The anti-apoptotic role of Ginkgolide B via mitochondrial permeability transition pore inhibition in retinal ischemia-reperfusion.

This research delves into the effectiveness of Ginkgolide B (GB), a compound from Ginkgo biloba, in combating cell death caused by glaucoma, with a focus on mitochondrial impairment and the mitochondrial permeability transition pore (mPTP). Utilizing models of high intraocular pressure and in vitro glaucoma simulations, the study investigates GB's impact on retinal progenitor cells (RPCs) under oxygen-glucose deprivation/reperfusion (OGD/R) and in a rat glaucoma model. The study methodologies included apoptosis assessment, apoptotic marker analysis via Western blot, and mitochondrial structure and function evaluation. The findings reveal that GB notably decreases apoptosis in RPCs exposed to OGD/R in vitro, and reduces ischemia-reperfusion damage in vivo. GB's protective role is attributed to its ability to preserve mitochondrial integrity, maintain membrane potential, regulate calcium levels, and inhibit mPTP opening. These results underscore GB's potential as a therapeutic agent for acute primary angle-closure glaucoma, highlighting its capability to alleviate mitochondrial damage and apoptosis in RPCs and retinal nerve fiber layer cells.

Melatonin antagonizes oxidative stress-induced apoptosis in retinal ganglion cells through activating the thioredoxin-1 pathway.

This study aimed to explore the role of melatonin in oxidative stress-induced injury on retinal ganglion cells and the underlying mechanisms. The immortalized RGC-5 cells were treated with H2O2 to induce oxidative injury. Cell viability was measured by Cell Counting Kit-8, and apoptosis was determined by flow cytometry and western blot assays. Reactive oxygen species (ROS), lactate dehydrogenase (LDH), and malondialdehyde (MDA) levels were examined to evaluate oxidative stress levels. In addition, Thioredoxin-1 (Trx1) was silenced in RGC-5 cells using small interfering RNA followed by signaling pathway examination to explore the underlying mechanisms of melatonin in alleviating oxidative injury. Melatonin pre-treatment significantly alleviated H2O2-induced apoptosis in RGC-5 cells. Melatonin also markedly reversed the upregulation of cleaved-caspase 3, cleaved-caspase 9, and Bax expression and downregulation of Bcl-2 expression induced by H2O2. Further analyses presented that melatonin significantly attenuated the increase of ROS, LDH, and MDA levels in RGC-5 cells after H2O2 treatment. Melatonin also abolished the downregulated expression of Superoxide dismutase type 1, Trx1, and Thioredoxin reductase 1, and the reduced activity of thioredoxin reductase in RGC-5 cells after H2O2 treatment. Notably, Trx1 knockdown significantly mitigated the protective effect of melatonin in alleviating H2O2-induced apoptosis and oxidative stress, while administration of compound C, a common inhibitor of c-Jun N-terminal kinase (JNK) signaling, partially reversed the effect of Trx1 silencing, thereby ameliorating the apoptosis and oxidative injury induced by H2O2 in RGC-5 cells. Melatonin could significantly alleviate oxidative stress-induced injury of retinal ganglion cells via modulating Trx1-mediated JNK signaling pathway.

Viability of mitochondria-labeled retinal ganglion cells in organotypic retinal explant cultures by two methods.

Retinal explant cultures provide a valuable system to study retinal function in vitro. This study established a new retinal explant culture method to prolong the survival of retinal ganglion cells (RGCs). Explants were prepared in two different ways: with or without optic nerve. Retinas from newborn mice that had received an injection of MitoTracker Red into the contralateral superior colliculus to label axonal mitochondria were cultured as organotypic culture for 7 days in vitro. At several time points during the culture, viability of RGCs was assessed by multi-electrode array recording, and morphology by immunohistochemical methods. During the culture, the thickness of the retinal tissue in both groups gradually decreased, however, the structure of the layers of the retina could be identified. Massive apoptosis in the retinal ganglion cell layer (GCL) appeared on the first day of culture, thereafter the number of apoptotic cells decreased. Glial activation was observed throughout the culture, and there was no difference in morphology between the two groups. RGCs loss was exacerbated on 3rdday of culture, and RGCs loss in retinal explants with preserved optic nerve was significantly lower than in retinas that did not preserve the optic nerve. More and longer-lasting mitochondrial signals were observed in the injured area of the optic nerve-preserving explants. Retinal explants provide an invaluable tool for studying retinal function and developing treatments for ocular diseases. The optic nerve-preserving culture helps preserve the integrity of RGCs. The higher number of mitochondria in the nerve-preserving cultures may help maintain viability of RGCs.

