Research on the morphological structure of partial fracture healing process in diabetic mice based on synchrotron radiation phase-contrast imaging computed tomography and deep learning.

The prevalence of diabetes mellitus has exhibited a notable surge in recent years, thereby augmenting the susceptibility to fractures and impeding the process of fracture healing. The primary objective of this investigation is to employ synchrotron radiation phase-contrast imaging computed tomography (SR-PCI-CT) to examine the morphological and structural attributes of different types of callus in a murine model of diabetic partial fractures. Additionally, a deep learning image segmentation model was utilized to facilitate both qualitative and quantitative analysis of callus during various time intervals. A total of forty male Kunming mice, aged five weeks, were randomly allocated into two groups, each consisting of twenty mice, namely, simple fracture group (SF) and diabetic fracture group (DF). Mice in DF group were intraperitoneally injected 60 mg/kg 1 % streptozotocin(STZ) solution for 5 consecutive days, and the standard for modeling was that the fasting blood glucose level was ≥11.1 mmol /l one week after the last injection of STZ. The right tibias of all mice were observed to have oblique fractures that did not traverse the entire bone. At three, seven, ten and fourteen days after the fracture occurred, the fractured tibias were extracted for SR-PCI-CT imaging and histological analysis. Furthermore, a deep learning image segmentation model was devised to automatically detect, categorize and quantitatively examine different types of callus. Image J software was utilized to measure the grayscale values of different types of callus and perform quantitative analysis. The findings demonstrated that:1)SR-PCI-CT imaging effectively depicted the morphological attributes of different types of callus of fracture healing. The grayscale values of different types of callus were significantly different(P < 0.01).2)In comparison to the SF group, the DF group exhibited a significant reduction in the total amount of callus during the same period (P < 0.01). Additionally, the peak of cartilage callus in the hypertrophic phase was delayed.3)Histology provides the basis for training algorithms for deep learning image segmentation models. The deep-learning image segmentation models achieved accuracies of 0.69, 0.81 and 0.733 for reserve/proliferative cartilage, hypertrophic cartilage and mineralized cartilage, respectively, in the test set. The corresponding Dice values were 0.72, 0.83 and 0.76, respectively. In summary, SR-PCI-CT images are close to the histological level, and a variety of cartilage can be identified on synchrotron radiation CT images compared with histological examination, while artificial intelligence image segmentation model can realize automatic analysis and data generation through deep learning, and further determine the objectivity and accuracy of SR-PCI-CT in identifying various cartilage tissues. Therefore, this imaging technique combined with deep learning image segmentation model can effectively evaluate the effect of diabetes on the morphological and structural changes of callus during fracture healing in mice.

Psychological stress induces hair regenerative disorders through corticotropin-releasing hormone-mediated autophagy inhibition.

Psychosocial stress is increasing, causing a growing number of people to suffer from hair loss. Stress-related corticotropin-releasing hormone (CRH) is associated with hair loss, but the mechanism by which hair follicles respond to stress and CRH remain poorly understood. The aim of the study is to elucidate the association between CRH and stress-related hair regenerative disorders, and reveal the potential pathological mechanisms. A chronic unpredictable stress mouse model and a chronic social defeat stress mouse model were used to examine the role of CRH and stress-related hair regrowth. Chronic unpredictable stress and chronic social defeat stress increased the expression of CRH and CRH receptors (CRHRs), and contributed to the onset of hair-cycle abnormalities. Psychoemotional stress and stress-related CRH blocked hair follicle regrowth, which could be restored by astressin, a CRHR antagonist. Long-term exposure to either chronic unpredictable stress or CRH induced a decrease in autophagy, which could be partially rescued by astressin. Activating CRHR, by stress or CRH administration, decreased autophagy via the mTOR-ULK1 signaling pathway to mediate hair regenerative disorders, which could be partially reversed through enhancing autophagy by administration of brefeldin A. These findings indicate that CRH-mediated autophagy inhibition play an important role in stress-induced hair regenerative disorders. CRH regulates the local hypothalamic-pituitary-adrenal axis of hair follicles, but also plays an independent pathogenic role in stress-related hair regenerative disorders through CRH-mediated autophagy inhibition. This work contributes to the present understanding of hair loss and suggests that enhancing autophagy may have a therapeutic effect on stress-induced hair loss.

