Schizochytrium sp. Extracted Lipids Prevent Alopecia by Enhancing Antioxidation and Inhibiting Ferroptosis of Dermal Papilla Cells

Figures

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  Figure 1. SEL promoted the proliferation and antioxidant capacity of DPC. (a) Composition of SEL. MA, Myristic acid (C14H28O2); PA, palmitic acid (C16H32O2); SA, stearic acid (C18H36O2); EPA, eicosapentaenoic acid (C20H30O2); DPA, docosapentaenoic acid (C22H34O2); DHA, docosahexenoic acid (C22H32O2). (b–d) DPC cell proliferation exposed to SEL (12.5, 25, 50, and 100 µg mL−1) is shown at 12, 24, and 36 h. Minoxidil as a positive control and solvent (0.2% DMSO/ethanol (1:1)) as a negative control were used. (e–g) The survival ratio of DPC exposed to H2O2 (100 µM) for 1.5 h. (h) The summary graph of the relative intensity of fluorescence on oxidatively damaged DPCs. (i) The images of the DCFH-DA probe fluorescence of oxidatively damaged DPCs. Control, no treatment; damage, treated with 100 µM H2O2; minoxidil, treated by 12.5 µg mL−1 minoxidil + 100 µM H2O2; SEL, treated with 100 µg mL−1 SEL + 100 µM H2O2. Data are presented as the mean ± standard error of the mean (SEM) (n = 3). * p < 0.05; ** p < 0.01; *** p < 0.001; **** p < 0.0001 vs. control.

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  Figure 2. Quality assessment of RNA-seq data. (a) Pearson’s correlation analysis based on logarithm-transformed counts from the RNA-seq dataset of C, CS, CH, and CHS. (b) PCA analysis. The PCA axis percentage represents the proportional variance of each PC interpretation, and the upper–right side of the diagram represents the conditions. (c) Violin plot indicating gene expression and visualizing genes data density in each sample. (d–f) The volcano plot of DEGs in C vs. CS (d), C vs. CH (e), and CH vs. CHS (f). The gene expression fold change values on log2 (x-axis) and the significance of differential expression p-value on -log10 (y-axis). The vertical bar is twice the difference threshold, and the horizontal line is the p = 0.05 threshold. Red dots (upregulated), blue dots (downregulated DEGs), and black dots (non-DEG). (g) The summary graph of DEG in three groups. C, cell without treatment; CS, cell treated with 50 µg mL−1 SEL; CH, cell treated with 100 µM H2O2; CHS, cell treated with 50 µg mL−1 SEL + 100 µM H2O2.

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  Figure 3. Functional analysis by GO and KEGG enrichment. (a) GO classification of DEGs in C vs. CS. The abscissa is the GO classification, the ordinate is the percentage of genes on the left, and the right is the number of genes. Light colors indicate expression across the entire genome, and dark colors indicate expression in DEGs. (b) Enrichment of KEGG pathway in DEGs bubble patterns of C vs. CS. Each dot stands for one KEGG pathway. X-axis: enrichment factors; Y-axis: pathway. Enrichment factor = (annotated DEG ratio to overall DEGs)/(annotated gene ratio to overall genes). The dot color represents the q-value (the adjusted p-value). When the q value is smaller, enrichment is more reliable and significant. C, cell without treatment; CS, cell treated with 50 µg mL−1 SEL.

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  Figure 4. The gene expression level was verified at mRNA and protein levels. (a) The heatmap of transcription gene expression levels linked to ferroptosis in C vs. CS; (b) RT-qPCR validation of gene expression levels linked to ferroptosis in C vs. CS; C, cell without treatment; CS, cell treated with 50 µg mL−1 SEL. (c) The heatmap of gene expression levels related to antioxidation in CH vs. CHS; CH, cell treated with 100 µM H2O2; CHS, cell treated with 50 µg mL−1 SEL + 100 µM H2O2. (d) RT-qPCR validation of the transcript expression levels of genes related to antioxidation in CH vs. CHS. (e–k) The expression level of GCLM, ferritin, HMOX1, and NQO1 was assessed via the Western blotting technique. β-action is used as an internal parameter. The blue dot and the red box represent the protein expression level of the gene in the control group and SEL group, respectively. Each group has three biological replicates. Control, without treatment; SEL, cell treated with 50 µg mL−1 SEL for 36 h; HMOX1, heme oxygenase 1; SLC7A11, solute carrier family 7 member 11; FTL, ferritin L subunit; FTH1, ferritin heavy chain 1; NQO1, NAD(P)H quinone dehydrogenase 1; GCLM, glutamate–cysteine ligase modifier…

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  Figure 5. SEL promotes mouse hair growth and hair follicle growth. (a) The changes in mice hair after SEL administration. Vehicle (propylene glycol/ethanol (1:1, v/v)), 5% SEL, 10% SEL, feed containing 5% SEL, and 5% minoxidil were administered daily for two weeks. (n = 5). (b) Length of telogen in each group. When the skin of C57BL/6 mice is pink, the hair follicle is considered to be in the telogen period, and when the skin turns gray, it is thought to enter the growth period. (c) Length of new hair. (d) Weight of new hair. (e) The longest hair follicle length on mouse skin (magnification, 100×) (n = 3). (f) The HE-stained micrographs of skin tissue obtained on days seven and fourteen from mice treated with vehicle, SEL, or minoxidil (n = 3). Magnification: 100×. All data presented as the mean ± standard error of the mean (SEM) (n = 3). * p < 0.05, *** p < 0.001, and **** p < 0.0001 vs. control.

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  Figure 6. SEL inhibited the occurrence of ferroptosis in DPC and increased its antioxidant capacity. SEL reduces the ROS level in DPC by promoting the expression of SLC7A11, GCLM, HMOX1, NQO1, FTH1, and FTL, which can reduce the damage of lipid peroxidation, protein denaturation, and DNA damage caused by excess ROS. SEL: Schizochytrium sp. extracted lipids; SLC7A11: solute carrier family 7 member 11; SLC3A2: solute carrier family 3 member 2; GCLM: glutamate–cysteine ligase modifier subunit; GCLC: cysteine ligase catalytic subunit; GSH: glutathione; GS: glutathione synthetase; FTH1: ferritin heavy chain 1; FTL: ferritin light chain; HMOX1: heme oxygenase 1; NQO1: NAD(P)H quinone dehydrogenase 1.