Quantification of In Vivo Epidermal Keratinocyte Architecture Associated with the Signs of Skin Aging and the Skin Benefit Evaluation by Application of Galactomyces Ferment Filtrate (Pitera)-Containing Skin Care Product

Background: Aged skin exhibits visual alterations such as wrinkles, rough texture, pore dilation, and dull skin tone, as well as physiological aging, namely, decreased hydration and increased transepidermal water loss (TEWL). Recent advances in coherence tomography have also revealed that skin aging affects in vivo epidermal keratinocyte architecture. However, the interconnectivity between spatial architectural aging and visual/physiological aging parameters remains largely unknown. Purpose: To elucidate whether the tomographic keratinocyte architectural aging is correlated with visual and physiological skin aging parameters and to quantitatively evaluate the improvements of the architectural, visual, and physiological aging parameters by the daily treatment of the skin care formula containing Galactomyces Ferment Filtrate (GFF, 8X PiteraTM). Method: We measured the in vivo keratinocyte cellular architecture with two-photon stereoscopic tomography obtaining by-layer epidermal section images in 78 Asian females of various ages. Visual aging parameters were analyzed using a portable image capture system. Hydration and TEWL were also assessed. The anti-aging effects of GFF-containing skin moisturizer (SK-II LXP CreamTM) were also examined in two studies after twice-daily application for 2 (N = 35) and 4 (N = 32) weeks. Results: As for the keratinocyte cellular architecture, skin aging was significantly associated with decreased cell density and increased cell uniformity. These architectural aging parameters were significantly correlated with visual and physiological aging parameters, namely, rough texture, wrinkles, pore dilation, dull skin tone, dehydration, and increased TEWL. The strong interconnectivity allowed us to develop formulae to estimate the keratinocyte architecture from visual aging parameters. Moreover, twice-daily application of SK-II significantly improved the keratinocyte architecture associated with multiple skin aging visual and physiological parameters. Conclusion: Skin aging is a process involving mutual interconnections among epidermal keratinocyte cellular architecture, visual, and physiological parameters. The GFF-containing moisturizer SK-II effectively improves spatial architecture of keratinocytes in epidermis and these evaluated skin aging parameters in a new trajectory over the course of treatment. .

Chemerin Exacerbates Psoriasis by Stimulating Keratinocyte Proliferation and Cytokine Production

Objective Psoriasis is often combined with metabolic abnormalities,such as obesity and diabetes.The upregulation of chemerin,which is an essential protein produced primarily from white fat,is strongly correlated to the development of psoriasis.However,there is no clarification on its exact function and mechanism in disease pathogenesis.The present study aims to determine its function and mechanism in disease pathogenesis.Methods The present study used a psoriasislike inflammatory cell model and imiquimod(IMQ)-induced mouse model to confirm whether chemerin is upregulated in psoriasis patients.Results Chemerin enhanced the keratinocyte proliferation,inflammatory cytokine secretion,and activation of the MAPK signaling pathway.Crucially,the intraperitoneal injection of neutralizing anti-chemerin antibody(ChAb)diminished the epidermal proliferation and inflammation in the IMQ-induced mouse model.Conclusion The present results indicate that chemerin promotes keratinocyte proliferation,and enhances the production of inflammatory cytokines,thereby aggravating the psoriasis.Thus,chemerin can be a prospective target for the treatment of psoriasis.

Transplantation of human induced pluripotent stem cell derived keratinocytes accelerates deep second-degree burn wound healing

