Limbal Stem Cell Dysfunction Induced by Severe Dry Eye via Activation of the p38 MAPK Signaling Pathway.

Severe dry eye (SDE) can cause grievous damage to the ocular surface and result in vision impairment and even blindness. To investigate the fate of limbal stem cells in SDE and the underlying mechanism, the current study established an SDE rat model by removing the extraorbital and infraorbital lacrimal glands and maintaining them in a low-humidity environment. One month after the surgery, aqueous tear secretion was reduced dramatically, blood vessels invaded into the central cornea, and inflammatory cells infiltrated into the limbal stroma. The expressions of keratin 12 and paired box gene 6 were down-regulated dramatically, while those of keratin 10, small proline-rich protein 1b, and mucin 5AC were up-regulated in the corneal epithelium of the SDE rats. Cell proliferation in the limbal epithelium was up-regulated, while the stem/progenitor marker adenosine 5'-triphosphate-binding cassette member 2 and the limbal epithelial colony-forming efficiency were decreased in the SDE condition. Furthermore, the p38 mitogen-activated protein kinase signaling pathway was activated in the limbal corneal epithelium of SDE rats. The abnormal differentiation and stemness loss in the corneal epithelium could be reversed upon treatment with a p38 inhibitor in a SDE in vivo model and in vitro hyperosmolar corneal epithelial culture conditions. These data suggest that SDE can lead to limbal stem cell dysfunction, and p38 mitogen-activated protein kinase signaling pathway activation plays an essential role in this process.

Exploration and validation of a combined Hypoxia and m6A/m5C/m1A regulated gene signature for prognosis prediction of liver cancer.

BACKGROUND: It is widely acknowledged that hypoxia and m6A/m5C/m1A RNA modifications promote the occurrence and development of tumors by regulating the tumor microenvironment. This study aimed to establish a novel liver cancer risk signature based on hypoxia and m6A/m5C/m1A modifications. METHODS: We collected data from The Cancer Genome Atlas (TCGA-LIHC), the National Omics Data Encyclopedia (NODE-HCC), the International Cancer Genome Consortium (ICGC), and the Gene Expression Omnibus (GEO) databases for our study (GSE59729, GSE41666). Using Cox regression and least absolute shrinkage and selection operator (LASSO) method, we developed a risk signature for liver cancer based on differentially expressed genes related to hypoxia and genes regulated by m6A/m5C/m1A modifications. We stratified patients into high- and low-risk groups and assessed differences between these groups in terms of gene mutations, copy number variations, pathway enrichment, stemness scores, immune infiltration, and predictive capabilities of the model for immunotherapy and chemotherapy efficacy. RESULTS: Our analysis revealed a significantly correlated between hypoxia and methylation as well as m6A/m5C/m1A RNA methylation. The three-gene prognosis signature (CEP55, DPH2, SMS) combining hypoxia and m6A/m5C/m1A regulated genes exhibited strong predictive performance in TCGA-LIHC, NODE-HCC, and ICGC-LIHC-JP cohorts. The low-risk group demonstrated a significantly better overall survival compared to the high-risk group (p < 0.0001 in TCGA, p = 0.0043 in NODE, p = 0.0015 in ICGC). The area under the curve (AUC) values for survival at 1, 2, and 3 years are all greater than 0.65 in the three cohorts. Univariate and Multivariate Cox regression analyses of the three datasets indicated that the signature could serve as an independent prognostic predictor (p < 0.001 in the three cohorts). The high-risk group exhibited more genome changes and higher homologous recombination deficiency scores and stemness scores. Analysis of immune infiltration and immune activation confirmed that the signature was associated with various immune microenvironment characteristics. Finally, patients in the high-risk group experienced a more favorable response to immunotherapy, and various common chemotherapy drugs. CONCLUSION: Our prognostic signature which integrates hypoxia and m6A/m5C/m1A-regulated genes, provides valuable insights for clinical prediction and treatment guidance for liver cancer patients.

