Non‐invasive evaluation techniques to quantify the efficacy of cosmetic anti‐cellulite products1

Background: The majority of women suffer from the unattractive sight of dimpling skin on the thighs and buttocks, globally known as cellulite. Cellulite can be regarded as the most investigated non‐disease, because, from the cosmetic viewpoint, most women desire a reduction in cellulite severity. Despite investigations made, cellulite is still not well understood at the cellular level, which leads to controversy regarding the investigative methods for cellulite reduction as well as the development of products to treat cellulite skin. Objective: The aim of our work was to improve the set up of macrophotography for making images of dimpled skin and to automatize image analysis of 20 MHz ultrasound imaging – these two methods being just two of a variety of available methods for investigating cellulite skin. Methods: Macrophotography was standardized on the aspects of volunteer's positioning, skin illumination, background used, and camera position. It was performed before, during and after a 3‐month‐treatment of a cosmetic product. Scoring assessments of the generated images were made by the volunteers themselves as well as by six trained experts. Ultrasound imaging was performed at the baseline visit in order to correlate the newly developed analysis with the visually rated cellulite score. A second study is also presented showing a variety of parameters that can be used for cosmetic testing of cellulite products: skin firmness, blood circulation and circumferential thigh measurements. Results: Standardization of macrophotography minimized differences in image features between assessment times, therefore, enabling follow‐up rating assessments of the images. A custom‐made rating program simplified the scoring procedure by presenting images as blind and randomized, and by implementing computer‐based analysis using an online rating scale. Volunteers and experts scored significant improvement of skin appearance over the course of a 3‐month cosmetic treatment. Image analysis of ultrasound imaging was automatized, and a modification of the commonly known roughness parameter Ra was implemented to characterize cellulite severity. Comparison with the visually rated cellulite score showed an existing correlation between the score and the modified parameter Ra_m. Further parameters investigated in an exemplary study, as mentioned above, demonstrated a significant improvement of skin appearance after treatment with a cosmetic product. Conclusions: Macrophotography and ultrasound imaging can be regarded as important tools for determining and quantifying the aspects of cellulite. With a gold standard missing for investigating cellulite severity, these two methods may not determine cellulite at the cellular level, but they do characterize the skin appearance so typical for cellulite. Combined with a variety of other methods, macrophotography and ultrasound imaging can very well define cellulite‐reducing efficacy from the cosmetic point of view.     

Dexamethasone decreases temozolomide-induced apoptosis in human gliobastoma T98G cells

Human glioblastoma is a deadly brain tumor that is often treated with a combination of drugs. A new alkylating agent, temozolomide (TMZ), has recently been found efficacious in the clinical trials for glioblastoma. Steroids, such as dexamethasone (DXM), are often used concomitantly as a supportive therapy to treat cerebral edema. However, any possible modulatory effect of the steroids on the efficacy of TMZ has not yet been evaluated experimentally. In this study, we have examined whether DXM provides synergistic or antagonistic effect on TMZ-induced apoptosis in human glioblastoma T98G cells. T98G cells were pretreated with various doses of DXM followed by TMZ. The cell viability was assessed by the trypan blue dye exclusion test. Wright staining and the TdT-mediated dUTP nick-end labeling (TUNEL) assay were used to evaluate apoptotic cell death based on the morphological and biochemical (DNA fragmentation) features, respectively. More biochemical features of apoptotic death, such as upregulation of Bax:Bcl-2 ratio, calpain activity, and caspase-3 activity, were assessed by Western blot analysis. A significant number of T98G cells committed apoptosis after treatment with 200 microM TMZ. However, a pretreatment with 100 microM or 200 microM DXM protected T98G cells against TMZ-induced apoptosis, concomitantly decreasing Bax:Bcl-2 ratio, calpain activity, and caspase-3 activity. These experimental results indicate that DXM works as an antagonistic agent in combination with TMZ. Therefore, our investigation strongly implies that the combination of DXM and TMZ may be counteractive in treating human glioblastoma.     

