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Rationale: Corneal neovascularization (CoNV) is a severe complication of various types of corneal diseases, that leads to permanent visual impairment. Current treatments for CoNV, such as steroids or anti-vascular endothelial growth factor agents, are argued over their therapeutic efficacy and adverse effects. Here, we demonstrate that transforming growth factor-β (TGF-β)-activated kinase 1 (TAK1) plays an important role in the pathogenesis of CoNV. Methods: Angiogenic activities were assessed in ex vivo and in vitro models subjected to TAK1 inhibition by 5Z-7-oxozeaenol, a selective inhibitor of TAK1. RNA-Seq was used to examine pathways that could be potentially affected by TAK1 inhibition. A gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol was developed as the eyedrop to treat CoNV in a rodent model. Results: We showed that 5Z-7-oxozeaenol reduced angiogenic processes through impeding cell proliferation. Transcriptome analysis suggested 5Z-7-oxozeaenol principally suppresses cell cycle and DNA replication, thereby restraining cell proliferation. In addition, inhibition of TAK1 by 5Z-7-oxozeaenol blocked TNFα-mediated NFκB signalling, and its downstream genes related to angiogenesis and inflammation. 5Z-7-oxozeaenol also ameliorated pro-angiogenic activity, including endothelial migration and tube formation. Furthermore, topical administration of the gelatin-nanoparticles-encapsulated 5Z-7-oxozeaenol led to significantly greater suppression of CoNV in a mouse model compared to the free form of 5Z-7-oxozeaenol, likely due to extended retention of 5Z-7-oxozeaenol in the cornea. Conclusion: Our study shows the potential of TAK1 as a therapeutic target for pathological angiogenesis, and the gelatin nanoparticle coupled with 5Z-7-oxozeaenol as a promising new eyedrop administration model in treatment of CoNV.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) system provides a groundbreaking genetic technology that allows scientists to modify genes by targeting specific genomic sites. Due to the relative simplicity and versatility of the CRISPR/Cas system, it has been extensively applied in human genetic research as well as in agricultural applications, such as improving crops. Since the gene editing activity of the CRISPR/Cas system largely depends on the efficiency of introducing the system into cells or tissues, an efficient and specific delivery system is critical for applying CRISPR/Cas technology. However, there are still some hurdles remaining for the translatability of CRISPR/Cas system. In this review, we summarized the approaches used for the delivery of the CRISPR/Cas system in mammals, plants, and aquacultures. We further discussed the aspects of delivery that can be improved to elevate the potential for CRISPR/Cas translatability.

Aberrant growth of blood vessels (neovascularization) is a key feature of severe eye diseases that can cause legal blindness, including neovascular age-related macular degeneration (nAMD) and diabetic retinopathy (DR). The development of anti-vascular endothelial growth factor (VEGF) agents has revolutionized the treatment of ocular neovascularization. Novel proangiogenic targets, such as angiopoietin and platelet-derived growth factor (PDGF), are under development for patients who respond poorly to anti-VEGF therapy and to reduce adverse effects from long-term VEGF inhibition. A rapidly advancing area is gene therapy, which may provide significant therapeutic benefits. Viral vector-mediated transgene delivery provides the potential for continuous production of antiangiogenic proteins, which would avoid the need for repeated anti-VEGF injections. Gene silencing with RNA interference to target ocular angiogenesis has been investigated in clinical trials. Proof-of-concept gene therapy studies using gene-editing tools such as CRISPR-Cas have already been shown to be effective in suppressing neovascularization in animal models, highlighting the therapeutic potential of the system for treatment of aberrant ocular angiogenesis. This review provides updates on the development of anti-VEGF agents and novel antiangiogenic targets. We also summarize current gene therapy strategies already in clinical trials and those with the latest approaches utilizing CRISPR-Cas gene editing against aberrant ocular neovascularization.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

Retinal neovascularization is a severe complication of proliferative diabetic retinopathy (PDR). MicroRNAs (miRNAs) are master regulators of gene expression that play an important role in retinal neovascularization. In this study, we show that miR-143-3p is significantly downregulated in the retina of a rat model of oxygen-induced retinopathy (OIR) by miRNA-sequencing. Intravitreal injection of synthetic miR-143 mimics significantly ameliorate retinal neovascularization in OIR rats. miR-143 is identified to be highly expressed in the neural retina particularly in the ganglion cell layer and retinal vasculature. In miR-143 treated cells, the functional evaluation showed a decrease in cell migration and delayed endothelial vessel-like tube remodeling. The multiomics analysis suggests that miR-143 negatively impacts endothelial cell activity through regulating cell-matrix adhesion and mediating hypoxia-inducible factor-1 signaling. We predict hub genes regulated by miR-143 that may be involved in mediating endothelial cell function by cytoHubba. We also demonstrate that the retinal neovascular membranes in patients with PDR principally consist of endothelial cells by CIBERSORTx. We then identify 2 hub genes, thrombospondin 1 and plasminogen activator inhibitor, direct targets of miR-143, that significantly altered in the PDR patients. These findings suggest that miR-143 appears to be essential for limiting endothelial cell-matrix adhesion, thus suppressing retinal neovascularization.

Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-β-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-β1 and other proinflammatory cytokines. TAK1 is also a key mediator of proinflammatory signals and plays an important role in maintaining vascular integrity upon proinflammatory cytokine stimulation such as TNFα. However, its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. Here, we investigate the regulatory role of TAK1 in human endothelial cells responding to inflammatory stimuli and in a rat model of oxygen-induced retinopathy (OIR) featured retinal neovascularization. Using TAK1 knockout human endothelial cells that subjected to inflammatory stimuli, transcriptome analysis revealed that TAK1 is required for activation of NFκB signaling and mediates its downstream gene expression related to endothelial activation and angiogenesis. Moreover, pharmacological inhibition of TAK1 by 5Z-7-oxozeaenol attenuated angiogenic activities of endothelial cells. Transcriptome analysis also revealed enrichment of TAK1-mediated NFκB signaling pathway in the retina of OIR rats and retinal neovascular membrane from patients with proliferative diabetic retinopathy. Intravitreal injection of 5Z-7-oxozeaenol significantly reduced hypoxia-induced inflammation and microglial activation, thus attenuating aberrant retinal angiogenesis in OIR rats. Our data suggest that inhibition of TAK1 may have therapeutic potential for the treatment of retinal neovascular pathologies.

Age-related macular degeneration (AMD) is the leading cause of low vision and blindness for which there is currently no cure. Increased matrix metalloproteinase-9 (MMP-9) was found in AMD and potently contributes to its pathogenesis. Resident microglia also promote the processes of chronic neuroinflammation, accelerating the progression of AMD. The present study investigates the effects and mechanisms of the natural compound theissenolactone B (LB53), isolated from Theissenia cinerea, on the effects of RPE dysregulation and microglia hyperactivation and its retinal protective ability in a sodium iodate (NaIO

Multiple myeloma (MM) is one of the most common tumors of the hematological system and remains incurable. Recent studies have shown that long noncoding RNA NORAD is a potential oncogene in a variety of tumors. However, the general biological role and clinical value of NORAD in MM remains unknown. In this study, we measured NORAD expression in bone marrow of 60 newly diagnosed MM, 30 post treatment MM and 17 healthy donors by real-time quantitative polymerase chain reaction (qPCR). The NORAD gene was knockdown by lentiviral transfection in MM cell lines, and the effects of NORAD on apoptosis, cell cycle and cell proliferation in MM cells were examined by flow cytometry, CCK8 assay, EDU assay and Western blot, and the differential genes after knockdown of NORAD were screened by mRNA sequencing, followed by in vivo experiments and immunohistochemical assays. We found that knockdown of NORAD promoted MM cell apoptosis, induced cell cycle G1 phase arrest, and inhibited MM cell apoptosis in in vivo and in vitro experiments. Mechanistically, NORAD plays these roles through the BMP6/P-ERK1/2 axis. We discuss a novel mechanism by which NORAD acts pro-tumorigenically in MM via the BMP6/P-ERK1/2 axis.