Novel prognostic gene signature for pancreatic ductal adenocarcinoma based on hypoxia.

BACKGROUND: Currently, there is lack of marker to accurately assess the prognosis of patients diagnosed with pancreatic ductal adenocarcinoma (PDAC). This study aims to establish a hypoxia-related risk scoring model that can effectively predict the prognosis and chemotherapy outcomes of PDAC patients. METHODS: Using unsupervised consensus clustering algorithms, we comprehensively analyzed The Cancer Genome Atlas (TCGA) data to identify two distinct hypoxia clusters and used the weighted gene co-expression network analysis (WGCNA) to examine gene sets significantly associated with these hypoxia clusters. Then univariate Cox regression, the least absolute shrinkage and selection operator (LASSO) Cox regression and multivariate Cox regression were used to construct a signature and its efficacy was evaluated using the International Cancer Genome Consortium (ICGC) PDAC cohort. Further, the correlation between the risk scores obtained from the signature and carious clinical, pathological, immunophenotype, and immunoinfiltration factors as well as the differences in immunotherapy potential and response to common chemotherapy drugs between high-risk and low-risk groups were evaluated. RESULTS: From a total of 8 significantly related modules and 4423 genes, 5 hypoxia-related signature genes were identified to construct a risk model. Further analysis revealed that the overall survival rate (OS) of patients in the low-risk group was significantly higher than the high-risk group. Univariate and multivariate Cox regression analysis showed that the risk scoring signature was an independent factor for prognosis prediction. Analysis of immunocyte infiltration and immunophenotype showed that the immune score and the anticancer immune response in the high-risk were significantly lower than that in the low-risk group. CONCLUSION: The constructed hypoxia-associated prognostic signature demonstrated could be used as a potential risk classifier for PDAC.

NirS-type denitrifying bacteria in aerobic water layers of two drinking water reservoirs: Insights into the abundance, community diversity and co-existence model.

The nirS-type denitrifying bacterial community is the main drivers of the nitrogen loss process in drinking water reservoir ecosystems. The temporal patterns in nirS gene abundance and nirS-type denitrifying bacterial community harbored in aerobic water layers of drinking water reservoirs have not been studied well. In this study, quantitative polymerase chain reaction (qPCR) and Illumina Miseq sequencing were employed to explore the nirS gene abundance and denitrifying bacterial community structure in two drinking water reservoirs. The overall results showed that the water quality parameters in two reservoirs had obvious differences. The qPCR results suggested that nirS gene abundance ranged from (2.61 ± 0.12) × 105 to (3.68 ± 0.16) × 105 copies/mL and (3.01 ± 0.12) × 105 to (5.36 ± 0.31) × 105 copies/mL in Jinpen and Lijiahe reservoirs, respectively. The sequencing results revealed that Paracoccus sp., Azoarcus sp., Dechloromonas sp. and Thauera sp. were the dominant genera observed. At species level, Cupriavidus necator, Dechloromonas sp. R-28400, Paracoccus denitrificans and Pseudomonas stutzeri accounted for more proportions in two reservoirs. More importantly, the co-occurrence network analysis demonstrated that Paracoccus sp. R-24615 and Staphylococcus sp. N23 were the keystone species observed in Jinpen and Lijiahe reservoirs, respectively. Redundancy analysis indicated that water quality (particularly turbidity, water temperature, pH and Chlorophyll a) and sampling time had significant influence on the nirS-type denitrifying bacterial community in both reservoirs. These results will shed new lights on exploring the dynamics of nirS-type denitrifying bacteria in aerobic water layers of drinking water reservoirs.

Real-time confocal microscopy imaging of corneal cytoarchitectural changes induced by different stresses.

