RNAseq analysis of treatment-dependent signaling changes during inflammation in a mouse cutaneous wound healing model

Background Despite proven therapeutic effects in inflammatory conditions, the specific mechanisms of phytochemical therapies are not well understood. The transcriptome effects of Traumeel (Tr14), a multicomponent natural product, and diclofenac, a non-selective cyclooxygenase (COX) inhibitor, were compared in a mouse cutaneous wound healing model to identify both known and novel pathways for the anti-inflammatory effect of plant-derived natural products. Methods Skin samples from abraded mice were analyzed by single-molecule, amplification-free RNAseq transcript profiling at 7 points between 12 and 192 h after injury. Immediately after injury, the wounds were treated with either diclofenac, Tr14, or placebo control (n = 7 per group/time). RNAseq levels were compared between treatment and control at each time point using a systems biology approach. Results At early time points (12–36 h), both control and Tr14-treated wounds showed marked increase in the inducible COX2 enzyme mRNA, while diclofenac-treated wounds did not. Tr14, in contrast, modulated lipoxygenase transcripts, especially ALOX12/15, and phospholipases involved in arachidonate metabolism. Notably, Tr14 modulated a group of cell-type specific markers, including the T cell receptor, that could be explained by an overarching effect on the type of cells that were recruited into the wound tissue. Conclusions Tr14 and diclofenac had very different effects on the COX/LOX synthetic pathway after cutaneous wounding. Tr14 allowed normal autoinduction of COX2 mRNA, but suppressed mRNA levels for key enzymes in the leukotriene synthetic pathway. Tr14 appeared to have a broad ‘phytocellular’ effect on the wound transcriptome by altering the balance of cell types present in the wound.

Curcumin upregulates transforming growth factor-β1, its receptors, and vascular endothelial growth factor expressions in an in vitro human gingival fibroblast wound healing model

Background Curcumin accelerates healing of oral wounds; however, the responsible mechanisms remain underexplored. Our hypothesis is curcumin regulates the expression of wound healing-related genes in human gingival fibroblasts (hGFs). This study investigated whether curcumin regulates transforming growth factor (TGF)-β1, type I TGF-β receptor (TGF-βRI), type II TGF-β receptor (TGF-βRII), and vascular endothelial growth factor (VEGF) expression in unwounded hGFs and an in vitro hGF wound healing model. Methods The cytotoxicity of curcumin was evaluated using the MTT assay. Unwounded hGFs were treated with non-cytotoxic concentrations of curcumin for 24 h. Gene expression was determined by quantitative polymerase chain reaction. Then, hGFs were treated with 1 µM curcumin in an in vitro wound healing model. PD98059 pretreatment was performed to determine whether extracellular signal-regulated kinase (ERK) signaling was required for regulation of gene expression by curcumin. Results Curcumin at 0.1–20 µM caused no significant change in cell viability. In unwounded hGFs, curcumin had no significant effect on TGF-β1, TGF-βRI, TGF-βRII, or VEGF expression. Conversely, curcumin significantly upregulated the expression of these genes in the in vitro wound healing model. PD98059 significantly attenuated the curcumin-stimulated TGF-βRI, TGF-βRII, and VEGF expression, whereas it had no effect on TGF-β1 expression. Conclusions Curcumin upregulated TGF-β1, TGF-βRI, TGF-βRII, and VEGF expression in an in vitro hGF wound healing model. The ERK pathway is required for TGF-βRI, TGF-βRII, and VEGF induction by curcumin. Our findings support the development of curcumin as a therapeutic agent for gingival ulcers.

A Comparative Analysis of the Structure and Biological Properties of Films and Microfibrous Scaffolds Based on Silk Fibroin

A comparative analysis of the structure and biological properties of silk fibroin constructions was performed. Three groups of constructions were obtained: films obtained by casting an aqueous solution of silk fibroin and electrospun microfibrous scaffolds based on silk fibroin, with the addition of 30% gelatin per total protein weight. The internal structures of the films and single fibers of the microfibrous scaffolds consisted of densely packed globule structures; the surface area to volume ratios and volume porosities of the microfibrous scaffolds were calculated. All constructions were non-toxic for cells and provide high levels of adhesion and proliferation. The high regenerative potential of the constructions was demonstrated in a rat full-thickness skin wound healing model. The constructions accelerated healing by an average of 15 days and can be considered to be promising constructions for various tasks of tissue engineering and regenerative medicine.

