Anti-Inflammatory Activity of No-Ozone Cold Plasma in Porphyromonas gingivalis Lipopolysaccharide-Induced Periodontitis Rats.

Periodontitis is an inflammatory disease caused by Porphyromonas gingivalis (P. gingivalis) in the oral cavity. This periodontal disease causes damage to the periodontal ligament and alveolar bone and can cause tooth loss, but there is no definite treatment yet. In this study, we investigated the possibility of using no-ozone cold plasma to safely treat periodontitis in the oral cavity. First, human gingival fibroblasts (HGFs) were treated with P. gingivalis-derived lipopolysaccharide (PG-LPS) to induce an inflammatory response, and then the anti-inflammatory effect of NCP was examined, and a study was conducted to identify the mechanism of action. Additionally, the anti-inflammatory effect of NCP was verified in rats that developed an inflammatory response similar to periodontitis. When NCP was applied to PG-LPS-treated HGFs, the activities of inflammatory proteins and cytokines were effectively inhibited. It was confirmed that the process of denaturing the medium by charged particles of NCP is essential for the anti-inflammatory effect of NCP. Also, it was confirmed that repeated treatment of periodontitis rats with NCP effectively reduced the inflammatory cells and osteoclast activity. As a result, this study suggests that NCP can be directly helpful in the treatment of periodontitis in the future.

No-ozone cold plasma induces apoptosis in human neuroblastoma cell line via increased intracellular reactive oxygen species (ROS).

BACKGROUND: This study aimed to evaluate the effect of argon-based No-ozone Cold Plasma (NCP) on neuroblastoma cancer cell apoptosis. METHODS: Experiments were performed with SK-N-SH and HS 68. Cell cultures were treated with NCP for 1, 3, and 5 min. NCP was applied using three different strategies: direct NCP application to cell cultures, to only media, and to only cells. Evaluation of cell viability and the level of the reactive oxygen species (ROS) was performed. N-acetyl-L-cysteine (NAC) was also used to antagonize intracellular ROS. Cleaved caspase 3, PARP, aquaporin (AQP) 3 and 8 were detected. RESULTS: NCP induced a gradual decrease in the SK-N-SH cell viability. In contrast, the viability of HS 68 cells did not change. SK-N-SH cells viability was reduced the most when the only media-NCP application strategy was employed. Intracellular ROS levels were significantly increased with time. Cleaved caspase 3 and PARP were increased at 6 h after NCP application. SK-N-SH cells remained viable with NAC after NCP application. AQP 3 and 8 were over-expressed in SK-N-SH cells. CONCLUSION: These findings demonstrate the anti-cancer effect of NCP on neuroblastoma cells. NCP enhanced the selective apoptosis of neuroblastoma cells due to the increased intracellular ROS.

Topical Application of No-Ozone Cold Plasma in Combination with Vitamin C Reduced Skin Redness and Pigmentation of UV-Irradiated Mice.

Ultraviolet (UV) is the main cause of sunburn on the skin as it induces erythema and accelerates pigmentation. Vitamin C is one of the most frequently used compounds to reduce UV-induced skin pigmentation, but it has limitations in absorption through the skin. In this study, we tested whether a no-ozone cold plasma (NCP) treatment can improve UV-irradiated skin by helping the action of Vitamin C. For this, among five groups of HRM-2 hairless mice, four groups of mice were subjected to UVB irradiation, and three groups of UVB-treated mice were treated with NCP, Vitamin C, and NCP + Vitamin C, respectively. For evaluating the effect of each treatment, the melanin and erythema index was measured during animal experiments. Histological changes were monitored by performing H&E and MTS and IHC against tyrosinase and melanin. As a result, the naturally recovered mice showed a 28-point decrease in the melanin index, whereas a decrease of around 88, 74.3, and 106 points was detected in NCP-, Vitamin C-, and NCP + vitamin C-treated mice, respectively. Likewise, only a 39-point reduction in the erythema index was monitored in naturally recovered mice, but the NCP-, vitamin C-, and NCP + vitamin C-treated mice showed a 87.3-, 77-, and 111-point reduction, respectively. Interestingly, the skin tissues of the mice treated with NCP in combination with Vitamin C mostly recovered from UVB-induced damage. Altogether, this study elucidated the beneficial effect of the treatment of NCP in combination with Vitamin C on the UVB-irradiated skin, which might be helpful for treating sunburn on the skin.

