Autophagy-related 7 proteindependent autophagy mediates resveratrol-caused upregulation of mitochondrial biogenesis and steroidogenesis in aged Leydig cell.

BACKGROUND: Mitochondrial dysfunction may contribute to decreased testosterone synthesis in aged Leydig cells. Resveratrol (RSV) as an antioxidant has been shown to exhibit multiple positive effects on mitochondrion, where steroidogenesis takes place. Whether RSV can improve steroidogenesis in aged testis is still unknown. This study investigates the effect of RSV on testosterone production during aging and corresponding changes in mitochondrial biogenesis and autophagy activity, which are closely associated with steroidogenesis. Whether ATG7, an important autophagy-related protein, functions in RSV-treated aged Leydig cells will also be explored. METHODS AND RESULTS: Two-month-old male C57BL/6 mice were fed for 16 months by customized regular diet with or without RSV as diet supplement. Leydig cell line TM3 cells were treated with D-galactose to induce senescence, followed with or without RSV treatment. Results found that RSV supplement increased testosterone production in both aged mice and D-galactose-induced senescent Leydig cells. Western blot results revealed that RSV treatment elevated levels of steroidogenic rate-limiting enzymes StAR and 3ß-HSD, as well as autophagy-related proteins LC3II, Beclin1, ATG5 and ATG7 and mitochondrial function-related proteins mtTFA and COXIV. However, after Atg7 was knocked down in senescent Leydig cells, even though RSV was added, levels of these proteins declined significantly, accompanied by decreased levels of mitochondrial transcript factors PGC-1α, mtTFA and NRF-1 and more fragmented mitochondria, demonstrating that Atg7 knockdown wrecked the protective effects of RSV on steroidogenesis in senescent Leydig cells. CONCLUSION: ATG7-dependent autophagy plays a key role in RSV-brought testosterone production increase through regulating mitochondrial biogenesis in senescent Leydig cells.

Melatonin ameliorates diabetic hyperglycaemia-induced impairment of Leydig cell steroidogenic function through activation of SIRT1 pathway.

BACKGROUND: Diabetes mellitus (DM)-related complications are important health problems worldwide. The underlying mechanisms for diabetic male subfertility/infertility are considerably complicated and need to be unveiled for therapeutic intervention. Melatonin treatment was investigated to assess the beneficial effects on injured steroidogenic function in DM due to its regulatory roles in mitochondria and autophagy. METHODS: Diabetic hyperglycaemia was induced in rats injected with streptozotocin (STZ, 55 mg/kg/d) or simulated in TM3 Leydig cell line cultured with medium containing 30 mM D-glucose. Then, diabetic rats or the TM3 cells under high glucose were treated with melatonin. The diabetic rats were randomly divided into diabetes mellitus group (DM group), insulin treatment group (DM + INS group) and melatonin treatment group (DM + MT group). The TM3 Leydig cells were divided into a normal glucose control group (NG group), a high glucose treatment group (HG group), and a melatonin treatment group (HG + MT group). Then, Sirt1 (silent mating type information regulation 2 homologue) 1 expression was knocked down by siRNA. RESULTS: The results showed that hyperglycaemia induced a decline in steroidogenesis, accompanied by autophagy defects, mitochondrial dysfunction and oxidative stress, in rats in the DM group or TM3 Leydig cells in the HG group. Furthermore, reduced SIRT1 expression levels and hyperacetylation were found in Leydig cells of DM group. Melatonin treatment ameliorated hyperglycaemia-induced impairment of Leydig cell function with simultaneous stimulation of 5'-adenosine monophosphate activated protein kinase (AMPK)/SIRT1 activity and the expression of autophagy-related genes. With regards to mitochondrial function, it promoted mitochondrial biogenesis with elevated PGC-1α, NRF1 and mtTFA, improved mitochondrial morphology, increased BNIP3L-related mitophagy and alleviated oxidative stress. Further results revealed that knockdown of Sirt1 in Leydig cells prevented the protective effects provided by melatonin against high glucose treatment, and interestingly, neutralization of reactive oxygen species (ROS) by N-acetyl-L-cysteine pretreatment abolished the stimulatory effect of melatonin on AMPK/SIRT1 activity in Leydig cells and prevented the induction of autophagy and mitochondrial biogenesis in the context of high glucose, indicating that modulation of SIRT1 pathway by melatonin was closely linked to ROS levels and oxidative stress. CONCLUSIONS: These findings suggest that SIRT1 pathway plays essential roles in the pleiotropic actions of melatonin on Leydig cells and in the prevention of hyperglycaemia-induced steroidogenic dysfunction. The stimulatory action of melatonin on SIRT1 pathway is related to oxidative stress and its antioxidant property. Our data provide new evidence for the relationship of melatonin and SIRT1 pathway in the context of hyperglycaemia, and melatonin as a combination therapy may be useful to combat DM-related complications, especially male reproductive system injury.