Dermal papilla cell-secreted biglycan regulates hair follicle phase transit and regeneration by activating Wnt/ß-catenin.

Alopecia is a prevalent problem of cutaneous appendages and lacks effective therapy. Recently, researchers have been focusing on mesenchymal components of the hair follicle, i.e. dermal papilla cells, and we previously identified biglycan secreted by dermal papilla cells as the key factor responsible for hair follicle-inducing ability. In this research, we hypothesized biglycan played an important role in hair follicle cycle and regeneration through regulating the Wnt signalling pathway. To characterize the hair follicle cycle and the expression pattern of biglycan, we observed hair follicle morphology in C57BL/6 mice on Days 0, 3, 5, 12 and 18 post-depilation and found that biglycan is highly expressed at both mRNA and protein levels throughout anagen in HFs. To explore the role of biglycan during the phase transit process and regeneration, local injections were administered in C57BL/6 and nude mice. Results showed that local injection of biglycan in anagen HFs delayed catagen progression and involve activating the Wnt/ß-catenin signalling pathway. Furthermore, local injection of biglycan induced HF regeneration and up-regulated expression of key Wnt factors in nude mice. In addition, cell analyses exhibited biglycan knockdown inactivated the Wnt signalling pathway in early-passage dermal papilla cell, whereas biglycan overexpression or incubation activated the Wnt signalling pathway in late-passage dermal papilla cells. These results indicate that biglycan plays a critical role in regulating HF cycle transit and regeneration in a paracrine and autocrine fashion by activating the Wnt/ß-catenin signalling pathway and could be a potential treatment target for hair loss diseases.

N-n-Butyl haloperidol iodide ameliorates liver fibrosis and hepatic stellate cell activation in mice.

N-n-Butyl haloperidol iodide (F2) is a novel compound that has antiproliferative and antifibrogenic activities. In this study we investigated the therapeutic potential of F2 against liver fibrosis in mice and the underlying mechanisms. Two widely used mouse models of fibrosis was established in mice by injection of either carbon tetrachloride (CCl4) or thioacetamide (TAA). The mice received F2 (0.75, 1.5 or 3 mg·kg-1·d-1, ip) for 4 weeks of fibrosis induction. We showed that F2 administration dose-dependently ameliorated CCl4- or TAA-induced liver fibrosis, evidenced by significant decreases in collagen deposition and c-Jun, TGF-ß receptor II (TGFBR2), α-smooth muscle actin (α-SMA), and collagen I expression in the liver. In transforming growth factor beta 1 (TGF-ß1)-stimulated LX-2 cells (a human hepatic stellate cell line) and primary mouse hepatic stellate cells, treatment with F2 (0.1, 1, 10 µM) concentration-dependently inhibited the expression of α-SMA, and collagen I. In LX-2 cells, F2 inhibited TGF-ß/Smad signaling through reducing the levels of TGFBR2; pretreatment with LY2109761 (TGF-ß signaling inhibitor) or SP600125 (c-Jun signaling inhibitor) markedly inhibited TGF-ß1-induced induction of α-SMA and collagen I. Knockdown of c-Jun decreased TGF-ß signaling genes, including TGFBR2 levels. We revealed that c-Jun was bound to the TGFBR2 promoter, whereas F2 suppressed the binding of c-Jun to the TGFBR2 promoter to restrain TGF-ß signaling and inhibit α-SMA and collagen I upregulation. In conclusion, the therapeutic benefit of F2 against liver fibrosis results from inhibition of c-Jun expression to reduce TGFBR2 and concomitant reduction of the responsiveness of hepatic stellate cells to TGF-ß1. F2 may thus be a potentially new effective pharmacotherapy for human liver fibrosis.

