Anti-inflammatory effects of Polygonum minus (Huds) extract (Lineminus™) in in-vitro enzyme assays and carrageenan induced paw edema.

BACKGROUND: The study was aimed to evaluate the anti-inflammatory activity of ethanolic and aqueous extracts of Polygonum minus (Huds) using in vitro and in vivo approaches. METHODS: The in vitro tests used to evaluate ethanolic extract are cyclooxygenase-1 (COX-1), cyclooxygenase-2 (COX-2), lipooxygenase (5-LOX), secretory phospholipase-A2 (sPLA2) inhibition assay whilst the in-vivo effect was measured by the ability of aqueous extracts to reduce paw edema induced by λ-carrageenan, in rats. RESULTS: The ethanolic extract inhibited the activities of 5-LOX and COX-1(p < 0.05) whilst the inhibitory effect on COX-2 was only moderate. A marked inhibition of 5-LOX was observed at 30 µg/ ml. The extract did not inhibit the activity of sPLA2. The ability of the ethanolic extracts of Polygonum minus to inhibit both 5-LOX and COX, prompted a study to evaluate the effects of using an aqueous extract of Polygonum minus(LineminusTM); as this would be more suitable for future clinical testing. The anti-inhibitory activity of the aqueous extract from this plant was evaluated using a rat model where inflammation was induced in the paws by injection of λ-carrageenan. The aqueous extracts from Polygonum minus administered at doses of 100 and 300 mg/kg body weight (b.w.), significantly (p < 0.01) reduced paw edema induced by λ-carrageenan in the experimental model, at 4 h compared to the vehicle control. Furthermore, administration of 100 mg/kg b.w. or 300 mg/kg b.w. completely reduced inflammation of the paw 4 h after injection. CONCLUSION: These findings suggest that aqueous extract of Polygonum minus possesses potent anti-inflammatory activities.

Stiffening of human skin fibroblasts with age.

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Stiffening of human skin fibroblasts with age.

Changes in mechanical properties are an essential characteristic of the aging process of human skin. Previous studies attribute these changes predominantly to the altered collagen and elastin organization and density of the extracellular matrix. Here, we show that individual dermal fibroblasts also exhibit a significant increase in stiffness during aging in vivo. With the laser-based optical cell stretcher we examined the viscoelastic biomechanics of dermal fibroblasts isolated from 14 human donors aged 27 to 80. Increasing age was clearly accompanied by a stiffening of the investigated cells. We found that fibroblasts from old donors exhibited an increase in rigidity of ∼60% with respect to cells of the youngest donors. A FACS analysis of the content of the cytoskeletal polymers shows a shift from monomeric G-actin to polymerized, filamentous F-actin, but no significant changes in the vimentin and microtubule content. The rheological analysis of fibroblast-populated collagen gels demonstrates that cell stiffening directly results in altered viscoelastic properties of the collagen matrix. These results identify a new mechanism that may contribute to the age-related impairment of elastic properties in human skin. The altered mechanical behavior might influence cell functions involving the cytoskeleton, such as contractility, motility, and proliferation, which are essential for reorganization of the extracellular matrix.

Inhibition of TRPV1 for the treatment of sensitive skin.

During the past years, the topic sensitive skin became one of the most important fields in dermatology. The tremendous interest is based on several studies showing that about 50% of the population declares to have sensitive skin. The human thermoreceptor hTRPV1 was previously identified to contribute to this skin condition while facilitating neurogenic inflammation leading to hyperalgesia. Furthermore, skin sensitivity towards capsaicin, a natural activator of TRPV1, was shown to correlate with sensitive skin. In a screening campaign based on recombinant HEK293-cells stably transfected with hTRPV1, the selective antagonist trans-4-tert-butylcyclohexanol was identified. This antagonist is able to inhibit capsaicin-induced hTRPV1 activation with an IC(50) value of 34 ± 5 µm tested in HEK293-cells as well as in electrophysiological recordings performed in oocytes expressing hTRPV1. Strikingly, in a clinical study with 30 women using topical treatment with o/w emulsions containing 31.6 ppm capsaicin, we were able to show that 0.4% of this inhibitor significantly reduces capsaicin-induced burning (P < 0.0001) in vivo. Thus trans-4-tert-butylcyclohexanol has the potential as a novel bioactive for the treatment of sensitive skin.