Maintenance of the corneal refractive power and tissue transparency is essential for normal vision. Real-time characterization of changes in corneal cells during suffering stresses or wound healing may provide a way to identify novel targets, whose therapeutic manipulation can improve the outcome of this response induced by injury. Here we describe a novel user friendly and effective confocal real-time confocal microscopy attachment that monitors the effects of anisoosmotic stress on cell morphology and corneal thickness in situ. Corneal epithelial nuclei gradually became highly reflective in the isotonic group and the corneal stroma was slightly thickened as compared with that seen prior to 60 min exposure to a hypotonic solution. After 30 min of exposure to hypertonic stress, the corneal stromal cells became crenate and shriveled. The hyper-reflective area of the corneal stroma in the hypo-osmotic group was significantly larger than that in the other two groups, as demonstrated by 3D reconstruction imaging. The hypotonic fresh chlorinated pool water was observed to cause atrophy of corneal epithelial nuclei, while the isosmotic bee venom solution caused high reflection of the corneal stroma layer and corneal endothelial cell damage. With the microscopic attachment, the inward movement of corneal epithelial cells toward the denuded central region was detected in the serum-treated group. The microscopy attachment is an effective system for obtaining a more detailed understanding of the time dependent losses in the corneal cell structure and tissue architecture of full thickness corneas induced by osmotic stress or cytotoxic agents.

Biogeographic distribution patterns of algal community in different urban lakes in China: Insights into the dynamics and co-existence.

Urban lake ecosystems are significant for social development, but currently we know little about the geographical distribution of algal community in urban lakes at a large-scale. In this study, we investigated the algal community structure in different areas of urban lakes in China and evaluated the influence of water quality parameters and geographical location on the algal community. The results showed that obvious differences in water quality and algal communities were observed among urban lakes in different geographical areas. Chlorophyta was the dominant phylum, followed by cyanobacteria in all areas. The network analysis indicated that algal community composition in urban lakes of the western and southern area showed more variations than the eastern and northern areas, respectively. Redundancy analysis and structural equation model revealed that nutrients and pH were dominant environmental factors that affected the algal community, and they showed higher influence than that of iron, manganese and COD Mn concentration. Importantly, algal community and density exhibited longitude and latitude relationship. In general, these results provided an ecological insight into large-scale geographical distributions of algal community in urban lakes, thereby having potential applications for management of the lakes.

The role of autophagy in the pathogenesis of exposure keratitis.

Incomplete tear film spreading and eyelid closure can cause defective renewal of the ocular surface and air exposure-induced epithelial keratopathy (EK). In this study, we characterized the role of autophagy in mediating the ocular surface changes leading to EK. Human corneal epithelial cells (HCECs) and C57BL/6 mice were employed as EK models, respectively. Transmission electron microscopy (TEM) evaluated changes in HCECs after air exposure. Each of these models was treated with either an autophagy inhibitor [chloroquine (CQ) or 3-methyladenine (3-MA)] or activator [Rapamycin (Rapa)]. Immunohistochemistry assessed autophagy-related proteins, LC3 and p62 expression levels. Western blotting confirmed the expression levels of the autophagy-related proteins [Beclin1 and mammalian target of rapamycin (mTOR)], the endoplasmic reticulum (ER) stress-related proteins (PERK, eIF2α and CHOP) and the PI3K/Akt/mTOR signalling pathway-related proteins. Real-time quantitative PCR (qRT-PCR) determined IL-1ß, IL-6 and MMP9 gene expression levels. The TUNEL assay detected apoptotic cells. TEM identified autophagic vacuoles in both EK models. Increased LC3 puncta formation and decreased p62 immunofluorescent staining and Western blotting confirmed autophagy induction. CQ treatment increased TUNEL positive staining in HCECs, while Rapa had an opposite effect. Similarly, CQ injection enhanced air exposure-induced apoptosis and inflammation in the mouse corneal epithelium, which was inhibited by Rapa treatment. Furthermore, the phosphorylation status of PERK and eIF2α and CHOP expression increased in both EK models indicating that ER stress-induced autophagy promoted cell survival. Taken together, air exposure-induced autophagy is indispensable for the maintenance of corneal epithelial physiology and cell survival.

Potential New Target for Dry Eye Disease-Oxidative Stress.

