Carteolol triggers senescence via activation of ß-arrestin-ERK-NOX4-ROS pathway in human corneal endothelial cells in vitro.

Carteolol is a commonly-used topical medication for primary open-angle glaucoma. However, long-term and frequent ocular application of carteolol entails its residuals at low concentration in the aqueous humor for a long duration and may exert latent toxicity in the human corneal endothelial cells (HCEnCs). Here, we treated the HCEnCs in vitro with 0.0117% carteolol for 10 days. Thereafter, we removed the cartelolol and normally cultured the cells for 25 days to investigate the chronical toxicity of carteolol and the underlying mechanism. The results exhibited that 0.0117% carteolol induces senescent features in the HCEnCs, such as increased senescence-associated ß-galactosidase positive rates, enlarged relative cell area and upregulated p16INK4A and senescence-associated secretory phenotypes, including IL-1α, TGF-ß1, IL-10, TNF-α, CCL-27, IL-6 and IL-8, as well as decreased Lamin B1 expression and cell viability and proliferation. Thereby, further exploration demonstrated that the carteolol activates ß-arrestin-ERK-NOX4 pathway to increase reactive oxygen species (ROS) production that imposes oxidative stress on energetic metabolism causing a vicious cycle between declining ATP and increasing ROS production and downregulation of NAD+ resulting in metabolic disturbance-mediated senescence of the HCEnCs. The excess ROS also impair DNA to activate the DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with diminished poly(ADP-Ribose) polymerase (PARP) 1, a NAD+-dependent enzyme for DNA damage repair, resulting in cell cycle arrest and subsequent DDR-mediated senescence. Taken together, carteolol induces excess ROS to trigger HCEnC senescence via metabolic disturbance and DDR pathway.

Ultraviolet B irradiation induces senescence of human corneal endothelial cells in vitro by DNA damage response and oxidative stress.

The human corneal endothelial cells (HCEnCs) play a vital role in the maintenance of corneal transparency and visual acuity. In our daily life, HCEnCs are inevitably exposed to ultraviolet B (UVB) radiation leading to decreases of visual acuity and corneal transparency resulting in visual loss eventually. Therefore, understanding the UVB-induced cytotoxicity in HCEnCs is of importance for making efficient strategies to protect our vision from UVB-damage. However, in-depth knowledge about UVB-induced cytotoxicity in HCEnCs is missing. Herein, we pulse-irradiated the HCEnCs in vitro with 150 mJ/cm2 UVB (the environmental dose) at each subculture for 4 passages to explore the insights into UVB-induced phototoxicity. The results showed that the UVB-treated HCEnCs exhibit typical senescent characteristics, including significantly enlarged relative cell area, increased senescence-associated ß-galactosidase positive staining, and upregulated p16INK4A and senescence associated secretory phenotypes (SASPs) such as CCL-27, IL-1α/6/8/10, TGF-ß1 and TNF-α, as well as decreased cell proliferation and Lamin B1 expression, and translocation of Lamin B1. Furthermore, we explored the causative mechanisms of senescence and found that 150 mJ/cm2 UVB pulse-irradiation impairs DNA to activate DNA damage response (DDR) pathway of ATM-p53-p21WAF1/CIP1 with downregulated DNA repair enzyme PARP1, leading to cell cycle arrest resulting in DDR-mediated senescence. Meanwhile, UVB pulse-irradiation also elicits a consistent increase of ROS production to aggravate DNA damage and impose oxidative stress on energy metabolism leading to metabolic disturbance resulting in metabolic disturbance-mediated senescence. Altogether, the repeated pulse-irradiation of 150 mJ/cm2 UVB induces HCEnC senescence via both DDR pathway and energy metabolism disturbance.

Chitosan/PLGA shell nanoparticles as Tylotoin delivery platform for advanced wound healing.

Wound treatment remains one of the most prevalent healthcare issues. Tylotoin is a skin repair peptide identified from salamander (Tylototriton verrucosus) and exhibits skin wound healing properties. Noticeably, the easy degradation and frequent administration limit its application in wound healing. Chitosan (CS) -PLGA-Tylotoin nanoparticles (CPT NPs) were prepared to circumvent this limitation and deliver Tylotoin for the promotion of the healing of skin wounds. Results showed that optimized CPT NPs particle size, zeta potential, encapsulation efficiency and drug loading were 297.80 ± 5.37 nm, 20.37 ± 0.83 mV, 81.00 % and 1.74 %, respectively. In vitro, CPT NPs exhibited good antibacterial properties and biocompatibility and persistently promoted the cell migration of HaCaT cells and HUVECs due to the long-term sustained release of Tylotoin within 14 days (64.81 %). In vivo, the scarless healing of skin wound promotion was evaluated in mouse back full-thickness wound models. We demonstrated that mouse back full-thickness wounds topically treated with CPT NPs once every two weeks exhibited better scarless healing than those treated with Tylotoin once daily. We envision that CPT NPs, as a Tylotoin delivery platform might, may be potentially utilized to in skin wounds healing in clinics in the future.

