A novel H­tert immortalized human sebaceous gland cell line (XL-i-20) for the investigation of photodynamic therapy.

BACKGROUND: Acne vulgaris is a species-specific human disease. To date, there has been no established human sebocyte cell line of Asian origin. Our previous study has demonstrated the efficacy of 5-aminolevulinic acid photodynamic therapy (ALA-PDT) in the treatment of acne vulgaris, primarily attributed to its cytotoxic properties; however, its regulatory mechanism remains largely unknown. OBJECTIVES: To establish an immortalized human sebocyte cell line derived from Chinese population and investigate the underlying mechanism of ALA-PDT. METHODS: Human primary sebocytes were transfected with the human tert gene (h­tert). The biological characteristics, including cell proliferation, cell markers, and sebum secretion function, were compared between primary sebocytes and the immortalized sebocytes (XL-i-20). Stimulations such as ALA-PDT, were applied respectively to both primary sebocytes and XL-i-20 cells to assess changes in their cellular functions. The transcriptome differences between primary sebocytes and XL-i-20 sebocytes were investigated using RNA-seq analysis. The XL-i-20 cell line was used to establish a sebaceous gland (SG) organoid culture, serving as a representative model of SG for the investigation of ALA-PDT. RESULTS: The h­tert immortalized sebocyte cell line exhibited the ability to be consecutively cultured for more than fifty passages. Both primary and immortalized cells expressed sebocyte markers such as epithelial membrane antigens (EMA, or MUC-1), Cytokeratin 7 (CK7) and adipose differentiation-related protein associated antigens (ADRP), and maintained sebum secretion function. The proliferative capacity of XL-i-20 was found to be significantly higher than that of primary sebocytes. The responses of XL-i-20 to ALA-PDT were indistinguishable from those elicited by primary sebocytes. Cell viability and sebum secretion were decreased after ALA-PDT in both two cell lines, and lipid-related proteins (SREBP-1/PPARγ) were down-regulated. The transcriptome data consistently demonstrated upregulation of genes related to inflammatory responses and downregulation of genes involved in lipid metabolism in both cell types following PDT. The analysis of common differential genes of primary sebocytes and XL-i-20 sebocytes post ALA-PDT showed that TNF signaling pathways, MAPK signaling pathways and JAK-STAT signaling pathways were activated. The SG organoids were spherical, which expressed markers of FANS and PLET1. Ki-67 was down-regulated after ALA-PDT. CONCLUSIONS: We have developed an h­tert immortalized sebocyte cell line from an Asian population. The cell line, XL-i-20, maintains the essential characteristics of its parent primary sebocytes. Moreover, XL-i-20 sebocyte exhibited a significant respond to ALA-PDT, demonstrating comparable phenotypic and molecular changes to primary sebocytes. Therefore, XL-i-20 and its derived SG organoid serve as appropriate in vitro models for investigating the efficacy and mechanisms of ALA-PDT in SG-related diseases.

Tricholoma matsutake polysaccharides suppress excessive melanogenesis via JNK-mediated pathway: Investigation in 8- methoxypsoralen induced B16-F10 melanoma cells and clinical study.

Skin hyperpigmentation is a worldwide condition associated with augmented melanogenesis. However, conventional therapies often entail various adverse effects. Here, we explore the safety range and depigmentary effects of polysaccharides extract of Tricholoma matsutake (PETM) in an in vitro model and further evaluated its efficacy at the clinical level. An induced-melanogenesis model was established by treating B16-F10 melanoma cells with 8-methoxypsoralen (8-MOP). Effects of PETM on cell viability and melanin content were examined and compared to a commonly used depigmentary agent, α-arbutin. Expressions of key melanogenic factors and upstream signaling pathway were analysed by quantitative PCR and western blot. Moreover, a placebo-controlled clinical study involving Chinese females with skin hyperpigmentation was conducted to measure the efficacy of PETM on improving facial pigmented spots, melanin index, and individual typology angle (ITA°). Results demonstrated that PETM (up to 0.5 mg/mL) had little effect on the viability and motility of B16-F10 cells. Notably, it significantly suppressed the melanin content and expressions of key melanogenic factors induced by 8-MOP in B16-F10 melanoma cells. Western blotting results revealed that PETM inhibited melanogenesis by inactivating c-Jun N-terminal kinase (JNK), and this inhibitory role could be rescued by JNK agonist treatment. Clinical findings showed that PETM treatment resulted in a significant reduction of facial hyperpigmented spot, decreased melanin index, and improved ITA° value compared to the placebo-control group. In conclusion, these in vitro and clinical evidence demonstrated the safety and depigmentary efficacy of PETM, a novel polysaccharide agent. The distinct mechanism of action of PETM on melanogenic signaling pathway positions it as a promising agent for developing alternative therapies.

