Transcription Factor DLX5 Promotes Hair Follicle Stem Cell Differentiation by Regulating the c-MYC/microRNA-29c-3p/NSD1 Axis.

INTRODUCTION: Adult stem cell function has been one of the most intensively explored areas of biological and biomedical research, with hair follicle stem cells serving as one of the best model systems. This study explored the role of the transcription factor DLX5 in regulating hair follicle stem cell (HFSC) differentiation. METHODS: HFSCs were isolated, characterized, and assessed for their expression of DLX5, c-MYC, NSD1, and miR-29c-3p using RT-qPCR, Western blot analysis, or immunofluorescence. Next, the ability of HFSCs to proliferate as well as differentiate into either sebaceous gland cells or epidermal cells was determined. The binding of DLX5 to the c-MYC promoter region, the binding of c-MYC to the miR-29c-3p promoter region, and the binding of miR-29c-3p to the 3'-UTR of NSD1 mRNA were verified by luciferase activity assay and ChIP experiments. RESULTS: DLX5 was highly expressed in differentiated HFSCs. DLX5 transcriptionally activated c-MYC expression to induce HFSC differentiation. c-MYC was able to bind the miR-29c-3p promoter and thus suppressed its expression. Without miR-29c-3p mediated suppression, NSD1 was then able to promote HFSC differentiation. These in vitro experiments suggested that DLX5 could promote HFSC differentiation via the regulation of the c-MYC/miR-29c-3p/NSD1 axis. DISCUSSION: This study demonstrates that DLX5 promotes HFSC differentiation by modulating the c-MYC/miR-29c-3p/NSD1 axis and identifies a new mechanism regulating HFSC differentiation.

RNA sequence analysis of dermal papilla cells' regeneration in 3D culture.

It is well known that dermal papilla cells (DPCs) are crucial for hair follicle growth and regeneration. However, dermal papilla cells in 2D culture could lose their ability of regeneration after several passage intervals. As opposed to DPCs in 2D culture, the DPCs in 3D culture could passage extensively. However, the molecular mechanisms of DPCs' regeneration in 3D culture remain unclear. Accordingly, gene sequencing is recommended for the investigation of hair regeneration between 2D and 3D culture, the three groups were established including DPCs in passage 2 in 2D culture, DPCs in passage 8 in 2D culture and DPCs in passage 8 in 3D culture. The differentially expressed genes (DEGs) were identified using the Venn diagram of these three groups, which included 1642 known and 359 novel genes, respectively. A total of 1642 known genes were used for Gene Ontology (GO), Kyoto Gene, Genomic Encyclopedia (KEGG) pathway enrichment and protein-protein interaction (PPI) analyses, respectively. The functions and pathways of DEGs were enriched in biological regulation, signal transduction and immune system, etc. The key module and the top 10 hub genes (IL1B, CXCL12, HGF, EGFR, APP, CCL2, PTGS2, MMP9, NGF and SPP1) were also identified using the Cytoscape application. Furthermore, the qRT-PCR results of the three groups validated that the hub genes were crucial for hair growth. In conclusion, the ten identified hub genes and related pathways in the current study can be used to understand the molecular mechanism of hair growth, and those provided a possibility for hair regeneration.

Rapid gelation of oxidized hyaluronic acid and succinyl chitosan for integration with insulin-loaded micelles and epidermal growth factor on diabetic wound healing.

In this work, poly(ethylene glycol)-b-poly[3-acrylamidophenylboronic acid-co-styrene] (PEG-b-P(PBA-co-St) has been firstly synthesized for loading of insulin to form insulin-loaded micelles. Insulin-loaded micelles (ILM) and epidermal growth factor (EGF) are further embedded into the composite hydrogels that can be rapidly gelled by mixing of oxidized hyaluronic acid (OHA) and succinyl chitosan (SCS). Then, the morphology, rheology, degradation, swelling and cytotoxicity properties of the as-prepared composite hydrogels are further investigated to evaluate their physical properties and biocompatibility of as the wound dressing. The as-prepared composite hydrogels show the excellent cell compatibility and low toxicity. To evaluate the wound healing ability of as-prepared composite hydrogels, the tests of wound healing in vivo are conducted on streptozotocin-induced rat models. And the as-prepared composite hydrogels with ILM and EGF show an excellent wound healing performance for promotion of fibroblast proliferation and tissue internal structure integrity, as well as the deposition of collagen and myofibrils. These results suggest that the as-prepared composite hydrogels with loading of ILM and EGF could be a promising candidate for wound healing applications.

