MicroRNA-146a-5p protects retinal ganglion cells through reducing neuroinflammation in experimental glaucoma.

Neuroinflammation plays important roles in retinal ganglion cell (RGC) degeneration in glaucoma. MicroRNA-146 (miR-146) has been shown to regulate inflammatory response in neurodegenerative diseases. In this study, whether and how miR-146 could affect RGC injury in chronic ocular hypertension (COH) experimental glaucoma were investigated. We showed that in the members of miR-146 family only miR-146a-5p expression was upregulated in COH retinas. The upregulation of miR-146a-5p was observed in the activated microglia and Müller cells both in primary cultured conditions and in COH retinas, but mainly occurred in microglia. Overexpression of miR-146a-5p in COH retinas reduced the levels pro-inflammatory cytokines and upregulated the levels of anti-inflammatory cytokines, which were further confirmed in the activated primary cultured microglia. Transfection of miR-146a-5p mimic increased the percentage of anti-inflammatory phenotype in the activated BV2 microglia, while transfection of miR-146a-5p inhibitor resulted in the opposite effects. Transfection of miR-146a-5p mimic/agomir inhibited the levels of interleukin-1 receptor associated kinase (IRAK1) and TNF receptor associated factor 6 (TRAF6) and phosphorylated NF-κB subunit p65. Dual luciferase reporter gene assay confirmed that miR-146a-5p could directly target IRAK1 and TRAF6. Moreover, downregulation of IRAK1 and TRAF6 by siRNA techniques or blocking NF-κB by SN50 in cultured microglia reversed the miR-146a-5p inhibitor-induced changes of inflammatory cytokines. In COH retinas, overexpression of miR-146a-5p reduced RGC apoptosis, increased RGC survival, and partially rescued the amplitudes of photopic negative response. Our results demonstrate that overexpression of miR-146a-5p attenuates RGC injury in glaucoma by reducing neuroinflammation through downregulating IRAK1/TRAF6/NF-κB signaling pathway in microglia.

Morphological alterations of intrinsically photosensitive retinal ganglion cells after ablation of mouse photoreceptors with selective photocoagulation.

In this work, we investigated changes in the morphology of intrinsically photosensitive retinal ganglion cells (ipRGCs), M1 subtype, and pupillary light reflex following local and selective ablation of photoreceptors in mice. Laser photocoagulation was used to selectively destroy four patches of photoreceptors per eye at around 4 papillary diameters from the optic disc and at the 3, 6, 9, and 12 o'clock positions between the retinal vessels in the adult mouse retina, leaving cells in the inner retina intact. Morphological parameters of individual M1 cells specifically labeled by the antibody against melanopsin (PA1-780), including dendritic field size, total dendritic length, and dendritic branch number, were examined 1, 2, 4, and 8 weeks after photocoagulation with Neurolucida software. A considerable reduction in these parameters in M1 cells in the "lesioned areas" was found at all the four time points after photocoagulation, as compared with those in the "unlesioned areas". Although M1 cells in the lesioned areas showed significant changes as early as 1 week after laser treatment and the changes gradually increased, reaching a peak value at 2 weeks, morphological restoration was clearly seen in these cells over time. However, no difference in the morphological parameters of M1 cells was observed between the unlesioned areas of laser-treated mice and the corresponding areas of age-matched normal mice without laser lesions. Fluorescence intensity of the somata of melanopsin-positive M1 cells located inside the lesioned areas was significantly decreased at all the four time points after photocoagulation, whereas no changes in pupillary light reflex were detected at different light irradiations, indicating that photocoagulation-induced local photoreceptor loss and alterations of ipRGCs may be insufficient to cause abnormalities in non-image-forming (NIF) visual functions. The results suggest that intact photoreceptors could be crucial for maintaining the expression levels of melanopsin and normal morphology of M1 cells.

Neuroprotection of the P2X7 receptor antagonist A740003 on retinal ganglion cells in experimental glaucoma.