Polymorphisms in TRIB2 and CAPRIN2 Genes Contribute to the Susceptibility to High Myopia-Induced Cataract in Han Chinese Population.

BACKGROUND Myopia has been shown to be associated with many pathological complications including cataracts, and previous evidence supported that high myopia facilitates the formation of cataracts. However, no studies have identified a link between the genetic susceptibility of high myopia-induced cataracts (HMC) and the underlying genetic mechanisms. Our study aimed to determine how the TRIB2 and CAPRIN2 genes correlate to the risk of HMC. MATERIAL AND METHODS In total, we successfully recruited 3162 participants, including 1026 participants with high myopia and cataracts and 2136 controls with high myopia only. For genotyping, 22 tag single nucleotide polymorphisms (SNPs) in TRIB2 and CAPRIN2 genes were chosen. Single marker association analysis and functional effects of significant SNPs were carried out. RESULTS Strong correlation signals were captured for SNP rs890069 (χ²=22.13, P=2.55×10-6) in TRIB2 and SNP rs17739338 (χ²=16.07, P=6.10×10-5) in CAPRIN2. In patients with high myopia, the C allele at SNP rs890069 was strongly linked to cataract risk (OR [95% CI]=1.36 [1.20-1.55]). In patients with high myopia, the T allele at SNP rs17739338 was significantly related to a lower risk of cataract (OR [95% CI]=0.54 [0.40-0.74]). In different types of human tissues, SNPs rs890069 and rs17739338 were found to be significantly correlated to the levels of TRIB2 and CAPRIN2 gene expression. CONCLUSIONS Our study indicated that both TRIB2 and CAPRIN2 genes conferred the susceptibility to cataract in patients with high myopia and Chinese Han ancestry. Future research remains necessary for fully understanding the pathogenic mechanisms and genetic characteristics of cataract.

Promotion of corneal angiogenesis by sensory neuron-derived calcitonin gene-related peptide.

The normal cornea has no blood vessels but has abundant innervation. There is emerging evidence that sensory nerves, originated from the trigeminal ganglion (TG) neurons, play a key role in corneal angiogenesis. In the current study, we examined the role of TG sensory neuron-derived calcitonin gene-related peptide (CGRP) in promoting corneal neovascularization (CNV). We found that CGRP was expressed in the TG and cultured TG neurons. In the cornea, minimal CGRP mRNA was detected and CGRP immunohistochemical staining was exclusively co-localized with corneal nerves, suggesting corneal nerves are likely the source of CGRP in the cornea. In response to intrastromal suture placement and neovascularization in the cornea, CGRP expression was increased in the TG. In addition, we showed that CGRP was potently pro-angiogenic, leading to vascular endothelial cell (VEC) proliferation, migration, and tube formation in vitro and corneal hemangiogenesis and lymphangiogenesis in vivo. In a co-culture system of TG neurons and VEC, blocking CGRP signaling in the conditioned media of TG neurons led to decreased VEC migration and tube formation. More importantly, subconjunctival injection of a CGRP antagonist CGRP8-37 reduced suture-induced corneal hemangiogenesis and lymphangiogenesis in vivo. Taken together, our data suggest that TG sensory neuron and corneal nerve-derived CGRP promotes corneal angiogenesis.

Effects of VEGF levels on anti-VEGF therapy for patients with idiopathic choroidal neovascularization.