Effect of No-ozone Cold Plasma on Regeneration after Crushed Mental Nerve Injury in rats.

Background: This experimental research aimed to determine whether No-ozone Cold Plasma (NCP) has regenerative effect on crushed injured sensory nerves in a rat model (Wistar A) and to evaluate whether NCP can be used as an alternative treatment method for sensory nerve injury in the oral-maxillofacial region. Methods: A total of 10 Wistar A rats were used for this experiment. They were divided into three groups according to whether the mental nerve of the left mandible was injured and NCP was applied or not: group 1 (n=3) (non-mental nerve damage, non-MD) - the left mental nerve was exposed and non-damaged; group 2 (n=3) (mental nerve damage, MD) - the left mental nerve was exposed and damaged, NCP was not applied; and group 3 (n=4) (mental nerve damage and NCP, MD-NCP) - the left mental nerve was exposed and damaged, NCP was applied with regular intervals (three times a week). Results: For the behavior analysis, von Frey test was used. Furthermore, the nerve tissues were examined with hematoxylin and eosin (H&E) staining, and the extent of neurorecovery was evaluated with the immunofluorescence staining of certain markers. The behavioral analysis showed that the function recovery sensory nerve was faster in group 3 (MD-NCP). In the histomorphologic and immunofluorescence analyses, the expression of the factors involved in neurorecovery was much higher in group 3 than in group 2 (MD). Conclusions: The expeditious recovery of sensory nerve function as well as the higher expression of the factors indicating nerve function recovery in the NCP-treated group suggest that NCP has a positive effect on regeneration after sensory nerve crushing injury. Therefore, in the case of sensory impairment of the oral-maxillofacial region, no-ozone cold plasma can be applied for therapeutic effect.

Anti-Cancer Activity of the Combinational Treatment of Noozone Cold Plasma with p-FAK Antibody-Conjugated Gold Nanoparticles in OSCC Xenograft Mice.

Oral squamous cell cancer (OSCC) is the most common type of oral cancer (about 80-90% of cases) and various research is being done to cure the disease. This paper aims to verify whether treatment with no-ozone cold plasma (NCP), which is designed for safe usage of the plasma on oral cavities, in combination with gold nanoparticles conjugated with p-FAK antibody (p-FAK/GNP) can trigger the selective and instant killing of SCC-25 cells both in vitro and in vivo. When SCC25 and HaCaT cells are exposed to p-FAK/GNP+NCP, the instant cell death was observed only in SCC25 cells. Such p-FAK/GNP+NCP-mediated cell death was observed only when NCP was directly treated on SCC25 harboring p-FAK/GNP. During NCP treatment, the removal of charged particles from NCP using grounded electric mesh radically decreased the p-FAK/GNP+NCP-mediated cell death. This p-FAK/GNP+NCP-mediated selective cell death of OSCC was also observed in mice xenograft models using SCC25 cells. The mere treatment of p-FAK/GNP and NCP on the xenograft tumor slowly decreased the size of the tumor, and only about 50% of the tumor remained at the end of the experiment. On the other hand, 1 week of p-FAK/GNP+NCP treatment was enough to reduce half of the tumor size, and most of tumor tissue had vanished at the end. An analysis of isolated tissues showed that in the case of individual treatment with p-FAK/GNP or NCP, the cancer cell population was reduced due to apoptotic cell death. However, in the case of p-FAK/GNP+NCP, apoptotic cell death was unobserved, and most tissues were composed of collagen. Thus, this paper suggests the possibility of p-FAK/GNP+NCP as a new method for treating OSCC.

No-ozone cold plasma can kill oral pathogenic microbes in H2O2-dependent and independent manner.

To apply the sterilisation effect of low-temperature plasma to the oral cavity, the issue of ozone from plasma must be addressed. In this study, a new technology for generating cold plasma with almost no ozone is developed and is named Nozone (no-ozone) Cold Plasma (NCP) technology. The antimicrobial efficacy of the NCP against four oral pathogens is tested, and its specific mechanism is elucidated. The treatment of NCP on oral pathogenic microbes on a solid medium generated a growth inhibition zone. When NCP is applied to oral pathogens in a liquid medium, the growth of microbes decreased by more than 105 colony forming units, and the bactericidal effect of NCP remained after the installation of dental tips. The bactericidal effect of NCP in the liquid medium is due to the increase in hydrogen peroxide levels in the medium. However, the bactericidal effect of NCP in the solid medium depends on the charged elements of the NCP. Furthermore, the surface bactericidal efficiency of the dental-tip-installed NCP is proportional to the pore size of the tips and inversely proportional to the length of the tips. Overall, we expect this NCP device to be widely used in dentistry in the near future.