The aim of this study was to explore the neuroprotective effects of the P2X7 receptor antagonist A740003 on retinal ganglion cells (RGCs) in chronic intraocular hypertension (COH) experimental glaucoma mouse model. Bioinformatics was used to analyze the glaucoma-related genes. Western blot, real-time fluorescence quantitative PCR, and immunofluorescence staining techniques were employed to explore the mechanisms underlying the neuroprotective effects of A740003 on RGCs in COH retinas. Bioinformatic analysis revealed that oxidative stress, neuroinflammation, and cell apoptosis were highly related to the pathogenesis of glaucoma. In COH retinas, intraocular pressure elevation significantly increased the levels of translocator protein, a marker of microglial activation, which could be reversed by intravitreal preinjection of A740003. A740003 also suppressed the increased mRNA levels of proinflammatory cytokines interleukin (IL) 1ß and tumor necrosis factor α in COH retinas. In addition, although the mRNA levels of anti-inflammatory cytokine IL-4 and IL-10 were kept unchanged in COH retinas, administration of A740003 could increase their levels. The mRNA and protein levels of Bax and cleaved caspase-3 were increased in COH retinas, which could be partially reversed by A740003, while the levels of Bcl-2 kept unchanged in COH retinas with or without the injections of A740003. Furthermore, A740003 partially attenuated the reduction in the numbers of Brn-3a-positive RGCs in COH mice. A740003 could provide neuroprotective roles on RGCs by inhibiting the microglia activation, attenuating the retinal inflammatory response, reducing the apoptosis of RGCs, and enhancing the survival of RGCs in COH experimental glaucoma.

Topical administration of GLP-1 eyedrops improves retinal ganglion cell function by facilitating presynaptic GABA release in early experimental diabetes.

ABSTRACT: Diabetic retinopathy is a prominent cause of blindness in adults, with early retinal ganglion cell (RGC) loss contributing to visual dysfunction or blindness. In the brain, defects in y-aminobutyric acid (GABA) synaptic transmission are associated with pathophysiological and neurodegenerative disorders, whereas glucagon-like peptide-1 (GLP-1) has demonstrated neuroprotective effects. However, it is not yet clear whether diabetes causes alterations in inhibitory input to RGCs and whether and how GLP-1 protects against neurodegeneration in the diabetic retina through regulating inhibitory synaptic transmission to RGCs. In the present study, we used the patch-clamp technique to record GABA subtype A receptor-mediated miniature inhibitory postsynaptic currents (mIPSCs) in RGCs from streptozotocin-induced diabetes model rats. We found that early diabetes (4 weeks of hyperglycemia) decreased the frequency of GABAergic mIPSCs in RGCs without altering their amplitude, suggesting a reduction in the spontaneous release of GABA to RGCs. Topical administration of GLP-1 eyedrops over a period of 2 weeks effectively countered the hyperglycemia-induced downregulation of GABAergic mIPSC frequency, subsequently enhancing the survival of RGCs. Concurrently, the protective effects of GLP-1 on RGCs in diabetic rats were eliminated by topical administration of exendin-9-39, a specific GLP-1 receptor antagonist, or SR95531, a specific antagonist of the GABA subtype A receptor. Furthermore, extracellular perfusion of GLP-1 was found to elevate the frequencies of GABAergic mIPSCs in both ON- and OFF-type RGCs. This elevation was shown to be mediated by activation of the phosphatidylinositol-phospholipase C/inositol 1,4,5-trisphosphate receptor/Ca2+/protein kinase C signaling pathway downstream of GLP-1 receptor activation. Moreover, multielectrode array recordings revealed that GLP-1 functionally augmented the photoresponses of ON-type RGCs. Optomotor response tests demonstrated that diabetic rats exhibited reductions in visual acuity and contrast sensitivity that were significantly ameliorated by topical administration of GLP-1. These results suggest that GLP-1 facilitates the release of GABA onto RGCs through the activation of GLP-1 receptor, leading to the de-excitation of RGC circuits and the inhibition of excitotoxic processes associated with diabetic retinopathy. Collectively, our findings indicate that the GABA system has potential as a therapeutic target for mitigating early-stage diabetic retinopathy. Furthermore, the topical administration of GLP-1 eyedrops represents a non-invasive and effective treatment approach for managing early-stage diabetic retinopathy.