LncRNA-PCAT1 maintains characteristics of dermal papilla cells and promotes hair follicle regeneration by regulating miR-329/Wnt10b axis.

BACKGROUND: The failure of hair follicle regeneration is the major cause of alopecia, which is a highly prevalent disease worldwide. Dermal papilla (DP) cells play important role in the regulation of hair follicle regeneration. However, the molecular mechanism of how dermal papilla cells direct follicle regeneration is still to be elucidated. METHODS: In vitro DP 3D culturing and in vivo nude mice DP sphere implanted models were used to examine the molecular regulation of DP cells and follicle regeneration. qRT-PCR and Western blotting were used to detect gene and protein expression, respectively. Immunofluorescence was used to detect the expression level of Wnt10b, Ki-67 and ß-catenin. Luciferase assay was used to examine the relationship among PCAT1, miR-329 and Wnt10b. ALP activity was measured by ELISA. H&E staining was used to measure follicle growth in skin tissues. RESULTS: Up-regulation of PCAT1 and Wnt10b, however, down-regulation of miR-329 were found in the in vitro 3D dermal papilla. Bioinformatics analysis and luciferase assays demonstrated that PCAT1 promoted Wnt10b expression by sponging miR-329. Knockdown of PCAT1 suppressed the proliferation and activity, as well as ALP and other DP markers of DP cells by targeting miR-329. Knockdown of PCAT1 regulated miR-329/Wnt10b axis to attenuate ß-catenin expression and nucleus translocation to inhibit Wnt/ß-catenin signaling. Furthermore, knockdown of PCAT1 suppressed DP sphere induced follicle regeneration and hair growth in nude mice. CONCLUSION: PCAT1 maintains characteristics of DP cells by targeting miR-329 to activating Wnt/ß-catenin signaling pathway, thereby promoting hair follicle regeneration.

LncRNA-XIST promotes dermal papilla induced hair follicle regeneration by targeting miR-424 to activate hedgehog signaling.

BACKGROUND: Alopecia is a highly prevalent disease characterizing by the loss of hair. Dermal papilla (DP) cells are the inducer of hair follicle regeneration, and in vitro three-dimensional (3D) culturing DP cells have been proven to induce hair follicle regeneration. However, the molecular mechanisms behind the regulation of 3D culturing DP cells remain unclear. METHODS: 3D-cultivated DP cells were used as in vitro cell model. DP sphere xenograft to nude mice was performed for in vivo study of hair follicle regeneration. qRT-PCR, Western blotting, and immunofluorescence were used for detecting the level of XIST, miR-424 and Hedgehog pathway-related proteins, respectively. H&E staining was used to examine hair neogenesis. Cell viability, proliferation and ALP activity were measured by MTT, CCK-8 and ELISA assays, respectively. Luciferase assays were used for studying molecular regulation between XIST, miR-424 and Shh 3'UTR. RESULTS: XIST and Shh were up-regulated, and miR-424 was down-regulated in 3D DP cells. Molecular regulation studies suggested that XIST sponged miR-424 to promote Shh expression. Knockdown of XIST suppressed DP cell activity, cell proliferation, ALP activity and the expression of other DP markers by sponging miR-424. Knockdown of XIST suppressed Shh mediated hedgehog signaling by targeting miR-424. Moreover, the knockdown of XIST inhibited DP sphere induced in vivo hair follicle regeneration and hair development. CONCLUSION: XIST sponges miR-424 to promote Shh expression, thereby activating hedgehog signaling and facilitating DP mediated hair follicle regeneration.

Fabrication of separable microneedles with phase change coating for NIR-triggered transdermal delivery of metformin on diabetic rats.

