Topically applied hypericin exhibits skin penetrability on nude mice.

Hypericin, a powerful natural photosensitizer in photodynamic therapy (PDT), is suitable for treating skin diseases involving excess capillary proliferation. In the present study, we aimed to evaluate the skin penetrability of topically applied hypericin, expecting a reduced risk of prolonged skin photosensitivity, which often occurs after systemic administration. Firstly, the Franz diffusion cell assays were performed to evaluate the penetration effects of different enhancers, including menthol, propylene glycol, camphanone, azone, and carbamide. In view of above evaluation results, we selected menthol as the enhancer in the subsequent in vivo studies. The setting groups were as follows: the blank control group, the light-exposure control group, the gel-base control group, the hypericin gel group, and a hypericin gel-containing menthol group. Except for the blank control, all other animals were irradiated by a LED light. Then, fluorescence microscopy was performed to examine the distribution of hypericin in the skin of nude mouse. Macroscopic and microscopic analyses were also carried out to detect pathological changes in the skin after topical hypericin-PDT treatment. Immunohistochemistry was used to determine the expression change of PECAM-1. As shown in the results, menthol facilitated hypericin penetrate the skin of nude mice most. The results of in vivo assays revealed that hypericin penetrated nude mouse skin, spread to the dermis, and resulted in obvious photosensitivity reaction on the dermal capillaries. Moreover, skin injured by the photosensitive reaction induced by hypericin-PDT treatment was replaced by normal skin within 7 days. We concluded that topical applied hypericin could penetrate nude mouse skin well and has a great potential in PDT treatment of skin diseases.

The Cellular and Molecular Mechanisms Underlying Silver Nanoparticle/Chitosan Oligosaccharide/Poly(vinyl alcohol) Nanofiber-Mediated Wound Healing.

Wound healing is a complex pathophysiological process that occurs frequently in everyday pathology and remains a challenge during the treatment of trauma. Previously, we prepared silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) (PVA/COS-AgNP) nanofibers via an electrospinning technique. These nanofibers promoted the proliferation of human skin fibroblasts (HSFs) and the expression of transforming growth factor TGF-ß1 in the early stage of wound repair, although the specific mechanisms remain unclear. Therefore, considering that TGF-ß1 has emerged as a major modulator of wound healing, the objective of this study was to further understand whether the molecular mechanisms responsible for PVA/COS-AgNP nanofiber-mediated wound healing include the TGF-ß1/Smad signal transduction pathway. In this study, we used human skin fibroblasts (HSFs) to investigate the molecular and cellular mechanisms underlying PVA/COSAgNP nanofiber-mediated wound healing. Cell adhesion and proliferation experiments, immunofluorescence staining, hydroxyproline content measurements, flow cytometry, quantitative real-time PCR (qRT-PCR), and western blotting (WB) were used to analyze the wound healing mechanisms of human skin fibroblasts treated with various concentrations of PVA/COS-AgNP nanofibers and the combined application of silver nanofibers and SB431542 (an inhibitor of the TGF-ß1 receptor kinase). Our study showed that PVA/COS-AgNP nanofibers markedly promoted fibroblast proliferation, collagen synthesis, and cell adherence. We also found that treating fibroblasts with PVA/COS-AgNP nanofibers stimulated cell cycle progression from G1 into the S and G2 phases, reducing the proportion of cells in the G0/G1 phase and inducing S and G2/M arrest. Importantly, the cell factors associated with the TGF-ß1/Smad signal transduction pathway, such as TGF-ß1, TGFßRI, TGFßRII, pSmad2, pSmad3, collagen I, collagen III, and fibronectin were also up-regulated. Moreover, this enhancing effect was markedly inhibited by the TGFßRI receptor inhibitor, SB431542. Therefore, the PVA/COS-AgNP nanofibers used to accelerate wound healing do so by activating the TGF-ß1/Smad signal transduction pathway.