Background Elevated intraocular pressure (IOP) is a major risk factor for glaucoma that has been found to induce matrix metalloproteinase-9 (MMP-9) activation and result in eventual retinal dysfunction. Proinflammatory cytokines such as monocyte chemoattractant protein-1 (MCP-1) and interleukin-1β (IL-1β) were also found to be involved in disease progression by mediating MMP-9 production. We previously reported that fungal derivative theissenolactone C (LC53) could exert ocular protective effects by suppressing neuroinflammation in experimental uveitis. Purpose The aim of this study was to investigate the retinoprotective effects of natural compound LC53 on the high IOP-induced ischemia/reperfusion (I/R)-injury model of glaucoma and its cellular mechanisms. Methods A high IOP-induced I/R-injury model was manipulated by normal saline injection into the anterior chamber of the rat eye. MCP-1-stimulated monocytes and IL-1β-activated primary astrocytes were used to investigate the cellular mechanisms of LC53. Retinal function was evaluated with the scotopic threshold response (STR) and combined rod–cone response by electroretinography (ERG). As a positive control, rats were treated with memantine. MMP-9 gelatinolysis, mRNA expression and protein expression were analyzed by gelatin zymography, RT-PCR, and Western Blot, respectively. The phosphorylation levels of MAPKs and NF-κB p65 were tested by Western Blot. Additionally, the levels of inflammatory MCP-1 and IL-1β were determined by ELISA. Results The present study revealed that LC53 preserved the retina functional deficiency assessed by scotopic threshold response (STR) and combined rod–cone response of ERG after high IOP-induced I/R injury. These retinal protective effects of LC53 were positively correlated with inhibitory activities in I/R injury-elicited ocular MMP-9 activation and expression. The increased level of MCP-1 was not affected, and the enhanced IL-1β production was partially reduced by LC53 in the retina after I/R injury. According to cellular studies, LC53 significantly and concentration-dependently abrogated MMP-9 activation and expression in MCP-1-stimulated THP-1 monocytes. We found the inhibitory activities of LC53 were through the ERK- and NF-κB-dependent pathways. In addition, LC53 dramatically suppressed IL-1β-induced MMP-9 activation and expression in primary astrocytes. The phosphorylation of 65-kD protein (p65) of NF-κB was substantially blocked by LC53 in IL-1β-stimulated primary astrocytes. Conclusion LC53 exerted a retinal protective effect through NF-κB inhibition and was highly potent against MMP-9 activities after high IOP-induced I/R injury, suggesting that LC53 would be a promising drug lead for glaucoma or related medical conditions attributed to retinal ischemia.

Age-related macular degeneration (AMD) occurs due to an abnormality of retinal pigment epithelium (RPE) cells that leads to gradual degeneration of the macula. Currently, AMD drug pipelines are endowed with limited options, and anti-VEGF agents stand as the dominantly employed therapy. Despite the proven efficacy of such agents, the evidenced side effects associated with their use underscore the need to elucidate other mechanisms involved and identify additional molecular targets for the sake of therapy improvement. The previous literature provided us with a solid rationale to preliminarily explore the potential of selective HDAC6 and HSP90 inhibitors to treat wet AMD. Rather than furnishing single-target agents (either HDAC6 or HSP90 inhibitor), this study recruited scaffolds endowed with the ability to concomitantly modulate both targets (HDAC6 and HSP90) for exploration. This plan was anticipated to accomplish the important goal of extracting amplified benefits via dual inhibition (HDAC6/HSP90) in wet AMD. As a result, G570 (indoline-based hydroxamate), a dual selective HDAC6-HSP90 inhibitor exerting its effects at micromolar concentrations, was pinpointed in the present endeavor to attenuate blue light-induced cell migration and retinal neovascularization by inhibiting VEGF production. In addition to the identification of a potential chemical tool (G570), the outcome of this study validates the candidate HDAC6-HSP90 as a compelling target for the development of futuristic therapeutics for wet AMD.

Intracerebral hemorrhage (ICH) is a devastating neurological disorder characterized by an exacerbation of neuroinflammation and neuronal injury, for which few effective therapies are available at present. Inhibition of excessive neuroglial activation has been reported to alleviate ICH-related brain injuries. In the present study, the anti-ICH activity and microglial mechanism of ergosta-7,9(11),22-trien-3β-ol (EK100), a bioactive ingredient from Asian medicinal herb Antrodia camphorate, were evaluated. Post-treatment of EK100 significantly attenuated neurobehavioral deficit and MRI-related brain lesion in the mice model of collagenase-induced ICH. Additionally, EK100 alleviated the inducible expression of cyclooxygenase (COX)-2 and the activity of matrix metalloproteinase (MMP)-9 in the ipsilateral brain regions. Consistently, it was shown that EK100 concentration-dependently inhibited the expression of COX-2 protein in Toll-like receptor (TLR)-4 activator lipopolysaccharide (LPS)-activated microglial BV-2 and primary microglial cells. Furthermore, the production of microglial prostaglandin E2 and reactive oxygen species were attenuated by EK100. EK100 also attenuated the induction of astrocytic MMP-9 activation. Among several signaling pathways, EK100 significantly and concentration-dependently inhibited activation of c-Jun N-terminal kinase (JNK) MAPK in LPS-activated microglial BV-2 cells. Consistently, ipsilateral JNK activation was markedly inhibited by post-ICH-treated EK100 in vivo. In conclusion, EK100 exerted the inhibitory actions on microglial JNK activation, and attenuated brain COX-2 expression, MMP-9 activation, and brain injuries in the mice ICH model. Thus, EK100 may be proposed and employed as a potential therapeutic agent for ICH.