Modification of vimentin: a general mechanism of nonenzymatic glycation in human skin.

In a recent study, we were able to show that the intermediate filament protein vimentin aggregates in human dermal fibroblasts because of modification by the advanced glycation endproduct carboxymethyllysine (CML). In this work, we investigated the formation of intracellular CML in relation to the concentration of glucose in the culture medium. The natural degradation product of glucose, methylglyoxal, was able to induce the aggregation of vimentin. This dicarbonyl leads to the formation of the modifications MG-H1 and carboxyethyllysine (CEL) as a result of the reaction with arginine and lysine residues of proteins. Furthermore, we found that the protein vimentin was modified, not only by CML and CEL, but also by pentosidine and pyrraline. These findings underline the special position of vimentin as a preferential target of the Maillard reaction in human skin.

Inhibition of cytosolic and mitochondrial creatine kinase by siRNA in HaCaT- and HeLaS3-cells affects cell viability and mitochondrial morphology.

The creatine kinase (CK) system is essential for cellular energetics in tissues or cells with high and fluctuating energy requirements. Creatine itself is known to protect cells from stress-induced injury. By using an siRNA approach to silence the CK isoenzymes in human keratinocyte HaCaT cells, expressing low levels of cytoplasmic CK and high levels of mitochondrial CK, as well as HeLa cancer cells, expressing high levels of cytoplasmic CK and low levels of mitochondrial CK, we successfully lowered the respective CK expression levels and studied the effects of either abolishing cytosolic brain-type BB-CK or ubiquitous mitochondrial uMi-CK in these cells. In both cell lines, targeting the dominant CK isoform by the respective siRNAs had the strongest effect on overall CK activity. However, irrespective of the expression level in both cell lines, inhibition of the mitochondrial CK isoform generally caused the strongest decline in cell viability and cell proliferation. These findings are congruent with electron microscopic data showing substantial alteration of mitochondrial morphology as well as mitochondrial membrane topology after targeting uMi-CK in both cell lines. Only for the rate of apoptosis, it was the least expressed CK present in each of the cell lines whose inhibition led to the highest proportion of apoptotic cells, i.e., downregulation of uMi-CK in case of HeLaS3 and BB-CK in case of HaCaT cells. We conclude from these data that a major phenotype is linked to reduction of mitochondrial CK alone or in combination with cytosolic CK, and that this effect is independent of the relative expression levels of Mi-CK in the cell type considered. The mitochondrial CK isoform appears to play the most crucial role in maintaining cell viability by stabilizing contact sites between inner and outer mitochondrial membranes and maintaining local metabolite channeling, thus avoiding transition pore opening which eventually results in activation of caspase cell-death pathways.

Vimentin is the specific target in skin glycation. Structural prerequisites, functional consequences, and role in skin aging.

Until now, the glycation reaction was considered to be a nonspecific reaction between reducing sugars and amino groups of random proteins. We were able to identify the intermediate filament vimentin as the major target for the AGE modification N(epsilon)-(carboxymethyl)lysine (CML) in primary human fibroblasts. This glycation of vimentin is neither based on a slow turnover of this protein nor on an extremely high intracellular expression level, but remarkably it is based on structural properties of this protein. Glycation of vimentin was predominantly detected at lysine residues located at the linker regions using nanoLC-ESI-MS/MS. This modification results in a rigorous redistribution of vimentin into a perinuclear aggregate, which is accompanied by the loss of contractile capacity of human skin fibroblasts. CML-induced rearrangement of vimentin was identified as an aggresome. This is the first evidence that CML-vimentin represents a damaged protein inside the aggresome, linking the glycation reaction directly to aggresome formation. Strikingly, we were able to prove that the accumulation of modified vimentin can be found in skin fibroblasts of elderly donors in vivo, bringing AGE modifications in human tissues such as skin into strong relationship with loss of organ contractile functions.