Enhancement of Osteoblast Differentiation Using No-Ozone Cold Plasma on Human Periodontal Ligament Cells.

Periodontitis is an inflammatory disease that leads to periodontal tissue destruction and bone resorption. Proliferation and differentiation of cells capable of differentiating into osteoblasts is important for reconstructing periodontal tissues destroyed by periodontitis. In this study, the effects of the nozone (no-ozone) cold plasma (NCP) treatment on osteoblastic differentiation in periodontal ligament (PDL) cells were investigated. To test the toxicity of NCP on PDL cells, various NCP treatment methods and durations were tested, and time-dependent cell proliferation was analyzed using a water-soluble tetrazolium salts-1 assay. To determine the effect of NCP on PDL cell differentiation, the cells were provided with osteogenic media immediately after an NCP treatment to induce differentiation; the cells were then analyzed using alkaline phosphatase (ALP) staining, an ALP activity assay, real time PCR, and Alizarin Red S staining. The NCP treatment without toxicity on PDL cells was the condition of 1-min NCP treatment immediately followed by the replacement with fresh media. NCP increased ALP, osteocalcin, osteonectin, and osteopontin expression, as well as mineralization nodule formation. NCP treatment promotes osteoblastic differentiation of PDL cells; therefore, it may be beneficial for treating periodontitis.

Protective effects of non-thermal plasma on triethylene glycol dimethacrylate-induced damage in odontoblast-like MDPC-23 cells.

AIM: To evaluate whether the use of non-thermal plasma (NTP) could reduce triethylene glycol dimethacrylate (TEGDMA)-mediated damage in MDPC-23 cells. METHODOLOGY: The effects of NTP and TEGDMA on MDPC-23 cell proliferation were tested using WST-1 assays after pretreatment with NTP for 1 min and exposure to TEGDMA. Live/Dead assays were used to visualize cell death. To monitor the effects of NTP and TEGDMA on the cell cycle and apoptotic cell death, flow cytometry was performed. Western blotting was used to assess changes in protein levels mediated by NTP and TEGDMA treatment, and enzyme-linked immunosorbent assays were performed to evaluate the effects of NTP and TEGDMA on prostaglandin E2 (PGE2 ) expression. One-way analysis of variance and Duncan's post hoc tests were used for statistical analysis. RESULTS: NTP treatment effectively protected cells from TEGDMA-mediated cell damage and blocked TEGDMA-mediated cell growth inhibition (p < .05). NTP appeared to protect cells from death (p < .05) and blocked TEGDMA-mediated apoptotic cell death. Additionally, NTP reduced TEGDMA-mediated apoptotic activation of poly (ADP) ribose polymerase-1 and caspase-3 (p < .05). Furthermore, NTP effectively reduced TEGDMA-mediated expression of cyclooxygenase-2 and PGE2 proteins by inhibiting nuclear factor-κB protein expression (p < .05). CONCLUSIONS: NTP alleviated TEGDMA-mediated adverse effects by reducing cytotoxicity and inflammatory reactions in cells exposed to TEGDMA.

Gold nanoparticles conjugated with programmed death-ligand 1 antibodies induce apoptosis of SCC-25 oral squamous cell carcinoma cells via programmed death-ligand 1/signal transducer and transcription 3 pathway.