Exendin-4 promotes retinal ganglion cell survival and function by inhibiting calcium channels in experimental diabetes.

Progressive damage of retinal ganglion cells (RGCs) is observed in early diabetic retinopathy. Intracellular Ca2+ overload mediated by Ca2+ influx through voltage-gated Ca2+ channels (VGCCs) is involved in neurodegeneration, whereas glucagon-like peptide-1 (GLP-1) provides neuroprotection. However, whether GLP-1 plays a neuroprotective role in diabetic retinas by modulating VGCCs remains unknown. We found that eye drops of exendin-4, a long-acting GLP-1 receptor (GLP-1R) agonist, prevented the increase of L-type Ca2+ current (ILCa) densities of RGCs induced by 4-week hyperglycemia and promoted RGC survival by suppressing L-type VGCC (L-VGCC) activity in streptozotocin-induced diabetic rats. Moreover, exendin-4-induced suppression of ILCa in RGCs may be mediated by a GLP-1R/Gs/cAMP-PKA/ryanodine/Ca2+/calmodulin/calcineurin/PP1 signaling pathway. Furthermore, exendin-4 functionally improved the light-evoked spiking ability of diabetic RGCs. These results suggest that GLP-1R activation enhances cAMP to PP1 signaling and that PP1 inactivates L-VGCCs by dephosphorylating them, thereby reducing Ca2+ influx, which could protect RGCs against excitotoxic Ca2+ overload.

Phylogeny-Related Variations in Venomics: A Test in a Subset of Habu Snakes (Protobothrops).

We conducted a comparative analysis to unveil the divergence among venoms from a subset of Old World habu snakes (Protobothrops) in terms of venomic profiles and toxicological and enzymatic activities. A total of 14 protein families were identified in the venoms from these habu snakes, and 11 of them were shared among these venoms. The venoms of five adult habu snakes were overwhelmingly dominated by SVMP (32.56 ± 13.94%), PLA2 (22.93 ± 9.26%), and SVSP (16.27 ± 4.79%), with a total abundance of over 65%, while the subadult P. mangshanensis had an extremely low abundance of PLA2 (1.23%) but a high abundance of CTL (51.47%), followed by SVMP (22.06%) and SVSP (10.90%). Apparent interspecific variations in lethality and enzymatic activities were also explored in habu snake venoms, but no variations in myotoxicity were found. Except for SVSP, the resemblance of the relatives within Protobothrops in other venom traits was estimated to deviate from Brownian motion evolution based on phylogenetic signals. A comparative analysis further validated that the degree of covariation between phylogeny and venom variation is evolutionarily labile and varies among clades of closely related snakes. Our findings indicate a high level of interspecific variation in the venom proteomes of habu snakes, both in the presence or absence and the relative abundance of venom protein families, and that these venoms might have evolved under a combination of adaptive and neutral mechanisms.

Signaling Mechanism for Modulation by GLP-1 and Exendin-4 of GABA Receptors on Rat Retinal Ganglion Cells.

Glucagon-like peptide-1 (GLP-1) is expressed in retinal neurons, but its role in the retina is largely unknown. Here, we demonstrated that GLP-1 or the GLP-1 receptor (GLP-1R; a G protein-coupled receptor) agonist exendin-4 suppressed γ-aminobutyric acid receptor (GABAR)-mediated currents through GLP-1Rs in isolated rat retinal ganglion cells (GCs). Pre-incubation with the stimulatory G protein (Gs) inhibitor NF 449 abolished the exendin-4 effect. The exendin-4-induced suppression was mimicked by perfusion with 8-Br-cAMP (a cAMP analog), but was eliminated by the protein kinase A (PKA) inhibitor Rp-cAMP/KT-5720. The exendin-4 effect was accompanied by an increase in [Ca2+]i of GCs through the IP3-sensitive pathway and was blocked in Ca2+-free solution. Furthermore, when the activity of calmodulin (CaM) and CaM-dependent protein kinase II (CaMKII) was inhibited, the exendin-4 effect was eliminated. Consistent with this, exendin-4 suppressed GABAR-mediated light-evoked inhibitory postsynaptic currents in GCs in rat retinal slices. These results suggest that exendin-4-induced suppression may be mediated by a distinct Gs/cAMP-PKA/IP3/Ca2+/CaM/CaMKII signaling pathway, following the activation of GLP-1Rs.