Dry eye disease (DED) is a multifactorial condition affecting the ocular surface. It is characterized by loss of tear film homeostasis and accompanied by ocular symptoms that may potentially result in damage to the ocular surface and even vision loss. Unmodifiable risk factors for DED mainly include aging, hormonal changes, and lifestyle issues such as reduced sleep duration, increased screen exposure, smoking, and ethanol consumption. As its prevalence continues to rise, DED has garnered considerable attention, prompting the exploration of potential new therapeutic targets. Recent studies have found that when the production of ROS exceeds the capacity of the antioxidant defense system on the ocular surface, oxidative stress ensues, leading to cellular apoptosis and further oxidative damage. These events can exacerbate inflammation and cellular stress responses, further increasing ROS levels and promoting a vicious cycle of oxidative stress in DED. Therefore, given the central role of reactive oxygen species in the vicious cycle of inflammation in DED, strategies involving antioxidants have emerged as a novel approach for its treatment. This review aims to enhance our understanding of the intricate relationship between oxidative stress and DED, thereby providing directions to explore innovative therapeutic approaches for this complex ocular disorder.

Relation Between Corneal Dendritic Cell Density and Tear Film Stability in Patients with Epidemic Keratoconjunctivitis Associated Dry Eye.

PURPOSE: To clarify the ocular surface features of patients with recent history of epidemic keratoconjunctivitis (EKC) and the relation between corneal dendritic cells (DCs) and ocular discomfort. METHODS: Normal controls (NC) and dry eye (DE) patients without EKC were recruited. Patients with recent EKC history (onset >4 weeks, but <20 weeks) were recruited as EKC + DE group (with dry eye) or EKC-DE group (without dry eye). Ocular surface disease index (OSDI) questionnaire, tear film parameters including lipid layer thickness, first tear break-up time (fBUT), average tear break-up time (aBUT), tear meniscus height and Schirmer I test, meibomian gland parameters, and in vivo corneal confocal microscopy were evaluated. RESULTS: 50 subjects in the NC group, 83 patients in the DE group, 76 patients in the EKC + DE group, and 38 patients in the EKC-DE group were included. Compared with the NC, DE, and EKC-DE groups, the EKC + DE group represented higher OSDI, lid margin, and meibum score (p < 0.05). In the EKC + DE group, the tear volume (10.5 ± 3.7 mm) was significantly higher than in the DE group (8.1 ± 2.8 mm, p < 0.001). The DC density in the EKC + DE group (29.98 ± 15.38 cells/image) was significantly higher than in NC, DE, and EKC-DE groups (4.68 ± 4.05 cells/image) (p < 0.001). The DC density was positively correlated with OSDI, lid margin, and meibum score (all p < 0.01) while inversely correlated with fBUT, aBUT (all p < 0.001) in the EKC + DE group. CONCLUSIONS: Corneal DC density significantly correlates to ocular discomfort and tear film instability in patients with recent EKC history who suffer from DE without aqueous tear deficiency.

A Novel Pancreatic Cancer Hypoxia Status Related Gene Signature for Prognosis and Therapeutic Responses.

Pancreatic cancer (PAC) is a highly fatal and aggressive type of cancer. Hypoxia is a common feature of PAC. The aim of this study was to develop a hypoxia status-related prognostic model for predicting the survival outcomes in PAC. The data sets of PAC from The Cancer Genome Atlas and the International Cancer Genome Consortium were used to construct and validate the signature. A 6 hypoxia status-related differential expression genes prognostic model for predicting the survival outcomes was established. The Kaplan-Meier analysis and Received operating characteristic curve indicated the good performance of the signature at predicting overall survival. Univariate and Multivariate Cox regression revealed that the signature was an independent prognostic factor in PAC. Weighted Gene Co-expression Network Analysis and immune infiltration analysis indicated that Immune-related pathways and immune cell infiltration was mostly enriched in the low-risk group, which presented a better prognosis. We also evaluated the predictive of the signature for immunotherapy and chemoradiotherapy. Risk gene LY6D may be a potential prognostic predictor of PAC. This model can be used as an independent prognostic factor for predicting clinical outcomes and a possible classifier for response to chemotherapy.

