A Novel Role of ARA70 in Regulating Ferritinophagy of RGCs During Retinal Ischemia Reperfusion.

Although the contribution of ferroptosis, an iron-dependent cell death, to ischemia reperfusion (IR)-induced retinal injury has been reported before, to optimize therapeutic strategy, there is still an urgent need to identify potential candidates involved in this process. Androgen Receptor-Associated Protein of 70 kDa (ARA70) is a cargo receptor for ferritinophagy, and its role in retinal ferroptosis has not been revealed yet. Herein, we explored the role of ARA70 in IR-associated retinal lesions by in vivo (C57BL/6 J mice with intraocular pressure of 90-100 mmHg) and in vitro (retinal ganglion cells (RGCs) stimulated with tert-butyl hydroperoxide (tBH)) experiments. It was found that IR upregulated ARA70 expression and accelerated lipid peroxidation in retinal tissues. We first confirmed that two ferroptosis inhibitors, deferiprone or ferrostatin-1 (Fer-1), suppressed ferritin degradation, restrained apoptosis and inflammation, and protected mouse retinas against IR stress. Next, primary mouse RGCs were treated with tBH to simulate IR environment in vitro. ARA70 expression was decreased at lower concentrations of tBH (5-20 µM), but increased at higher concentrations (40-80 µM). Interestingly, the expression of ferritin-related proteins (ferritin heavy chain, FTH; ferritin light chain, FTL) showed an opposite alteration. Knockdown of ARA70 protected RGCs from tBH-induced damage. It inhibited the delivery of ferritin to lysosomes for ferritinophagy and thus reducing cellular Fe2+ concentration. Besides, ARA70 knockdown suppressed autophagy and inflammation of tBH-treated RGCs. These findings provide novel insights into the pathogenesis of retinal IR, and may be helpful for treatment of retinal diseases.

MicroRNA-5195-3p alleviates high glucose­induced injury in human ARPE-19 cells by targeting GMFB.

Hyperglycemia is generally considered to be an important cause of diabetic retinopathy (DR). The aim of the present study was to investigate the role of miR-5195-3p in high glucose (HG)-induced human retinal pigment epithelial ARPE-19 cell injury. Here, we first found that the expression level of miR-5195-3p was significantly downregulated in HG-stimulated ARPE-19 cells using reverse transcription quantitative PCR. Overexpression of miR-5195-3p attenuated the impaired cell viability, increased apoptosis and pro-inflammatory cytokines secretion in ARPE-19 cells under HG condition using CCK-8 assay, flow cytometry and ELISA assay, respectively. Luciferase reporter assay showed that miR-5195-3p could specifically bind to the 3'UTR of glia maturation factor-ß (GMFB). GMFB overexpression reversed, while knockdown enhanced the protective effects of miR-5195-3p overexpression against HG-induced ARPE-19 cell injury. In summary, miR-5195-3p targeting GMFB might be a potential therapeutic target for DR.

Synthesis of the Proposed Structures of Parvistemoamide and Their Transformations to Stemoamide Derivatives.

The proposed structures of parvistemoamide have been achieved by macrolactamization, but none of the characterization data of synthetic samples matched with those of the natural sample. The transformation of the highly strained 10-membered lactam ring in parvistemoamide into the pyrrolo[1,2-a]-azepine nucleus in stemoamide is accomplished for the first time by either transannular cyclization or Pilli's transformation. This research may promote the total synthesis of other more complex stemoamide-type or medium-sized-ring-containing Stemona alkaloids.

Identification of epigenetic factor KAT2B gene variants for possible roles in congenital heart diseases.

Congenital heart disease (CHD) is a group of anatomic malformations in the heart with high morbidity and mortality. The mammalian heart is a complex organ, the formation and development of which are strictly regulated and controlled by gene regulatory networks of many signaling pathways such as TGF-ß. KAT2B is an important histone acetyltransferase epigenetic factor in the TGF-ß signaling pathway, and alteration in the gene is associated with the etiology of cardiovascular diseases. The aim of this work was to validate whether KAT2B variations might be associated with CHD. We sequenced the KAT2B gene for 400 Chinese Han CHD patients and evaluated SNPs rs3021408 and rs17006625. The statistical analyses and Hardy-Weinberg equilibrium tests of the CHD and control populations were conducted by the software SPSS (version 19.0) and PLINK. The experiment-wide significance threshold matrix of LD correlation for the markers and haplotype diagram of LD structure were calculated using the online software SNPSpD and Haploview software. We analyzed the heterozygous variants within the CDS region of the KAT2B genes and found that rs3021408 and rs17006625 were associated with the risk of CHD.

Asymmetric Total Syntheses and Biological Studies of Tuberostemoamide and Sessilifoliamide A.