Collagen-based biocomposites inspired by bone hierarchical structures for advanced bone regeneration: ongoing research and perspectives.

Bone is a hard-connective tissue composed of matrix, cells and bioactive factors with a hierarchical structure, where the matrix is mainly composed of type I collagen and hydroxyapatite. Collagen fibers assembled by collagen are the template for mineralization and make an important contribution to bone formation and the bone remodeling process. Therefore, collagen has been widely clinically used for bone/cartilage defect regeneration. However, pure collagen implants, such as collagen scaffolds or sponges, have limitations in the bone/cartilage regeneration process due to their poor mechanical properties and osteoinductivity. Different forms of collagen-based composites prepared by incorporating natural/artificial polymers or bioactive inorganic substances are characterized by their interconnected porous structure and promoting cell adhesion, while they improve the mechanical strength, structural stability and osteogenic activities of the collagen matrix. In this review, various forms of collagen-based biocomposites, such as scaffolds, sponges, microspheres/nanoparticles, films and microfibers/nanofibers prepared by natural/synthetic polymers, bioactive ceramics and carbon-based materials compounded with collagen are reviewed. In addition, the application of collagen-based biocomposites as cytokine, cell or drug (genes, proteins, peptides and chemosynthetic) delivery platforms for proangiogenesis and bone/cartilage tissue regeneration is also discussed. Finally, the potential application, research and development direction of collagen-based biocomposites in future bone/cartilage tissue regeneration are discussed.

Homogeneous deacetylation and degradation of chitin in NaOH/urea dissolution system.

Since being discovered, alkali/urea has been widely used in the dissolution of natural polysaccharides and the preparation of functional materials such as hydrogels, fibers, films and nanoparticles. This work will focus on verifying the structural stability, homogeneous degradation and deacetylation of chitin in alkali-soluble systems. The chitin was dissolved in NaOH/urea solution and stored at different temperature. At the specific time, the structure, viscosity, acetylation degree (DA) and biocompatibility of chitin and prepared chitosan were determined. The results indicated that dissolution process did not affect the structure and bioactivity of chitin. However, with the increase of storage time and temperature, chitin undergone significant homogeneous deacetylation (DA from 99.5% to 33.2%) and degradation (viscosity from 9284 cP to 1538 cP), accompanying by changes in crystalline structure and thermal stability. Moreover, the processed chitins were no-toxic for the biomedicine applications. This work will provide new ideas for the application of alkali-soluble systems.

Establish an In Vitro Cell Model to Explore the Impacts of UVA on Human Corneal Endothelial Wound Healing.

PURPOSE: To provide scientific data for clinical practice in making strategies for accelerating corneal endothelial wound healing, we investigated the impact of UVA on the corneal endothelial wound healing process and the underlying mechanism using an in vitro cell model. MATERIALS AND METHODS: An in vitro cell model for corneal endothelial wound healing was established by scratching the in vitro cultured human corneal endothelial cell (HCEnC) confluent layer. Then, we investigated the impacts of UVA irradiation and Ascorbic acid-2-phosphate (Asc-2p) on the wound healing process of the in vitro HCEnC model by examining wound-healing index, F-actin+ rate, Ki-67+ rate, and ROS production. RESULTS: After scratching, the Ki-67+ and F-actin+ HCEnCs occupied the scratching gap. Furthermore, the F-actin+ rates were significantly higher than Ki-67+ rates in the wound closure area. After irradiated with UVA, the wound-healing indexes, Ki-67+ rates and F-actin+ rates of the wound-healing model significantly reduced, whereas the ROS production significantly increased in a dose-dependent manner. Pretreatment with Asc-2p significantly reduced the ROS production as well as increased the wound-healing indexes, Ki-67+rates and F-actin+ rates of the UVA irradiated wound-healing model. CONCLUSION: The migration of HCEnC plays a major role in the wound healing process of the established cell model, which is like the wound healing process in vivo. UVA decreases the wound closure of the in vitro HCEnC model dose-dependently, while antioxidant Asc-2p can attenuate the damage to UVA to HCEnCs probably via reducing ROS to improve their migration.

Phenylephrine induces necroptosis and apoptosis in corneal epithelial cells dose- and time-dependently.