Idiopathic Epiretinal Membrane Surgery in Patients Aged Over 80 Years: Efficacy and Safety.

Purpose: To evaluate the efficacy and safety of idiopathic epiretinal membrane (ERM) surgery in patients aged over 80 years. Methods: Consecutive patients who underwent pars plana vitrectomy (PPV) combined with ERM and internal limiting membrane (ILM) peeling with retrobulbar anesthesia were recruited. Based on age, the patients were divided into the elderly group (≥ 80 years of age) and the control group (< 80 years of age). The best-corrected visual acuity (BCVA) and surgical complications were regarded as the main measurement indicators. Results: This study included 43 eyes from 43 patients aged 80 to 91 years and 86 eyes from 86 patients aged 54 to 79 years. Surgical intervention substantially improved BCVA both in the elderly and control groups (p = 0.005 and p < 0.001, respectively). Statistical analyses showed no significant difference in the incidence of surgical complications between the two groups (p = 0.631). The operations in either group were not delayed or canceled for the reason of complications of retrobulbar anesthesia, severe anxiety, or physical discomfort in the perioperative period. Moreover, no patient required a second operation. Also, no stroke, myocardial infarction, or death occurred during the follow-up period. All the surgical complications were treated satisfactorily. Conclusion: Our outcomes indicate that PPV combined with ERM and ILM peeling with retrobulbar anesthesia is effective and safe in elderly patients aged 80 years or older. Based on age alone, we believe that advancing age should not be the risk factor for idiopathic ERM surgery.

Retinal thickness and fundus blood flow density changes in chest pain subjects with dyslipidemia.

AIM: To assess the retinal thickness and fundus blood flow density changes in chest pain patients with dyslipidemia using optical coherence tomography angiography (OCTA). METHODS: All subjects with chest pain as the main symptom accepted a comprehensive ophthalmological examination. According to the serum lipid levels, the participants were divided into the control group and the dyslipidemia group. The retina thickness and fundus blood flow density were determined using OCTA. RESULTS: The study enrolled 87 left eyes from 87 adults with dyslipidemia and 87 left eyes from age- and sex-matched participants without dyslipidemia. The retina of dyslipidemia subjects was significantly thinner than that of the controls in the inferior (P=0.004 and P=0.014, respectively) and temporal (P=0.015 and P=0.019, respectively) regions, both inner and outer layers. In terms of blood flow density in the macula or optic disk, there was a decreasing trend in the dyslipidemia group compared with the control group, especially in the inferior and temporal regions. CONCLUSION: Dyslipidemia may contribute to the decrease in retinal thickness and fundus blood flow density. Further validation of the association between abnormal lipid metabolism and fundus microcirculation alterations needs to be carried out in chest pain patients.

The role of white spot syndrome virus (WSSV) VP466 protein in shrimp antiviral phagocytosis.

Widespread evidence indicates that the structural proteins of virus play very important roles in virus-host interactions. However, the effect of viral proteins on host immunity has not been addressed. Our previous studies revealed that the host shrimp Rab6 (termed as PjRab previously), tropomyosin, ß-actin and the white spot syndrome virus (WSSV) envelope protein VP466 formed a complex. In this study, the VP466 protein was shown to be able to bind host Rab6 protein and increase its GTPase activity in vivo and vitro. Thus, VP466 could function as a GTPase-activating protein (GAP) of Rab6. In the VP466-Rab-actin pathway, the increase of the Rab6 activity induced rearrangements of the actin cytoskeleton, resulting in the formation of actin stress fibers which promoted the phagocytosis against virus. Therefore our findings revealed that a viral protein could be employed by host to initiate the host immunity, representing a novel molecular mechanism in the virus-host interaction. Our study would help to better understand the molecular events in immune response against virus infection in invertebrates.