The opposing roles of the mTOR signaling pathway in different phases of human umbilical cord blood-derived CD34+ cell erythropoiesis.

As an indispensable, even lifesaving practice, red blood cell (RBC) transfusion is challenging due to several issues, including supply shortage, immune incompatibility, and blood-borne infections since donated blood is the only source of RBCs. Although large-scale in vitro production of functional RBCs from human stem cells is a promising alternative, so far, no such system has been reported to produce clinically transfusable RBCs due to the poor understanding of mechanisms of human erythropoiesis, which is essential for the optimization of in vitro erythrocyte generation system. We previously reported that inhibition of mammalian target of rapamycin (mTOR) signaling significantly decreased the percentage of erythroid progenitor cells in the bone marrow of wild-type mice. In contrast, rapamycin treatment remarkably improved terminal maturation of erythroblasts and anemia in a mouse model of ß-thalassemia. In the present study, we investigated the effect of mTOR inhibition with rapamycin from different time points on human umbilical cord blood-derived CD34+ cell erythropoiesis in vitro and the underlying mechanisms. Our data showed that rapamycin treatment significantly suppressed erythroid colony formation in the commitment/proliferation phase of erythropoiesis through inhibition of cell-cycle progression and proliferation. In contrast, during the maturation phase of erythropoiesis, mTOR inhibition dramatically promoted enucleation and mitochondrial clearance by enhancing autophagy. Collectively, our results suggest contrasting roles for mTOR in regulating different phases of human erythropoiesis.

LncRNA H19 Overexpression Activates Wnt Signaling to Maintain the Hair Follicle Regeneration Potential of Dermal Papilla Cells.

Androgenetic alopecia (AGA) is a common hair loss disorder resulting in seriously abnormal social interaction and psychological disorders. Transplantation with autologous dermal papilla cells represents a prospective therapy. However, the ability of dermal papilla cells to induce hair follicle development is lost upon cell culturing. Long non-coding RNAs (lncRNAs) are an important class of genes involved in various biological functions, are aberrantly expressed in disease and may play roles in the regulation of Wnt signaling, a critical pathway in maintaining the hair follicle-inducing capability of dermal papilla cells. Examination of dermal papilla cells by lncRNA microarray revealed that H19 was highly expressed in early passage dermal papilla cells compared with late-passage dermal papilla cells. In this study, we constructed H19-overexpressing dermal papilla cells to examine the role of H19 on hair follicle inductivity. Dermal papilla cells infected with lentivirus encoding H19 maintained their cell shape, and continued to display both multiple-layer aggregation and hair follicle-inducing ability upon prolonged culture. H19 exerted these effects through inducing miR-29a to activate Wnt signaling by directly downregulating the expression of Wnt suppressors, including DKK1, Kremen2, and sFRP2, thereby forming a novel regulatory feedback loop between H19 and miR-29a to maintain hair follicle- inducing potential. These results suggest that lncRNA H19 maintains the hair follicle-inducing ability of dermal papilla cells through activation of the Wnt pathway and could be a target for treatment of androgenetic alopecia.

miR-195-5p Regulates Hair Follicle Inductivity of Dermal Papilla Cells by Suppressing Wnt/ß-Catenin Activation.

Dermal papilla (DP) cells play a vital role in hair follicle (HF) development and postnatal hair cycling. However, the abilities are lost on further culture. Recent studies have demonstrated significant influences of posttranscriptional regulation by microRNA (miRNA) on HF development. The current study aims to investigate how miRNAs regulate Wnt/ß-catenin to control HF inductivity of DP cells by performing microarray analysis in early- and late-passage DP cells and transfecting with miRNAs inhibitor or mimic. Results showed early-passage DP cells strongly expressed miRNAs related to inhibition of noncanonical Wnt pathways. In late-passage DP cells, miRNAs capable of inhibiting the canonical Wnt/ß-catenin pathway were upregulated, in addition to the miRNAs targeting the noncanonical Wnt pathway. Moreover, we verified that ß-catenin expression was downregulated by miR-195-5p overexpression in dose manner. Meanwhile LRP6 expression was downregulated in both protein and mRNA as well as the genes involved in the hair inductivity of DP cells. These results suggest that the appearance of miRNAs that suppress the Wnt/ß-catenin pathway may be responsible for the loss of ability of DP cells in culture and miR-195-5p is the potential key factor involved in regulating HF inductivity of DP cells.