A chlorin e6 derivative-mediated photodynamic therapy inhibits cutaneous squamous cell carcinoma cell proliferation via Akt/mTOR signaling pathway.

BACKGROUND AND OBJECTIVES: Although most cutaneous squamous cell carcinoma (cSCC) cases are generally nonlethal and manageable with surgical excision, there ares till significant hazards for patients who are ineligible for surgical resection. We sought to find a suitable and effective treatment for cSCC. METHODS: We modified chlorin e6 by adding a hydrogen chain with a six-carbon ring to the benzene ring and named this new photosensitizer as STBF. We first investigated the fluorescence characteristics, cellular uptake of STBF and subcellular localization. Next, cell viability was detected by CCK-8 assay and the TUNEL staining was performed. Akt/mTOR-related proteins were examined by western blot. RESULTS: STBF-photodynamic therapy (PDT) inhibits cSCC cells viability in a light dose dependent manner. The antitumor mechanism of STBF-PDT might be due to the suppression of the Akt/mTOR signaling pathway. Further animal investigation determined that STBF-PDT led to a marked reduction in tumor growth. CONCLUSIONS: Our results suggest that STBF-PDT exerts significant therapeutic effects in cSCC. Thus, STBF-PDT is expected to be a promising method for the treatment of cSCC and the photosensitizer STBF may be destined for a wider range of applications in photodynamic therapy.

ALA-PDT augments intense inflammation in the treatment of acne vulgaris by COX2/TREM1 mediated M1 macrophage polarization.

Severe acne vulgaris is a common chronic inflammatory skin disease worldwide. 5-Aminolaevulinic acid photodynamic therapy (ALA-PDT) is effective and safe for severe acne. However, the mechanism is not fully understood. Intense acute inflammatory response at 24 h after ALA-PDT is reported positively correlated to the effectiveness. Inflammation regulation influence the progression or outcome of diseases. ALA-PDT may exert its therapeutic effect by augmenting intense inflammation and break the chronic inflammation. This study was set out to explore the mechanism of ALA-PDT augmenting intense acute inflammation in the treatment of acne. As a result, transcriptome microarrays analysis of severe acne patients showed that ALA-PDT significantly up-regulated expression of various inflammation-related genes, especially TREM1 and PTGS2, which were further confirmed by a C.acnes induced acne-like mouse ear model. The subsequent experiments demonstrated that ALA-PDT could trigger pro-inflammatory M1 polarization of macrophages in vitro and in vivo. Additionally, the crosstalk between keratinocytes and macrophages studied by a transwell co-culture system indicated that PGE2 secreted by ALA-PDT treated HaCaT cells could promote THP-1 macrophages M1 polarization by COX2/PGE2/TLR4/TREM1 axis to augment inflammation. Our study provides a novel insight that ALA-PDT could amplify inflammation by COX2/TREM1 mediated macrophages M1 polarization for the treatment of acne. It is hoped that this research will decipher the mechanism of ALA-PDT for the treatment of acne and provide a theoretical basis for optimizing the clinical ALA-PDT management.

A 1-min double embedding method for small tissue specimens preserves comedone histology and eliminates the need for punch biopsies.

BACKGROUND: It is difficult to preserve the structure and microbial distribution inside comedonal plugs during routine processing. OBJECTIVE: The objective of this study is to determine the optimal method to preserve the comedonal corneum plug structure and inherent microorganisms thereby eliminating the need to perform punch biopsies in relevant studies. METHODS: Corneum plugs were extracted from comedones of acne vulgaris patients. Primary embedding using either a 2% agarose, 2% agar, 25% gelatin, or 2% agar + 2.5% gelatin solution was subsequently performed and the results compared. The specimens were then fixed, waxed, sectioned, and examined by light, fluorescence, and scanning electron microscopies to observe the structures and microorganisms within the plugs. RESULTS: Both the 25% gelatin and 2% agarose solutions successfully preserved the structural integrity of corneum plugs and the inherent microorganisms. When considering other factors such as thermostability, reusability, and convenience, the 25% gelatin solution was the superior choice among the four materials. CONCLUSION: We report a simple and effective method for double embedding comedonal plugs and other small tissue specimens. The technique preserves the structure and microbial distribution in situ within comedonal corneum plugs, eliminates the need for punch biopsies. This method may also be applied to other tiny and fragile tissue specimens, thereby enabling a potentially wide array of future large-scale investigations and alleviated patients' pain.

5-Aminolaevulinic acid photodynamic therapy suppresses lipid secretion of primary sebocytes through AMPK/SREBP-1 pathway.