Fighting against Skin Aging: The Way from Bench to Bedside.

As the most voluminous organ of the body that is exposed to the outer environment, the skin suffers from both intrinsic and extrinsic aging factors. Skin aging is characterized by features such as wrinkling, loss of elasticity, laxity, and rough-textured appearance. This aging process is accompanied with phenotypic changes in cutaneous cells as well as structural and functional changes in extracellular matrix components such as collagens and elastin. In this review, we summarize these changes in skin aging, research advances of the molecular mechanisms leading to these changes, and the treatment strategies aimed at preventing or reversing skin aging.

Expansion of Hair Follicle Stem Cells Sticking to Isolated Sebaceous Glands to Generate in Vivo Epidermal Structures.

Hair follicle stem cells (HFSCs) are considered one of the useful donor cell types for skin regenerative medicine owing to their robust proliferative capacity and multipotency. However, methods for easily and effectively obtaining HFSCs from a limited skin biopsy are still lacking. Here we report a novel approach for obtaining a subpopulation of HFSCs from a small skin sample from the rat tail, which uses the sebaceous glands (SGs) to capture the adjacent HFSCs. By means of organ culture, keratinocytes were expanded from the detached SGs, which also included adherent HFSCs from the hair follicle that could be passaged at the single-cell level. These SG-captured keratinocytes strongly expressed the basal layer markers K14, integrin α6, and p63; the bulge stem cell marker K15; and the upper isthmus stem cell marker Plet1. Furthermore, we reconstituted new epidermis, hair follicles, and SGs from the SG-captured keratinocytes using an easily operated, modified skin reconstitution assay based on silicone gel sheeting. This study suggests that the SGs could be an accessible capturer to harvest the adjacent HFSC subpopulation, particularly when the donor tissue is limited.

Ovine Hair Follicle Stem Cells Derived from Single Vibrissae Reconstitute Haired Skin.

Hair follicle stem cells (HFSCs) possess fascinating self-renewal capacity and multipotency, which play important roles in mammalian hair growth and skin wound repair. Although HFSCs from other mammalian species have been obtained, the characteristics of ovine HFSCs, as well as the methods to isolate them have not been well addressed. Here, we report an efficient strategy to obtain multipotent ovine HFSCs. Through microdissection and organ culture, we obtained keratinocytes that grew from the bulge area of vibrissa hair follicles, and even abundant keratinocytes were harvested from a single hair follicle. These bulge-derived keratinocytes are highly positive for Krt15, Krt14, Tp63, Krt19 and Itga6; in addition to their strong proliferation abilities in vitro, these keratinocytes formed new epidermis, hair follicles and sebaceous glands in skin reconstitution experiments, showing that these are HFSCs from the bulge outer root sheath. Taken together, we developed an efficient in vitro system to enrich ovine HFSCs, providing enough HFSCs for the investigations about the ovine hair cycle, aiming to promote wool production in the future.