The first asymmetric total syntheses of tuberostemoamide, sessilifoliamide A, and their epimers have been accomplished via the common intermediate ethylstemoamide. The stereochemistry control relationship at C8/C9/C10 of ethylstemoamide is clearly revealed for the first time, and a subtle difference of substituent at the C10 position between stemoamide and ethylstemoamide (Me vs Et) drastically changes the stereoselectivity, which is significantly valuable for syntheses of ethylstemoamide structurally related Stemona alkaloids. Biological studies reveal that the activities of each epimer show a significant difference. 11,13-Bis- epi-sessilifoliamide A is expected to be a selective and reversible BChE inhibitor for the treatment of neurodegenerative diseases, and sessilifoliamide A may be a part of the anti-inflammatory substances in Stemonaceae plants.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts.

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Effect of cytoglobin overexpression on extracellular matrix component synthesis in human tenon fibroblasts

BACKGROUND: Conjunctival filtering bleb scar formation is the main reason for the failure of glaucoma filtration surgery. Cytoglobin (Cygb) has been reported to play an important role in extracellular matrix (ECM) remodeling, fibrosis and tissue damage repairing. This study aimed to investigate the role of Cygb in anti-scarring during excessive conjunctival wound healing after glaucoma filtration surgery. METHODS: Cygb was overexpressed in human tenon fibroblasts (hTFs) by transfecting hTFs with lentiviral particles encoding pLenti6.2-FLAG-Cygb. Changes in the mRNA and protein levels of fibronectin, collagen I, collagen III, TGF-ß1, and HIF1α were determined by RT-PCR and western blotting respectively. RESULTS: After Cygb overexpression, hTFs displayed no significant changes in visual appearance and cell counts compared to controls. Whereas, Cygb overexpression significantly decreased the mRNA and protein expression levels of collagen I, collagen III and fibronectin compared with control (p < 0.01). There was also a statistically significant decrease in the mRNA and protein levels of TGF-ß1 and HIF-1α in hTFs with overexpressed Cygb compared with control group (p < 0.05). CONCLUSION: Our study provided evidence that overexpression of Cygb decreased the expression levels of fibronectin, collagen I, collagen III, TGF-ß1 and HIF-1α in hTFs. Therefore, therapies targeting Cygb expression in hTFs may pave a new way for clinicians to solve the problem of post-glaucoma surgery scarring.

Double 1,4-addition of (thio)salicylamides/thiosalicylic acids with propiolate derivatives: a direct, general synthesis of diverse heterocyclic scaffolds.

A simple and practical ring-closure procedure to prepare a range of diverse heterocycles has been developed. In this transformation, a variety of substituted (thio)salicylamides and thiosalicylic acids undergo a double 1,4-addition reaction with propiolate derivatives in the presence of an inorganic base (K3PO4), as a result benzothiazinones, benzoxazinones and benzoxathiinones were prepared in good to excellent yields, respectively, even in gram scales. In addition, further transformation towards more complex structures and oxicam drug analogues has also been successfully carried out.

The effects of combined menthol and borneol on fluconazole permeation through the cornea ex vivo.

The bioavailabilities of eye drops are very low, so it is eager to find a safe and effective penetration enhancer to improve drug bioavailability. In our study, the corneas of New Zealand albino rabbit were mounted in the improved Franz diffusion cells for the measurement of fluconazole diffusion across the corneal permeation barrier. The fluconazole concentrations and the cumulative osmolalities were calculated to investigate the changes of permeation parameters of fluconazole through the ex vivo cornea in the presence of different penetration enhancers. Compared with the control group, 0.05% and 0.1% menthol, as well as menthol combined with borneol were considered as the penetration enhancer significantly promoted the permeation of fluconazole through the cornea ex vivo (P<0.05), but the same trend was not found in borneol groups. Furthermore, the combination of borneol and menthol significantly enhanced the permeation rate in the same concentration, compared with their effects when used alone (P<0.01). In order to investigate the feasibility and safety of the mixture, the corneal hydration level or wink frequency over 5 min were detected. So there were no significant differences between the mixture group and the control one. Consequently, menthol combined with borneol can highly enhance fluconazole permeation through the ex vivo cornea. Because of its low irritation, it may be a new efficient and safe penetration enhancer with a good development and application potential.

Down-regulation of inducible co-stimulator (ICOS) by intravitreal injection of small interfering RNA (siRNA) plasmid suppresses ongoing experimental autoimmune uveoretinitis in rats.

