Salidroside can target both P4HB-mediated inflammation and melanogenesis of the skin

Abstract

Rationale: Many external factors can induce the melanogenesis and inflammation of the skin. Salidroside (SAL) is the main active ingredient of Rhodiola, which is a perennial grass plant of the Family Crassulaceae. This study evaluated the effect and molecular mechanism of SAL on skin inflammation and melanin production. It then explored the molecular mechanism of melanin production under ultraviolet (UV) and inflammatory stimulation. Methods: VISIA skin analysis imaging system and DermaLab instruments were used to detect the melanin reduction and skin brightness improvement rate of the volunteers. UV-treated Kunming mice were used to detect the effect of SAL on skin inflammation and melanin production. Molecular docking and Biacore were used to verify the target of SAL. Immunofluorescence, luciferase reporter assay, CO-IP, pull-down, Western blot, proximity ligation assay (PLA), and qPCR were used to investigate the molecular mechanism by which SAL regulates skin inflammation and melanin production. Results: SAL can inhibit the inflammation and melanin production of the volunteers. SAL also exerted a protective effect on the UV-treated Kunming mice. SAL can inhibit the tyrosinase (TYR) activity and TYR mRNA expression in A375 cells. SAL can also regulate the ubiquitination degradation of interferon regulatory factor 1 (IRF1) by targeting prolyl 4-hydroxylase beta polypeptide (P4HB) to mediate inflammation and melanin production. This study also revealed that IRF1 and upstream stimulatory factor 1 (USF1) can form a transcription complex to regulate TYR mRNA expression. IRF1 also mediated inflammatory reaction and TYR expression under UV- and lipopolysaccharide-induced conditions. Moreover, SAL derivative SAL-plus (1-(3,5-dihydroxyphenyl) ethyl-β-d-glucoside) showed better effect on inflammation and melanin production than SAL. Conclusion: SAL can inhibit the inflammation and melanogenesis of the skin by targeting P4HB and regulating the formation of the IRF1/USF1 transcription complex. In addition, SAL-plus may be a new melanin production and inflammatory inhibitor.

Keywords: Salidroside; interferon regulatory factor 1 (IRF1); prolyl 4-hydroxylase beta polypeptide (P4HB); tyrosinase; upstream stimulatory factor 1 (USF1).

Figure 1

Effect of Rhodiola extract on the skin of volunteers. A, B. Skin image detected by VISIA imaging system showing the speckle, red zone, UV spots, violet spots, and brown spots of volunteers. C, D. Statistical analyses of the improvement rate of speckle, red zone, UV spots, violet spots, and brown spots. E. Collagen content in the arm detected by the Dermalab instrument with skin test probes. F, G. Melanin reduction and brightness improvement rate detected by Dermalab. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).

Figure 2

Inhibitory effect of SAL on melanin biosynthesis in A375 cells. A. Inhibition rate of melanin production in A375 cells treated with different concentrations of SAL. B. Inhibition rate of the TYR activity of A375 cells treated with SAL. C. Changes in the melanin content of A375 cells treated with SAL. D. Western blot analysis of TYR expression in A375 cells treated with SAL. E. Effect of SAL on the skin of UV-treated mice as detected by HE staining of paraffin sections, IHC staining, and Fontana-Masson staining. F. Expression of TNF-α-, IL-6-, Cxcl9-, and Cxcl10-related mRNAs in the skin of SAL-treated UV irradiation mice. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).

Figure 3

SAL can bind to TYR and P4HB. A. Prediction of the target of SAL through SEA search server. B. SAL exhibited good binding activity to TYR as determined by molecular docking. C. SAL exhibited good binding activity to TYR as determined by Biacore assay. D. SAL exhibited good binding activity to P4HB as determined by molecular docking. E. SAL exhibited good binding activity to P4HB as determined by Biacore assay. F. Effect of SAL on the prolyl hydroxylase activity in A375 cells or P4HB knockdown cells. G, H. Effect of SAL on the proteomics of A375 cells (GO analysis) and protein-protein interaction network. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).