GPR39 marks specific cells within the sebaceous gland and contributes to skin wound healing.

G protein-coupled receptors (GPCRs) mediate multiple key biological processes in the body. The orphan receptor GPR39 has been reported to be involved in various pathophysiological events. However, the function of GPR39 in skin biology remains unknown. Using a genetically engineered mouse strain in which lacZ expression faithfully replaced endogenous Gpr39 expression, we discovered a unique expression pattern of Gpr39 in the sebaceous gland (SG). Using various methods, we confirmed that GPR39 marked a specific cell population at the opening of the SG and colocalised with the SG stem cell marker Blimp1. Further investigations showed that GPR39 was spatiotemporally expressed during skin wound repair. Although it was dispensable for skin development and homeostasis, GPR39 contributed positively to skin wound healing: its loss led to a delay in wound healing during the intermediate stage. The present study reveals a novel role of GPR39 in both dermatology and stem cell biology that has not been previously recognised.

Estrogen leads to reversible hair cycle retardation through inducing premature catagen and maintaining telogen.

Estrogen dysregulation causes hair disorder. Clinical observations have demonstrated that estrogen raises the telogen/anagen ratio and inhibits hair shaft elongation of female scalp hair follicles. In spite of these clinical insights, the properties of estrogen on hair follicles are poorly dissected. In the present study, we show that estrogen induced apoptosis of precortex cells and caused premature catagen by up-regulation of TGF ß2. Immediately after the premature catagen, the expression of anagen chalone BMP4 increased. The up-regulation of BMP4 may further function to prevent anagen transition and maintain telogen. Interestingly, the hair follicle stem cell niche was not destructed during these drastic structural changes caused by estrogen. Additionally, dermal papilla cells, the estrogen target cells in hair follicles, kept their signature gene expressions as well as their hair inductive potential after estrogen treatment. Retention of the characteristics of both hair follicle stem cells and dermal papilla cells determined the reversibility of the hair cycle suppression. These results indicated that estrogen causes reversible hair cycle retardation by inducing premature catagen and maintaining telogen.

Hair follicle stem cells derived from single rat vibrissa via organ culture reconstitute hair follicles in vivo.

Hair follicle stem cells (HFSCs) are potentially useful for the treatment of skin injuries and diseases. To achieve clinical application, a prerequisite must be accomplished: harvesting enough HFSCs from limited skin biopsy. The commonly used sorting approach for isolating HFSCs, however, suffers from its intrinsic disadvantages, such as requirement of large-scale skin biopsy. Here, we report an efficient organ culture method to isolate and expand rat HFSCs from limited skin biopsy and these HFSCs could reconstitute the epidermis and the hair follicles (HFs). Seventy-three percent of cultured HFs formed hair follicle stem cell colonies from the bulge, and a single hair follicle provided all the HFSCs used in this research, demonstrating the high efficiency of this method. Quantitative RT-PCR and immunofluorescent staining results revealed that these stem cells obtained from the bulge highly expressed basal layer markers K14 and alpha-6 integrin, epithelial stem cell marker P63, and bulge stem cell marker K15. After long-term culture in vitro, GFP-labeled hair follicle stem cells formed new hair follicles, epidermis, and sebaceous glands following xenotransplantation into the back of nude mice. This study indicated that multipotent hair follicle stem cells could be efficiently harvested through organ culture from limited skin material-even a single hair follicle-and reconstitute hair follicles in vivo after long-term expansion culture, providing the basis for future clinical applications.

NASA-approved rotary bioreactor enhances proliferation of human epidermal stem cells and supports formation of 3D epidermis-like structure.