BACKGROUND Current evidence shows that human induced pluripotent stem cells(hiPSCs)can effectively differentiate into keratinocytes(KCs),but its effect on skin burn healing has not been reported.AIM To observe the effects of hiPSCs-derived KCs transplantation on skin burn healing in mice and to preliminarily reveal the underlying mechanisms.METHODS An analysis of differentially expressed genes in burn wounds based on GEO datasets GSE140926,and GSE27186 was established.A differentiation medium containing retinoic acid and bone morphogenetic protein 4 was applied to induce hiPSCs to differentiate into KCs.The expression of KCs marker proteins was detected using immunofluorescence staining.A model of a C57BL/6 mouse with deep cutaneous second-degree burn was created,and then phosphate buffered saline(PBS),hiPSCs-KCs,or hiPSCs-KCs with knockdown of COL7A1 were injected around the wound surface.The wound healing,re-epithelialization,engraftment of hiPSCs-KCs into wounds,proinflammatory factor level,and the NF-κB pathway proteins were assessed by hematoxylin-eosin staining,carboxifluorescein diacetate succinimidyl ester(CFSE)fluorescence staining,enzyme linked immunosorbent assay,and Western blotting on days 3,7,and 14 after the injection,respectively.Moreover,the effects of COL7A1 knockdown on the proliferation and migration of hiPSCs-KCs were confirmed by immunohistochemistry,EdU,Transwell,and damage repair assays.RESULTS HiPSCs-KCs could express the hallmark proteins of KCs.COL7A1 was down-regulated in burn wound tissues and highly expressed in hiPSCs-KCs.Transplantation of hiPSCs-KCs into mice with burn wounds resulted in a significant decrease in wound area,an increase in wound re-epithelialization,a decrease in proinflammatory factors content,and an inhibition of NF-κB pathway activation compared to the PBS group.The in vitro assay showed that COL7A1 knockdown could rescue the inhibition of hiPSCs-KCs proliferation and migration,providing further evidence that COL7A1 speeds up burn wound healing by limiting cell proliferation and migration.CONCLUSION In deep,second-degree burn wounds,COL7A1 can promote KC proliferation and migration while also suppressing the inflammatory response.

Establishment and Application of A Human Primary Keratinocyte Inflammation Model for Cosmetic Raw Material Screening

Using inducers to induce cells to produce inflammatory response is a common in vitro experimental method to study inflammation.However,there are relatively few inflammatory models developed for the cosmetic industry,and there are also great differences in the control of model induction,the selection of inflammatory indicators,and the concentration of inducers.Therefore,in this paper,by systematically studying the effects of Lipopolysaccharide(LPS)on the cell viability,the levels of IL-1α,IL-8 and ROS of human primary keratinocytes,a skin inflammation model based human primary keratinocyte was developed.The results showed that 0.01~100μg/mL LPS had no significant effect on the cell viability of human primary keratinocytes,while 100μg/mL LPS could simultaneously induce human primary keratinocytes to produce large amounts of IL-1αand IL-8,and 0.01μg/mL LPS could induce plentiful ROS.Therefore,a skin inflammation model for differential induction of different inflammatory indicators was established,and the sample OSM2021041301 was tested with this model,it was found that sample OSM2021041301 could significantly inhibit LPS-induced elevated IL-1αand IL-8 levels,the inhibitory effect on LPS-induced elevated ROS level was weak.The results indicated that OSM2021041301 has certain anti-inflammatory effect on inflammation caused by the increase of IL-1α,IL-8 and ROS induced by LPS and its analogues.

Effect of a Thermal Spring Water on Carbohydrate-Protein Interactions in In-Vitro Models Implicating Normal Human Keratinocytes and Recombinant Lectins

Background: Sugar moiety of macromolecules is today very well known for its implications in many biological recognition mechanisms including cell-cell, extracellular matrix-cell and/or bacteria-cell interactions. In this context lectins, which are carbohydrate-binding proteins displaying a high affinity for sugar groups of other molecules, are of a great importance, notably in immune response involving bacteria, viruses and fungi. As protein-carbohydrate interactions are often mediated by ions such as calcium, zinc or magnesium, we were prompted to study the effect of a thermal spring water (which contains this type of component) on interactions existing between: 1) osidic receptors of human normal keratinocytes and 2) two lectins greatly implicated in the immune response mechanisms (i.e. the dectin-1 and the langerin), and their ligands. Materials and Methods: In a first series of experiments, we studied the effect of increasing concentrations of a thermal spring water on interactions existing between glycosylated molecules and the osidic receptors expressed at the normal human keratinocytes surface. In a second step, and in order to better understand the putative effect of our thermal spring water on the immune response, we analyzed its effect on the interactions existing between the dectin-1 (implicated in the recognition of bacteria, viruses and fungi) and the langerin (expressed by Langerhans cells, the immune cells of the cutaneous tissue), and their ligands in a model using recombinant human lectins and appropriate binding molecules. Results: We showed here that our thermal spring water was able to reinforce interactions between keratinocytes osidic receptors and some of their ligands, in a dose-related manner: From 8% to 55% of increase with 10% to 30% (v/v) of thermal spring water. In the second part of our studies, we also showed that our thermal spring water was able to modulate interactions between dectin-1 and langerin and their ligands through a biphasic effect: Interactions were enhanced by more than 40% and 20% respectively with 10% of thermal spring water, and return to their basal level or lower for higher concentrations. Conclusion: The tested thermal spring water, probably due to its ionic composition, could significantly affect interactions of osidic receptors with their ligands. This property could be of a great interest to help immune system to maintain an appropriate “vigilance state” by using the thermal water at up to a concentration of 10%, and by avoiding any runaway reaction in case of aggression, by using concentrations higher than 10%. .