To enhance the compliance of drug delivery for patients, the novel near-infrared (NIR) light-triggered and separable microneedles (MNs) have been developed in this work. Firstly, prussian blue nanoparticles (PB NPs) as the photo-thermal conversion factor and metformin as the hypoglycemic drug were embedded into the separable arrowheads, which consisted by poly (vinyl alcohol) and sucrose (PVA/Suc). The arrowheads of MNs were located on soluble solids supporting substrates that produced by poly(vinyl pyrrolidone) (PVP). Lauric acid (LA) as the phase transition coating covered on the surface of the MNs due to its lower phase transition temperature (~44 °C). Then, the separable arrowheads could be left into the skin because of the absorbing the interstitial fluid (IF) by the solid supporting substrates. With the irradiation of NIR light, LA could be melted due to the role of PB NPs in photo-thermal conversion, thus releasing the metformin from arrowheads. Compared with the traditional subcutaneous injections, the hypoglycemic effect was evaluated by the drug-release behaviors induced by NIR in vivo. The results showed that metformin could be allowed to on-demand release under the NIR irradiation. And the as-obtained MNs exhibited a good hypoglycemic effect, hypotoxicity and low inflammation reaction compared with those of traditional subcutaneous injections. The results indicate that the fabricated MNs have the potential treatment for diabetes due to their safety, convenience and painlessness.

Forward Wound Closure with Regenerated Silk Fibroin and Polylysine-Modified Chitosan Composite Bioadhesives as Dressings.

Wound dressing has been used for decades to be effective for accelerating skin wound healing. However, practical applications are still limited due to their lower cell affinity, tissue adhesiveness, and biocompatibility. Natural polymers are the important biomaterials because of their excellent biodegradability, biocompatibility, and low immunogenicity. In this work, the composite bioadhesives (PLS-CS/RSF) were prepared from regenerated silk fibroin (RSF) and polylysine-modified chitosan (PLS-CS) that were cross-linked by Ca2+ ions. The adhesion property tests showed that the PLS-CS/RSF exhibited excellent bonding potentials for various substrates, and the adhesive strength was up to 70 kPa for isolated porcine skin by the extension test. The as-prepared PLS-CS/RSF was nontoxic, displayed obvious antibacterial effects against Staphylococcus aureus and Escherichia coli in vitro, and their bacteriostasis rates were 100% after 120 min treatment. In addition, the PLS-CS/RSF exhibited favorable cytocompatibility by cell counting kit-8 assay. The animal model of wound closure results showed that PLS-CS/RSF can promote wound closure and the integrity of wound healing, inhibiting the secretion of inflammatory factor and tumor necrosis factor and stimulating vascular factor and α-smooth muscle actin to the release of vascular growth factor and promote angiogenesis during the process of wound healing by immunohistochemical assay.

The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells1.

PURPOSE: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. METHODS: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. RESULTS: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. CONCLUSION: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

Incorporation of ZnO/Bioactive Glass Nanoparticles into Alginate/Chitosan Composite Hydrogels for Wound Closure.

Development of proper skin wound dressing is a vital step for wound repair, especially for those patients with serious skin injuries. Herein, zinc-doped bioactive glass (ZBG)/succinyl chitosan (SCS)/oxidized alginate (OAL) composite hydrogels (Gel-ZBG) have been developed as wound dressings to accelerate wound closure. Schiff-based linkages have been introduced into the composite hydrogels, which provide a humid microenvironment for the proliferation of cells on wound sites. The amino groups from SCS and Zn2+ released from ZBG exhibited excellent antibacterial properties to composite hydrogels, confirmed by the antibacterial tests in vitro. Si4+ and Ca2+ ions are essential factors that stimulate fibroblasts to secrete beneficial factors for angiogenesis and wound closure. The epidermal growth factor (EGF) was further embedded into the hydrogels to improve cell proliferation and tissue remodeling in the wound bed. Finally, the formation of granulation tissue, deposition of collagen and myofibril, the release of anti-inflammatory factors, and angiogenesis have been investigated to determine the healing mechanism of the composite hydrogels as the wound dressings.

The effect of hirudin on antagonisting thrombin induced apoptosis of human microvascular endothelial cells