Retinal neovascularization, or pathological angiogenesis in the retina, is a leading cause of blindness in developed countries. Transforming growth factor-β-activated kinase 1 (TAK1) is a mitogen-activated protein kinase kinase kinase (MAPKKK) activated by TGF-β1 and other pro-inflammatory cytokines. TAK1 is also a key mediator of inflammation, innate immune responses, apoptosis and tissue homeostasis and plays an important role in physiological angiogenesis. Its role in pathological angiogenesis, particularly in retinal neovascularization, remains unclear. We investigated the regulatory role of TAK1 in pathological angiogenesis in the retina. Transcriptome analysis of human retina featuring retinal neovascularization revealed enrichment of known TAK1-mediated signaling pathways. Selective inhibition of TAK1 activation by 5Z-7-oxozeaenol attenuated aberrant retinal angiogenesis in rats following oxygen-induced retinopathy. Transcriptome profiling revealed that TAK1 activation in human microvascular endothelial cells under TNFα stimulation led to increase the gene expression related to cytokines and leukocyte-endothelial interaction, mainly through nuclear factor kappa B (NFκB) signaling pathways. These results reveal that inhibition of TAK1 signaling may have therapeutic value for the treatment of pathological angiogenesis in the retina.

Gene therapies that chronically suppress vascular endothelial growth factor (VEGF) represent a new approach for managing retinal vascular leakage and neovascularization. However, constitutive suppression of VEGF in the eye may have deleterious side effects. Here, we developed a novel strategy to introduce Flt23k, a decoy receptor that binds intracellular VEGF, fused to the destabilizing domain (DD) of Escherichia coli dihydrofolate reductase (DHFR) into the retina. The expressed DHFR(DD)-Flt23k fusion protein is degraded unless "switched on" by administering a stabilizer; in this case, the antibiotic trimethoprim (TMP). Cells transfected with the DHFR(DD)-Flt23k construct expressed the fusion protein at levels correlated with the TMP dose. Stabilization of the DHFR(DD)-Flt23k fusion protein by TMP was able to inhibit intracellular VEGF in hypoxic cells. Intravitreal injection of self-complementary adeno-associated viral vector (scAAV)-DHFR(DD)-Flt23k and subsequent administration of TMP resulted in tunable suppression of ischemia-induced retinal neovascularization in a rat model of oxygen-induced retinopathy (OIR). Hence, our study suggests a promising novel approach for the treatment of retinal neovascularization. Schematic diagram of the tunable system utilizing the DHFR(DD)-Flt23k approach to reduce VEGF secretion. a The schematic shows normal VEGF secretion. b Without the ligand TMP, the DHFR(DD)-Flt23k protein is destabilized and degraded by the proteasome. c In the presence of the ligand TMP, DHFR(DD)-Flt23k is stabilized and sequestered in the ER, thereby conditionally inhibiting VEGF. Green lines indicate the intracellular and extracellular distributions of VEGF. Blue lines indicate proteasomal degradation of the DHFR(DD)-Flt23k protein. Orange lines indicate the uptake of cell-permeable TMP. TMP, trimethoprim; VEGF, vascular endothelial growth factor; ER, endoplasmic reticulum.

Background: The present study investigated the effects of microvascular endothelial cells (MECs) on the chemotaxis, adhesion and proliferation of bone marrow hematopoietic stem cells (HSCs) ex vivo. Methods and Results: MECs were collected from the lung tissue of C57BL/6 mice, and HSCs were isolated with immunomagnetic beads from bone marrow of GFP mice. MECs and HSCs were co-cultured with or without having direct cell–cell contact in Transwell device for the measurement of chemotaxis and adhesion of MECs to HSCs. Experimental results indicate that the penetration rate of HSCs from the Transwell upper chamber to lower chamber in ‘co-culture’ group was significantly higher than that of ‘HSC single culture’ group. Also, the HSCs in co-culture group were all adherent at 24 h, and the co-culture group with direct cell–cell contact had highest proliferation rate. The HSC number was positively correlated with vascular endothelial growth factor (VEGF) and stromal cell-derived factor-1 (SDF-1) levels in supernatants of the culture. Conclusions: Our study reports that MECs enhance the chemotaxis, adhesion and proliferation of HSCs, which might be related to cytokines SDF-1 and VEGF secreted by MECs, and thus MECs enhance the HSC proliferation through cell–cell contact. The present study revealed the effect of MECs on HSCs, and provided a basis and direction for effective expansion of HSCs ex vivo.