Study of the efficacy of combination therapy of SiRNAs in HepG2.2.15 cells.

BACKGROUND/AIMS: Hepatitis B virus (HBV) infection is a world-wide health problem. The major obstacles for current anti-HBV therapy are the low efficacy and the occurrence of drug resistant HBV mutations. Recent studies have demonstrated that combination therapy can enhance antiviral efficacy and overcome the shortcomings. Here, the inhibitory effect mediated by combination of small interfering RNAs (siRNAs) targeting different sites of HBV nuclear localization signal (NLS) was monitored in HepG2.2.15 cells. METHODOLOGY: Recombinant plasmid psil-HBV was constructed and transfected into HepG2.2.15 cells. At 48, 72 and 96h after transfection, culture media were collected and cells were harvested for HBV replication assay. HBsAg and HBeAg in the cell culture medium were detected by enzyme-linked immunoadsorbent assay. Intracellular viral DNA and covalently closed circular DNA (cccDNA) was quantified by real-time PCR. HBV viral mRNA was measured by reverse-transcript PCR. RESULTS: Our data demonstrated that the three used siRNAs showed marked anti-HBV effects. Combination of siRNAs, compared with individual use of each siRNA, exerted a stronger inhibition on antigen expression and viral replication, even though the final concentration of siRNA in the therapy was the same. More importantly, we showed that combination therapy significantly suppressed HBV cccDNA amplification. CONCLUSION: Our results revealed that combination of siRNAs mediated a stronger inhibition on viral replication and antigen expression in HepG2.2.15 cells, especially, the amplification of cccDNA.

Emerging roles of circRNAs in the pathological process of myocardial infarction.

Myocardial infarction (MI) is defined as cardiomyocyte death in a clinical context consistent with ischemic insult. MI remains one of the leading causes of morbidity and mortality worldwide. Although there are a number of effective clinical methods for the diagnosis and treatment of MI, further investigation of novel biomarkers and molecular therapeutic targets is required. Circular RNAs (circRNAs), novel non-coding RNAs, have been reported to function mainly by acting as microRNA (miRNA) sponges or binding to RNA-binding proteins (RBPs). The circRNA-miRNA-mRNA (protein) regulatory pathway regulates gene expression and affects the pathological mechanisms of various diseases. Undoubtedly, a more comprehensive understanding of the relationship between MI and circRNA will lay the foundation for the development of circRNA-based diagnostic and therapeutic strategies for MI. Therefore, this review summarizes the pathophysiological process of MI and various approaches to measure circRNA levels in MI patients, tissues, and cells; highlights the significance of circRNAs in the regulation MI pathogenesis and development; and provides potential clinical insight for the diagnosis, prognosis, and treatment of MI.

Protective effect of edaravone against PrP106-126-induced PC12 cell death.

The prion protein peptide PrP106-126 induces cell apoptosis through mechanisms involving production of intracellular reactive oxygen species. The present study investigated the effects of edaravone, a potent free radical scavenger in clinical use, on cell cytotoxicity induced by PrP106-126. Results showed that PrP106-126 decreased PC12 cell viability in a dose- and time-dependent manner. Edaravone significantly antagonized the cytotoxic effects of PrP106-126. Mechanistically, PrP106-126 decreased PC 12 intracellular glutathione (GSH) concentrations, decreased superoxide dismutase (SOD) enzyme activity, increased concentrations of the oxidation end product malondialdehyde (MDA), depolarized the mitochondrial membrane, and increased caspase-3 activity. Edaravone alone did not affect GSH, SOD, or MDA but did effectively reverse all of the intracellular prooxidant effects induced by PrP106-126 and inhibit induced apoptosis in PC12 cells. In conclusion, edaravone may be a viable candidate for the treatment of oxidative stress-induced neurodegenerative disease.