Inhibition of the PI3K-Akt-mTOR signaling pathway in T lymphocytes in patients with active tuberculosis.

OBJECTIVES: To investigate PI3K-Akt-mTOR signaling pathway changes and the proliferation of FoxP3+Treg cells in patients with active tuberculosis. METHODS: We isolated PBMCs and CD4+CD25+FoxP3+Treg cells from peripheral blood collected from patients with active tuberculosis and healthy controls. We compared the proportion and MFI of PI3K-Akt-mTOR pathway components and PTEN by flow cytometry using specific cell-surface and intracellular markers. Moreover, we detected the specific secretory proteins ESAT-6 and Ag85B, cytokines IL-10, TGF-ß1 and IL-35 in serum by ELISA. RESULTS: Compared with healthy controls, the proportions of CD3+Akt+, CD3+p-Akt+, CD3+mTOR+, CD3+p-mTOR+ and CD3+PTEN+ cells, in the T lymphocyte population of patients with active tuberculosis, were decreased (p<0.05), while CD3+FoxP3+ cells were increased (p=0.013). Similarly, for CD4+CD25+FoxP3+Treg cells, the proportions of Akt+ cells, p-Akt+ cells, mTOR+ cells, p-mTOR+ cells and PTEN+ cells were decreased (p<0.05) in patients with active tuberculosis. Compared with healthy controls, the levels of ESAT-6 and Ag85B were higher in patients with active tuberculosis (p<0.001). Levels of IL-10 and TGF-ß1 were higher (p<0.001), whereas the level of IL-35 was lower (p<0.001). CONCLUSION: The PI3K-Akt-mTOR signaling pathway in T lymphocytes and CD4+CD25+FoxP3+Treg cells was inhibited, which could explain why M.tuberculosis can induce FoxP3+Treg cell to expand.

The protective effect of the natural compound hesperetin against fulminant hepatitis in vivo and in vitro.

BACKGROUND AND PURPOSE: Liver diseases are mostly accompanied by inflammation and hepatocyte death. Therapeutic approaches targeting both hepatocyte injury and inflammation are not available. Natural compounds are considered as potential treatment for inflammatory liver diseases. Hesperetin, a flavonoid component of citrus fruits, has been reported to have anti-inflammatory properties. The aim of this study was to evaluate the cytoprotective and anti-inflammatory properties of hesperetin both in vitro and in models of fulminant hepatitis. EXPERIMENTAL APPROACH: Apoptotic cell death and inflammation were induced in primary cultures of rat hepatocytes by bile acids and cytokine mixture respectively. Apoptosis was quantified by caspase-3 activity and necrosis by LDH release. The concanavalin A (ConA) and D-galactosamine/LPS (D-GalN/LPS) were used as models of fulminant hepatitis. Liver injury was assessed by alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, liver histology and TUNEL assay and inflammation by inducible NOS (iNOS) expression. KEY RESULTS: Hesperetin blocked bile acid-induced apoptosis and cytokine-induced inflammation in rat hepatocytes. Moreover, hesperetin improved liver histology and protected against hepatocyte injury in ConA- and D-GalN/LPS-induced fulminant hepatitis, as assessed by TUNEL assay and serum AST and ALT levels. Hesperetin also reduced expression of the inflammatory marker iNOS and the expression and serum levels of TNFα and IFN-γ, the main mediators of cell toxicity in fulminant hepatitis. CONCLUSION AND IMPLICATIONS: Hesperetin has anti-inflammatory and cytoprotective actions in models of acute liver toxicity. Hesperetin therefore has therapeutic potential for the treatment of inflammatory liver diseases accompanied by extensive hepatocyte injury, such as fulminant hepatitis.

iTRAQ-Based Quantitative Proteomic Comparison of Early- and Late-Passage Human Dermal Papilla Cell Secretome in Relation to Inducing Hair Follicle Regeneration.

