Resveratrol ameliorates lipid accumulation and inflammation in human SZ95 sebocytes via the AMPK signaling pathways in vitro.

BACKGROUND: Acne vulgaris is a prevalent skin disease lacking effective and well-tolerated treatment. An earlier study indicated that resveratrol (RVT) has therapeutic effects in acne patients through unknown mechanisms. OBJECTIVES: To evaluate the effects of RVT on linoleic acid (LA)-induced lipogenesis and peptidoglycan (PGN)-induced inflammation in cultured SZ95 sebocytes in vitro, and to investigate the underlying mechanisms. METHODS: RNA-sequencing was used to analyze the whole transcriptome. Nile red staining was used to detect intracellular neutral lipids, whereas lipidomics was used to investigate changes in the lipid profile in sebocytes. Interleukin (IL)-1ß and IL-6 mRNA and protein levels were assessed through quantitative real-time PCR and Enzyme-linked immunosorbent assay, respectively. Western blot was used to evaluate the expression of lipogenesis-related proteins, the inflammatory signaling pathway, and the AMP-activated protein kinase (AMPK) pathway. Further, specific small interfering RNA (siRNA) was used to knockdown sirtuin-1 (SIRT1) expression. RESULTS: RVT inhibited the lipogenesis-related pathway and nuclear factor-kappa B (NF-κB) signaling pathway in SZ95 sebocytes. It also downregulated LA-induced lipogenesis, the expression of lipid-related proteins, and the contents of unsaturated fatty acids. Besides, RVT promoted SIRT1 expression and deacetylation of the NF-κB p65 subunit, thereby lowering IL-1ß and IL-6 secretion under PGN induction. Furthermore, pretreatment with AMPK inhibitor Compound C abolished RVT-mediated sebosuppressive and anti-inflammation effects. Meanwhile, SIRT1 silencing abrogated the anti-inflammatory potential of RVT. CONCLUSION: In human SZ95 sebocytes, RVT exhibits sebosuppressive and anti-inflammatory effects partially through the AMPK pathway, which may justify the role of RVT treatment in acne vulgaris.

Formalin-killed Propionibacterium acnes activates the aryl hydrocarbon receptor and modifies differentiation of SZ95 sebocytes in vitro.

BACKGROUND: Acne vulgaris is a common pilosebaceous disease associated with Propionibacterium acnes (P. acnes). Resolution of comedones may occur in association with shrunken sebaceous glands (SGs) containing de-differentiated cells, however the role of P. acnes is unclear. OBJECTIVES: To investigate the effects of P. acnes on aryl hydrocarbon receptor (AhR) activation, lipogenesis and differentiation in cultured immortalized human SZ95 sebocytes. MATERIALS & METHODS: Cultured sebocytes were incubated with formalin-killed (f-) P. acnes (f-P. acnes) at different ratios of multiplicity of infection. The mRNA levels of the AhR downstream cytochrome P450 (CYP) genes were measured by quantitative RT-PCR, nuclear translocation of AhR by western blot and immunofluorescence, lipogenesis and keratinization by gene set enrichment analysis (GSEA), lipid related analysis by Oil red O staining and Nile red staining, and sebaceous differentiation-related gene expression by western blot. RESULTS: f-P. acnes upregulated CYPs mRNA levels and induced translocation of AhR protein from the cytoplasm into the nucleus. GSEA revealed downregulation of lipogenesis and upregulation of keratinization. f-P. acnes inhibited linoleic acid-induced neutral lipid synthesis and expression of sebocyte markers, keratin 7 and mucin1/EMA, but increased expression of keratinocyte markers, keratin 10 and involucrin, which were abolished by AhR gene silencing. Inhibition of lipogenesis-related genes, such as sterol response element-binding protein, was also observed. CONCLUSION: f-P. acnes inhibits lipogenesis and induces terminal differentiation of sebocytes, into keratinocyte-like cells, via activation of the AhR pathway in vitro, suggesting that follicular P. acnes is not only acnegenic but also promotes acne remission through feedback regulation of sebum production.

Comparison of responses of melanocyte lineages from p75(+) and p75(-) human scalp-derived neural crest stem cells under phototherapy.