The effect of keratinocyte growth factor-2(KGF-2) on the proliferation of human keratinocytes

Objective;In vitro studies have shown that KGF-2 has a proliferative effect on neonatal foreskin kerati-nocytes.Cells from adult donors have been shown to respond to KGF-1 to a lesser degree than neonatal keratino-cytes.The purpose of the study was to investigate the proliferative effect of KGF-2 on keratinocytes from an adultsubject.Methods;Standard medium was Keratinocyte Growth Medium without BPE,hydrocortisone and EGF.Ke-ratinocytes cultured from a 48-year-old subject were seeded at 2 10~4 in 32 mm ...

Cultured keratinocyte grafting on various biologic matrices

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Effect of lead on IL-8 production and cell proliferation in human oral keratinocytes

Objective:To investigate the effect of lead on the production of IL-8 and cell proliferation in normal human oral keratinocytes（NHKs）.Methods:NHKs were prepared as outgrowths from normal human buccal mucosa.The cells were treated with three concentrations of lead glutamate(4.5×10-5M,4.5×10-6M and 4.5×10-7M).NHKs grown in glutamic acid were used as control.The amounts of IL-8 secreted in the culture supernatants were evaluated at 12 and 24 h using enzyme-linked immunospecific assay（ELISA）.Cell proliferation was determined by the MTT colorimetric assay.Three cultures were used for each experiment,and three independent experiments were performed.Analysis of variance and Duncan’s multiple range tests were used for statistical analysis.Results:An elevation of IL-8 in culture supernatants of NHKs treated with lead at all concentrations at 12 and 24 h after exposure in a dose-dependent manner was revealed.A significant increase in cell numbers was observed only at 24 h exposed to 4.5×10- 5M lead glutamate.Conclusions:The capacity of NHKs,to secrete IL-8,enhanced by lead glutamate,is demonstrated here.Induction of cell proliferation is revealed only after exposure to high lead concentration.The elevation of secreted IL-8 is a probable initial sign for the acute inflammatory response and may be involved in the pathogenesis of lead stomatitis.

Lipoxin A4 Inhibits Lipopolysaccharide-induced Production of Inflammatory Cytokines in Keratinocytes by Up-regulating SOCS2 and Down-regulating TRAF6

Liopxin A4（LXA4） is considered to be a crucial modulator in the inflammatory responses. In the present study, we aimed to study the effect of LXA4 on the inflammatory cytokines production induced by lipopolysaccharide（LPS） and the possible mechanism in normal human epidermal keratinocytes（NHEKs）. NHEKs were isolated and cultured. The expression of toll-like receptor 4（TLR4）, LXA4 receptor（ALXR） and aryl hydrocarbon receptor（Ah R） in NHEKs was detected by reverse transcription polymerase chain reaction（RT-PCR）. The m RNA and protein levels of tumor necrosis factor-alpha（TNF-α） and interleukin-1β（IL-1β） were determined in NHEKs stimulated by LPS（10 μg/m L） with or without preincubation with LXA4（100 nmol/L） for 30 min by real-time quantitative PCR（real-time q PCR） and enzyme-linked immunosorbent assay（ELISA）, respectively. The expression levels of tumor necrosis factor receptor-associated factor 6（TRAF6） and suppressors of cytokine signaling 2（SOCS2） m RNAs and proteins, and nuclear translocation of NF-k B-p65 were measured by real-time q PCR and Western blotting, respectively. The results showed that NHEKs expressed TLR4, ALXR and Ah R. LXA4 significantly inhibited the m RNA and protein expression levels of TNF-α, IL-1β and TRAF6 induced by LPS in NHEKs, and LXA4 obviously increased the expression of SOCS2 at m RNA and protein levels. The nuclear NF-k B-p65 protein expression induced by LPS was inhibited after preincubation with LXA4 in NHEKs. It was concluded that LXA4 inhibits the LPS-induced production of TNF-α and IL-1β in NHEKs by up-regulating SOCS2 and down-regulating TRAF6.