The objective of this study is to investigate the levels of vascular endothelial growth factor (VEGF) and other cytokines in aqueous humor of patients with idiopathic choroidal neovascularization (CNV) and their effects together with central retinal thickness (CRT) on the response to intravitreal injection of anti-VEGF antibody ranibizumab. This clinical study recruited 32 eyes from 32 patients with CNV under or besides fovea. VEGF, interleukin (IL)-6, IL-8, and monocyte chemoattractant protein (MCP)-1 levels were detected in aqueous humor (0.1 ml) sampled during intravitreal injection. Aqueous humor controls were from nine cataract patients without any systemic disorders. The VEGF levels in aqueous humor were negatively related (r = -0.373, p = 0.035) to CRT, which was positively related (r = 0.743, p < 0.001) to the number of injections. The VEGF levels before treatment and during the third injection in four patients with three or more injections were 13.42 ± 8.50 and 5.75 ± 3.68 (p = 0.055), respectively. The average best corrected visual acuity (BCVA) before and 12 months after treatment were 57.03 ± 16.15 and 75.16 ± 11.78 (p < 0.001), and the average CRT before and 12 months after treatment were 352.09 ± 84.15 and 251.13 ± 63.96 (p < 0.001), respectively. The visual improvement was negatively related (r = -0.815, p < 0.001) to the visual baseline, and the vision 12 months after treatment was positively related (r = 0.581, p < 0.001) to that before treatment. No severe ocular or systemic complication appeared during treatment and follow-ups for all the patients. Intravitreal injection of anti-VEGF antibody ranibizumab is safe and effective for the treatment of idiopathic CNV through decreasing CRT. The patients with larger CRT baseline need more injections of ranibizumab.

[Effects of Gremlin on transdifferentiation and extracellular matrix synthesis in human lens epithelium cells].

OBJECTIVE: To explore the effects of Gremlin on transdifferentiation and extracellular matrix synthesis in cultured human lens epithelium cells (HLEC). METHODS: This is an experimental research. HLEC were incubated with different concentrations of Gremlin (0, 100, 200 and 400 µg/L) for 24 h. The morphological changes of HLEC were observed by inverted microscope. Real-time PCR and Western-Blot were used to evaluate the expression of α-smooth muscle actin (α-SMA) (as a landmark protein of epithelial mesenchymal transition), fibronectin (Fn) and collagen type 1 (COL-1) (as major components of extracellular matrix) after stimulation with different time (0, 12, 24, 48, 72 h) by 200 µg/L Gremlin. The same parameters were observed in Gremlin. siRNA transfected HLEC which treated with 1.0 µg/L TGF-ß2. α-SMA, Fn and COL-1 protein and mRNA expressions comparison with control group were analyzed using one-way and two-way ANOVA, while the difference between groups were compared using Turkey HSD and LSD-t test. RESULTS: The normal morphology of HLEC showed polygonal and anchorage-dependent. After the incubation of different concentrations of Gremlin for 24 h, morphological feature of HLEC were changed from monolayer and polygonal to multilayer and spindle fibroblast-like cells, and the intercellular space widened. The expression of α-SMA, Fn and COL-1 were increased with prolonging of Gremlin treatment time (α-SMA gene induction: 1.00 ± 0.00, 1.62 ± 0.57, 3.40 ± 0.83, 5.90 ± 0.49, 7.97 ± 0.91; F = 61.64, P < 0.05, q = 6.43, 13.13, 18.66, P < 0.05; Fn gene induction: 1.00 ± 0.00, 3.26 ± 0.23, 5.86 ± 0.90, 10.17 ± 2.16, 12.89 ± 1.63; F = 42.03, P < 0.05, q = 6.45, 12.18, 15.79, P < 0.05; COL-1 gene induction: 1.00 ± 0.00, 1.78 ± 0.88, 6.80 ± 0.44, 12.76 ± 2.46, 21.12 ± 3.66; F = 51.79, P < 0.05, q = 4.97, 10.08, 17.26, P < 0.05) (α-SMA protein expression: 0.13 ± 0.02, 0.26 ± 0.02, 0.29 ± 0.09, 0.47 ± 0.06, 0.68 ± 0.05; F = 45.14, q = 5.11, 10.67, 17.40, P < 0.05; Fn protein expression: 0.16 ± 0.04, 0.26 ± 0.07, 0.65 ± 0.03, 0.82 ± 0.04, 0.73 ± 0.02; F = 144.4, q = 20.09, 26.78, 23.12, P < 0.05; COL-1 protein expression: 0.11 ± 0.02, 0.23 ± 0.09, 0.41 ± 0.05, 0.61 ± 0.03, 0.74 ± 0.03; F = 75.47, q = 9.99, 16.60, 21.07, P < 0.05). Gremlin. siRNA transfection effectively suppressed TGF-ß2-induced expression of α-SMA, Fn, and COL-I (α-SMA gene induction: F = 81.89, P < 0.05, t = 3.234, 4.346, 10.35; t = 2.252, 7.272, 19.11, P < 0.05; Fn gene induction: F = 83.61, P < 0.05, t = 2.538, 8.202, 11.99; t = 6.316, 7.304, 14.80, P < 0.05; COL-1 gene induction: F = 73.64, P < 0.05, t = 3.385, 7.942, 11.64; t = 4.794, 9.006, 13.75, P < 0.05; Gremlin gene induction: F = 46.11, P < 0.05, t = 5.08, 7.24, 8.27; t = 6.27, 8.27, 12.14, P < 0.05) (α-SMA protein expression: F = 129.6, P < 0.05, t = 4.34, 4.85; 3.83, 4.34; 13.03, 14.82, P < 0.05; Fn protein expression: F = 26.18, P < 0.05, t = 5.68, 5.95; 3.10, 4.06; 4.19, 4.73, P < 0.05; COL-1 protein expression: F = 41.37, P < 0.05, t = 6.93, 5.51; 7.82, 6.93; 8.71, 7.64, P < 0.05; Gremlin protein expression: F = 59.52, P < 0.05, t = 2.24, 3.49; 5.74, 6.23; 6.98, 9.98, P < 0.05). CONCLUSIONS: Gremlin could induce HLEC to express α-SMA, Fn and COL-1 in a time-dependent manner and promote transdifferentiation and extracellular matrix synthesis. Specifically silencing the expression of Gremlin could effectively block the TGF-ß2-induced EMT and ECM synthesis in HLEC.