Phototherapy is an effective therapeutic option in the treatment of vitiligo; however, responses varied among the different types. The underlying mechanism has scarcely been investigated. To investigate and compare the effects of phototherapy on the mutation of melanocyte lineage differentiated from human scalp-derived neural crest stem cells (HS-NCSCs) with p75 neurotrophin receptor expression positive and p75 neurotrophin receptor expression negative group in vitro, the HS-NCSCs were isolated from fetal scalp tissue, which is identified by immunofluorescent staining. The p75(+) and p75(-) cells from HS-NCSCs were isolated by magnetic cell sorting, respectively. The embryonic neural crest stem cell biomarkers were detected by RT-PCR. Narrow-band UVB (NB-UVB) was used to irradiate the cells. Cell proliferation was evaluated by cell count. Tyrosinase, Tyrp1, and Tyrp2 gene expression were measured by quantitative RT-PCR. Tyrosinase and GRCR protein levels were investigated by Western blot analysis. The electrophoretic strip showed that Sox2, Oct4, Sox10, and Nestin of p75(+) HS-NCSCs were brighter than the p75(-) HS-NCSCs. After the same dose radiation with NB-UVB, the cell proliferation of p75(+) group showed less inhibitory rate compared with the p75(-) HS-NCSCs. The tyrosinase mRNA and protein expression of differentiated melanocytes increased significantly in the group of p75(+) HS-NCSCs compared with the p75(-) group. The melanocytic mutation of p75(+) HS-NCSCs increased significantly compared with the p75(-) HS-NCSCs under NB-UVB, which indicated there were more melanocyte precursors in the differentiated cells from p75(+) HS-NCSCs. This may provide new insights for the different repigmentation efficacy of segmental and non-segmental vitiligo.

Aryl Hydrocarbon Receptor Modulates the Expression of TNF-α and IL-8 in Human Sebocytes via the MyD88-p65NF-κB/p38MAPK Signaling Pathways.

Activation of Toll-like receptor (TLR)-2 and subsequent inflammatory response contribute to lesion development in acne vulgaris. A cross-talk between aryl hydrocarbon receptor (AhR), a cytosolic receptor protein that responds to environmental and physiological stress, and TLRs has recently been reported. In this study, we explored the possible role of AhR in the effects induced on cultured human SZ95 sebocytes by peptidoglycan (PGN), a classic TLR2 agonist. PGN-induced secretion of inflammatory factors TNF-α and IL-8 in human SZ95 sebocytes was suppressed after knockdown of AhR and pretreatment with the AhR antagonist CH223191. In addition, the AhR agonist 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) enhanced TNF-α and IL-8 secretion in PGN-pretreated sebocytes. Furthermore, PGN-induced expression of myeloid differentiation factor 88 (MyD88), phospho-p38MAPK (p-p38MAPK), and p-p65NF-κB was strengthened by TCDD and repressed by CH223191. AhR inhibition by transfecting shRNA blocked the ability of PGN to stimulate phosphorylation of p38MAPK and p65NF-κB in SZ95 sebocytes. Overall, these data demonstrate that AhR is able to modulate PGN-induced expression of TNF-α and IL-8 in human SZ95 sebocytes involving the MyD88-p65NF-κB/p38MAPK signaling pathway, which probably indicates a new mechanism in TLR2-mediated acne.

Activation and overexpression of the aryl hydrocarbon receptor contribute to cutaneous squamous cell carcinomas: an immunohistochemical study.

