Melatonin Promotes the Development of Secondary Hair Follicles in Adult Cashmere Goats by Activating the Keap1-Nrf2 Signaling Pathway and Inhibiting the Inflammatory Transcription Factors NFκB and AP-1

Abstract

Exogenous melatonin (MT) has been used to promote the growth of secondary hair follicles and improve cashmere fiber quality, but the specific cellular-level mechanisms involved are unclear. This study was carried out to investigate the effect of MT on the development of secondary hair follicles and on cashmere fiber quality in cashmere goats. The results showed that MT improved secondary follicle numbers and function as well as enhanced cashmere fiber quality and yield. The MT-treated goat groups had high secondary-to-primary ratios (S:P) for hair follicles, greater in the elderly group (p < 0.05). Antioxidant capacities of secondary hair follicles improved fiber quality and yield in comparison with control groups (p < 0.05/0.01). Levels of reactive oxygen and nitrogen species (ROS, RNS) and malondialdehyde (MDA) were lowered (p < 0.05/0.01) by MT. There was significant upregulation of antioxidant genes (for SOD-3; GPX-1; NFE2L2) and the protein of nuclear factor (Nrf2), and downregulation of the Keap1 protein. There were significant differences in the expression of genes for secretory senescence-associated phenotype (SASP) cytokines (IL-1β, IL-6, MMP-9, MMP-27, CCL-21, CXCL-12, CXCL-14, TIMP-1,2,3) plus their protein of key transcription factors, nuclear factor kappa B (NFκB) and activator protein-1 (AP-1), in comparison with the controls. We concluded that MT could enhance antioxidant capacity and reduce ROS and RNS levels of secondary hair follicles through the Keap1-Nrf2 pathway in adult cashmere goats. Furthermore, MT reduced the expression of the SASP cytokines genes by inhibiting the protein of NFκB and AP-1 in the secondary hair follicles in older cashmere goats, thus delaying skin aging, improving follicle survival, and increasing the number of secondary hair follicles. Collectively, these effects of exogenous MT enhanced the quality and yield of cashmere fibers, especially at 5-7 years old.

Keywords: AP-1; Keap1; NFκB; Nrf2; cashmere; melatonin; secondary hair follicles.

Figure 1

Cashmere fiber indices of cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Fiber diameter; (B) fiber length; (C) cashmere yield; (D) cashmere yield per unit live weight. All the values are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT.

Figure 2

Hair follicle traits of cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Photomicrographs of transverse sections of skin (40×, Sacpic staining); (B) photomicrographs of longitudinal sections of skin (40×, Sacpic staining); (C) primary hair follicle density (PFD); (D) secondary hair follicle density (SFD); (E) ratio secondary to primary follicles (S:P). All the values are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT. Scale bar = 200 μm.

Figure 3

Serum antioxidant capacity of cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Catalase (CAT); (B) total antioxidant capacity (T-AOC); (C) glutathione peroxidase (GSPH); (D) superoxide dismutase (SOD); (E) malondialdehyde (MDA). All the values are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT.

Figure 4

Skin antioxidant capacity of cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Catalase (CAT); (B) total antioxidant capacity (T-AOC); (C) glutathione peroxidase (GSPH); (D) superoxide dismutase (SOD); (E) malondialdehyde (MDA). All the values are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT.

Figure 5

Levels of reactive oxygen and nitrogen species (ROS, RNS) in the serum and skin of Cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) ROS levels in serum; (B) RNS levels in serum; (C) ROS levels in skin; (D) RNS levels in skin. All the values are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT.

Figure 6

mRNA expression in the skin of cashmere goats treated with melatonin (MT) or not treated (CK). Volcano plots indicating up and downregulated mRNA transcripts in the MT group compared with the control (CK) group at (A) 2 and (B) 7 years of age. Heat maps of mRNA transcripts showing hierarchical clustering of altered mRNA transcripts in the MT and CK groups at (C) 2 and (D) 7 years of age. Up and down-regulated genes are in red and blue, respectively.

Figure 7

mRNA expression in the skin of cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Gene ontology (GO) and (C) Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses of selected differential expression genes (DEGs) in skin between MT and control at 2 years of age. (B) GO and (D) KEGG) analyses of selected DEGs in skin between MT and control at 7 years of age.

Figure 8

Gene and protein expression in the skin of 2-year-old and 7-year-old cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Levels from qRT-PCR and RNAseq for: cyclooxygenase-1 (COX-1), cyclooxygenase-3 (COX-3), superoxide dismutase-3 (SOD-3), glutathione peroxidase-1 (GPX-1), nuclear factor Nrf2, numerals represent ages, 2 or 7 years. (B) Western blots showing Keap-1, Nrf2, and β-actin. All the values in A are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT (n = 3).

Figure 9

Gene and protein expression in the skin of 2-year-old and 7-year-old Cashmere goats treated with melatonin (MT) or not treated (CK) at different ages. (A) Levels from qRT-PCR and RNAseq for: interleukin-1β (IL-1β), IL-6, matrix metalloproteinase-9 (MMP-9), MMP-27, Tissue Inhibitor of metalloproteinase-1 (TIMP-1), TIMP-2, TIMP-3, chemokine ligand gene 21 (CCL-21), chemokine ligand gene-12 (CXCL-12), CXCL-14. Numerals represent ages 2 or 7 years. (B) Western blots showing C-jun, C-fos, P65, and β-actin. All the values are the mean ± standard deviation. * p < 0.05, ** p < 0.01, CK vs. MT (n = 3).

Figure 10

Schematic workflow of the experimental design of this study.

Figure 11

Schematic representation of the mode of action of melatonin on the promotion of secondary hair follicle growth and development. MT-melatonin, OS-oxidative stress, CAT-catalase, T-AOC-total antioxidant capacity, SOD-superoxide dismutase, GSHPX/GPx-glutathione peroxidase, MDA-malondialdehyde, SHF-secondary hair follicle, Keap1-Kelch-like ECH-associated protein 1, Nrf2-nuclear factor erythroid 2-related factor 2, NFκB-nuclear factor kappa B, SASP-senescence-associated phenotype, IL-interleukin, TIMP-tissue inhibitor of metallo proteinases, CXL/CCL-chemokine.