ON-Type Retinal Ganglion Cells are Preferentially Affected in STZ-Induced Diabetic Mice.

Purpose: We investigate morphologic and physiologic alterations of ganglion cells (GCs) in a streptozocin (STZ)-induced diabetic mouse model. Methods: Experiments were conducted in flat-mount retinas of mice 3 months after the induction of diabetes. Changes in morphology of four subtypes of GCs (ON-type RGA2 [ON-RGA2], OFF-type RGA2 [OFF-RGA2], ON-type RGC1 [ON-RGC1], and ON-OFF type RGD2 [ON-OFF RGD2]) were characterized in Thy1-YFP transgenic mice. Using whole-cell patch-clamp recording, passive membrane properties and action potential (AP) firing properties were further investigated in transient ON- and OFF-RGA2 cells. Results: Morphologic parameters were significantly altered in the dendrites branching in the ON sublamina of the inner plexiform layer (IPL) for ON-RGA2 cells and ON-OFF RGD2 cells. Much less significant changes, if any, were seen in those arborizing in the OFF sublamina of the IPL for OFF-RGA2 and ON-OFF RGD2 cells. No detectable changes in morphology were seen in RGC1 cells. Electrophysiologically, increased resting membrane potentials and decreased membrane capacitance were found in transient ON-RGA2 cells, but not in transient OFF-RGA2 cells. Similar alterations in AP firing properties, such as an increase in AP width and reduction in maximum spiking rate, were shared by these two subtypes. Furthermore, in response to depolarizing current injections, both cells generated more APs suggesting an enhanced excitability of these cells in diabetic conditions. Conclusions: These differential changes in morphology and electrophysiology in subtypes of GCs may be responsible for reduced contrast sensitivity known to occur during the early stage of diabetic retinopathy.

[The relationship between Fas/FasL and proliferating cell nuclear antigen expression and malignant degree in human melanoma].

OBJECTIVE: To study the expression of Fas, Fas-ligand (Fas/FasL), and proliferating cell nuclear antigen (PCNA) in malignant melanoma (MM), melanocytic nevus, and normal skin, and the relationship of Fas, Fas/FasL, and PCNA expressions to malignant degrees of MM. METHODS: Immunohistochemistry SP methods was used to detect the expressions of Fas, Fas-ligand (Fas/FasL), and PCNA in 35 samples of MM, 21 samples of melanocytic nevus, and 28 normal skin samples obtained by operation. RESULTS: The positive Fas rate of the MM group was 92.86% (26/28), significantly higher than those of the melanocytic nevus group (76.19%, 16/21) and normal skin group (45.71%, 16/35) (chi2 = 16.84, v =2, P < 0.01). The positive FasL rate of the MM group was 3.57% (1/28), significantly lower than those of the melanocytic nevus group (19.05%, 4/21) and normal skin group (62.86%, 22/35) (chi2 = 27.27, v = 2, P < 0. 01). The PCNA positive rate of the MM group was 100% (35/35), significantly higher than those of the melanocytic nevus group (33.33%, 7/21) and normal skin group (10.71%, 3/28) (chi2 = 54.4, v = 2, P < 0.01). The positive index (PI) of PCNA of the MM group was 55.0% +/- 14.8%, significantly higher than those of the melanocytic nevus group (6.2% +/- 3.0%, t = 19. 89, P < 0.01) and normal skin group (4.0% +/- 2.0%, t = 22.63, P < 0.01), however, there was no significant difference in the PCNA PI between the melanocytic nevus and normal skin groups (P > 0.05). The greater the infiltration depth of MM, the higher the PCNA PI. However, there was no direct dose-effect relationship between PI and pathological types. CONCLUSION: The expressions of Fas/FasL and PCNA reflect the malignant degree of human melanoma, and the more expression of PCNA, the more malignant degree of melanoma. The expressions of Fas/FasL and PCNA are closely associated with the development of MM.