BACKGROUND: RNA interference (RNAi) is now being exploited as a powerful tool for gene knockdown. Recently, we had shown that inducible co-stimulator (ICOS) was up-regulated in experimental autoimmune uveoretinitis (EAU). The aim of this study was to investigate whether intravitreal injection of small interfering RNA (siRNA) plasmid, targeting ICOS, suppresses the ongoing experimental autoimmune uveoretinitis (EAU) in rats. METHODS: Oligonucleotide targeting ICOS was cloned into linearized pRNAT-U6.1/Neo eukaryotic expression vector to construct the recombinant plasmid (pRNAT-U6.1/Neo-ICOS). After transfecting activated rat T cells with the recombinant plasmid, ICOS mRNA and protein expression levels were determined by real-time RT-PCR and Western blot analysis respectively. Rats were immunized with IRBP R16 peptide emulsified in complete Freund's adjuvant (CFA) and given an intravitreal injection of pRNAT-U6.1/Neo-ICOS on day 6 after immunization. After 13days of immunization, the ICOS protein expression and CD4(+) ICOS (+) T cells were identified in retinae through Western blot analysis and flow cytometry respectively. Intraocular inflammation was assessed by the scores of the clinical and histological appearances. Delayed-type hypersensitivity (DTH) and lymphocyte proliferation were detected to evaluate the systemic effect of intravitreal injection of pRNAT-U6.1/Neo-ICOS. RESULT: The recombinant plasmid (pRNAT-U6.1/Neo-ICOS) for the ICOS siRNA was successfully constructed. In vitro studies using the recombinant plasmid has showed the down-regulation of ICOS gene expression both at the mRNA and protein levels. Clinical and pathological scores showed that ocular inflammation of pRNAT-U6.1/Neo-ICOS-treated eyes was markedly less than that of vehicle-treated eyes. The expression of ICOS protein and the amount of CD4(+) ICOS(+) T cells in retinae significantly decreased by intravitreal injection of the recombinant plasmid, whereas delayed-type hypersensitivity response and lymphocyte proliferation were not impaired in rats treated with the recombinant plasmid. CONCLUSION: Intravitreal injection of siRNA plasmid targeting ICOS effectively down-regulated the expression of ICOS, and was highly effective in suppressing the ongoing process of EAU without any side-effects on systemic cellular immunity.

Polylactide-glycoli acid and rapamycin coating intraocular lens prevent posterior capsular opacification in rabbit eyes.

OBJECTIVES: Posterior capsular opacification (PCO) is caused by the proliferation and migration of residual lens epithelium cells (LECs) after extracapsular cataract extraction (ECCE). Rapamcin (RAPA) is known to be a potent immunosuppressive drug with anti-inflammatory and anti-proliferative effects. The aim of this study was to investigate the safety and efficacy of rapamycin sustained release from modified intraocular lens (IOLs) in the prevention of PCO in rabbits. METHODS: Three types of IOLs were used, including the original IOL without modification, IOL with polylactide-glycoli acid (PLGA) coating (PLGA-IOL), and RAPA-loaded PLGA-IOL (RAPA-PLGA-IOL). Sixty New Zealand albino rabbits undergoing phacoemulsification in left eyes were randomly and equally divided into three groups. Group A was implanted with the original IOLs, group B was implanted with the PLGA-IOLs, and group C was implanted with the RAPA-PLGA-IOLs. All of the 60 treated left eyes were examined by a slit-lamp microscope. The concentrations of RAPA in the aqueous humor and blood were determined by high-performance liquid chromatography (HPLC), indicating an vivo release of drug from the polymer carrier. Anterior segment tissue was histologically examined, and wet posterior capsules were weighed. Six months after intervention the PCO was graded. RESULTS: The mean concentrations of RAPA in the aqueous humor from group C at 2 h, 1 days, 3 days, and 7 days after operation were 12.81 +/- 1.27 microg/ml, 14.57 +/- 0.99 microg/ml, 6.39 +/- 0.95 microg/ml, and 1.10 +/- 0.32 microg /ml respectively. The concentrations of RAPA in blood were undetectable. During the early days after the operation, the reactions of the anterior chamber from groups A and B were more severe than from group C. Our findings showed that the initial appearance of PCO in group C was much later than in the other two groups. The wet posterior capsules were weighed to be 0.3735 +/- 0.0943 g (group A), 0.3754 +/- 0.1093 g (group B), and 0.0432 +/- 0.0089 g (group C). Histological observation showed a similar phenomenon, that there was remarkably less accumulation of lens materials on the posterior capsules in group C than in the other two groups. CONCLUSION: Our findings suggest that the designed RAPA-PLGA-IOL effectively prevented formation and development of PCO for a relatively long duration.

[Experimental study of the permeability of Fel Ursi's in eye].

OBJECTIVE: To determine whether Tauroursodeoxycholic acid (TUDCA) can penetrate the blood-ocular barrier after orally administration of Fel Ursi. METHODS: 56 rabbits were divided into two groups, 48 rabbits were used in experimental group, and other 8 rabbits were served as control. 100 mg/ml Fel Ursi were a fused into rabbits stomach. 2 ml blood from vein of auris-edge, aqueous humor from left eye and vitreous sample from right eye were obtained at 0.5, 1.0, 2.0, 4.0, 6.0 and 8.0 h after Fel Ursi administration. Concentration of TUDCA from all of samples was determined by HPLC. RESULTS: TUDCA Concentrations were (999.1 +/- 17.2) - (1300.6 +/- 78.2) microg/ml, (12.7 +/- 1.4) - (47.8 +/- 4.7) microg/ml, and (10.8 +/- 2.9) - (57.9 +/- 7.9) microg/ml in blood, aqueous humor and vitreous respectively. There was no significant differences in the concentration of TUDCA in samples of aqueous humor and vitreous (P > 0.05). However the concentration of TUDCA in rabbit blood was much higher compared with that in aqueous humor and vitreous (P < 0.01). CONCLUSION: Fel Ursi can reach intraocular tissue through penetrating blood-aqueous barrier and blood-vitreous barrier after orally application.