To investigate the effect of microvascular endothelial cells (MEC) on the proliferation of hematopoictic stem cells (HSC) under different culture conditions in vitro.The MEC from lung tissue of C57BL/6 mice and the HSC from bone marrow of GFP mice were used for non-contact co-culture, 2 D contact co-culture, at same time the single MEC and single HSC culture were seted up and were used as control group. The cell counting and CCK-8 method were used to detect and compare the proliferation levels of suspension cells in different groups on day 1, 3, 5 and 7.MEC presented adherent growth. In process of cell culture in vitro, the number of suspension cells in MEC and HSC co-culture group and single HSC culture group increased, the suspension cells in 2D contact and non-contact co-culture groups more early gated into logarithmic growth phase as compared with suspension cells in control group, the proliferation level of suspention cells in 2D contact culture group was higher than that in non-contact co-culture group and single HSC culture group (P0.05), the proliferation level of suspension cells in non-contact co-culture group was higher than that in single HSC culture group (P0.05).The culture of HSC in vitro can proliferate HSC, MEC can promote the proliferation of HSC, MEC also can promote the HSC proliferation by non-contact co-culture in vitro, which relates with the effect of cytokines secreted from MEC; the effect of MEC and HSC contact co-culture on the proliferation of HSC is stronger than that of non-contact co-culture, which relates with the regulation of cell-cell contact.在不同培养条件下微血管内皮细胞对骨髓造血干细胞增殖影响的比较.探讨在体外不同培养条件下微血管内皮细胞（microvascular endothelial cells, MEC）对骨髓造血干细胞(hematopoietic stem cells, HSC)增殖的作用.将来源于C57BL/6小鼠肺组织的MEC与来源于GFP小鼠骨髓的HSC用于非接触共培养、2 D接触共培养，并设置对照组HSC单独培养及MEC单独培养。采用细胞计数和CCK8实验比较各组悬浮细胞在1、3、5和7 d的增殖情况.MEC呈贴壁生长。HSC共培养组及单独培养组细胞在体外培养过程中悬浮细胞计数均有所增加；2 D接触培养组与非接触共培养组中悬浮细胞比对照组中悬浮细胞更早进入对数生长期；2 D接触培养组悬浮细胞增殖多于非接触共培养组及HSC单独培养组（P0.05）；非接触共培养组悬浮细胞增殖多于HSC单独培养组（P0.05）.体外培养HSC能使HSC增殖；MEC能促进HSC增殖；MEC还可以通过与HSC非接触共培养促进HSC增殖，这一效应与MEC分泌的细胞因子促HSC增殖作用有关；MEC与HSC直接接触培养对HSC的增殖作用比非接触共培养更强，这一效应与细胞-细胞接触对HSC增殖的调控有关.

Blue light-induced phototoxicity plays an important role in retinal degeneration and might cause damage as a consequence of smartphone dependency. Here, we investigated the effects of periodic exposure to blue light-emitting diode in a cell model and a rat retinal damage model. Retinal pigment epithelium (RPE) cells were subjected to blue light in vitro and the effects of blue light on activation of key apoptotic pathways were examined by measuring the levels of Bcl-2, Bax, Fas ligand (FasL), Fas-associated protein with death domain (FADD), and caspase-3 protein. Blue light treatment of RPE cells increased Bax, cleaved caspase-3, FasL, and FADD expression, inhibited Bcl-2 and Bcl-xL accumulation, and inhibited Bcl-2/Bax association. A rat model of retinal damage was developed with or without continuous or periodic exposure to blue light for 28 days. In this rat model of retinal damage, periodic blue light exposure caused fundus damage, decreased total retinal thickness, caused atrophy of photoreceptors, and injured neuron transduction in the retina.

To study the effect and mechanism of shh and mesenchymal stem cell（MSC）synergism on the proliferation of hematopoietic stem cells in noninvasive co-culture system in vitro.The mesenchymal stem cells were cultured in vitro，CD34The total number of cells，the total amount of RNA and the relative expression of ki67, Tie-2, VEGF and Ang-1 in non-contact co-culture group increased and showed the following trends on the 7th day：the above-mentioned indexes in group MSC + HSC, group shh + HSC were higher than those in group HSC, while those in MSC + shh + HSC Group was higher than those in MSC + HSC and shh + HSC group.Angiogenic factors help MSC to proliferate HSC and amplify the CD34