Effects of hydrogen peroxide on Scenedesmus obliquus: Cell growth, antioxidant enzyme activity and intracellular protein fingerprinting.

Hydrogen peroxide (H2O2) is an environmental-friendly algicide and it is widely used to control algal blooms in aquatic ecosystems. However, the response of algal cell metabolic characteristics and intracellular protein profile under H2O2 stress is still not well understood. In the present study, the green alga Scenedesmus obliquus was exposed to different concentrations of H2O2 (0, 2, 6, 8 and 10 mg L-1) to evaluate the changes in algal morphological, physiological, and proteomic features to H2O2 exposure. The results showed that 8 mg L-1 of H2O2 could effectively inhibit the cell growth and photosynthetic activity of S. obliquus including chlorophyll-a content and chlorophyll fluorescence parameters. The increased activities of superoxide dismutase (SOD) and catalase (CAT) observed in this study indicate that cells exposure to H2O2 caused oxidative stress. The metabolic activity of S. obliquus was significantly decreased by H2O2 treatment. In terms of proteomic analysis, 251 differentially expressed proteins (DEPs) were successfully identified. The Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis revealed significant protein enrichment in the metabolic pathways, photosynthesis, ascorbic acid, and alginate metabolism and phenylpropane biosynthesis of S. obliquus. The analysis of protein-protein interaction system shows that the pathways of photosynthesis and metabolic pathways of S. obliquus were essential to resist oxidative stress. Taking together, these results shed new lights on exploring the cell physiological metabolism and intracellular protein mechanisms of H2O2 inhibition on algal blooms.

Obstructive Sleep Apnea Affects Lacrimal Gland Function.

PURPOSE: To determine the effect of obstructive sleep apnea syndrome (OSA) on lacrimal gland function and its mechanism. METHODS: Male mice aged seven to eight weeks were housed in cages with cyclic intermittent hypoxia to mimic OSA, and the control group was kept in a normal environment. Slit-lamp observation, fluorescein staining, and corneal sensitivity detection are used to assess cornea changes. Tear secretion was detected by phenol red cotton thread, and the pathological changes of lacrimal gland were observed by hematoxylin and eosin staining, oil red O staining, cholesterol and triglyceride kits, immunofluorescence staining, immunohistochemical staining, real-time polymerase chain reaction, transmission electron microscopy, and Western blot. RESULTS: Studies revealed a decreased tear secretion, corneal epithelial defects and corneal hypersensitivity. Myoepithelial cell damage, abnormal lipid accumulation, reduced cell proliferation, increased apoptosis and inflammatory cell infiltration in the lacrimal gland were also seen. Hifα and NF-κB signaling pathways, moreover, were activated, while Pparα was downregulated, in the lacrimal glands of OSA mice. Fenofibrate treatment significantly alleviated pathological changes of the lacrimal gland induced by OSA. CONCLUSION: OSA disturbs the Hifα/Pparα/NF-κB signaling axis, which affects lacrimal gland structure and function and induces dry eye.

Ovalbumin-Induced Allergic Inflammation Diminishes Cross-Linked Collagen Structures in an Experimental Rabbit Model of Corneal Cross-Linking.