TGF-ß2 induces transdifferentiation and fibrosis in human lens epithelial cells via regulating gremlin and CTGF.

Transforming growth factor (TGF)-ß2, gremlin and connective tissue growth factor (CTGF) are known to play important roles in the induction of epithelial mesenchymal transition (EMT) and extracellular matrix (ECM) synthesis. However, the complex functional relationship among gremlin, CTGF and TGF-ß2 in the induction of EMT and ECM synthesis in human lens epithelial cells (HLECs) has not been reported. In this study, we found that TGF-ß2, CTGF and gremlin can individually induce the expression of α-smooth muscle actin (α-SMA), fibronectin (Fn), collagen type I (COL-I), Smad2 and Smad3 in HLECs. Blockade of CTGF and gremlin effectively inhibited TGF-ß2-induced expression of α-SMA, Fn, COL-I, Smad2, and Smad3 in HLECs. Furthermore blockade of Smad2 and Smad3 effectively inhibited CTGF and gremlin induced expression of α-SMA, Fn, COL-I in HLECs. In conclusion, TGF-ß2, CTGF and gremlin are all involved in EMT and ECM synthesis via activation of Smad signaling pathway in HLECs. Specifically silencing CTGF and gremlin can effectively block the TGF-ß2-induced EMT, ECM synthesis due to failure in activation of Smad signaling pathway in HLECs.

Mendelian randomization study shows no causal relationship between psychiatric disorders and glaucoma in European and East Asian populations.