OBJECTIVE: Objective of this study is to test the anti-cancer effect of the gold nanoparticles conjugated with programmed death-ligand 1 (PD-L1) specific antibodies (PDL1-GNP), on oral squamous cell carcinoma. DESIGN: To test the effect of PDL1-GNP on oral squamous cell carcinoma, SCC-25 cells, a type of human oral squamous cell carcinoma which were isolated from human tongue, and HaCaT human keratinocytes as normal cell control, were used. Cell viability was tested by the water-soluble tetrazolium-1 and live/dead assays, while apoptotic cell death of SCC-25 cells were monitored by immunofluorescent staining and flow cytometry. The molecular changes during PDL1-GNP-mediated apoptosis were analyzed using Western blot analysis. RESULTS: PDL1-GNP treatment effectively decreased the growth of SCC-25 cells but not HaCaT cells. The results of the confocal microscopic assay showed that PDL1-GNP specifically bound to the SCC-25 cell membrane. Furthermore, the results of the live/dead, cytochrome c release assays and flow cytometry indicated PDL1-GNP-mediated apoptotic cell death of SCC-25 cells. PDL1-GNP-treated SCC-25 cells showed a phenotype with increased apoptotic proteins, including cleaved form of caspase-3, caspase-9, and poly (ADP-ribose) polymerase 1 (PARP1). PDL1-GNP treatment also effectively decreased B-cell lymphoma 2 (Bcl-2) and PD-L1 protein expression. Phosphorylation of signal transducer and transcription 3 (STAT3) was significantly increased after PDL1-GNP treatment on SCC-25 cells. CONCLUSIONS: PDL1-GNP treatment induced SCC-25 cell apoptosis possibly by inhibiting the function of the PD-L1 protein, since PD-L1 blocks STAT3 phosphorylation, which promotes apoptotic cell death.

Non-thermal plasma accelerates the healing process of peripheral nerve crush injury in rats.

The objective of this study was to evaluate the effect of non-thermal plasma (NTP) on the healing process of peripheral nerve crush injuries, which can occur during dental implant procedures. For this, a rat model of sciatic nerve crush injury (SNCI) was adopted. The rats were divided into three groups: non-nerve damage (non-ND), nerve damage (ND), and ND+NTP group. To evaluate the sciatic nerve (SN) function, the static sciatic index was calculated, and the muscle and SN tissues were subjected to a histologic analysis. The results showed that NTP effectively accelerated the healing process of SNCI in rats. In contrast to the ND group, which showed approximately 60% recovery in the SN function, the NTP-treated rats showed complete recovery. Histologically, the NTP treatments not only accelerated the muscle healing, but also reduced the edema-like phenotype of the damaged SN tissues. In the ND group, the SN tissues had an accumulation of CD68-positive macrophages, partially destroyed axonal fibers and myelinated Schwann cells. Conversely, in the ND+NTP group, the macrophage accumulation was reduced and an overall regeneration of the damaged axon fibers and the myelin sheath was accomplished. The results of this study indicate that NTP can be used for healing of injured peripheral nerves.

Ethanol extract of asiasari radix preferentially induces apoptosis in G361 human melanoma cells by differential regulation of p53.

BACKGROUND: In Korea and China, asiasari radix (AR) is widely used as a traditional anti-inflammatory and analgesic agent. After its skin-regenerating and hair loss-preventing activities were identified, several types of AR extracts were used for aesthetic purposes. Nevertheless, the effect of ARE on various types of skin cancers was not fully studied yet. METHODS: In this study, we tested the effect of an ethanolic AR extract (ARE) on G361 human melanoma and HaCaT human keratinocyte cell lines. After ARE exposure, cell growth and the expression patterns of proteins and genes were monitored. RESULTS: The ARE-mediated cell growth inhibition was greater in G361 cells than in HaCaT cells due to differences in its cell growth regulation effects. Interestingly, ARE treatment induced caspase-3-mediated apoptosis in G361 cells, but not in HaCaT cells. Furthermore, ARE reduced the expression of p53 and p21 proteins in G361 cells, whereas it induced their expression in HaCaT cells. ARE induced cell death in G361 cells through the reactive oxygen species (ROS)-dependent regulation of p53 and p21 in G361 cells. Microarray analysis showed that ARE regulates Mouse double minute 2 homolog (MDM2) and CASP8 and FADD-like apoptosis regulator (CFLAR) gene expression in G361 and HaCaT cells differently. CONCLUSION: The treatment of ARE preferentially induces apoptosis in melanoma cells by the ROS-dependent differential regulation of p53 level. Therefore, ARE can be used as a new medicinal option for melanoma.

Plasma cupping induces VEGF expression in skin cells through nitric oxide-mediated activation of hypoxia inducible factor 1.