Background: Allergic conjunctivitis (AC) is one of the reported potential risk factors of progression in keratoconus patients after corneal cross-linking surgery; however, the causal relationship is still inconclusive. Recent studies have indicated that various inflammatory cytokines play a vital role in the development of primary keratoconus. It is still unclear whether these inflammatory mediators also trigger CXL failures. This study aimed to investigate the impact of AC on the rabbit corneas after trans-epithelial corneal cross-linking (TCXL). Methods: A total of six rabbits were kept untreated as the normal control (NC) group. A total of 18 rabbits were treated by TCXL and divided into three groups (six in each group), namely, no treatment (TCXL group); induction of AC (TCXL + AC group); and induction of AC plus topical prednisolone acetate (TCXL + AC + PA group), according to additional treatment. AC was induced by topical application of ovalbumin after intraperitoneal pre-sensitization with ovalbumin. Rabbits were evaluated by slit lamp, in vivo laser scanning confocal microscopy, anterior segment optical coherence tomography, and measurement of corneal biomechanics. The cornea specimens were collected for the transmission electron microscope, the collagenase I digestion test, and PCR assay for TNF-α, IL-6, IL-1ß, matrix metalloproteinase 9 (MMP-9), lysyl oxidase (LOX), and tissue inhibitor of metalloproteinases 1 (TIMP-1) on the day (D) 28. Results: On D28, the TNF-α, IL-6, IL-1ß, MMP-9, and LOX levels were significantly increased while the TIMP-1 was decreased in the TCXL + AC group when compared with the TCXL and TCXL + AC + PA groups. In vivo confocal microscopy revealed that at a depth of 150-210 µm, a trabecular patterned hyperdense structure surrounded by elongated needle-like processes could be observed in the TCXL and TCXL + AC + PA groups, but hardly seen in the TCXL + AC group. The demarcation lines were indistinct and blurred in the TCXL + AC group. An electron microscope demonstrated less interlacing fibril lamellae and higher interfibrillar spacing in the TCXL + AC group. The stability of corneal biomechanics and resistance to collagenase were decreased in the TCXL + AC group. Conclusion: The corneal microstructures induced by TCXL and biomechanical stability were diminished in rabbits with AC but could be maintained by topical anti-inflammatory treatment. Our results supported the causal relationship between altered cytokine profiles and corneal microstructure after primary corneal cross-linking.

Hyperglycemia Induces Meibomian Gland Dysfunction.

Purpose: Patients diagnosed with diabetes are inclined to have abnormalities on stability of tear film and disorder of meibomian gland (MG). This study aims to explore the pathological change of MG induced by diabetes in a rat model. Methods: Sprague-Dawley (SD) rats were intraperitoneally injected with streptozotocin (STZ) to establish a diabetic animal model. Lipid accumulation in MG was detected by Oil Red O staining and LipidTox staining. Cell proliferation status was determined by Ki67 and P63 immunostaining, whereas cell apoptosis was confirmed by TUNEL assay. Gene expression of inflammatory cytokines and adhesion molecules IL-1α, IL-1ß, ELAM1, ICAM1, and VCAM1 were detected by RT-PCR. Activation of ERK, NF-κB, and AMPK signaling pathways was determined by Western Blot analysis. Oxidative stress-related factors NOX4, 4HNE, Nrf2, HO-1, and SOD2 were detected by immunostaining or Western Blot analysis. Tom20 and Tim23 immunostaining and transmission electron microscopy were performed to evaluate the mitochondria functional and structure change. Results: Four months after STZ injection, there was acini dropout in MG of diabetic rats. Evident infiltration of inflammatory cells, increased expression of inflammatory factors, and adhesion molecules, as well as activated ERK and NF-κB signaling pathways were identified. Oxidative stress of MG was evident in 4-month diabetic rats. Phospho-AMPK was downregulated in MG of 2-month diabetic rats and more prominent in 4-month rats. After metformin treatment, phospho-AMPK was upregulated and the morphology of MG was well maintained. Moreover, inflammation and oxidative stress of MG were alleviated after metformin intervention. Conclusions: Long-term diabetes may lead to Meibomian gland dysfunction (MGD). AMPK may be a therapeutic target of MGD induced by diabetes.

Downregulation of p38 MAPK Signaling Pathway Ameliorates Tissue-Engineered Corneal Epithelium.