In the present study, the toxicity of phenylephrine, a selective α1-adrenergic receptor agonist, in corneal epithelial cells and its underlying mechanisms were investigated using an in vitro model of human corneal epithelial cells (HCEPCs) and an in vivo model of New Zealand white rabbit corneas. The HCEPCs treated with phenylephrine at concentrations from 10% to 0.078125% displayed abnormal morphology, decline of cell viability and elevation of plasma membrane permeability time- and dose-dependently. Moreover, 10%-1.25% phenylephrine induce necrosis characteristics of marginalization and uneven distribution of chromatin through up-regulation of RIPK1, RIPK3 and MLKL along with inactivation of caspase-8 and caspase-2, whereas 0.625% phenylephrine induced condensed chromatin, S phase arrest, phosphatidylserine externalization, DNA fragmentation and apoptotic body formation in the HCECs through activation of caspase-2, -8, -9 and -3 as well as down-regulation of Bcl-2, up-regulation of Bad, ΔΨm disruption and release of cytochrome c and AIF into cytosol. At last, 10% phenylephrine induced destruction of the corneal epithelia and apoptosis of corneal epithelial cells in rabbit corneas. In conclusion, 10% to 1.25% phenylephrine cause necroptosis via RIPK1-RIPK3-MLKL axis and 0.625% phenylephrine induce apoptosis via a mitochondrion-dependent and death receptor-mediated signal pathway in HCEPCs.

Expression pattern of Zinc finger protein 185 in mouse testis and its role in regulation of testosterone secretion.

Zinc finger protein 185 (ZNF185) belongs to the ZNF family and is involved in cell proliferation and differentiation. To the best of our knowledge, the association between ZNF185 and male reproduction is unknown. In the present study, the expression and localization of ZNF185 in mouse testis, as well as its role in testosterone secretion, cell cycle progression and apoptosis of mouse Leydig cells were investigated. The results of the immunofluorescence analysis indicated that ZNF185 was highly expressed in Leydig cells of the mouse testis, and primarily localized in the cytoplasm. The results of quantitative polymerase chain reaction and western blot analyses further validated that ZNF185 expression was significantly higher in Leydig cells and sperm compared with that in Sertoli cells. Subsequently, the expression pattern of ZNF185 in mouse testis was determined at different developmental stages. The results demonstrated that the expression of ZNF185 was highest in the testis of 10­week­old mice and lowest in 2­week­old mice. Furthermore, the role of ZNF185 in Leydig cells of the mouse testis was investigated. Different concentrations of luteinizing hormone (LH) were used to stimulate the Leydig cells and subsequently the expression of ZNF185, and testosterone concentration was detected. The results revealed that LH upregulated the expression of ZNF185 and testosterone secretion, and ZNF185 expression was significantly positively correlated with testosterone secretion. To further validate whether ZNF185 was involved in testosterone secretion, lentiviral­mediated RNA interference was used to knock down ZNF185 expression in Leydig cells. The results demonstrated that ZNF185 expression and testosterone secretion of Leydig cells were decreased significantly. In addition, the results demonstrated that the knockdown of ZNF185 expression did not significantly affect cell cycle progression or apoptosis. Taken together, the results of the present study revealed that ZNF185 was highly expressed in Leydig cells of the testis and involved in the secretion of testosterone. These results have contributed to the elucidation of the mechanism underlying male reproduction and may provide a novel target for the treatment of infertility, and the development of a contraceptive vaccine.

[Expression and localization of zinc finger protein 185 in sperm cells, Leydig cells and Sertoli cells of mouse testis].

Objective To investigate the expression of zinc finger protein 185 (ZNF185) in the sperm cells, Leydig cells and Sertoli cells of the mouse testis. Methods The localization of ZNF185 in the sperm cells, Leydig cells, Sertoli cells and mouse testis tissue was detected by immunofluorescence assay. The mRNA and protein expression levels of ZNF185 in the three kinds of cells were detected by quantitative real-time PCR (qRT-PCR) and Western blotting. Results Immunofluorescence assay showed that ZNF185 was expressed in sperm cells, Leydig cells and Sertoli cells, furthermore, ZNF185 was mainly distributed in the cytoplasm of Leydig cells and Sertoli cells, as well as the head and tail of sperm cells. The qRT-PCR and Western blotting showed that the mRNA and protein expression levels of ZNF185 in Sertoli cells were significantly lower than those in Leydig cells and sperm cells. Conclusion ZNF185 is distributed in sperm cells, Leydig cells and Sertoli cells of mouse testis, and the expression level was different between them.