Background: Glaucoma is a leading cause of blindness strongly associated with psychiatric disorders, but the causal association between glaucoma and psychiatric disorders remains uncertain because of the susceptibility of observational studies to confounding and reverse causation. This study aims to explore the potential causal association between glaucoma and three highly related psychiatric disorders (Depression, Insomnia, and Schizophrenia) in the European and East Asian populations using a two-sample Mendelian randomization analysis. Methods: Instrumental variables (IVs) of depression, insomnia, and schizophrenia in the European population were obtained after strict filtering. Summary-level data for glaucoma and glaucoma subtypes (primary open-angle glaucoma and primary closed-angle glaucoma) were obtained as outcomes. The inverse variance weighting (IVW) method was used as the primary method. Additionally, the causal effect was evaluated in the East Asian population using the same methods to validate analysis results. The robustness of these results was confirmed using heterogeneity, pleiotropy, and Steiger directionality test. Results: The primary MR results indicated that genetically driven psychiatric disorders were not causally associated with glaucoma (Depression: odds ratio (OR): 1.15, 95% confidence interval (CI): 0.93-1.42, p = 0.20; Insomnia: OR: 1.14, 95% CI: 0.63-2.05, p = 0.66; Schizophrenia: OR: 1.00, 95% CI: 0.93-1.08, p = 0.95), either with the risk of glaucoma subtypes in the European population. Meanwhile, results in the East Asian population were consistent with the results among the European population (Depression: OR = 1.38, CI 0.75-2.53, p = 0.30; Insomnia: OR = 0.99, CI 0.83-1.18, p = 0.93; Schizophrenia: OR = 1.06, CI 0.94-1.20, p = 0.34) with similar causal estimates in direction. Consistency was obtained by corroborating with other supporting methods. Besides, the robustness of the results was proved and the directionality test confirmed our estimation of potential causal direction (p < 0.001). Conclusion: This study found a non-causal association between psychiatric disorders and the risk of glaucoma in the European and East Asian populations, which contradicts many existing observational reports, indicating that increased psychiatric disorders in glaucoma patients were more likely modifiable rather not inheritable.

Septicemia caused by Leifsonia aquatica in a healthy patient after retinal reattachment surgery.

Leifsonia aquatica is an aquatic bacterium that is typically found in environmental water habitats. Infections due to L. aquatica are rare and commonly catheter associated in immunocompromised patients. We report the first case of an acute septicemia caused by L. aquatica in a healthy immunocompetent host after cryopexy in the absence of a catheter.

[Effects of transforming growth factor-ß2 on transdifferentiation, extracellular matrix synthesis and connective tissue growth factor expression of human lens epithelium cells].

OBJECTIVE: To explore the effects of transforming growth factor-ß2 (TGF-ß2) on the transdifferentiation, extracellular matrix synthesis and connective tissue growth factor (CTGF) expression in human lens epithelium cells (HLEC) in vitro. METHODS: HLEC were incubated with different concentrations of TGF-ß2 (0.0, 0.1, 1.0 and 10.0 µg/L) for 24 h in vitro. The morphological changes of HLEC were observed under inverted phase-contrast microscope. The expression of CTGF and α-smooth muscle actin (α-SMA, as a landmark protein of epithelial mesenchymal transition) in HLEC was measured by immunofluorescence method. Real-time PCR and Western blot were used to evaluate the expression of CTGF, α-SMA, fibronectin (Fn) and COL-I (as the major components of extracellular matrix) after stimulating by TGF-ß2. RESULTS: Normal HLEC presented polygonal shape and were anchorage-dependent. After incubated with different concentrations of TGF-ß2 for 24 h, the morphology of polygonal HLEC was changed into fibroblast-like shape and changed from monolayer and to multilayer cells, and the intercellular space became bigger. CTGF and α-SMA were expressed in the cytoplasm after induction of TGF-ß2. Expression of CTGF in HLEC was increased with increasing concentrations of TGF-ß2 (CTGF protein expression: 0.53 ± 0.03, 0.73 ± 0.01, 0.65 ± 0.03 in cells cultured with 0.1, 1.0 and 10 µg/L TGF-ß2, respectively; CTGF gene induction: 1.00 ± 0.00, 7.18 ± 0.41, 12.88 ± 0.45, 32.84 ± 1.61 in cells cultured with 0.0, 0.1, 1.0 and 10.0 µg/L TGF-ß2, respectively) (F = 77.55, P < 0.05; F = 379.0, P < 0.05). TGF-ß2 could induce HLEC transdifferentiation and accelerate. α-SMA expression was increased by TGF-ß2 dose-dependently (protein expression: 0.48 ± 0.01,0.78 ± 0.04, 0.69 ± 0.04; gene induction: 1.00 ± 0.00, 2.30 ± 0.22, 3.1 ± 0.21, 3.86 ± 0.10) (F = 62.73, P < 0.05; F = 80.22, P < 0.05). TGF-ß2 also promoted expression of Fn and COL-I in a dose-dependent manner (COL-I gene induction: 1.00 ± 0.00, 5.52 ± 0.96, 18.31 ± 1.2, 82.51 ± 1.45;COL-I protein expression: 0.78 ± 0.05, 1.15 ± 0.11, 2.16 ± 0.14; Fn gene induction: 1.00 ± 0.00, 2.36 ± 0.25, 3.27 ± 0.24, 4.25 ± 0.24; Fn protein expression: 0.64 ± 0.01,0.95 ± 0.02, 1.23 ± 0.14) (F = 1881.52, 105.30, P < 0.05; F = 64.44, 51.81, P < 0.05). CONCLUSION: TGF-ß2 induces the expression of CTGF by HLEC, promotes transdifferentiation of and extracellular matrix synthesis by HLEC.