Alopecia is an exceedingly prevalent problem that lacks effective therapy. Recently, research has focused on early-passage dermal papilla cells (DPCs), which have hair inducing activity both in vivo and in vitro. Our previous study indicated that factors secreted from early-passage DPCs contribute to hair follicle (HF) regeneration. To identify which factors are responsible for HF regeneration and why late-passage DPCs lose this potential, we collected 48-h-culture medium (CM) from both of passage 3 and 9 DPCs, and subcutaneously injected the DPC-CM into NU/NU mice. Passage 3 DPC-CM induced HF regeneration, based on the emergence of a white hair coat, but passage 9 DPC-CM did not. In order to identify the key factors responsible for hair induction, CM from passage 3 and 9 DPCs was analyzed by iTRAQ-based quantitative proteomic technology. We identified 1360 proteins, of which 213 proteins were differentially expressed between CM from early-passage vs. late-passage DPCs, including SDF1, MMP3, biglycan and LTBP1. Further analysis indicated that the differentially-expressed proteins regulated the Wnt, TGF-ß and BMP signaling pathways, which directly and indirectly participate in HF morphogenesis and regeneration. Subsequently, we selected 19 proteins for further verification by multiple reaction monitoring (MRM) between the two types of CM. These results indicate DPC-secreted proteins play important roles in HF regeneration, with SDF1, MMP3, biglycan, and LTBP1 being potential key inductive factors secreted by dermal papilla cells in the regeneration of hair follicles.

High Uric Acid Activates the ROS-AMPK Pathway, Impairs CD68 Expression and Inhibits OxLDL-Induced Foam-Cell Formation in a Human Monocytic Cell Line, THP-1.

BACKGROUND/AIMS: Hyperuricemia is part of the metabolic-syndrome cluster of abdominal obesity, impaired glucose tolerance, insulin resistance, dyslipidemia, and hypertension. Monocytes/macrophages are critical in the development of metabolic syndrome, including gout, obesity and atherosclerosis. However, how high uric acid (HUA) exposure affects monocyte/macrophage function remains unclear. In this study, we investigated the molecular mechanism of HUA exposure in monocytes/macrophages and its impact on oxidized low-density lipoprotein (oxLDL)-induced foam-cell formation in a human monocytic cell line, THP-1. METHODS: We primed THP-1 cells with phorbol-12-myristate-13-acetate (PMA) for differentiation, then exposed cells to HUA and detected the production of reactive oxygen species (ROS) and analyzed the level of phospho-AMPKα. THP-1 cells were pre-incubated with Compound C, an AMPK inhibitor, or N-acetyl-L-cysteine (NAC), a ROS scavenger, or HUA before PMA, to assess CD68 expression and phospho-AMPKα level. PMA-primed THP-1 cells were pre-treated with oxLDL before Compound C and HUA treatment. Western blot analysis was used to examine the levels of phospho-AMPKα, CD68, ABCG1, ABCA1, cyclooxygenase-2 (COX-2) and NF-κB (p65). Flow cytometry was used to assess ROS production and CD68 expression in live cells. Oil-red O staining was used to observe oxLDL uptake in cells. RESULTS: HUA treatment increased ROS production in PMA-primed THP-1 cells; NAC blocked HUA-induced oxidative stress. HUA treatment time-dependently increased phospho-AMPKα level in PMA-primed THP-1 cells. The HUA-induced oxidative stress increased phospho-AMPKα levels, which was blocked by NAC. HUA treatment impaired CD68 expression during cell differentiation by activating the AMPK pathway, which was reversed by Compound C treatment. Finally, HUA treatment inhibited oxLDL uptake in the formation of foam cells in THP-1 cells, which was blocked by Compound C treatment. HUA treatment significantly increased the expression of ABCG1 and reversed the oxLDL-reduced ABCG1 expression but did not affect the expression of ABCA1, NF-κB (p65) or COX-2. CONCLUSIONS: HUA exposure activated the ROS-AMPK pathway, impaired CD68 expression, and inhibited oxLDL-induced foam-cell formation in a human monocytic cell line, THP-1.

