorella pyrenoidosa only reached 39.71% at 2.00 mg mL −1 . 27Similarly, aqueous extracts of two microalgae Isochrysis galbana (PEA) and Nannochloropsis oculata (PEB) at concentration of 10.00 mg mL −1 exhibited an antioxidant activity of 41.45% and 59.07%, 28 but YT-WIE can achieve comparable results between 0.60 and 1.00 mg mL − . 29The soluble extract of Chlorella vulgaris was reported that can enhance mammalian cell growth. 30Meanwhile, aqueous microencapsulated extract of microalgae Phaeodactylum tricornutum can increase the cell viability. 31Our research results preliminarily indicate that YT-WIE contains various substances such as proteins, polysaccharides, and phenols, which may contain some key antioxidants.Phycocyanin contained in algae is a common antioxidant. 32,33 early as 1998, researchers highlighted phycocyanin's capability to effectively neutralize OH radicals (IC50 = 0.91 mg mL −1 ) and RO radicals (IC50 = 76 microg/mL).Its scavenging activity was found to be comparable to 0.125 mg mL −1 of dimethyl sulfoxide (DMSO) and 0.038 microg/mL of trolox, which are specific scavengers for these respective radicals. 34Alginate is a polysaccharide rich in sulfate groups, mainly found in brown algae. 35Research indicated that the fucoidan isolated from brown seaweed can effectively weaken UVB-induced light damage in vitro and in vivo, inhibit cell apoptosis by reducing intracellular ROS levels, and thereby increase the vitality of UVB irradiated human keratinocytes. 36In addition, phenolic substances such as phlorotannin contained in algae are also powerful antioxidants. 37According to reports, phlorotannin extracted from brown algae Ecklonia cava can clear ROS in zebrafish embryos, prevent lipid peroxidation, and reduce AAPH-induced cell death. 38According to the molecular size of the compounds, phenolic compounds such as phlorotannins, 39 polysaccharides such as fucoidan, 40 might can enter the cell membrane.For proteins, molecular weight below 50 kDa might also can enter the cell membrane. 41ese results indicates that YT-WIE is an efficient and nontoxic antioxidant with potential as a cosmetic additive.
Glutathione (GSH) is a tripeptide-based molecule that acts as an important cellular antioxidant, playing a crucial role in protecting cells against oxidative stress-induced damage. 42The synthesis of GSH is influenced by the activity of the rate-limiting enzyme, glutamate cysteine ligase (GCL), which consists of two subunits: the catalytic subunit (GCLC) and the modifier subunit (GCLM).The activity of GCL is a key determinant in the regulation of GSH synthesis. 43 (TXNRD1) is essential for controlling cell growth and transformation as well as shielding cells from oxidative damage. 44,45A widely present flavin protein called NAD(P)H Quinone Dehydrogenase 1 (NQO1) encourages the forced double electron reduction of quinone, quinone imines, nitroaromatics, and azo dyes, which lowers the formation of ROS. 46,47Heme oxygenase (HMOX1) is a cell protective enzyme that breaks down heme to produce carbon monoxide (CO), biliverdin, and molecular iron. 48The study has shown that HMOX1 can reduce endoplasmic reticulum stress and acute lung injury caused by sepsis. 49Research also suggested that cinnamaldehyde reduced UVB-induced photoaging by upregulating HMOX1 expression. 50The increased expression of these endogenous antioxidants helps maintain redox homeostasis 51 (Figure 7).In short, the antioxidant mechanism of YT-WIE may be through upregulating the expression of GCLC, GCLM, TXNRD1, NQO1, and HMOX1 to clear intracellular free radicals.
In summary, YT-WIE can efficiently eliminate free radicals, promote HSF proliferation, and protect HSF from UV damage.
Therefore, YT-WIE owns great potential in variety of applications such as antioxidants, nutrient substitutes, and food additives.

| CON CLUS IONS
The results of this study proved that YT-WIE has strong ability of scavenging free radical (DPPH, ABTS, •OH, O 2