Despite a long history, the clinical efficacy of cupping therapy is still under debate. This is likely due to the lack of direct evidence for the biological actions of cupping, since the short exposure of cells to vacuum condition rarely has affects cellular activity. In this study, the medicinal properties of a recent medical technology, non-thermal plasma, were added to classical cupping and designated as 'plasma cupping' (PC). In our results, the plasma-generating efficacy was increased under a cupping-like semi-vacuum condition (410 Torr) rather than normal atmospheric pressure (760 Torr). Notably, while cupping rarely affects the angiogenic factor vascular-endothelial growth factor (VEGF)-A, the PC treatment on HaCaT human keratinocytes significantly induced the expression of VEGF-A. The increased expression of the VEGF-A gene after the PC treatment was expected to be a result of PC-mediated ERK protein activation. The PC-mediated activation of ERK was essential for the activity of hypoxia inducible factor (HIF) 1 alpha, which is responsible for the PC-mediated expression of VEGF-A. The PC mediated increase of NO in the media was thought as a main reason for the elevated HIF-1 protein activity. In addition to the angiogenesis-promoting action of PC, it also showed anti-inflammatory activity by reducing TNF-α-mediated IL-1ß and IL-6 expression. Taken together, this study indicates the potential for PC that could enhance the clinical efficacy of cupping by adding the effects of non-thermal plasma to traditional cupping.

Increment of growth factors in mouse skin treated with non-thermal plasma.

Non-thermal plasma (NTP) has several beneficial effects, and can be applied as a novel instrument for skin treatment. Recently, many types of NTP have been developed for potential medical or clinical applications, but their direct effects on skin activation remain unclear. In this study, the effect of NTP on the alteration of mouse skin tissue was analyzed. After NTP treatment, there were no signs of tissue damage in mouse skin, whereas significant increases in epidermal thickness and dermal collagen density were detected. Furthermore, treatment with NTP increased the expression of various growth factors, including TGF-α, TGF-ß, VEGF, GM-CSF, and EGF, in skin tissue. Therefore, NTP treatment on skin induces the expression of growth factors without causing damage, a phenomenon that might be directly linked to epidermal expansion and dermal tissue remodeling.

Selective Killing of Melanoma Cells With Non-Thermal Atmospheric Pressure Plasma and p-FAK Antibody Conjugated Gold Nanoparticles.

Melanomas are fast growing high-mortality tumors, and specific treatments for melanomas are needed. Melanoma cells overexpress focal adhesion kinase (FAK) compared to normal keratinocytes, and we sought to exploit this difference to create a selectively lethal therapy. We combined gold nanoparticles (GNP) with antibodies targeting phosphorylated FAK (p-FAK). These conjugates (p-FAK-GNP) entered G361 melanoma cells and bound p-FAK. Treatment with p-FAK-GNP decreased the viability of G361 cells in a time dependent manner by inducing apoptosis. To maximize the preferential killing of G361 cells, non-thermal atmospheric pressure plasma was used to stimulate the GNP within p-FAK-GNP. Combined treatment with plasma and p-FAK-GNP showed much higher lethality against G361 cells than HaCaT keratinocyte cells. The p-FAK-GNP induced apoptosis over 48 hours in G361 cells, whereas plasma and p-FAK-GNP killed G361 cells immediately. This study demonstrates that combining plasma with p-FAK-GNP results in selective lethality against human melanoma cells.

The topical application of low-temperature argon plasma enhances the anti-inflammatory effect of Jaun-ointment on DNCB-induced NC/Nga mice.

BACKGROUND: Jaun-ointment (JO), also known as Shiunko in Japan, is one of the most popular medicinal formulae used in Korean traditional medicine for the external treatment of skin wound and inflammatory skin conditions. Since JO is composed of crude mixture of two herbal extracts (radix of Lithospermum erythrorhizon Siebold & Zucc and Angelica gigas Nakai), those been proved its anti-inflammatory activities in-vitro and in-vivo, JO has been expected as a good alternative treatment option for atopic dermatitis (AD). However, due to the lack of strategies for the penetrating methods of JO's various anti-inflammatory elements into the skin, an effective and safe transdermal drug delivery system needs to be determined. Here, low-temperature argon plasma (LTAP) was adopted as an ancillary partner of topically applied JO in a mice model of AD and the effectiveness was examined. METHODS: Dorsal skins of NC/Nga mice were challenged with DNCB (2,4-dinitrochlorobenzene) to induce AD. AD-like skin lesions were treated with JO alone, or in combination with LTAP. Inflammatory activity in the skin tissues was evaluated by histological analysis and several molecular biological tests. RESULTS: LTAP enhanced the effect of JO on AD-like skin lesion. Topical application of JO partially inhibited the development of DNCB-induced AD, shown by the moderate reduction of eosinophil homing and pro-inflammatory cytokine level. Combined treatment of JO and LTAP dramatically inhibited AD phenotypes. Interestingly, treatment with JO alone did not affect the activity of nuclear factor (NF)κB/RelA in the skin, but combined treatment of LTAP-JO blocked DCNB-mediated NFκB/RelA activation. CONCLUSIONS: LTAP markedly enhanced the anti-inflammatory activity of JO on AD-like skin lesions. The effect of LTAP may be attributed to enhancement of drug penetration and regulation of NFκB activity. Therefore, the combination treatment of JO and LTAP could be a potential strategy for the treatment of AD.