Figure 4

P4HB regulates the ubiquitination degradation of IRF1. A. Changes in TYR activity in P4HB overexpression or knockdown A375 cells. B. Changes in TYR mRNA expression in Sal treated, P4HB overexpression, and P4HB knockdown A375 cells. C. Western blot analysis of TYR expression in P4HB overexpression or knockdown A375 cells. D. Proteins have interactions with P4HB and USF1 analyzed by FpClass. E. Interaction of P4HB and IRF1 in A375 cells detected by PLA. F. Western blot analysis of the expression of IRF1 and P4HB in P4HB overexpression or knockdown A375 cells. G. Western blot analysis of IRF1 expression in the nucleus of A375 cells after P4HB overexpression or knockdown. H. Effect of SAL on the ubiquitination of IRF1 as determined by Western blot. I. Western blot analysis of IRF1 expression in whole cells and nucleus of SAL-treated A375 cells. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).

Figure 5

IRF1 and USF1 form a transcription complex to promote melanin production in A375 cells. A. Luciferase reporter assay of the transcriptional activation effect of IRF1 on TYR in A375 cells. B. Pull-down analysis of the proteins that interact with IRF1. Cellular extracts from Flag-IRF1 overexpression A375 cells were subjected to affinity purification with anti-Flag affinity columns and eluted with Flag peptide. Elutes were separated by SDS-PAGE and stained with silver. C. PLA analysis of the interaction between IRF1 and USF1 in A375 cells. D. Co-IP analysis of the interaction of IRF1 and USF1 in A375 cells. E. Interaction of IRF1 and USF1 verified by IF experiment. F, G. Changes in TYR mRNA and protein expression in IRF1 overexpression and IRF1 knockdown cells. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).

Figure 6

SAL inhibits LPS/UV-induced inflammation and melanin production in A375 cells. A. Effect of SAL on the prolyl hydroxylase activity in UV- and LPS-treated A375 cells. B. Interaction of IRF1 and USF1 in A375 cells induced by UV irradiation and LPS detected by immunofluorescence. C. Effect of SAL on TYR mRNA expression in UV- and LPS-treated A375 cells. D-F. Effect of SAL on the melanin content, TYR activity, and TYR mRNA expression of UV- or LPS-treated A375 cells. G, H. Effect of SAL on the expression of IRF1 and TYR proteins in UV- and LPS-treated A375 cells as detected by Western blot. I, J. Effects of SAL on the mRNA expression levels of Cxcl2, Cxcl9, Cxcl10, and Cxcl11 in UV- and LPS-treated A375 cells. K, L. Effect of SAL on the mRNA expression levels of Cxcl2, Cxcl9, Cxcl10, and Cxcl11 in UV- and LPS-treated HaCaT cells. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).

Figure 7

Synthesis and activity evaluation of SAL derivatives. A. Structural formula of synthetic SAL derivatives (SAL-plus). B. Inhibitory effect of SAL-plus on melanin production in A375 cells. C-E. Comparative detection of the TYR activity inhibition rate and melanin inhibition rate of SAL and SAL-plus in A375 cells. F, G. Western blot analysis of IRF1 and TYR protein expression changes in SAL- and SAL-plus-treated A375 cells. H. Analysis of the mRNA expression levels of TYR in SAL and SAL-plus (L: 50 µM, H: 100 µM) treated A375 cells. I, J. mRNA expression levels of related inflammatory factors Cxcl2, Cxcl9, Cxcl10, and Cxcl11 in UV, LPS, and SAL-plus treated A375 cells. K. Effect of SAL-plus on the skin of UV-treated mice as detected by HE staining, IHC staining, and Fontana-Masson staining of paraffin sections. Data are expressed as mean ± SD (^*P < 0.05, ^**P < 0.01).