Calpain activation in apoptosis of ventral spinal cord 4.1 (VSC4.1) motoneurons exposed to glutamate: calpain inhibition provides functional neuroprotection

Glutamate toxicity has been implicated in cell death in neurodegenerative diseases and injuries. Glutamate-induced Ca2+ influx may mediate activation of calpain, a Ca2+-dependent cysteine protease, which in turn may degrade key cytoskeletal proteins. We investigated glutamate-mediated apoptosis of VSC4.1 motoneurons and functional neuroprotection by calpain inhibition. Exposure of VSC4.1 cells to 10 microM glutamate for 24 hr caused significant increases in intracellular free [Ca2+], as determined by fura-2 assay. Pretreatment of cells with 10 or 25 microM calpeptin (a cell-permeable calpain-specific inhibitor) for 1 hr prevented glutamate-induced Ca2+ influx. Western blot analyses showed an increase in Bax:Bcl-2 ratio, release of cytochrome c from mitochondria, and calpain and caspase-3 activities during apoptosis. Cell morphology, as evaluated by Wright staining, indicated predominantly apoptotic features following glutamate exposure. ApopTag assay further substantiated apoptotic features morphologically as well as biochemically. Our data showed that calpeptin mainly prevented calpain-mediated proteolysis and apoptosis and maintained whole-cell membrane potential, indicating functional neuroprotection. The results imply that calpeptin may serve as a therapeutic agent for preventing motoneuron degeneration, which occurs in amyotrophic lateral sclerosis and spinal cord injury. In this investigation, we also examined glutamate receptor subtypes involved in the initiation of apoptosis in VSC4.1 cells following exposure to glutamate. Our results indicated that the N-methyl-D-aspartate (NMDA) receptors contributed more than alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA) receptors to glutamate-mediated Ca2+ influx and cell death mechanism. Inhibition of the activities of both NMDA and AMPA receptors protected VSC4.1 cells from glutamate toxicity and preserved whole-cell membrane potential.     

A macromolecular complex of beta 2 adrenergic receptor,CFTR, and ezrin/radixin/moesin-binding phosphoprotein 50 is regulated by PKA

It has been demonstrated previously that both the cystic fibrosis transmembrane conductance regulator (CFTR) and beta(2) adrenergic receptor (beta(2)AR) can bind ezrinradixinmoesin-binding phosphoprotein 50 (EBP50, also referred to as NHERF) through their PDZ motifs. Here, we show that beta(2) is the major adrenergic receptor isoform expressed in airway epithelia and that it colocalizes with CFTR at the apical membrane. beta(2)AR stimulation increases CFTR activity, in airway epithelial cells, that is glybenclamide sensitive. Deletion of the PDZ motif from CFTR uncouples the channel from the receptor both physically and functionally. This uncoupling is specific to the beta(2)AR receptor and does not affect CFTR coupling to other receptors (e.g., adenosine receptor pathway). Biochemical studies demonstrate the existence of a macromolecular complex involving CFTR-EBP50-beta(2)AR through PDZ-based interactions. Assembly of the complex is regulated by PKA-dependent phosphorylation. Deleting the regulatory domain of CFTR abolishes PKA regulation of complex assembly. This report summarizes a macromolecular signaling complex involving CFTR, the implications of which may be relevant to CFTR-dysfunction diseases.     

Tannic acid treatment enhances biostability and reduces calcification of glutaraldehyde fixed aortic wall

Progressive degeneration and calcification of glutaraldehyde (Glut) fixed tissues used in cardiovascular surgery restrict their long-term clinical performance. This limited biological stability may be attributable to the inability of Glut to adequately protect certain tissue components such as elastin from enzymatic attack. The aim of our studies was to develop novel tissue-processing techniques targeted specifically at elastin stabilization by using tannic acid (TA), a plant polyphenol capable of protecting elastin from digestion by specific enzymes. In present studies we demonstrated that Glut does not adequately protect porcine aorta from elastase-mediated degradation in vitro. The addition of TA to the Glut fixation process increased the stability of Glut-fixed aorta to elastase digestion by 15-fold and also decreased calcification in the rat subdermal model by 66%. TA was found to be chemically compatible with Glut fixation and did not hinder collagen crosslinking as shown by minor changes in thermal denaturation temperatures, resistance to collagenase and mechanical properties. In vitro and in vivo studies also revealed that TA binding to aortic wall was stable over an extended period of time. TA-mediated elastin stabilization in Glut-fixed cardiovascular implants may significantly extend the clinical durability of these tissue replacements.     