Wound Healing Activities of Hydromethanolic Crude Extract and Solvent Fractions of Bersama abyssinica Leaves in Mice

Background. Bersama abyssinica leaves are traditionally used for management of wounds in several communities of Ethiopia, despite no scientifically approved studies done on wound healing. Our study planned to work out the wound healing effects of B. abyssinica leaves extract in mice. Methods. B. abyssinica leaves were extracted with 80% hydromethanol and fractioned with chloroform, hexane, and water. The acute dermal toxicity of the crude extract was evaluated in mice. The crude extract formulated at 5% and 10% w/w ointment was investigated in excision, incision, and burn wound models and solvent fractions in the excision model using simple ointment (negative control) and nitrofurazone 0.2% w/v (positive control). We evaluated histopathological analysis, wound contraction rate, complete epithelialization period, and skin durability. One-way ANOVA followed by the post hoc Tukey HSD test with IBM SPSS software version 23.0 was used for data analysis, and p < 0.05 was considered statistically significant. Results. Hydromethanolic crude extract produced 5% (99.5%) and 10% (100%) wound contraction on the 16th day of the treatment and 5% (18.8) and 10% (28.2) percent reduction in the epithelization period on the excision wound healing model. Hydromethanolic crude extract produced 5% (47.5) and 10% (61.17) percent durability on the incision wound healing model. Hydromethanolic crude extract produced 5% (99.82%) and 10% (100%) wound contraction on the 20th day of treatment and 5% (13%) and 10% (21.7%) reduction in the epithelization period on the burn wound healing model. The chloroform fraction produced 5% (90.17%) and 10% (91.01%), hexane fraction produced 5% (85.81%) and 10% (86.78%), and aqueous fraction produced 5% (99.17%) and 10% (99.38%) wound contraction on the 14th day of the treatment and 5% (18.8) and 10% (28.2) percent reduction in the epithelization period on the excision wound healing model. Both hydromethanolic crude extract and solvent fractions at 5% and 10% (w/w) were significant ( p < 0.001) compared with negative control. Conclusion. The results of this study showed that both 5% w/w and 10% w/w of 80% hydromethanolic crude extract and solvent fractions of B. abyssinica leaves have wound healing effects.

RNAseq Analysis of Treatment-dependent Signaling Changes During Inflammation in a Mouse Cutaneous Wound Healing Model.

Background: Despite proven therapeutic effects in inflammatory conditions, the specific mechanisms of phytochemical therapies are not well understood. The transcriptome effects of Tr14 (Traumeel), a multicomponent natural product, and diclofenac, a non-selective cyclooxygenase (COX) inhibitor, were compared in a mouse cutaneous wound healing model to identify both known and novel pathways for the anti-inflammatory effect of plant-derived natural products. Methods: Skin samples from abraded mice were analyzed by single-molecule, amplification-free RNAseq transcript profiling at 7 points between 12-192 hours after injury. Immediately after injury, the wounds were treated with either diclofenac, Tr14, or placebo control (n=7 per group/time). RNAseq levels were compared between treatment and control at each time point using a systems biology approach. Results: At early time points (12-36 hours), both control and Tr14-treated wounds showed marked increase in the inducible COX2 enzyme mRNA, while diclofenac-treated wounds did not. Tr14, in contrast, modulated lipoxygenase transcripts, especially ALOX12/15, and phospholipases involved in arachidonate metabolism. Notably, Tr14 modulated a group of cell-type specific markers, including the T cell receptor, that could be explained by an overarching effect on the type of cells that were recruited into the wound tissue. Conclusions: Tr14 and diclofenac had very different effects on the COX/LOX synthetic pathway after cutaneous wounding. Tr14 allowed normal autoinduction of COX2 mRNA, but suppressed mRNA levels for key enzymes in the leukotriene synthetic pathway. Tr14 appeared to have a broad ‘phytocellular’ effect on the wound transcriptome by altering the balance of cell types present in the wound.

Force-FAK signaling coupling at individual focal adhesions coordinates mechanosensing and microtissue repair

How adhesive forces are transduced and integrated into biochemical signals at focal adhesions (FAs) is poorly understood. Using cells adhering to deformable micropillar arrays, we demonstrate that traction force and FAK localization as well as traction force and Y397-FAK phosphorylation are linearly coupled at individual FAs on stiff, but not soft, substrates. Similarly, FAK phosphorylation increases linearly with external forces applied to FAs using magnetic beads. This mechanosignaling coupling requires actomyosin contractility, talin-FAK binding, and full-length vinculin that binds talin and actin. Using an in vitro 3D biomimetic wound healing model, we show that force-FAK signaling coupling coordinates cell migration and tissue-scale forces to promote microtissue repair. A simple kinetic binding model of talin-FAK interactions under force can recapitulate the experimental observations. This study provides insights on how talin and vinculin convert forces into FAK signaling events regulating cell migration and tissue repair.