BACKGROUND: In vitro studies showed that the aryl hydrocarbon receptor (AHR) contributed to the development of cutaneous squamous cell carcinomas, but supporting clinical data are lacking. METHODS: Immunohistochemical analysis was used to detect the expression of AHR, CYP1A1, EGFR, and Ki-67 in 10 actinic keratosis (AK) cases, 10 Bowen disease (BD) cases, 20 cutaneous squamous cell carcinoma (cSCC) cases and 20 normal skin samples. H-scores were used to assess the immunoreactivity. RESULTS: Weak positive AHR immunoreactivity was found in all normal skin samples, while strong positive AHR immunoreactivity was found in atypical squamous proliferation (AK, BD and cSCC) cases. H-scores and the rate of strong immunostaining of the atypical squamous proliferation cases were higher than those of normal controls (p < 0.01). Nuclear expression of AHR was higher in atypical squamous proliferation cases than in normal controls (p < 0.01). H-scores and the nuclear expression rate of AHR were significantly higher in AK and BD cases than cSCC cases (p < 0.01). CYP1A1 expression was low and showed no differences among the four studied groups (p > 0.05). The H-score of AHR was positively correlated with EGFR expression (r = 0.54, p < 0.01) in atypical squamous proliferation cases but was not correlated with CYP1A1 (r = - 0.17, p = 0.295) and Ki-67 (r = - 0.48, p = 0.222) expression. CONCLUSION: AHR plays a vital role in cSCC pathogenesis. The overexpression and activation of AHR are involved in the early development of skin cancers. AHR expression correlates with EGFR expression and may influence cell proliferation. AHR is a valuable therapeutic target for skin cancers.

Efficacy and Safety of Targeted High-Intensity Medium-Band (304-312 nm) Ultraviolet B Light in Pediatric Vitiligo.

BACKGROUND/OBJECTIVES: Phototherapy is a commonly used treatment for vitiligo that has demonstrated safety and efficacy. High-intensity targeted ultraviolet B (UVB) light (304-312 nm) delivered using a phototherapy device is a useful therapeutic option because it can induce repigmentation in a short time without global exposure to radiation, but information regarding this device in children is limited. METHODS: We performed a retrospective analysis of 95 patches of vitiligo in 27 children treated using a targeted phototherapy device. Phototherapy was administered twice a week. RESULTS: After the first 10 treatment sessions, 82 (86.3%) patches demonstrated some repigmentation and 36.8% achieved 50% or more repigmentation. After a mean of 20.4 treatment sessions, 86 patches (90%) demonstrated some repigmentation and 53.7% achieved 50% or more repigmentation. Responses varied depending on the anatomic location of the lesions. Better responses were usually observed on the face and trunk, whereas the extremities typically showed little response. Repigmentation was better in patients with active vitiligo than in those with stable vitiligo, with responses better with a disease duration of 1 year or less than in those with a duration of more than 1 year. There was no statistically significant difference in repigmentation between those with segmental and generalized vitiligo. The only short-term local side effect was mild erythema that required a decrease in dosage in six patients. CONCLUSION: Targeted high-intensity medium-band UVB phototherapy alone can produce clinical improvement in pediatric vitiligo and is well tolerated.

Clinical and histopathological characteristics in patients with scarring folliculitis type of acne inversa.

Objective: This study was designed to study the clinical and histopathological characteristics of patients with the scarring folliculitis type acne inversa in Chinese population. Methods: A total of 21 patients with acne inversa and 6 controls without known dermatological disease were recruited from outpatient department of dermatology and orthopedic surgery. Two-millimeter punch biopsies were taken from 8 patients with acne inversa and 6 controls, fixed in formalin, embedded in paraffin and stained with haematoxylin and eosin prior to histopathological analysis. Results: There were 12 patients (57.14%) belonging to the scarring folliculitis type presented with double comedones, papules, nodules, depressed scars, and were mainly Hurley stage I (66.67%). Many of the scarring folliculitis type were smokers (58.33%), some had a history of occupational exposure (41.67%) and some were overweight (50%), the mean BMI of which is 25.18±3.16 kg/m2. Histopathological changes such as perifollicular inflammation can be observed in scarring folliculitis type of acne inversa and controls as well. However, epidermal hyperplasia, follicular hyperplasia, sebaceous gland disappearance, destruction of hair follicle and sebaceous gland, collagen hyperplasia, perivascular inflammation, granulomatous inflammation, Micro thrombus were only seen in scarring folliculitis type. The mean surface area in patients (8073.36±15798.43 µm2) was smaller than that in controls (302059.08±502813.78 µm2), with statistically significant difference. (P = 0.024). Conclusion: The scarring folliculitis type in acne inversa in Chinese population could be characterized by depressed scars, double-ended comedones, epidermal cysts and had high proportion of smokers, or occupational exposure with lower Hurley stage, as well as diminished sebaceous gland. Further studies are needed to clarify the relations between the clinical subtypes of acne inversa and their corresponding genetypes.