Flow Cytometric Analysis of the Toxicity of Nitrofen in Cultured Keratinocytes

Lactate dehydrogenase (LDH) release test, 3 H-thymidine (3 H-TdR) and 3 H-leucine (3 H-Leu) incoopration tests and flow cytometric analysis (FCM) of cell cycle were empoyed to elucidate cellular and molecular mechanism of nitrofen-induced toxicity in cultured keratinocytes.The results showed that cell morphologic damages were observed after exposure to 1.0 mmol/L and 10.0 mmol/L nitrofen. LDH release increased in a dose- and time-dependent manner. Depressions in 3H -TdR and 3 H-Leu incorpration were found even at 0.01 mmol/L, and increased with the exposure dose. Cell cycle was analyzed from the DNA- histogram with propidium iodde stain. The results showed that there was no pronounced alteration in cell cycle after cells exposed to 0.01 and 0.1 mmol/L nitrofen. At dose of 1.0 mmol/L, S phase cells increased 2 times of that of control. With the increase of dose, G2/M phase cells became to increase about 5 times of that of the control. At 1 .0 mmol/L, time course of cell cycle after exposure was observed. At the beginning of exposure, cells in S phase and G2/M phase were about 8 .7 % and 11 %. Following 24 h incubation with nitrofen, cells in S phase increased to 18.0% with almost no change in G2/M. 72 h after exposure, G2/M phase cells increased to 63 .3%. The forve results demonstrated that S phase and G2/M phase blockage in cultured keratinocytes after exposed to nitrofen seems of importance in the mechanism of nitrofen-induced toxicity.

Activated protein C: A regulator of human skin epidermal keratinocyte function

Activated protein C(APC) is a physiological anticoagulant, derived from its precursor protein C(PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor(EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

Effects of RNA Interference Combined with Ultrasonic Irradiation and SonoV ue Microbubbles on Expression of STAT3 Gene in Keratinocytes of Psoriatic Lesions

The most effective sequence of small interfering RNA（si RNA） silencing STAT3 of psoriatic keratinocytes（KCs） was screened out,and the effects of the most effective si RNA combined with ultrasonic irradiation and Sono Vue microbubbles on the expression of STAT3 of KCs and the dose-and time-response were investigated.Three chemically-synthetic si RNAs targeting STAT3 carried by Lipofectamine 3000 were transfected into KCs,and the effects on STAT3 expression were detected,then the most effective si RNA was selected for the subsequent experiments.The negative controls of siR NA（si RNA-NC） labeled with Cy3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles were transfected into KCs,then the optimal parameters of ultrasonic irradiation were determined.The most effective si RNA carried by Lipofectamine 3000 combined with ultrasonic irradiation at the optimal parameters and Sono Vue microbubbles was transfected into KCs,and the dose-and time-response of RNA interference was determined.The effect of RNA interference by the most effective si RNA at the optimal time and dose carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles（LUS group） was compared with that only carried by Lipofectamine 3000（L group）.The results showed that si RNA-3 achieved the highest silencing efficacy.0.5 W/cm2 and 30 s were selected as the parameters of ultrasonic irradiation.The si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and Sono Vue microbubbles could effectively knock down the STAT3 expression at m RNA and protein levels in dose-and time-dependent manners determined at 100 nmol/L with maximum downregulation on m RNA at 48 h,and on protein at 72 h after transfection.The LUS group achieved the highest silencing efficacy.It was concluded that si RNA-3 carried by Lipofectamine 3000 combined with ultrasonic irradiation and SonoV ue microbubbles could effectively knock down the STAT3 expression in psoriatic KCs,and the optimized transfection condition and the sequence of si RNA-3 could serve for further research on gene therapy of psoriasis.