To determine the toxic effects of long-term topical usage of fluorometholone (FLM) on ganglion cells using a direct in vivo retinopathological Brown Norway (BN) rat model. The BN rat retinal model was investigated with a minimum of 3 rats and a maximum of 4 rats per group. Rats received vehicle and 0.02% FLM suspension via topical administration 3 times a day for 28 days. The fundus images and retinal vessels were detected on days 1, 14, and 28 using Micron III retinal imaging microscope and fundus fluorescein angiography (FFA). For retinal structures, spectral-domain optical coherence tomography (SD-OCT) images were taken after FFA on days 1, 14, and 28 using an SD-OCT Imaging System. For retinal function, electrical signal transduction of photoreceptors and bipolar cells was determined by electroretinographic (ERG) recording on days 1 and 28 and IOP detection. At the end of the experiment on day 28, immunohistochemistry and TUNEL assay were performed to investigate apoptosis in ganglion cells. Total retina and nerve fiber layer (NFL) to the inner plexiform layer (IPL) were significantly thinner following 28 days of FLM treatment. Hematoxylin and eosin stain showed that there were NFL and ganglion cell layer deformations in the FLM group. With FLM treatment, TUNEL assay showed approximately a 4.68-fold increase in apoptotic cells. Moreover, FLM decreased ERG b-wave amplitude by about 56%. Using ophthalmofundoscopy devices, after 28 days of topical administration, FLM decreased NFL-IPL and total retina thickness. This suggests that long-term FLM induces adverse effects with respect to ganglion cell apoptosis.

Much evidence has indicated that matrix metalloproteinases (MMPs) participate in the progression of neuroinflammatory disorders. The present study was undertaken to investigate the inhibitory effect and mechanism of the antipsychotic haloperidol on MMP activation in the stimulated THP-1 monocytic cells. Haloperidol exerted a strong inhibition on tumor necrosis factor- (TNF-) α-induced MMP-9 gelatinolysis of THP-1 cells. A concentration-dependent inhibitory effect of haloperidol was observed in TNF-α-induced protein and mRNA expression of MMP-9. On the other hand, haloperidol slightly affected cell viability and tissue inhibition of metalloproteinase-1 levels. It significantly inhibited the degradation of inhibitor-κB-α (IκBα) in activated cells. Moreover, it suppressed activated nuclear factor-κB (NF-κB) detected by a mobility shift assay, NF-κB reporter gene, and chromatin immunoprecipitation analyses. Consistent with NF-κB inhibition, haloperidol exerted a strong inhibition of lipopolysaccharide- (LPS-) induced MMP-9 gelatinolysis but not of transforming growth factor-β1-induced MMP-2. In in vivo studies, administration of haloperidol significantly attenuated LPS-induced intracerebral MMP-9 activation of the brain homogenate and the in situ in C57BL/6 mice. In conclusion, the selective anti-MMP-9 activation of haloperidol could possibly involve the inhibition of the NF-κB signal pathway. Hence, it was found that haloperidol treatment may represent a bystander of anti-MMP actions for its conventional psychotherapy.

Retinitis pigmentosa (RP) is an inherited photoreceptor-degenerative disease, and neuronal degeneration in RP is exacerbated by glial activation. Cassia seed (Jue-ming-zi) is a traditional herbal medicine commonly used to treat ocular diseases in Asia. In this report, we investigated the retina-protective effect of chrysophanol, an active component of Cassia seed, in an N-methyl-N-nitrosourea (MNU)-induced mouse model of RP. We determined that chrysophanol inhibited the functional and morphological features of MNU-induced retinal degeneration using scotopic electroretinography (ERG), optical coherence tomography (OCT), and immunohistochemistry analysis of R/G opsin and rhodopsin. Furthermore, TUNEL assays revealed that chrysophanol attenuated MNU-induced photoreceptor cell apoptosis and inhibited the expression of the apoptosis-associated proteins PARP, Bax, and caspase-3. In addition, chrysophanol ameliorated reactive gliosis, as demonstrated by a decrease in GFAP immunolabeling, and suppressed the activation of matrix metalloproteinase (MMP)-9-mediated gelatinolysis. In vitro studies indicated that chrysophanol inhibited lipopolysaccharide (LPS)-induced iNOS and COX-2 expression in the BV2 mouse microglia cell line and inhibited MMP-9 activation in primary microglia. Our results demonstrate that chrysophanol provided neuroprotective effects and inhibited glial activation, suggesting that chrysophanol might have therapeutic value for the treatment of human RP and other retinopathies.