Aryl hydrocarbon receptor negatively regulates lipid synthesis and involves in cell differentiation of SZ95 sebocytes in vitro.

The aryl hydrocarbon receptor (AhR) is the receptor for 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), benzo(a)pyrene (BaP) and other exogenous compounds. In human sebocytes, TCDD and BaP were found to activate the expression of multiple genes, including cytochrome P450 1A1 (CYP1A1), and inhibit lipid synthesis via AhR, while little is known about endogenous functions of the AhR. In order to expand this knowledge, we analyzed the impact of AhR knockdown on lipid synthesis as well as on cell differentiation of SZ95 sebocytes in vitro and observed that lipid synthesis was significantly induced in AhR silenced SZ95 sebocytes. In line with this result, expression of lipogenesis-associated genes, such as peroxisome proliferator activated receptor (PPAR) δ and PPARγ, was increased. Morphological changes with smaller cells in size but more abundant cytoplasmic lipids were observed in AhR silenced SZ95 sebocytes compared with the AhR activated cells. Besides, the expression of keratin 7, an early sebaceous differentiation marker, was increased, while the expression of the terminal sebocyte differentiation marker epithelial membrane antigen (EMA) was reduced. Moreover, the terminal keratinocyte differentiation markers keratin 10 and involucrin, and the AhR downstream protein CYP1A1 were reduced after AhR silencing. To the best of our knowledge, we provide evidence that in the absence of exogenous ligands, the AhR inhibits lipid synthesis and involves in cell differentiation of human SZ95 sebocytes, which indicates the physiological function of this receptor in human sebocytes.

Benzo(a)pyrene induces interleukin (IL)-6 production and reduces lipid synthesis in human SZ95 sebocytes via the aryl hydrocarbon receptor signaling pathway.

In this study, we determined the effects of benzo(a)pyrene (BaP) on the expression of aryl hydrocarbon receptor (AhR) and cytochrome P450 1A1 (CYP1A1), and assessed the action of BaP on inflammatory cytokine expression and lipid synthesis in SZ95 sebocytes in vitro. BaP (10(-8), 10(-7), 10(-6) and 10(-5)M) was not cytotoxic for SZ95 sebocytes after 24h exposure. Expression of AhR was promoted in mRNA lever, while was inhibited in protein lever after BaP (10(-5)M) exposure. CYP1A1 expression was up-regulated in both mRNA and protein levels. BaP (10(-5)M) exerted a stimulatory action on interleukin (IL)-6 secretion, while a dose-dependently inhibitory effect on lipid synthesis from 10(-8)M to 10(-5)M in SZ95 sebocytes. Both actions were partly antagonized in AhR-knockdowned SZ95 sebocytes. This study demonstrates that BaP can activate AhR signaling pathway, and exhibits pro-inflammatory effects and inhibitory effects on sebum production in human sebocytes.

The assessment of pulsed dye laser treatment of port-wine stains with reflectance confocal microscopy.

BACKGROUND: Reflectance confocal microscopy (RCM) is a noninvasive technique for evaluating cutaneous lesions with cellular level resolution close to conventional histopathology. The aim of this study is to observe the vascular changes in Port-wine (PWS) lesions and assess the clinical efficacy of Pulsed Dye Laser (PDL) treatment by examining vessel diameter and density with RCM. MATERIALS AND METHODS: Eleven adult patients with PWS, each had four test patches carried out with different pulse durations (1.5, 3, 6, and 10 ms), respectively; fluences of 9-12 J/cm²; and a spot size of 7 mm. The PDL treatment was repeated 3-5 times at a 2-month interval. Photographs and measurements with RCM were taken before each treatment and 2 months after the last treatment. RESULTS: The PDL treatment exhibited increasing clearance with reducing pulse durations. Vessel diameters and densities were significantly decreased in the same pulse-duration groups after treatment. There was significant difference between 1.5 ms pulse-duration group and other pulse-duration groups in reducing blood vessel diameter at the depth of 150 µm. CONCLUSIONS: RCM can be used to assess the clinical efficacy of PDL treatment.