Inhibition of inflammatory reactions in 2,4-Dinitrochlorobenzene induced Nc/Nga atopic dermatitis mice by non-thermal plasma.

Non-thermal plasma (NTP) has recently been introduced and reported as a novel tool with a range of medicinal and biological roles. Although many studies using NTP have been performed, none has investigated the direct relationship between NTP and immune responses yet. Especially, the effects of NTP on atopic dermatitis (AD) were not been explored. Here, NTP was tested whether it controls immune reactions of AD. NTP treatment was administered to pro-inflammatory cytokine-stimulated keratinocytes and DNCB (2,4-Dinitrochlorobenzene)-induced atopic dermatitis mice, then the immune reactions of cells and skin tissues were monitored. Cells treated with NTP showed decreased expression levels of CCL11, CCL13, and CCL17 along with down-regulation of NF-κB activity. Repeated administration of NTP to AD-induced mice reduced the numbers of mast cells and eosinophils, IgE, CCL17, IFNγ levels, and inhibited NF-κB activity in the skin lesion. Furthermore, combined treatment with NTP and 1% hydrocortisone cream significantly decreased the immune responses of AD than that with either of these two treatments individually. Overall, this study revealed that NTP significantly inhibits several immune reactions of AD by regulating NF-κB activity. Therefore, NTP could be useful to suppress the exaggerated immune reactions in severe skin inflammatory diseases such as AD.

Scutellariae radix induces apoptosis in chemoresistant SCC-25 human tongue squamous carcinoma cells.

Scutellariae radix is one of the most widely used anticancer herbal medicines in several Asian countries, including Korea, Japan, and China. Squamous cell carcinoma (SCC) is one of the most common head and neck carcinomas, which is highly invasive and metastatic, and can potentially develop chemoresistance. Therefore, new effective treatment methods are urgently needed. We determined the effects of Scutellariae radix on SCC-25 cells using the WST-1 assay, F-actin staining, flow cytometry analysis, immunofluorescence staining, and western blot analysis. Scutellariae radix treatment inhibited SCC-25 cell growth in a dose- and time-dependent manner, but it did not inhibit HaCaT (human keratinocyte) cell growth. Changes in cell morphology and disruption of filamentous (F)-actin organization were observed. Scutellariae radix-induced apoptosis as indicated by the translocation of cytochrome c and apoptosis-inducing factor (AIF) into the nucleus and cytosol. Scutellariae radix-induced an increase in cells with sub-G1 DNA content, and increased Bax, cleaved caspase-3, caspase-7, caspase-9, DNA fragmentation factor 45 (DFF 45), and poly(ADP-ribose) polymerase-1 (PARP-1) expression levels. Furthermore, increased expression of phosphorylated mitogen-activated protein kinase (MAPK)-related proteins was detected. The antitumor effect of Scutellariae radix was due to decreased cell proliferation, changes in cell morphology, and the activation of caspase and MAPK pathways. Taken together, the findings of this study highlight the anticancer activity of Scutellariae radix in chemoresistant SCC-25 oral squamous carcinoma cells.

Treatment with low-temperature atmospheric pressure plasma enhances cutaneous delivery of epidermal growth factor by regulating E-cadherin-mediated cell junctions.