Glial activation and neuroinflammatory processes play important roles in the pathogenesis of brain abscess and neurodegenerative diseases. Activated glial cells can secrete various proinflammatory cytokines and neurotoxic mediators, which contribute to the exacerbation of neuronal cell death. The inhibition of glial activation has been shown to alleviate neurodegenerative conditions. The present study was to investigate the specific HDAC8 inhibitor WK2-16, especially its effects on the neuroinflammatory responses through glial inactivation. WK2-16 significantly reduced the gelatinolytic activity of MMP-9, and expression of COX-2/iNOS proteins in striatal lipopolysaccharide (LPS)-induced neuroinflammation in C57BL/6 mice. The treatment of WK2-16 markedly improved neurobehavioral deficits. Immunofluorescent staining revealed that WK2-16 reduced LPS-stimulated astrogliosis and microglial activation in situ. Consistently, cellular studies revealed that WK2-16 significantly suppressed LPS-induced mouse microglia BV-2 cell proliferation. WK2-16 was proven to concentration-dependently induce the levels of acetylated SMC3 in microglial BV-2 cells. It also reduced the expression of COX-2/iNOS proteins and TNF-&alpha, production in LPS-activated microglial BV-2 cells. The signaling studies demonstrated that WK2-16 markedly inhibited LPS-activated STAT-1/-3 and Akt activation, but not NF-&kappa, B or MAPK signaling. In summary, the HADC8 inhibitor WK2-16 exhibited neuroprotective effects through its anti-neuroinflammation and glial inactivation properties, especially in microglia in vitro and in vivo.

Aberrant remodeling of the extracellular matrix occurs in many pathological processes, and its breakdown is mainly accomplished by matrix metalloproteinases (MMPs), which participate in the course of inflammation and tumor invasion. Nuclear factor-kappaB (NF-kappaB), a key transcription factor for the production of MMP-9, can be activated by various proinflammatory cytokines and promotes inflammation. In the present study, we investigated the intracellular mechanism for the inhibitory effects of an analogue of N-hydroxycinnamoylphenalkylamides, N-2-(4-bromophenyl) ethyl caffeamide (EK5), on tumor necrosis factor (TNF)-alpha stimulated expression of MMP-9 in a human monocytic cell line, THP-1. Our results show that TNF-alpha-induced expression of MMP-9 at both mRNA and protein levels was completely blocked by EK5 in a concentration-dependent (1-20microM) manner. We also found that EK5 markedly suppressed NF-kappaB signaling as detected by the NF-kappaB reporter gene assay but had no effects on the degradation of IkappaBalpha or translocation of NF-kappaB. Interestingly, chromatin immunoprecipitation results revealed that the association between p65 and MMP-9 promoter gene was completely abrogated by EK5, but the p65 phosphorylation was not affected. Overall, our findings suggest that EK5 inhibits MMP-9 production through the nuclear-targeted down-regulation of NF-kappaB signaling in human monocytic cells and this may provide a novel molecular basis of EK5 activity. Further studies are needed to verify its anti-inflammatory effects.

Objects are strictly divided into clusters in the conventional algorithms;however,most of the time,the object boundary cannot be strictly classified.The rough set based k-means clustering algorithm and leader clustering algorithm divide the data object into a cluster's upper-bound or lower-bound using rough set,which provides a new perspective of dealing with uncertainty and solve the problem of uncertain boundary region.The problem is that both of the two algorithms cannot deal with mixed valued data,and clustering results significantly depend on the initial value.A definition of the distance for mixed valued data was introduced in this paper,an improved method was put forward for the selection of the initial value,and a clustering algorithm for mixed valued data based on rough set was given.Finally,a simulation experiment was carried out.Simulation results show,under the uncertain situation of cluster number,the clustering accuracy of the algorithm is significantly improved than the traditional k-means algorithm.

To re-evaluate and compare the research design and the use of statistical methods in Chinese Journal of Cardiology.Summary the research design and statistical methods in all of the original papers in Chinese Journal of Cardiology all over the year of 2011, and compared the result with the evaluation of 2008.(1) There is no difference in the distribution of the design of researches of between the two volumes. Compared with the early volume, the use of survival regression and non-parameter test are increased, while decreased in the proportion of articles with no statistical analysis. (2) The proportions of articles in the later volume are significant lower than the former, such as 6(4%) with flaws in designs, 5(3%) with flaws in the expressions, 9(5%) with the incomplete of analysis. (3) The rate of correction of variance analysis has been increased, so as the multi-group comparisons and the test of normality. The error rate of usage has been decreased form 17% to 25% without significance in statistics due to the ignorance of the test of homogeneity of variance.Many improvements showed in Chinese Journal of Cardiology such as the regulation of the design and statistics. The homogeneity of variance should be paid more attention in the further application.

