Figure 1.
Wound healing progress in WHM.
Effect of TE and betulin on reepithelialization in the porcine ex-vivo WHM. (A) Reepithelialization in WHM treated with 10% TE in sunflower oil (oleogel) compared to sunflower oil alone, sunflower oil with ethylcellulose and untreated control 48 h after wounding. (B) Reepithelialization in WHM treated with 10 µg/ml TE and betulin in the concentration as it occurs in 10 µg/ml TE in PBS compared to PBS 48 h after wounding. Reepithelialization of the various models was normalized to untreated control (A) or PBS (B), respectively. Mean ± SEM. (C) Effect of TE and betulin in PBS on barrier regeneration in WHM normalized to barrier regeneration in WHM treated with PBS alone. WHM were treated 72 h after wounding for 24 h. Mean ± SEM. A: n = 7; B: n = 5–7; C: n = 4–5. *p<0.05 and ***p<0.001.
Figure 2.
Birch bark (TE) and betulin differently influence mRNA of COX-2, IL-6 and IL-8 in human primary keratinocytes.
COX-2 (A), IL-6 (B) and IL-8 (C) mRNA are upregulated in response to birch bark (TE) and betulin (Bet), but not to lupeol (Lup) and betulinic acid (BA). Time course of mRNA expression in response to TE (1 and 5 µg/mL) measured by qRT-PCR. The isolated triterpenes (Bet, Lup, BA) were measured in that concentration in which they occur in 5 µg/mL TE extract. Values represent means of at least three independent experiments ± SEM. *p<0.05, **p<0.01, ***p<0.001 versus control (DMSO).
Figure 3.
TE upregulates mRNA of IL-6 and COX-2 in an ex-vivo wound healing model 6 h after wounding and treatment with 10 µg/ml TE, after 48 h the levels decreased to normal levels.
Values represent means of at least three independent experiments ± SEM. *p<0.05, **p<0.01, ***p<0.001 as indicated.
Figure 4.
TE and betulin (Bet) enhance release of IL-6 (A) and IL-8 (B), as well as formation of COX-2 protein (C) in primary human keratinocytes.
Measurement of IL-6 (A) and IL-8 (B) release in response to TE (1 and 5 µg/mL) and betulin (0.87 µg/mL, which is in 1 µg/mL TE) measured by ELISA. Values represent means of at least three independent experiments ± s.d. (*p<0.05, **p<0.01 and ***p<0.001 versus control). (C) COX-2 protein was measured by Western blot analysis after 24 h treatment with TE (1 and 5 µg/mL) and the respective betulin concentration (0.87 and 4.34 µg/mL). DMSO (0.1%, v/v) served as a solvent control. A representative Western blot is shown, n = 3.
Figure 5.
TE and betulin enhance the production of various cytokines, chemokines and growth factors in human keratinocytes after 24 h of incubation.
Cells were treated either with TE (1 µg/mL) or betulin (0.87 µg/mL, which is in 1 µg/mL TE) for 24 h. Protein levels of the indicated mediators were determined in the supernatant with the Bio-Plex® Cytokine Assay. Values represent means of two independent experiments ± s.d. *p<0.05, **p<0.01 and ***p<0.001 versus control.
Figure 6.
TE and betulin increase the mRNA half-life time of COX-2 (A, B) and IL-6 (C, D) by modulating RNA stability involving p38 MAPK.
Primary human keratinocytes were treated either with TE (1 µg/mL) (A, C) or betulin (0.87 µg/mL) (B, D) for 24 h or left untreated followed by ActD (5 µg/mL) for the indicated times with or without the p38 MAPK inhibitor LN950 (100 nM). COX-2 and IL-6 mRNA levels were quantified by qRT-PCR. Results are expressed as % of initial (0 h) mRNA, decay curves are applied and RNA half-life times were calculated (E). Values represent means of at least three independent experiments ± s.d. (F) TE and betulin lead to increased phospho-p38 MAPK levels in Western blot. Primary human keratinocytes were treated either with TE (1 and 5 µg/mL) or betulin (0.87 and 4.34 µg/mL, which is in 1 and 5 µg/mL TE, respectively) for 0.5 and 1 h. IL-1β (20 ng/mL) was used as a positive control. Phosphorylated p38 MAPK (p-p38 MAPK) and total p38 MAPK levels were analyzed. The result of the Western blot was reproduced and one representative Western blot is shown.
Figure 7.
TE increases the amount of cytosolic HuR determined by Western blot analysis.
(A). Treatment of human primary keratinocytes with 1 µg/mL TE for 1 to 3 h showed a time dependent increase in cytosolic HuR. IL-1β (20 ng/mL) was used as a positive control, the hyphen indicates untreated cells. Actin was used as a loading control. The result of the Western blot was reproduced and one representative Western blot is shown. (B) TE increases HuR mRNA analysed by qRT-PCR. Primary human keratinocytes were treated with TE (1 µg/mL) for various times. Values represent means of at least two independent experiments ± SEM. **p<0.01 and ***p<0.001 versus control or DMSO.
Figure 8.
TE (1 µg/mL) induces phosphorylation of the transcription factor STAT3 ( = pSTAT3).
The amount of total STAT3 and actin served as loading controls. The result of the Western blot was reproduced and one representative Western blot is shown.
Figure 9.
TE enhances migration of primary human keratinocytes in the scratch assay.
Cells were incubated with 1 µg/mL TE, 10 ng/mL HGF or treated with DMSO, as control. Images were taken immediately after wounding (0 h) and after 8 h incubation (A). The scale bar is of 100 µm width. Representative images of repeated experiments are shown. (B) Closed area in % after 8 h (1 µg/mL TE, 10 ng/mL HGF or untreated control) compared to the scratch area at time point zero (0 h). Values represent mean of closed areas of 4 independent experiments ± s.d. ***p<0.001 versus control.
Figure 10.
TE, betulin and lupeol influence the actin cytoskeleton of primary human keratinocytes.
Cells were incubated on glass coverslips for 2/mL TE and 1, 10 and 100 nM betulin and lupeol, respectively. The actin cytoskeleton was stained with phalloidin-rhodamine. 0.1% (v/v) DMSO was used as solvent control and CNF1 and CNFY as positive controls. Rows labeled with F show the impact on filopodia and lamellipodia and S the impact on stress fiber formation. A white arrow indicates the leading edge of the cell. Representative pictures of repeated experiments (n = 5) are shown.
Figure 11.
Influence of TE (5.1 or 51 ng/mL), betulin (10 or 100 nM) and lupeol (10 or 100 nM) on the activity of the Rho GTP-binding proteins RhoA (A), Cdc42 (B) and Rac1 (C) after 3 h incubation measured by pulldown experiments in primary human keratinocytes.
CNFY and CNF1 were used as positive controls. (D) Influence of the calcium channel blocker verapamil (100 µM) on the activation of RhoA. Verapamil was added 10 min prior to treatment with TE (5.1 ng/mL) and lupeol (10 nM). Each experiment was reproduced and a representative Western blot is shown.