Abstract Purpose: To investigate whether hirudin exerts its antithrombin action to decrease the ratio of Human Microvascular Endothelial Cells (HMVECs) apoptosis. Methods: Human microvascular endothelial cells (HMVECs) cultured in the third and fifth generations were used. HMVECs were divided into normal group, thrombin group (T group), natrual hirudin group (H group), thrombin + natrual hirudin group (T + H group), AG490 group, thrombin + AG490 group (T + AG490 group), natrual hirudin + AG490 group (H + AG490 group), thrombin + natural hirudin + AG490 (T + H + AG490 group).Apart from the normal group, the other groups were exposed to the relevant drugs for 24 hours.HMVEC apoptosis was assessed by flow cytometric and double Immunofluorescence of phosphorylation of JAK (P-JAK2) and TUNEL assay. Results: Compared with the normal group, in thrombin group the HMVECs apoptosis rate were significantly increased (P<0.05).The results indicated that the index of apoptosis and the apoptosis rate were improved in cultures treated by natural hirudin (T + H group), relative to cultures with thrombin only (T group). We found that the index of apoptosis and the apoptosis rate in the AG490 + thrombin group were higher than that in the hirudin + thrombin group (P<0.05). Double Immunofluorescence of p-JAK2 and TUNEL assays showed that cells were double positive for P-JAK2 uptake and TUNEL detection liquid binding. Conclusion: The natural hirudin and JAK2/STATs signal inhibitor AG490 could block the effects of thrombin. Natural hirudin could attenuate HMVECs apoptosis via antagonizing thrombin and it is suggested that this effect may occur by blocking the JAK2/STATs signaling pathway and this signaling pathways appears to be not the only pathway.

[Effect of natural hirudin on angiogenesis of human microvascular endothelial cells].

Objective: To explore the effect of natural hirudin on proliferation of human microvascular endothelial cells (HMVECs) and its preliminary mechanism of promoting angiogenesis. Methods: Three-dimensional culture models of HMVECs were established in vitro and observed by inverted phase contrast microscopy after 24 hours of culturing. Then, the three-dimensional culture models of HMVECs were treated with different concentrations (1, 4, and 7 ATU/mL) of the natural hirudin, respectively, and Dulbecco's modified Eagle's medium containing 10% fetal bovine serum as control. The cell proliferations of 4 groups were detected by cell counting kit 8 (CCK-8) method at 24, 48, and 72 hours; the angiogenesis of 4 groups were observed by tube formation assay at 24 hours; the expressions of vascular endothelial growth factor (VEGF) and Notch1 of HMVECs in 4 groups were observed by immunofluorescence staining at 24 hours. Results: The observation of cells in three-dimensional culture models showed that HMVECs attached to Matrigel well, and the cells formed tube structure completely after 24 hours. The results of CCK-8 test showed that the absorbance ( A) value of 1 and 4 ATU/mL groups were higher than that of control group at each time point ( P<0.05), and A value of 4 ATU/mL group was the highest. The A value of 7 ATU/mL group was significantly lower than those of 1 and 4 ATU/mL groups and control group ( P<0.05). The tube formation assay showed that the tube structure was more in 1 and 4 ATU/mL groups than in 7 ATU/mL group and control group, and in 4 ATU/mL group than in 1 ATU/mL group, showing significant differences ( P<0.05). There was no significant difference between 7 ATU/mL group and control group ( P>0.05). The results of immunofluorescence staining showed that compared with control group, the Notch1 expression was higher in 1 and 4 ATU/mL groups and lower in 7 ATU/mL group; and there was significant difference between 4 and 7 ATU/mL groups and control group ( P<0.05). The VEGF expression was higher in 1, 4, and 7 ATU/mL groups than in control group, in 4 ATU/mL group than in 1 and 7 ATU/mL groups, showing significant differences ( P<0.05). Conclusion: Natural hirudin can promote angiogenesis at low and medium concentrations, but suppress angiogenesis at high concentrations. Its mechanism may be related to the VEGF-Notch signal pathway.

Polymeric Microneedles Integrated with Metformin-Loaded and PDA/LA-Coated Hollow Mesoporous SiO2 for NIR-Triggered Transdermal Delivery on Diabetic Rats.

Herein, an NIR-responsive polymeric microneedle (MN) system incorporated with metformin-loaded and polydopamine/lauric-acid-coated (PDA/LA-coated) hollow mesoporous SiO2 has been developed for transdermal delivery of antidiabetic drug (metformin). First, an antidiabetic drug was loaded within hollow mesoporous SiO2 nanoparticles (HMSNs) by a diffusion method. Then, PDA as photothermal conversion agent and lauric acid (LA) as phase change material (PCM) were coated onto the HMSN to form NIR-responsive drug nanocarriers. Finally, these metformin-loaded and PDA/LA-coated HMSNs were encapsulated into poly(vinylpyrrolidone) (PVP) MNs. After insertion into skin tissue, LA could melt with the photothermal conversion of PDA under NIR light, thus enabling release of encapsulated metformin from MNs. The in vivo release behavior of metformin from MNs into skin was further studied to investigate its hypoglycemic effect on diabetic rats. Compared with the subcutaneous injection of metformin, the bioavailability of MN-NIR groups was 95.8 ± 2.7%. The antidiabetic drug can be precisely released by adjustment of exposure time and power densities of NIR light.