Metformin inhibits estrogen-dependent endometrial cancer cell growth by activating the AMPK-FOXO1 signal pathway.

Metformin is an oral biguanide commonly used for treating type II diabetes and has recently been reported to possess antiproliferative properties that can be exploited for the prevention and treatment of a variety of cancers. The mechanisms underlying this effect have not been fully elucidated. Our study shows a marked loss of AMP-activated protein kinase (AMPK) phosphorylation and nuclear human Forkhead box O1 (FOXO1) protein in estrogen-dependent endometrial cancer (EC) tumors compared to normal control endometrium. Metformin treatment suppressed EC cell growth in a time-dependent manner in vitro; this effect was cancelled by cotreatment with an AMPK inhibitor, compound C. Metformin decreased FOXO1 phosphorylation and increased FOXO1 nuclear localization in Ishikawa and HEC-1B cells, with non-significant increase in FOXO1 mRNA expression. Moreover, compound C blocked the metformin-induced changes of FOXO1 and its phosphorylation protein, suggesting that metformin upregulated FOXO1 activity by AMPK activation. Similar results were obtained after treatment with insulin. In addition, transfection with siRNA for FOXO1 cancelled metformin-inhibited cell growth, indicating that FOXO1 mediated metformin to inhibit EC cell proliferation. A xenograft mouse model further revealed that metformin suppressed HEC-1B tumor growth, accompanied by downregulated ki-67 and upregulated AMPK phosphorylation and nuclear FOXO1 protein. Taken together, these data provide a novel mechanism of antineoplastic effect for metformin through the regulation of FOXO1, and suggest that the AMPK-FOXO1 pathway may be a therapeutic target to the development of new antineoplastic drugs.

Comparison of Calcium and Barium Microcapsules as Scaffolds in the Development of Artificial Dermal Papillae.

This study aimed to develop and evaluate barium and calcium microcapsules as candidates for scaffolding in artificial dermal papilla. Dermal papilla cells (DPCs) were isolated and cultured by one-step collagenase treatment. The DPC-Ba and DPC-Ca microcapsules were prepared by using a specially designed, high-voltage, electric-field droplet generator. Selected microcapsules were assessed for long-term inductive properties with xenotransplantation into Sprague-Dawley rat ears. Both barium and calcium microcapsules maintained xenogenic dermal papilla cells in an immunoisolated environment and induced the formation of hair follicle structures. Calcium microcapsules showed better biocompatibility, permeability, and cell viability in comparison with barium microcapsules. Before 18 weeks, calcium microcapsules gathered together, with no substantial immune response. After 32 weeks, some microcapsules were near inflammatory cells and wrapped with fiber. A few large hair follicles were found. Control samples showed no marked changes at the implantation site. Barium microcapsules were superior to calcium microcapsules in structural and mechanical stability. The cells encapsulated in hydrogel barium microcapsules exhibited higher short-term viability. This study established a model to culture DPCs in 3D culture conditions. Barium microcapsules may be useful in short-term transplantation study. Calcium microcapsules may provide an effective scaffold for the development of artificial dermal papilla.

Canonical and non-canonical Wnt signaling control the regeneration of amputated rodent vibrissae follicles.

Although mammals are notoriously poor at regeneration compared with many lower-order species, the hair follicle, particular to mammals, is capable of regeneration following partial amputation. The detailed internal mechanism of this phenomenon is still unclear. Development and regrowth of the hair follicle depends on dermal-epidermal interaction within the hair follicle. Previous studies have shown that Wnt/ß-catenin, Shh, Bmp, PDGF, TGF and Notch signals all take part in the development and growth of the hair follicle, and the Wnt/ß-catenin signaling additionally plays an indispensable role in hair follicle morphogenesis and regrowth. In this study, we investigated the localization, as well as, protein levels of Wnt/ß-catenin signaling molecules during amputated whisker follicle regeneration.