Topically applied hypericin exhibits skin penetrability on nude mice / 中国药理学与毒理学杂志

OBJECTIVE Hypericin, a powerful naturally photosensitizer in photodynamic therapy (PDT), is suitable for treating skin diseases involving excess capillary proliferation. In the present study, we aimed to evaluate the skin penetrability of a topically applied hypericin, expecting reducing the risk of prolonged skin photosensitivity, which often occurs after systemic administration. METHODS The Franz diffusion cell assay was performed to evaluate different penetration enhancers. In vivo studies, fluorescence microscopy was performed to examine the distribution of hypericin in the skin, macroscopic and microscopic analyses were also carried out to detect pathological changes in the skin after topical hypericin-PDT treatment. Immunohistochemistry was used to determine the expression of PECAM-1 in the treated skin. RESULTS 5% menthol facilitated hypericin penetrate the skin of nude mice most. The results of in vivo assays revealed that hypericin penetrated nude mice skin, spread to the dermis, and resulted in obvious photosensitivity reaction on the dermal capillaries. Moreover, skin injured by the photosensitive reaction induced by hypericin was replaced by normal skin 7 d after hypericin-PDT treat?ment. CONCLUSION Topical hypericin could penetrate nude mouse skin well and be great potential in PDT treatment of skin diseases.

Silver nanoparticles/chitosan oligosaccharide/poly(vinyl alcohol) nanofiber promotes wound healing by activating TGFß1/Smad signaling pathway.

Wound healing occupies a remarkable place in everyday pathology and remains a challenging clinical problem. In our previous study, we prepared a silver nanoparticle/chitosan oligosaccharide/poly(vinyl alcohol) (PVA/COS-AgNPs) nanofiber via electrospinning and revealed that it could promote wound healing; however, the healing mechanism remained unknown. Therefore, we aimed to clarify the mechanism underlying the accelerated healing effect of the PVA/COS-AgNPs nanofiber. The TGFß1/Smad signaling pathway is actively involved in wound healing. Considering the key role of this signaling pathway in wound healing, our preliminary study showed that the TGFß1 level was significantly increased during the early stage of wound healing. Thus, in this study, hematoxylin-eosin, Masson's trichrome, immunofluorescent staining, hydroxyproline content, quantitative real-time polymerase chain reaction, and Western blot analyses were used to analyze the wound healing in a rat model treated with gauze, the PVA/COS-AgNPs nanofiber, and the nanofiber plus SB431542 (an inhibitor of TGFß1 receptor kinase). The results showed that the PVA/COS-AgNPs nanofiber promoted wound healing and upregulated the expression levels of cytokines associated with the TGFß1/Smad signaling pathway such as TGFß1, TGFßRI, TGFßRII, collagen I, collagen III, pSmad2, and pSmad3. Inhibiting this pathway with SB431542 resulted in prevention of the PVA/COS-AgNPs nanofiber-associated salutary effects on the early stage of wound healing and relative cytokines expression. In conclusion, the wound healing effect of the PVA/COS-AgNPs nanofiber involves activation of the TGFß1/Smad signaling pathway.

Hypericin-photodynamic therapy induces human umbilical vein endothelial cell apoptosis.

The conventional photosensitizers used in photodynamic therapy (PDT), such as haematoporphyrin (HP), have not yet reached satisfactory therapeutic effects on port-wine stains (PWSs), due largely to the long-term dark toxicity. Previously we have showed that hypericin exhibited potent photocytotoxic effects on Roman chicken cockscomb model of PWSs. However, the molecular mechanism of hypericin-mediated photocytotoxicity remains unclear. In this study, we employed human umbilical vein endothelial cells (HUVECs) to investigate the hypericin-photolytic mechanism. Our study showed that hypericin-PDT induced reactive oxygen species (ROS), resulting in cell killings and an activation of the inflammatory response. Importantly, we have also discovered that photoactivated hypericin induced apoptosis by activating the mitochondrial caspase pathway and inhibiting the activation of the vascular endothelial growth factor-A (VEGF-A)-mediated PI3K/Akt pathway. Notably, we found that hypericin exhibited a more potent photocytotoxic effect than HP, and largely addressed the inconvenience issue associated with the use of HP. Thereby, hypericin may be a better alternative to HP in treating PWSs.

Hypericin damages the ectatic capillaries in a Roman cockscomb model and inhibits the growth of human endothelial cells more potently than hematoporphyrin does through induction of apoptosis.