Separable Microneedles for Near-Infrared Light-Triggered Transdermal Delivery of Metformin in Diabetic Rats.

Near-infrared (NIR) light-triggered and separable segmented microneedles (MNs), consisting of lauric acid and polycaprolactone (LA/PCL) arrowheads and poly(vinyl alcohol) and polycaprolactone (PVA/PVP) supporting bases, have been fabricated. A hypoglycemic drug (metformin) and photothermal conversion factor (Cu7S4 nanoparticles) are encapsulated into LA/PCL arrowheads. Due to the dissolution of soluble supporting bases after the absorption of tissue fluid, the separable MNs arrowheads can be embedded into skin after insertion. Under the NIR-light irradiation, the LA/PCL arrowheads exhibit an excellent thermal-ablation change with a low amount of Cu7S4 nanoparticles (0.1 wt %) due to the low melting point of LA and PCL, thus enabling the release behavior of the encapsulated model drug to be photothermally triggered. Compared to the hypodermic injection of metformin, the thermal ablation of separable MNs triggered by NIR irradiation in the current research exhibit an excellent hypoglycemic effect in vivo. It suggests that the NIR-induced thermal-ablation MNs comprise a prospective transdermal drug-delivery system for the precise control of the timing and dosage of a drug that is dependent on NIR administration.

Microneedles Integrated with ZnO Quantum-Dot-Capped Mesoporous Bioactive Glasses for Glucose-Mediated Insulin Delivery.

A self-responsive insulin delivery system is highly desirable because of its high sensitivity dependent on blood glucose levels. Herein, a smart pH-triggered and glucose-mediated transdermal delivery system, insulin-loaded and ZnO quantum dots (ZnO QDs) capped mesoporous bioactive glasses (MBGs) integrated with microneedles (MNs), was developed to achieve control and painless administration. ZnO QDs as a promise pH-responsive switch were employed to cap the nanopores of MBGs via electrostatic interaction. The drug (insulin) and glucose-responsive factor (glucose oxidase/catalase, GOx/CAT) were sealed into the pores of MBGs. GOx/CAT in the MBGs could catalyze glucose to form gluconic acid, resulting decrease in the local pH. The ZnO QDs on the surface of the MBGs could be dissolved in the acidic condition, leading to disassembly of the pH-sensitive MBGs and then release of preloaded insulin from the MBGs. As a result of administration in a diabetic model, an excellent hypoglycemic effect and lower hypoglycemia risk were obtained. These results indicate that as-prepared pH-triggered and glucose-mediated transdermal delivery systems have hopeful applications in the treatment of diabetes.

[Expression of Egr-1, Nab2 and Cav-1 and its relationship with scar hyperplasia].

Objective To investigate the expression of early growth response protein 1 (Egr-1),NGFI-A binding protein 2 (Nab2) and caveolin 1 (Cav-1) in normal skin,flat-cicatrix and hypertrophic scar,and explore its role in the formation of hypertrophic scar.Methods The expression of Egr-1,Nab2 and Cav-1 protein in 9 normal skin tissues,8 flat-cicatrix tissues and 9 hypertrophic scar tissues were examined with immunohistochemistry SP method and were analyzed statistically.Results The expression of Egr-1 in epidermal cells of hypertrophic scar was significantly higher than that in normal skin and flat scar tissue.The expression of Egr-1 increased in the course of scar proliferation.The distribution patterns of Nab2 were different from Egr-1.The expression of Egr-1 was increased,while expression of Nab2 was decreased.The expression of Cav-1 in normal skin and flat-cicatrix was significantly higher than that in hypertrophic scar.Conclusions The expression of Egr-1,Nab2 and Cav-1 is closely related to the formation of hypertrophic scar,and the up-regulated expression of Egr-1 and the deficient expression of Nab2 and Cav-1 may be the indicators of the progress of formation of hypertrophic scar.