Tissue-engineered corneal epithelium transplantation is effective treatment for severe limbal stem cell deficiency (LSCD), while epithelial terminal differentiation, tans-differentiation, and insufficient stem cell during construction affect the quality of tissue-engineered corneal epithelium. In this study, we applied SB203580 in the culture medium to downregulate the p38 mitogen-activated protein kinase (MAPK) signaling pathway during construction of tissue-engineered corneal epithelium. With application of SB203580, tissue-engineered corneal epithelium showed enhanced strength and condensed structure. The expression of progenitor cell markers ATP-binding cassette sub-family G member 2, tumor protein p63, keratin 14, and Wnt family member 7A was increased, differentiation markers keratin 12, paired box 6, keratin 10, and keratin 13 and trans-differentiation markers actin alpha 2, smooth muscle and snail family transcriptional repressor 1 was decreased, while cell junction markers claudin 1 and cadherin 1 was increased in the tissue-engineered corneal epithelium. The Wnt/catenin beta 1 signaling pathway was upregulated in the epithelium after p38 MAPK inhibition. Transplantation of tissue-engineered corneal epithelium treated with SB203580 to rabbit LSCD model showed faster wound healing and improved epithelial quality. We conclude that downregulation of p38 MAPK signaling pathway helps maintain the stemness and prevent terminal differentiation and abnormal differentiation of corneal epithelial cells during epithelium construction process, and thus can improve the quality of tissue-engineered corneal epithelium. Impact statement Downregulation of p38 MAPK signaling pathway helps maintain the self-renewal of limbal stem cells and prevents terminal differentiation and abnormal differentiation of corneal epithelial cells. Small molecules modulating p38 MAPK signaling pathway ameliorate tissue-engineered corneal epithelium.

Modulation of the canonical Wnt pathway by Benzalkonium Chloride in corneal epithelium.

Benzalkonium Chloride (BAC) is commonly used in eyedrops. Although the cytotoxicity of BAC has been reported, the mechanism underlying its toxic effect has not been elucidated. The present study investigated the role of the Wnt signaling pathway in the cytotoxicity of BAC in corneal epithelial cells and in the rat cornea. We demonstrated that phosphorylation of ß-catenin, a downstream effector of the canonical Wnt pathway, was down-regulated by a short exposure to BAC in both cultured human corneal epithelial cells (HCE) and a cultured mouse corneal epithelial progenitor cell line (TKE2), suggesting an activation of the Wnt pathway. The activation of Wnt pathway is correlated with the decrease of cell viability induced by BAC. On the other hand, a specific Wnt pathway inhibitor, secreted frizzled-related protein-1 (sFRP1), reversed BAC-induced down-regulating effects on the level of phosphorylation of ß-catenin and ameliorated cell viability in cells treated with BAC. In the rat cornea, the levels of total ß-catenin were significantly up-regulated 8 h after the topical administration of BAC. Taken together, these results provided novel evidence suggesting that the cytotoxicity of BAC may be mediated through modulation of the Wnt pathway.

Autophagy positively regulates Wnt signaling in mice with diabetic retinopathy.

Diabetic retinopathy (DR) is a microvascular complication of diabetes. Aberrant Wnt signaling activation plays a pathological role in DR. However, the underlying mechanisms of aberrant Wnt signaling in DR remain unknown. Autophagy has been reported to be involved in the pathophysiology of DR. The present study aimed therefore to investigate the regulatory effects of autophagy on Wnt signaling in DR. Wnt signaling was activated in the retina of db/db mice combined with an increase in the expression of the autophagic proteins microtubule-associated protein 1A/1B-light chain 3 and beclin-1 and a decrease in the expression of the autophagic protein P62. Inhibition of autophagy by 3-methyladenin decreased Wnt signaling in diabetic retinas, indicating a potential association between Wnt signaling and autophagy. Rapamycin, an autophagy inducer, upregulated Wnt signaling in the retina of normal C57BL/6J mice. In cultured Müller cells, rapamycin induced autophagy and activated Wnt signaling, while chloroquine, an autophagy inhibitor, inhibited autophagy and downregulated Wnt signaling, suggesting that autophagy could regulate Wnt signaling in mice retina and retinal cells. In summary, this study demonstrated that autophagy may positively regulate Wnt signaling in diabetic retinas, indicating a potential mechanism of Wnt signaling upregulation in DR and a possible novel therapeutic target of DR.