In vivo reflectance confocal microscopy of Basal cell carcinoma: feasibility of preoperative mapping of cancer margins.

BACKGROUND: Reflectance confocal microscopy (RCM) images skin at cellular resolution and has shown utility for the diagnosis of nonmelanoma skin cancer in vivo. It has the potential to define lesion margins before surgical therapy. OBJECTIVES: To investigate the feasibility of RCM in defining the margins of basal cell carcinoma before surgery. METHODS: The margins of 10 lesions were evaluated using RCM. Biopsies of the margins were used to confirm the results. A protocol was constructed to define margins. RCM was used to delineate preoperative surgical margins in 13 patients. Intraoperative frozen biopsy was used to confirm the margins. RESULTS: In seven of 10 (70.0%) cases, the margins of the cancer were identified suing RCM. The tumor island was the critical feature in identifying the margins. In 12 of 13 (92.3%) cases, frozen biopsy corroborated that the surgical margins delineated by RCM were clear. CONCLUSION: RCM imaging of the margins is feasible and demonstrates the possibility of preoperative mapping of cancer margins.

In vivo reflectance confocal microscopy of extramammary Paget disease: diagnostic evaluation and surgical management.

BACKGROUND: Extramammary Paget disease (EMPD) is a diagnostic challenge. In vivo reflectance confocal microscopy (RCM) has been reported to be useful for in vivo skin tumor evaluation. It may also assist in the surgical management of EMPD lesions. OBJECTIVE: We sought to describe confocal features of EMPD and correlate them with histopathologic findings. The potential of RCM to map the lesions for subsequent surgical management was also investigated. METHODS: A total of 23 lesions from 14 recruited patients were evaluated by RCM and histopathologic examination. RCM was used to delineate preoperative surgical margins in two patients. RESULTS: Erythematous, hyperpigmented, and hypopigmented lesions were evaluated by RCM and results were confirmed by histopathologic examination. Paget cells were observed throughout the epidermis. Typical Paget cells on RCM were characterized by a mild bright nucleus and dark cytoplasm, frequently twice the size of keratinocytes or larger. At the dermoepidermal junction, tumor nests were seen as dark glandular structures. A high density of dendritic cells was observed in pigmented lesions and a low density in erythematous lesions. Dilated vessels and inflammatory cells were seen in pigmented and erythematous lesions. Paget cells within the epidermis and nest structures at the dermoepidermal junction were seen in most lesions. These two features were useful for delineating the margins. Histologic examination corroborated the surgical margins found by RCM. LIMITATIONS: The sensitivity and specificity of these diagnostic features have not been fully studied, and differential diagnostic features require exploration. CONCLUSION: Features correlating well to histopathology are observed on the RCM of EMPD lesions. RCM may be used as an auxiliary diagnostic tool for the diagnosis and management of EMPD.

The application of ZnO luminescent nanoparticles in labeling mice.

Photoluminescent ZnO@polymer core-shell nanoparticles were used in mouse imaging through intradermal injections and intravenous injections, and the results proved that such ZnO fluorescence probes are nontoxic to live mice and have great potential in in vivo applications.

In vivo reflectance confocal microscopy for the differential diagnosis between vitiligo and nevus depigmentosus.

BACKGROUND: Nevus depigmentosus (ND) is frequently confused with vitiligo. Differential diagnosis can be difficult. In vivo reflectance confocal microscopy (RCM) is a noninvasive technique for real-time en face imaging of the superficial layers of the skin down to the superficial dermis with cellular level resolution close to conventional histopathology. In this study, we tried to use this new technology to study the features of the distribution of pigment cells of these two hypopigmentation disorders and then concluded the differential features. METHODS: Sixty vitiligo patients and 62 ND patients were enrolled in the study. Three points in each patient (lesional, margin of the lesions and adjacent non- lesional points) were examined with RCM. The gray value of image was quantified using software, and we calculated the relative gray value. RESULTS: The RCM image feature was different between vitiligo and ND patients. The differential diagnosis was made based on the following four RCM features: complete absence of pigment cells; the distribution of pigment cells; the margins; and the relative gray value. CONCLUSION: RCM can be used as an auxiliary diagnostic tool for the differential diagnosis between vitiligo and ND.