Sublethal vancomycin-induced ROS mediating antibiotic resistance in Staphylococcus aureus.

Staphylococcus aureus is the leading cause of many human infectious diseases. Besides infectious dangers, S. aureus is well-known for the quickly developed drug resistance. Although great efforts have been made, mechanisms underlying the antibiotic effects of S. aureus are still not well clarified. Recently, reports have shown that oxidative stress connects with bactericidal antibiotics [Dwyer et al. (2009) Curr. Opin. Microbiol. 12: , 482-489]. Based on this point, we demonstrate that reactive oxygen species (ROS) induced by sublethal vancomycin may be partly responsible for the antibiotic resistance in heterogeneous vancomycin resistant S. aureus (hVRSA). Sublethal vancomycin treatment may induce protective ROS productions in hVRSA, whereas reduction in ROS level in hVRSA strains may increase their vancomycin susceptibility. Moreover, low dose of ROS in VSSA (vancomycin susceptible S. aureus) strains may promote their survival under vancomycin conditions. Our findings reveal that modest ROS generation may be protective for vancomycin resistance in hVRSA. These results recover novel insights into the relationship between oxidative stress and bacterial resistance, which has important applications for further use of antibiotics and development of therapeutics strategies for hVRSA.

Rifapentine and isoniazid once a week versus rifampicin and isoniazid twice a week for treatment of drug-susceptible pulmonary tuberculosis in HIV-negative patients: a randomised clinical trial.

BACKGROUND: Rifapentine has a long half-life in serum, which suggests a possible treatment once a week for tuberculosis. We aimed to compare rifapentine and isoniazid once a week with rifampicin and isoniazid twice a week. METHODS: We did a randomised, multicentre, open-label trial in the USA and Canada of HIV-negative people with drug-susceptible pulmonary tuberculosis who had completed 2 months of a 6-month treatment regimen. We randomly allocated patients directly observed treatment with either 600 mg rifapentine plus 900 mg isoniazid once a week or 600 mg rifampicin plus 900 mg isoniazid twice a week. Primary outcome was failure/relapse. Analysis was by intention to treat. FINDINGS: 1004 patients were enrolled (502 per treatment group). 928 successfully completed treatment, and 803 completed the 2-year 4-month study. Crude rates of failure/relapse were 46/502 (9.2%) in those on rifapentine once a week, and 28/502 (5.6%) in those given rifampicin twice a week (relative risk 1.64, 95% CI 1.04-2.58, p=0.04). By proportional hazards regression, five characteristics were independently associated with increased risk of failure/relapse: sputum culture positive at 2 months (hazard ratio 2.8, 95% CI 1.7-4.6); cavitation on chest radiography (3.0, 1.6-5.9); being underweight (3.0, 1.8-4.9); bilateral pulmonary involvement (1.8, 1.0-3.1); and being a non-Hispanic white person (1.8, 1.1-3.0). Adjustment for imbalances in 2-month culture and cavitation diminished the association of treatment group with outcome (1.34; 0.83-2.18; p=0.23). Of participants without cavitation, rates of failure/relapse were 6/210 (2.9%) in the once a week group and 6/241 (2.5%) in the twice a week group (relative risk 1.15; 95% CI 0.38-3.50; p=0.81). Rates of adverse events and death were similar in the two treatment groups. INTERPRETATION: Rifapentine once a week is safe and effective for treatment of pulmonary tuberculosis in HIV-negative people without cavitation on chest radiography. Clinical, radiographic, and microbiological data help to identify patients with tuberculosis who are at increased risk of failure or relapse when treated with either regimen.