Hypericin (HY) is a promising photosensitizer (PS) for use in photodynamic therapy (PDT). Port-wine stains (PWSs) are congenital superficial dermal capillary malformations. In this study, we evaluated the photocytotoxic effects of HY for PDT in human vascular endothelial cells and a chicken cockscomb model. HY significantly inhibited the growth of human umbilical vein endothelial cells (HUVECs), as determined by colorimetric assays and morphological observation, and flow cytometry assays indicated induction of apoptosis and collapse of the mitochondrial membrane potential. In addition, HY more effectively inhibited growth of and induced apoptosis in HUVECs compared with hematoporphyrin (HP). Further experiments performed in a Roman chicken cockscomb model also showed a clear photocytotoxic effect on the cockscomb dermal capillary upon intravenous injection of HY. This effect may be due to the role of HY in the induction of apoptosis. Transmission electron microscopical analysis showed mitochondrial morphological changes such as incomplete ridges and swelling, and immunohistochemical assays showed an increase in the release of cytochrome c. In conclusion, HY exhibited a greater photocytotoxic activity than did HP toward the growth of endothelial cells and may thus represent a potent PS for PWS PDT.

Optimization of extraction and purification of arctiin from Fructus arctii and its protection against glucose-induced rat aortic endothelial cell injury.

To develop an efficient method for extracting and purifying the active ingredient, arctiin, from Fructus arctii and to investigate the protective effect of arctiin against glucose-induced rat aortic endothelial cell (RAEC) injury was investigated. Using a L9 (34) orthogonal array and two-step column chromatography (with AB-8 macroporous resin) arctiin extraction was optimized using a reflux method with 70% ethanol. The RAECs were then treated with different concentrations of arctiin (1, 10, or 100 µg/ml). The effects of arctiin on cell viability in a high glucose medium, malondialdehyde (MDA) levels, and lactate dehydrogenase were measured using commercially available assays. After extraction, the purity of arctiin reached 95.7%. In rats, arctiin was shown to stimulate the proliferation of RAECs in a high glucose medium in a dose-dependent manner. Exposure of RAECs to high glucose resulted in a significant increase in MDA and release of lactate dehydrogenase. This was accompanied by significant increase in nitric oxide release and expression of antiendothelial nitric oxide synthase. This technique resulted in relatively pure arctiin extraction. Furthermore, the results from this study suggest that arctiin could potentially function as a protector against vascular endothelial cell injury and further investigation is warranted.

Effects of arctiin on streptozotocin-induced diabetic retinopathy in Sprague-Dawley rats.

Diabetic retinopathy is one of the most common and severe complications of diabetes mellitus. Arctiin, a bioactive compound isolated from the dry seeds of Arctium lappa L., has been reported to have antidiabetic activity. In this study, we investigated the effect of arctiin on the serum glucose and HBA1c levels, the blood viscosity, and VEGF expression in the retinal tissues of rats with diabetic retinopathy. We first extracted arctiin from Fructus Arctii and then investigated its chemopreventive effect on streptozotocin-induced diabetic retinopathy in male Sprague-Dawley rats. After the induction of diabetes using streptozotocin (30 mg/kg, i. p.), the rats were randomly divided into five groups (n = 20 per group) and treated with intragastric doses of 30, 90, or 270 mg/kg/d wt of arctiin, 100 mg/kg/d wt of calcium dobesilate, or 0.5 % CMC-Na. Twenty nondiabetic sham-treated rats were treated with 0.5 % CMC-Na. The occurrence of diabetic retinopathy did not differ dramatically among the groups. However, at week 16, the glycosylated haemoglobin (HBA1c) level was significantly decreased in all of the arctiin-treated groups when compared with the control group, and the serum glucose level was also decreased in the rats treated with the highest dose of arctiin. In addition, treatment with arctiin ameliorated retinal oedema, detachment of the retina, and VEGF expression in the retina, as detected using histological and immunochemical examinations. Finally, arctiin increased the viability of retinal microvascular endothelial cells in vitro. Together, these findings demonstrate that arctiin decreases the severity of diabetic complications, demonstrating the importance of this compound as an inhibitor of diabetic retinopathy.