Suppressing cyclooxygenase-2 prevents nonalcoholic and inhibits apoptosis of hepatocytes that are involved in the Akt/p53 signal pathway.

Cyclooxygenase-2 (COX-2) can exert pro-inflammatory effects in nonalcoholic steatohepatitis (NASH). The aim of this study was to determine if the inhibition of COX-2 attenuates hepatocyte apoptosis in steatohepatitis and to examine the underlying molecular mechanism. Male wild type C57BL6/J mice and COX-2 knock out (COX-2-/-) mice were fed a methionine choline deficient (MCD) diet for 3 weeks. The wild type mice were also treated with celecoxib or a combination of celecoxib and a Akt specific inhibitor, miltefosine (MTF). After that, liver histology, serum alanine aminotransferase (ALT) levels, hepatic triglyceride (TG) levels, hepatocyte apoptosis, phosphorylated Akt (Ser473, pAkt) and p53 protein levels in mice livers were assessed. Celecoxib attenuated the severity of liver steatohepatitis and reduced the number of apoptotic cells, accompanied by increasing the activity of Akt and decreasing expression of p53. On the contrary, MTF can abrogate the effects of celecoxib on anti-apoptosis and anti-steatohepatitis. Moreover, the effects on the COX-2-/- mice that were fed the MCD diet were similar to that for celecoxib. The findings suggested that suppressing COX-2 can improve steatohepatitis by inhibiting hepatocyte apoptosis in mice via regulating the Akt/p53 pathway. Celecoxib treatment may be a favorable treatment option for NASH.

Expression of Wnt/ß-catenin signaling, stem-cell markers and proliferating cell markers in rat whisker hair follicles.

The rat whisker hair follicle (HF) is a model for studying the reconstruction of the HF or dermal papilla (DP), and involves the Wnt/ß-catenin signaling pathway, which is a key pathway in HF development and HF cycling after birth. It has been reported that Wnt/catenin signaling plays an indispensable role in human or rat pelages development and postnatal growth. However, the distribution of some Wnt/ß-catenin signaling pathway factors and their relationship with the epithelial stem cell markers in whisker follicles has not been characterized. In this study, we investigated the immunolocalization of Wnt/catenin signaling pathway members, including Wnt10b, Wnt10a, Wnt5a, ß-catenin, and downstream lymphoid enhancer-binding factor 1 (LEF1) and transcription factor 3 (TCF3), as well as, HF stem-cell markers CD34, CK15 and proliferating cell nuclear antigen (PCNA) protein, in rat anagen phase whisker follicles. ß-catenin, Wnt5a, Wnt10b, Wnt10a, LEF1, and TCF3 were expressed in the outer root sheath (ORS), inner root sheath, matrix and hair shaft of anagen follicles. ß-catenin, Wnt10b, LEF1, and TCF3 were highly expressed and Wnt5a and Wnt10a weakly expressed in DP and dermal sheath (DS) regions. The expression of α-smooth muscle actin was strong in the lower DS and it was also detected in some DP cells. CD34, CK15 and PCNA were all expressed in the ORS; and CD34 and PCNA were also detected in the matrix, however CD34 was extensively expressed in DP and DS regions. Our studies located the position of Wnts, downstream LEF1 and TCF3 and stem cell marker proteins, which provide new information in understanding the role of the Wnt singaling pathway in whisker follicles' growth.

Long noncoding RNA expression in dermal papilla cells contributes to hairy gene regulation.

