Dendrobium officinale polysaccharide promotes angiogenesis as well as follicle regeneration and hair growth through activation of the WNT signaling pathway

Abstract

Introduction: Hair loss is one of the common clinical conditions in modern society. Although it is not a serious disease that threatens human life, it brings great mental stress and psychological burden to patients. This study investigated the role of dendrobium officinale polysaccharide (DOP) in hair follicle regeneration and hair growth and its related mechanisms.

Methods: After in vitro culture of mouse antennal hair follicles and mouse dermal papilla cells (DPCs), and mouse vascular endothelial cells (MVECs), the effects of DOP upon hair follicles and cells were evaluated using multiple methods. DOP effects were evaluated by measuring tentacle growth, HE staining, immunofluorescence, Western blot, CCK-8, ALP staining, tube formation, scratch test, and Transwell. LDH levels, WNT signaling proteins, and therapeutic mechanisms were also analyzed.

Results: DOP promoted tentacle hair follicle and DPCs growth in mice and the angiogenic, migratory and invasive capacities of MVECs. Meanwhile, DOP was also capable of enhancing angiogenesis and proliferation-related protein expression. Mechanistically, DOP activated the WNT signaling and promoted the expression level of β-catenin, a pivotal protein of the pathway, and the pathway target proteins Cyclin D1, C-Myc, and LDH activity. The promotional effects of DOP on the biological functions of DPCs and MVECs could be effectively reversed by the WNT signaling pathway inhibitor IWR-1.

Conclusion: DOP advances hair follicle and hair growth via the activation of the WNT signaling. This finding provides a mechanistic reference and theoretical basis for the clinical use of DOP in treating hair loss.

Keywords: Angiogenesis; Dendrobium officinale polysaccharide; Follicle regeneration; Hair loss; LDH; WNT signaling pathway.

Fig. 1

DOP advances hair follicle growth. A. Freshly isolated tentacle hair follicle tissues of C57BL/6 mice were subjected to 100 μg/ml DOP treatment with the same volume of PBS as a control, and the gross morphological structure of the hair follicles was observed under the microscope on days 0 and 6, respectively. Scale bar = 500 μm. B. Quantitative analysis of tentacle growth length in mice. N = 6 (biological replicates). ∗∗P < 0.01 compared to PBS group.

Fig. 2

DOP enhances the expression of vascularization and proliferation factors in tentacle follicles. The tentacle hair follicles of mice were treated for 6 days with DOP and its control PBS. A. The pathological structural changes of hair follicles were observed by HE staining. Scale bar = 100 μm. B. CD31 expression within hair follicles was evaluated using Immunofluorescence staining. Scale bar = 50 μm. C-D. Western blot was conducted to determine the protein expression levels of proliferation factors Ki-67 and p-S6 (C) and vascular growth factors CD31 and VEGFA (D) in hair follicles. N = 6 (biological replicates). ∗∗P < 0.01 compared to PBS group.

Fig. 3

DOP strengthens the viability of DPCs. A. Cell morphology of DPCs cultured at day 1, 5, and 7 days was observed under the microscope. Scale bar = 100 μm. B. Cytokeratin 8, Versican and α-SMA expression levels within DPCs were determined by immunofluorescence staining. Scale bar = 20 μm. C. ALP levels and the active level of cell proliferation in DPCs were evaluated by ALP staining and toluidine blue staining. Scale bar = 100 μm. D. CCK-8 assay was performed to measure the viability of DPCs subjected to treatments with various concentrations of DOP (10, 50, 100, and 200 μg/ml). N = 3 (biological replicates). ∗P < 0.05, ∗∗P < 0.01 compared to PBS group.

Fig. 4

DOP promotes DPC cell proliferation by activating the WNT signaling pathway. DPCs were subjected to 48-h treatment with 100 μg/ml of DOP. A. DPC proliferation was assessed using EdU staining. Scale bar = 50 μm. B. ALP expression levels in DPCs were measured by ALP staining. Scale bar = 100 μm. C. LDH activity in DPCs was evaluated by LDH kit. D. Ki-67, Cyclin D1, MMP-3, and β-catenin proteins within DPCs were detected using Western blot. N = 3 (biological replicates). ∗P < 0.05, ∗∗P < 0.01 compared to PBS group.

Fig. 5

DOP enhances the angiogenesis, migration, and invasion ability of MVECs. MVECs were subjected to 48-h treatment with 100 μg/ml of DOP. A. The tubule formation assay was carried out to examine MVECs vascularization level. B. The migration ability of MVECs was detected using Scratch test. C. MVECs invasion was examined using Transwell assay. D. Western blot was implemented to detect the expression levels of key proteins of angiogenesis (MMP-2, MMP-9, VEGFA, and CD31) and proteins associated with the WNT pathway (C-Myc, Cyclin D1, and β-catenin). All scale bar = 100 μm. N = 3 (biological replicates). ∗P < 0.05, ∗∗P < 0.01 compared to PBS group.

Fig. 6

WNT pathway inhibitor reverses the effects of DOP in DPCs. After treating DPCs with 100 μg/ml DOP for 24 h, 2.5 μM IWR-1 was given for a total of 24 h. A. CCK-8 assay was conducted to assess DPC viability. B. ALP expression levels in DPCs were measured by ALP staining. Scale bar = 100 μm. C. LDH activity in DPCs was evaluated by LDH kit. D. Ki-67, Cyclin D1, MMP-3, and β-catenin protein levels within DPCs were detected using Western blot. N = 3 (biological replicates). ∗∗P < 0.01 compared to PBS group; #P < 0.05; ##P < 0.01 compared to DOP group.

Fig. 7

WNT pathway inhibitor reverses the effects of DOP in MVECs. A. The level of vascularization of MVECs was examined by tubule formation assay. B. Scratch test was utilized to detect the migration ability of MVECs. C. MVECs invasion was examined using Transwell assay. D. The expression levels of key proteins of angiogenesis (MMP-2, MMP-9, VEGFA, and CD31) and proteins related to the WNT signaling pathway (C-Myc, Cyclin D1, and β-catenin) were determined using Western blot. All scale bar = 100 μm. N = 3 (biological replicates). ∗∗P < 0.01 compared to PBS group; ##P < 0.01 compared to DOP group.