To evaluate the effect of pitavastatin on blood glucose in patients with hypercholesterolemia, and to investigate the efficacy of pitavastatin in diabetic patients combined with hypercholesterolemia.This study was a 12-week, multi-center, open-label, without parallel-group comparison, phase IV clinical trail.Contrasting to baseline, the prevalences at week 4 and 12 post-treatment of abnormal fasting plasma glucose (FPG) and glycosylated hemoglobin A1c (HbA1c) (FPG: 14.2%vs 14.1% and 11.0%; HbA1c: 14.3% vs 15.1% and 16.1%) in the safety set subjects without diabetes mellitus (DM), as well as in those with DM but not taking glucose-lowering drugs (FPG: 7/7 vs 4/7 and 5/7; HbA1c: 5/5 vs 4/4 and 5/5) had no significant changes (all P values0.05). Contrasting to baseline, the levels of TC [(6.51 ± 0.94) mmol/L vs (5.12 ± 0.93) mmol/L and (4.54 ± 1.00) mmol/L], LDL-C [(4.11 ± 0.79) mmol/L vs (3.02 ± 0.81) mmol/L and (2.51 ± 0.70) mmol/L] and TG [2.10 (1.53, 2.54) mmol/L vs 1.62 (1.26, 2.00) mmol/L and 1.35 (1.10, 1.86) mmol/L]at week 4 and 12 post-treatment in the per protocol set 55 subjects with DM were significantly reduced (all P values0.05); 33.3% of subjects at high risk and 10.0% of subjects at very high risk had achieved a TC target value; 55.6% of subjects at high risk and 40.0% of subjects at very high risk had achieved a LDL-C target value.Pitavastatin has a safe effect on blood glucose and it could be used to treat diabetic patients combined with hypercholesterolemia in China.

To evaluate the effect of pitavastatin on high sensitivity C-reactive protein (hsCRP) in patients with hypercholesterolemia, and determine risk factors for the effect.This study was a 12-week, multicenter, open-label, without parallel-group comparison, phase IV clinical trail.There were 330 subjects in the per protocol set. Contrast to the baseline, the average levels of hsCRP in all of subjects and the group without a history of receiving previous statin medication at week 12 post-treatment decreased respectively 26.4% (1.20 mg/L vs 1.68 mg/L) and 27.5% (1.21 mg/L vs 1.97 mg/L, all P0.05). The results of multilevel models indicated that the average levels of hsCRP reduced with the passage of treatment time, the time-varying rate of per-visit was 0.97 mg/L (95% confidence interval 0.96 - 0.98). Controlled individual background covariates, the model predicted that pulse pressure and white blood cell count on the baseline had the significant positive effects on hsCRP (P0.01).Pitavastatin decreases hsCRP in patients with hypercholesterolemia. The main risk factors for the effect are pulse pressure and white blood cell count on the baseline.

To evaluate the safety and efficacy of pitavastatin in patients with hypercholesterolemia in China under conditions of extensive usage.This was a 12-week, multicenter, open-label, without parallel-group comparison, phase IV clinical trial.There were 427 subjects in the safety set. The adverse events mainly included vomiting, myalgia and the elevations of aspartate transaminase (AST), alanine transaminase (ALT) and creatine kinase (CK), etc. The incidence of drug-related adverse events was 4.22%. There were no significant differences between pre-exposure and post-exposure average levels of renal function indicators and blood routine examination item (all P0.05). None of them had a high AST/ALT value, i.e.3 times upper limits of normal (ULN), or had a high CK value, i.e.10 times ULN. There were 397 subjects in the per protocol set. At week 12 post-treatment, the blood levels of total cholesterol and low density lipoprotein cholesterol (LDL-C) in subjects without previous treatment decreased 24.6% and 31.0% respectively, that of high density lipoprotein cholesterol (HDL-C) in subjects with HDL-C1.04 mmol/L increased 60.1% while that of triglyceride (TG) in subjects with TG1.70 mmol/L decreased 22.5% (P0.05). And 207 (92.3%) subjects were at a low risk, 46 (76.1%) subjects at an intermediate risk, 134 (47.8%) subjects at a high risk and 10 (40.0%) of subjects at a very high risk had achieved a LDL-C target value; the LDL-C goal achievement rate after switching from previous medication to pitavastatin was significant higher than that of pre-switching.Pitavastatin demonstrates positive safety and efficacy. It may be used for the treatment of patients with hypercholesterolemia in China.

Based on the analysis of the fundamental framework and the applied levels of the Service Oriented Architecture (SOA), combined with the demands of the automobile industry chain, a kind of framework of the automobile industry chain cooperation platform based on SOA is presented, its key supporting technologies are discussed. Meanwhile, a flow model of coarse granular business is set up on the basis of the cooperative cases of the chain,. Besides, a particular explanation on how to realize the material share and business cooperation between corporations are made, and systematic operating examples is given.

The access control service has been an important orientation in the security research. In this paper, the essentiality of access control service in the E-Commerce Web was analyzed; the relative technologies were introduced at the same time. And through describing a concrete collaborative e-commerce platform, a detailed description of how to design and implementation of access control systems were explained in detail.