Dermal papilla (DP) cells may be the source of dermal-derived signaling molecules involved in hair-follicle development and postnatal hair cycling. Early-passage DP cells can induce hair growth in vivo, but, on further culture, this ability is lost. The cellular mechanisms underlying the hair-follicle induction property of early-passage DP cells are unclear. Long noncoding RNAs (lncRNAs) are an important class of genes involved in various biological functions. They are aberrantly expressed and play roles in the regulation of the Wnt signaling pathway, a critical point in maintaining hair-induction activity. LncRNA microarray revealed 1683 upregulated and 1773 downregulated lncRNAs in passage-4 DP cells compare with passage-10 DP cells. To investigate the relation between lncRNAs and coding genes in WNT signaling, we constructed a coding-noncoding gene co-expression network using lncRNAs and coding genes that were differentially expressed between the passage-4 and -10 DP cells. RP11-766N7.3, H19 and HOTAIR are specific lncRNAs that were aberrantly expressed in DP cells and played an important role in regulating Wnt signaling. This study may provide potential targets for discovering the hair-follicle induction mechanism of early-passage DP cells.

Metformin suppresses hepatocellular carcinoma cell growth through induction of cell cycle G1/G0 phase arrest and p21CIP and p27KIP expression and downregulation of cyclin D1 in vitro and in vivo.

Metformin is used as a first-line therapy for type 2 diabetes, with reports of its usefulness for the prevention and control of several types of cancers. This study investigated the effects of metformin on hepatocellular carcinoma (HCC). The human HCC cell lines HepG2 and PLC/PRF/5 were cultured and treated with metformin or 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), an activator of adenosine monophosphate (AMP)-activated protein kinase. Changes in cell viability and cell cycle distribution were evaluated by MTT and flow cytometry, respectively. Apoptosis was assessed by fluorescent-dye staining. An HCC model was established in 6- to 8-week-old BALB/c-nu mice by subcutaneous injection of PLC/PRF/5 cells. After 1 week, mice were treated intragastrically with metformin or vehicle. Tumor xenograft tissues were examined using immunohistochemistry for evaluation of the the expression of cyclin D1, p21CIP and p27KIP. HCC cells and tissues from the in vitro and in vivo experiments, respectively, were subjected to protein extraction and western blotting. We found that metformin treatment reduced HCC cell viability in a dose-dependent manner similar to AICAR treatment. In addition, metformin treatment induced HCC cell cycle arrest at G1/G0 phase and apoptosis. Intragastric treatment of the mouse PLC/PRF/5 cell xenograft model with metformin showed that metformin not only blocked tumor progression, but also reduced tumor morbidity. Treatment with metformin upregulated the expression of p21CIP and p27KIP, but downregulated cyclin D1 levels, both in vitro and in vivo. Metformin treatment also upregulated the expression of phosphorylated AMPK protein in xenograft tissues. These findings indicate that metformin warrants further evaluation as a novel therapeutic and preventive strategy against HCC.

Uric acid induces oxidative stress and growth inhibition by activating adenosine monophosphate-activated protein kinase and extracellular signal-regulated kinase signal pathways in pancreatic ß cells.

Hyperuricaemia is a disorder of purine metabolism, and is strongly associated with insulin resistance and abnormal glucose metabolism. As the producer of insulin, pancreatic ß cells might be affected by elevated serum uric acid levels and contribute to the disregulated glucose metabolism. In this study, we investigated the effect of high uric acid on rat pancreatic ß cell function. Under high uric acid condition, proliferation of pancreatic ß cells was inhibited, production of reactive oxygen species increased, and glucose stimulated insulin secretion was also compromised. Further examination on signal transduction pathways revealed that uric acid-induced ROS is involved in the activation of adenosine monophosphate-activated protein kinase (AMPK), and extracellular signal-regulated kinase (ERK). Pharmacological inhibition of ERK activation rescued ß cells from growth inhibition. More importantly, activation of ERK induced by uric acid is significantly diminished by AMPK inhibitor, indicating ERK as a downstream target of AMPK in response to high uric acid condition. We also investigated the transportation channel for uric acid into pancreatic ß cells. While major urate transporter URAT1 is not expressed in ß cells, organic anion transporter (OAT) inhibitor successfully blocked the activation of ERK by uric acid. Our data indicate that high uric acid levels induce oxidative damage and inhibit growth of rat pancreatic ß cells by activating the AMPK and ERK signal pathways. Hyperuricemia may contribute to abnormal glucose metabolism by causing oxidative damage and function inhibition of pancreatic ß cells.