The skin is susceptible to different injuries and diseases. One major obstacle in skin tissue engineering is how to develop functional three-dimensional (3D) substitute for damaged skin. Previous studies have proved a 3D dynamic simulated microgravity (SMG) culture system as a "stimulatory" environment for the proliferation and differentiation of stem cells. Here, we employed the NASA-approved rotary bioreactor to investigate the proliferation and differentiation of human epidermal stem cells (hEpSCs). hEpSCs were isolated from children foreskins and enriched by collecting epidermal stem cell colonies. Cytodex-3 micro-carriers and hEpSCs were co-cultured in the rotary bioreactor and 6-well dish for 15 days. The result showed that hEpSCs cultured in rotary bioreactor exhibited enhanced proliferation and viability surpassing those cultured in static conditions. Additionally, immunostaining analysis confirmed higher percentage of ki67 positive cells in rotary bioreactor compared with the static culture. In contrast, comparing with static culture, cells in the rotary bioreactor displayed a low expression of involucrin at day 10. Histological analysis revealed that cells cultured in rotary bioreactor aggregated on the micro-carriers and formed multilayer 3D epidermis structures. In conclusion, our research suggests that NASA-approved rotary bioreactor can support the proliferation of hEpSCs and provide a strategy to form multilayer epidermis structure.

Correlation analysis of liver tumor-associated genes with liver regeneration.

AIM: To study at transcriptional level the similarities and differences of the physiological and biochemical activities between liver tumor (LT) and regenerating liver cells. METHODS: LT-associated genes and their expression changes in LT were obtained from databases and scientific articles, and their expression profiles in rat liver regeneration (LR) were detected using Rat Genome 230 2.0 array. Subsequently their expression changes in LT and LR were compared and analyzed. RESULTS: One hundred and twenty one LT-associated genes were found to be LR-associated. Thirty four genes were up-regulated, and 14 genes were down-regulated in both LT and regenerating liver; 20 genes up-regulated in LT were down-regulated in regenerating liver; 21 up-regulated genes and 16 down-regulated genes in LT were up-regulated at some time points and down-regulated at others during LR. CONCLUSION: Results suggested that apoptosis activity suppressed in LT was still active in regenerating liver, and there are lots of similarities and differences between the LT and regenerating liver at the aspects of cell growth, proliferation, differentiation, migration and angiogenesis.

Expression patterns and action analysis of genes associated with hepatitis virus infection during rat liver regeneration.

AIM: To study the action of hepatitis virus infection-associated genes at transcription level during liver regeneration (LR). METHODS: Hepatitis virus infection-associated genes were obtained by collecting the data from databases and retrieving the correlated articles, and their expression changes in the regenerating rat liver were detected with the rat genome 230 2.0 array. RESULTS: Eighty-eight genes were found to be associated with liver regeneration. The number of genes initially and totally expressed during initial LR [0.5-4 h after partial hepatectomy (PH)], transition from G0 to G1 (4-6 h after PH), cell proliferation (6-66 h after PH), cell differentiation and reorganization of structure-function (66-168 h after PH) was 37, 8, 48, 3 and 37, 26, 80, 57, respectively, indicating that the genes were mainly triggered at the early stage of LR (0.5-4 h after PH), and worked at different phases. These genes were classified into 5 types according to their expression similarity, namely 37 up-regulated, 9 predominantly up-regulated, 34 down-regulated, 6 predominantly down-regulated and 2 up/down-regulated genes. Their total up- and down-regulation frequencies were 359 and 149 during LR, indicating that the expression of most genes was enhanced, while the expression of a small number of genes was attenuated during LR. According to time relevance, they were classified into 12 groups (0.5 and 1 h, 2 and 4 h, 6 h, 8 and 12 h, 16 and 96 h, 18 and 24 h, 30 and 42 h, 36 and 48 h, 54 and 60 h, 66 and 72 h, 120 and 144 h, 168 h), demonstrating that the cellular physiological and biochemical activities during LR were fluctuated. According to expression changes of the genes, their expression patterns were classified into 23 types, suggesting that the cellular physiological and biochemical activities during LR were diverse and complicated. CONCLUSION: The anti-virus infection capacity of regenerating liver can be enhanced and 88 genes play an important role in LR.