•-
).Furthermore, we confirmed that YT-WIE significantly reduce the level of ROS induced with UV in HSF.By promoting the expression of endogenous antioxidative genes GCLC, GCLM, TXNRD1, HMOX1, NQO1, YT-WIE can promote HSF viability under oxidative stress.Therefore, YT-WIE are potentially valuable to be employed in cosmetic formulations to protect skin against oxidative stress caused aging.
Microalgal strains were offered by Innova Bay (Shenzhen) Technology Co. Ltd. (Shenzhen, China).Based on morphological features and 18s rDNA sequences, they were identified as Desmodesmus sp.YT.The strains were cultured in Blue-Green Medium (BG11) with a salinity of 16‰.BG-11 is one of the best culture media for cultivating and maintaining cyanobacteria.The incubator maintained a stable temperature of 25 ± 1°C, a 12-h light-dark cycle, and continuous aeration for 24 h.Cell morphology of Desmodesmus sp.YT was observed by Fluorescence microscope (BX51, Olympus, Tokyo, Japan).A preweighed 0.45 μm filter paper was used to filter the 50 mL of algal liquid.The filter was dried in a freeze-drying machine for 24 h before being reweighed.The biomass can be obtained by subtracting the two weighing results.

F I G U R E 1
Cell identification of Desmodesmus sp.YT and the main components of YT-WIE.(A) The cell micrograph of Desmodesmus sp.YT under fluorescence microscope (BX51).The scale error bar was 20.0 μm.(B) The proportion of proteins, carbohydrates, and phenols in YT-WIE.The data were expressed as mean ± SD.

F I G U R E 2
The capacity of scavenging free radicals and antioxidant enzyme activities of YT-WIE at different concentrations.(A-D) The activity of scavenging DPPH, ABTS, •OH, O 2 •− .(E-H) IC50 of DDPH, ABTS, •OH, O 2 •− whether this function is related to the difference.In addition, a total of 576 DEGs were annotated in the KEGG pathway.The three most significantly enriched pathways of DEGs are cancer pathway (64 DEGs), cell cycle (39 DEGs), and PI3K-Akt signaling pathway (31 DEGs; Figure5B).

F I G U R E 3
The effect of YT-WIE on the cell viability and antioxidant capacity of HSF.(A) The viability of HSF was assessed after treatment with various concentrations of YT-WIE for 24 h.*p < 0.05, **p < 0.01 versus 0 μg mL −1 .(B) The cell viability of HSF treated with different concentrations of YT-WIE and vitamin C for 24 h and irradiated by UV at 9999 mJ cm −2 for 240 s. *p < 0.05, **p < 0.01 versus Con.(C) The images of DCFH-DA probe fluorescence of HSF treated by different ways under invert microscope (10 × 10 times) and irradiated by UV crosslinker under 9999 mJ cm −2 for 240 s. (D) The average fluorescence intensity of HSF cells treated with different methods is depicted in Figure 3C.*p < 0.05, **p < 0.01 versus Damage.The data were expressed as mean ± SD.Control: no treatment; Damage: HSF was only subjected to UV exposure to induce oxidative damage; VC: Vitamin C, a common antioxidant, was used here as a positive control.respectively (Figure 6A).Besides, the protein content of NQO1 and HMOX1 in YT-WIE-treated group were 1.23 and 1.49 times higher than the control group (Figure 6B-D).Our results indicated that YT-WIE enhanced the antioxidant capacity of HSF by promoting the expression of these antioxidant genes.
1 , indicating that YT-WIE is more efficient for scavenging free radicals.To further assess the antioxidant potential of YT-WIE, we conducted cytotoxicity detected and antioxidant studies on HSF.Comparing with the UV damage group, 400 ug mL −1 YT-WIE could increase HSF viability by 44.63% and reduce ROS level by 89.15%.The ability of microalgae extracts to promote cell growth has also been confirmed in other reports.For example, astaxanthin-rich extracts from Haematococcus pluvialis can promote human dermal fibroblasts cell viability by inhibiting the expression of MMP1 and MMP3 and enhancing the expression of TIMP1

F I G U R E 4
Statistics of DEGs.(A) Cluster heatmap of all DEGs in B versus Y. (B) Volcano plot of all DEGs in B versus Y. (C) Number of DEGs in B versus Y. B: control.Y: HSF that had been treated with YT-WIE (400 μg mL −1 ) for 24 h.

1 F I G U R E 5
Therefore, promoting the expression of GCLM and GCLC contributes to the synthesis of GSH.As a crucial NADPH-dependent enzyme that catalyzes the reduction of disulfide at the active sites of thioredoxin 1 (Trx1) and a number of other substrates, thioredoxin reductase Gene function and metabolic pathways of DEGs.(A) GO classification of DEGs.(B) KEGG classification diagram of DEGs.