HPLC法测定蒙成药三子散中没食子酸的含量

目的:建立蒙成药三子散中没食子酸的含量测定方法.方法:采用HPLC法,采用Phenomenex C18色谱柱(250mm×4.6 mm,5μm),流动相为甲醇-0.1％磷酸水溶液(5:95),流速1.0 mL(min-1,柱温35℃,进样量为10μL,检测波长为273nm.结果:没食子酸进样量范围在6.357～158....     

Estrogen attenuates oxidative stress-induced apoptosis in C6 glial cells

We examined the mechanism of 17beta-estradiol (estrogen)-mediated inhibition of apoptosis in C6 (rat glioma) cells following exposure to hydrogen peroxide (H(2)O(2)). Cells were preincubated with 4 microM estrogen for 2 h and then exposed to 100 microM H(2)O(2) for 24 h. Exposure to H(2)O(2) caused significant increases in intracellular calcium (Ca(2+)), as determined by fura-2, which was attenuated by preincubation with estrogen. H(2)O(2) and ionomycin caused cell death in a dose-dependent manner, as measured by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Preincubation with estrogen restored viability in cells exposed to H(2)O(2) but not in cells exposed to ionomycin. Western blot analysis showed an increase in Bax/Bcl-2 ratio, calpain activity, and caspase-3 activity following treatment with H(2)O(2), and estrogen pretreatment decreased levels of all three. Cell morphology, as evaluated by Wright staining, indicated apoptosis in cells treated with H(2)O(2), and pretreatment with estrogen reduced apoptosis. Results from MTT and Wright staining were further supported by the terminal deoxyribonucleotidyl transferase (TdT)-mediated dUTP Nick End Labeling (TUNEL) assay. These results indicate a role for estrogen in preventing apoptosis in C6 glial cells exposed to H(2)O(2). Our results suggest that estrogen may have a protective role in minimizing glial cell apoptosis in neurological diseases such as demyelinating disease or central nervous system trauma.     

Estrogen attenuated markers of inflammation and decreased lesion volume in acute spinal cord injury in rats

Spinal cord injury (SCI) is a devastating neurologic injury with functional deficits for which the only currently recommended pharmacotherapy is high-dose methylprednisolone, which has limited efficacy. Estrogen is a multi-active steroid that has shown antiinflammatory and antioxidant effects, and estrogen may modulate intracellular Ca(2+) and attenuate apoptosis. For this study, male rats were divided into three groups. Sham group animals received a laminectomy at T12. Injured rats received both laminectomy and 40 g x cm force SCI. Estrogen-group rats received 4 mg/kg 17beta-estradiol (estrogen) at 15 min and 24 hr post-injury, and vehicle-group rats received equal volumes of dimethyl sulfoxide (vehicle). Animals were sacrificed at 48 hr post-injury, and 1-cm-long segments of the lesion, rostral penumbra, and caudal penumbra were excised. Inflammation was assessed by examining tissue edema, infiltration of macrophages/microglia, and levels of cytosolic and nuclear NFkappaB and inhibitor of kappa B (IkappaBalpha). Myelin integrity was examined using Luxol fast blue staining. When compared to sham, vehicle-treated animals revealed increased tissue edema, increased infiltration of inflammatory cells, decreased cytosolic levels of NFkappaB and IkappaBalpha, increased levels of nuclear NFkappaB, and increased myelin loss. Treatment of SCI rats with estrogen reduced edema and decreased inflammation and myelin loss in the lesion and penumbral areas, suggesting its potential as a therapeutic agent. Further work needs to be done, however, to elucidate the neuroprotective mechanism of estrogen.