Growth dynamics of individual clones of normal human keratinocytes: observations and theoretical considerations

The life histories of 429 individual epidermal keratinocyte clones picked at random were studied. Individual basal keratinocytes were derived from asynchronous rapidly proliferating subconfluent cultures propagated in either a low calcium (0.1mM) or a high calcium (2mM) serum-free medium. Single-celled clones were isolated by seeding trypsin-EDTA dissociated cells into a Petri dish containing cloning chips. Chips with only one cell per chip were transferred into dishes containing either low calcium or high calcium growth factor replete serum-free medium. Clone formation was monitored microscopically and the number of cells in each colony tallied at least twice daily for further analysis. A total of 369 clones were established from seven different neonatal foreskin cell strains (A-F), and 60 clones were derived from one adult human skin cell strain (G). During a five-day culture interval, among 32 clones of strain A, 83% divided at least once, 50% divided once in 24 hours, 86% divided at least three times within three days, and more than 50% divided at least four to five times in five days. Of 231 clones amongst the other five cell strains (B-F), an average of 63% (±12 S,E) divided more than three times in an eight day period, the remainder divided either once, twice or not at all. Of the 106 clones of strain G, reared in high calcium serum-free medium, 67% divided more than three times in a six-day period, and 55% divided five or more times in 6 days. Clones derived from adult skin strain H had a lower clone forming potential with 70% dividing at least once in seven days, and only 30% dividing three or more times. By contrast, the average generation time (AvGT) for second and third passage keratinocytes derived from neonatal foreskin cultures was 24 hrs. Detailed dendrograms were constructed for many of the proliferating clones. The majority of clones expressed a synblastic division pattern with every cell dividing at least once per day. A fraction of clones either exceeded this circadian division rate or displayed a biphasic division pattern with all cells initially dividing once a day and then abruptly slowing to once every other day or to an intermediate rate. A minority of clones was committed to a few terminal divisions. The division patterns of the non-synblastic clones fit an alternating bifurcated branching mode of clonal expansion expressed by the Fibonacci sequence for numbers of accumulated cells per clone per day. These results were analyzed in terms of deterministic, probabilistic and a limit cycle oscillator models of cell division timing.

Royal Jelly Extract Accelerates Keratinocyte Proliferation, and Upregulates Laminin α3 and Integrin β1 mRNA Expression, via Akt/mTOR/HIF-1α Pathway

Background: In the previous decade, various benefits and biological activities of royal jelly, applied in alternative and modern medicine, and cosmetics, have been reported. However, the effects of royal jelly extract (RJ) on keratinocytes have not been fully elucidated. Objective: The primary objectives of this study were to reveal the effects of RJ on keratinocytes and explore the underlying mechanism. Methods: HaCaT cells, an immortal human epidermis-derived keratinocyte cell line, were used in this study. Laminin α3 (LAMA3), integrin β1 (ITGB1), and hypoxia-inducible factor-1α (HIF-1α) mRNA expression levels were determined using real-time PCR. Cell proliferation rate was measured using a bromodeoxyuridine uptake assay. Results: RJ treatment upregulated LAMA3, ITGB1 and HIF-1α mRNA expression, and accelerated HaCaT cell proliferation. Akt and mTOR inhibitors suppressed the RJ-induced HIF-1α expression and cell proliferation. HIF-1α silencing abrogated RJ-induced LAMA3 and ITGB1 mRNA expression and cell proliferation, whereas LAMA3 silencing and antibody-mediated ITGB1 blockade did not affect the effects of RJ. Conclusion: RJ upregulates LAMA3 and ITGB1 mRNA expression levels by HIF-1α expression enhancement. In addition, RJ accelerates keratinocyte proliferation via Akt/mTOR/HIF-1α/NF-κB signaling pathway. These suggest that RJ is beneficial for anti-aging, as a skin care product ingredient.

KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

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Biological properties of differently-aged human keratinocytes:population doubling time growth curve and cell cycle analysis

Objective To explore the biological properties of keratinocytes from differently-aged healthy human beings. Methods Keratinocytes from fetus,teenager and middle-aged groups were separated and cultured. The population doubling time (PDT) and cell growth curve in different cells were compared,and the cell cycles were analyzed by flow cytometry. Results ① In primary culture of keratinocytes,the adherence time in middle-aged group was longer than that in fetus and teenager groups. However,all cell morphology showed no obvious differences. In subculture of keratinocytes,with donator’s age increasing,time of cell adherence prolonged,passage number decreased and differences in cell morphology were obvious. ② The average PDT of keratinocytes was shorter in fetus group than in teenager and middle-aged groups. But difference in cell growth curve between different passages was not observed. ③ Keratinocytes showed G2/M period in fetus group but G0/G1 period in teenager and middle-aged groups mainly. Conclusion As age increases,the biological properties of keratinocytes change obviously.