The barrier system of the skin not only defends against antigens and harmful substances, but also hinders the permeation of medicines and cosmetics into the dermis. Several strategies have been developed to enhance the absorption ability of skin, including the use of chemicals and skin ablation devices. However, the cost and inconvenience of these strategies highlights the need for a novel and safe method for increasing skin absorption. In this study, we examined the effect of low temperature atmospheric pressure plasma (LTAPP) on the efficiency of drug penetration through the skin, as well as its mechanism of action. HaCaT human keratinocytes and hairless mice were exposed to LTAPP treatment, and the cellular and tissue gene expression, and morphological changes were monitored. We found that the LTAPP exposure reduced the expression of E-cadherin in skin cells and led to the loss of cell-cell contacts. The exposure of mouse skin to LTAPP also reduced the expression of E-cadherin and prevented intercellular junction formation within the tissue, leading to enhanced absorption of hydrophilic agents, eosin and epidermal growth factor. The reduction in E-cadherin expression and reduced skin barrier function recovered completely within 3 h of LTAPP exposure. Taken together, these data show that LTAPP can induce a temporal decrease in the skin barrier function by regulating E-cadherin-mediated intercellular interactions, leading to the enhanced transdermal delivery of drugs and cosmetics.

Reactive oxygen species-mediated activation of the Akt/ASK1/p38 signaling cascade and p21(Cip1) downregulation are required for shikonin-induced apoptosis.

Shikonin derivatives exert powerful cytotoxic effects, induce apoptosis and escape multidrug resistance in cancer. However, the diverse mechanisms underlying their anticancer activities are not completely understood. Here, we demonstrated that shikonin-induced apoptosis is caused by reactive oxygen species (ROS)-mediated activation of Akt/ASK1/p38 mitogen-activated protein kinase (MAPK) and downregulation of p21(Cip1). In the presence of shikonin, inactivation of Akt caused apoptosis signal-regulating kinase 1 (ASK1) dephosphorylation at Ser83, which is associated with ASK1 activation. Shikonin-induced apoptosis was enhanced by inhibition of Akt, whereas overexpression of constitutively active Akt prevented apoptosis through modulating ASK1 phosphorylation. Silencing ASK1 and MKK3/6 by siRNA reduced the activation of MAPK kinases (MKK) 3/6 and p38 MAPK, and apoptosis, respectively. Antioxidant N-acetyl cysteine attenuated ASK1 dephosphorylation and p38 MAPK activation, indicating that shikonin-induced ROS is involved in the activation of Akt/ASK1/p38 pathway. Expression of p21(Cip1) was significantly induced in early response, but gradually decreased by prolonged exposure to shikonin. Overexpression of p21(Cip1) have kept cells longer in G1 phase and attenuated shikonin-induced apoptosis. Depletion of p21(Cip1) facilitated shikonin-induced apoptosis, implying that p21(Cip1) delayed shikonin-induced apoptosis via G1 arrest. Immunohistochemistry and in vitro binding assays showed transiently altered localization of p21(Cip1) to the cytoplasm by shikonin, which was blocked by Akt inhibition. The cytoplasmic p21(Cip1) actually binds to and inhibits the activity of ASK1, regulating the cell cycle progression at G1. These findings suggest that shikonin-induced ROS activated ASK1 by decreasing Ser83 phosphorylation and by dissociation of the negative regulator p21(Cip1), leading to p38 MAPK activation, and finally, promoting apoptosis.

Low-temperature atmospheric plasma increases the expression of anti-aging genes of skin cells without causing cellular damages.

Efforts to employ various types of plasma in the field of skin care have increased consistently because it can regulate many biochemical reactions that are normally unaffected by light-based therapy. One method for skin rejuvenation adopted a high-temperature plasma generator to remove skin epithelial cells. In this case, the catalyzing effects of the plasma were rarely used due to the high temperature. Hence, the benefits of the plasma were not magnified. Recently, many types of low-temperature plasma devices have been developed for medical applications but their detailed functions and working mechanisms are unclear. The present study examined the effect of low-temperature microwave plasma on skin cells. Treatment with low-temperature plasma increased the expression of anti-aging genes in skin cells, including collagen, fibronectin and vascular endothelial growth factor. Furthermore, the plasma treatment did not cause cell death, but only induced slight cell growth arrest at the G2 phase. Although the cells treated with low-temperature plasma showed moderate growth arrest, there were no signs of thermal or genetic damage of skin cells. Overall, this low-temperature microwave plasma device induces the expressions of some anti-aging-related genes in skin cells without causing damage.