BACKGROUND: Acne vulgaris is a chronic inflammatory skin disease around pilosebaceous unit. 5-Aminolaevulinic acid photodynamic therapy (ALA-PDT) is an effective therapy for severe acne vulgaris. However, its specific treatment mechanism remains unclear. In the present study, we investigated the potential mechanism of how ALA-PDT induced lipid secretion inhibition in acne vulgaris. METHODS: Primary human sebocytes and sebaceous gland of golden hamster were treated with/without ALA-PDT. Cell viability was evaluated by Live/Dead Cell assay. Fluorescence microscope was used to observe lipids secretion in sebocytes after Nile red staining. The expression of SREBP-1 after ALA-PDT was evaluated by qRT-PCR. Regulation of ALA-PDT on AMPK/SREBP-1 was evaluated by western blot. RESULTS: The results showed that ALA-PDT suppressed lipid secretion of primary human sebocytes. In addition, ALA-PDT could inhibit the expression of SREBP-1 in vitro. We also found that ALA-PDT activated AMPK pathway, down-regulating the expression of SREBP-1 in sebocytes after ALA-PDT. CONCLUSIONS: These findings elucidate that ALA-PDT suppresses lipid secretion through AMPK/SREBP-1 pathway in treatment of acne vulgaris.

5-Aminolaevulinic acid photodynamic therapy amplifies intense inflammatory response in the treatment of acne vulgaris via CXCL8.

Acne vulgaris is a chronic inflammatory cutaneous disease. 5-Aminolaevulinic acid photodynamic therapy (ALA-PDT) is a novel and effective approach for severe acne vulgaris treatment. However, its specific treatment mechanism still remains unclear. In the present study, we investigated the potential mechanism of how ALA-PDT regulated intense inflammatory response in acne vulgaris. It appeared that ALA-PDT suppresses proliferation and lipid secretion of primary human sebocytes. Besides, ALA-PDT could up-regulate the expression of CXCL8 in vivo and in vitro, amplifying the inflammatory response by recruiting T cells, B cells, neutrophils and macrophages. We also found that ALA-PDT elevated the expression of CXCL8 via p38 pathway. SB203580, a p38 pathway inhibitor, decreased the expression of CXCL8 in sebocytes after ALA-PDT. These findings indicate that ALA-PDT amplifies the intense inflammatory response in the treatment of acne vulgaris via CXCL8. Our data decipher the mechanism of intense inflammatory response after ALA-PDT for acne vulgaris.

Is Human Sebum the Source of Skin Follicular Ultraviolet-Induced Red Fluorescence? A Cellular to Histological Study.

BACKGROUND: The ultraviolet-induced red fluorescence (UVRF) from human skin follicles was suggested to be a result of Propionibacterium acnes and was used for the monitoring of acne. More recent studies suggested that the UVRF may be more related to sebum rather than to microorganisms. OBJECTIVE: To clarify whether human sebum or follicular microorganisms are the source of UVRF. METHODS: We examined the fluorescence of human-derived SZ95 sebocytes, human sebaceous glands, sebum extracted from the sebaceous glands, and bacteria isolated from human hair follicles under ultraviolet light. RESULTS: SZ95 sebocytes, human sebaceous glands, and sebum do not emit UVRF. Two types of UVRF peaking at about 635 nm and at about 620 nm were detected in P. acnes and Staphylococcus epidermidis, respectively. This is the first report that S. epidermidis emits UVRF when it is anaerobically cultured and then exposed to air. CONCLUSION: Human follicular UVRF is emitted by resident bacteria, not by sebum. Therefore, UVRF may be used to monitor certain species of skin microorganisms.

Quantifying facial skin erythema more precisely by analyzing color channels of The VISIA Red images.

BACKGROUND: The VISIA Red images were developed to document and measure facial skin erythema, but diffuse erythema cannot be fully segmented by the VISIA system due to the automatic thresholding segmentation method. Moreover, topical area analysis is not available in the system. MATERIALS AND METHODS: Erythema severity degrees of 20 simulated Red images were designated 1-20 with 1-20 inflammatory lesions for each, respectively. The RGB channel mean values of each simulated image were acquired by ImageJ and relative intensity of red values calculated. RESULTS: The relative intensity of red values positively correlate to erythema severity with a coefficient of 0.999345 (p < 0.001). We also proposed a method for calibration when pustules were present in the erythema area. The method was proved by mathematical reasoning and verified by certified dermatologists. CONCLUSION: We demonstrated a simple and more precise method to quantify and compare facial skin erythema by analyzing the RGB channel values of the VISIA Red images. Our method brings convenience for erythema evaluation in dermatological studies.