Investigation on etretin effects on expression of Fas/FasL ligand and apoptosis in cultured human keratinocytes

Objective:To further illuminate a possibme mechanism of Fas/FasL in the treatment of psoriasis, the expression of it and apoptosis of KC were investigated. Methods: With cell culture,immunocytochemistry, the expression of Fas/FasL protein after the treatment with etretin was observed in cultured human normal keratinocytes. Then, the state of apoptosis in cultured keratinocyte after stimulation with etretin was detected with FACS(Fluorescence Activited Cell Sortor). Results:① There was no expression of Fas/FasL protein in the cultured normal human KC. ②After the treatment with etretin, the strongest expression of FasL protein appeared after 16h,so did Fas after 40h.③ The higher the concentration of etretin,the stronger the expression of Fas/FasL protien.Under the same condition,the expression of Fas is stronger than that of FasL.④ Keratinocytes' apoptosis occurred after stimulation with etretin. Conclusion: Fas/FasL system wasn't involved in apoptosis in cultured normol human keratinocytes.But during limited period, the apoptosis of KC could be induced by (etretin),thus it can antagonize benign proliferate of keratinocytes.Our data showed up-regulation of the expression of Fas/FasL and apoptosis in cultured human keratinocytes stimulated by etretin, and its function may be involved in the therapeutic machanism of psoriasis.

The expression of P63 protein in some keratinocyte original tissues and cells

Objective： To examine the expression patterns of p63 in tissues of particular keratinocyte original hyperproliferate diseases and variety cell types for determining if P63 is the marker of proliferative potential keratinocytes. Methods： P63 protein was detected and analyzed by immunoreactivity method and Western blot in biopsy specimens of keratinocyte original disorders including squamous cell carcinomas SCC, basal cell carcinomas BCC, Bowen＇ s disease and other tissues or cells, such as psoriasis vulgaris, normal skin tissues, primary cultured keratinocytes, immortal HaCaT cells, and epidermoid carcinoma cells A431. Results： P63 protein was expressed in the nuclei of basal and suprabasal layer of the epidermis, germinative cells of sebaceous glands in normal epidermal. P63 was strongly and diffusely detected in the majority of tumor cells in BCC and poorly-differentiated SCC. In Bowen＇ s disease, p63 expresses are remarkable in all cell layers. In the psoriasis plaque epidermal, p63 expressed mainly in basal cells and part of spinous cells. P63 expressed more strongly in primary cultured keratinocytes than in A431 cells or HaCaT cells. Conclusion： P63 is a nuclei marker of undifferentiated keratinocytes with the proliferative potential and may disrupt the terminal differentiation. The overexpression of p63 reflects immaturity of the tumor cells. The immunohistochemical staining of p63 may be useful for investigating the origin and differentiation of tumor cells.

NPS31807, a Standardized Extract from <i>Sphaeranthus indicus</i>, Inhibits Inflammatory, Migratory and Proliferative Activity in Keratinocytes and Immune Cells

Chronic inflammation induced hyper-proliferation, and migration of keratinocytes are pathological hallmarks of psoriasis. Extracts from Sphaeranthus spp. demonstrate pharmacological activity in-vitro and in-vivo. However, the activity in modulating disease relevant pathways in psoriasis has not been reported. In the current study, a standardized herbal extract from Sphaeranthus indicus (NPS31807) was used to study the mechanistic activity under conditions of inflammation, keratinocyte proliferation and migration using cell based and gene expression assays. NPS31807 treatment reduced levels of pro-inflammatory cytokines from human macrophages and activated epidermal keratinocytes in a dose dependent manner. Treatment with NPS31807 diminished NFκB and AP-1 transcription activity in human macrophages. Lowered nuclear translocation of p65 sub-unit in macrophages by treatment confirmed reduced activity of NFκB. Gene expression profiling showed attenuated expression of genes involved with inflammation such as TNF signaling, and angiogenesis by NPS31807. Inhibition of angiogenesis and matrix metalloproteinase production in keratinocytes was confirmed using RTq-PCR assays. Pretreatment with NPS31807 led to significant reduction of STAT3 phosphorylation and mitogen induced cellular migration. NPS31807 induced inhibition of proliferative genes and BrdU uptake in epidermal keratinocytes. In summary, our study provides novel molecular insights into the anti-inflammatory, anti-migratory and anti-proliferative properties of NPS31807. In summary, NPS31807, an extract from Sphaeranthus indicus can be used as therapeutic option in inflammatory and auto-immune conditions such as psoriasis.