Clinical research of EX-PRESS drainage device and modified trabeculectomy combined with intravitreal conbercept treatment for neovascular glaucoma.

AIM: To evaluate the efficacy and safety of modified trabeculectomy (experimental group) and implantation of EX-PRESS drainage device (control group), combined with intravitreal conbercept injection for neovascular glaucoma (NVG). METHODS: Totally 30 patients with NVG were selected from June 2014 to June 2017, and randomly divided into experimental group and control group. All patients were underwent intravitreal conbercept (0.5 mg/0.05 mL) treatment before surgery. Modified trabeculectomy was performed in MT group, while EX-PRESS drainage device implantation was performed in EX group. The success rates, best corrected visual acuity (BCVA), intraocular pressure (IOP), filtering bleb and complications were observed and compared. RESULTS: The differences of success rate, BCVA and filtering bleb were not statistically significant 12mo after the surgery (P>0.05), however, the difference of IOP at 1d, 1wk, 1, 3, and 6mo after surgery was statistically significant (F time=390.64, P time<0.0001) between two groups. The interactions between two groups in the given time showed no significant difference (F intergroup×time=0.181, P intergroup×time=0.57), and also there was no significant difference in IOP between the two groups (F=3.16, P=0.09). The results of pairwise comparison at each time point showed no significant difference in IOP between 1d and 1wk, 3 and 6, 3mo and 12mo after surgery (P>0.05), while the results at other time point indicate statistical differences (P<0.05). CONCLUSION: The modified trabeculectomy and the implantation of EX-PRESS drainage device have clinical application value in reducing IOP and postoperative complications of refractory NVG.

Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri-infarct angiogenesis after focal cerebral ischemia.

Neurogenesis and angiogenesis are two important processes that may contribute to the repair of brain injury after stroke. This study was designed to investigate whether transplantation of human embryonic neural stem cells (NSCs) into cortical peri-infarction 24h after ischemia effects cell proliferation in the subventricular zone (SVZ) and angiogenesis in the peri-infarct zone. NSCs were prepared from embryonic human brains at 8 weeks gestation. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. Animals were randomly divided into two groups (n=30, each) at 24h after ischemia: NSC-grafted and medium-grafted groups. Toluidine blue staining and 5'-bromo-2'-deoxyuridine (BrdU) or von Willebrand factor (vWF) immunohistochemistry were performed at 7, 14 and 28 days after transplantation. NSC transplantation increased the number of BrdU-positive cells in the ischemic ipsilateral SVZ compared with the medium control at 7 days (P<0.01). This difference in SVZ cell proliferation persisted at 14 days (P<0.01), but was not significant at 28 days (P>0.05). In addition, angiogenesis, as indicated by BrdU and vWF staining in cortical peri-infarct regions, was augmented by 46% and 65% in NSC-grafted rats versus medium-grafted rats at 7 and 14 days, respectively (P<0.05). However, this increase became non-significant at 28 days (P>0.05). Our results indicate that NSC transplantation enhances endogenous cell proliferation in the SVZ and promotes angiogenesis in the peri-infarct zone, even if it is performed in the acute phase of ischemic injury.

LncRNA AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cell through regulating microRNA-1199-5p/HSPA5 axis.

AIMS: Dysregulated long non-coding RNA (lncRNA) expression is closely related to neuroinflammation, leading to multiple neurodegenerative diseases. In this study, we investigated the function and regulation of lncRNA AK148321 in neuroinflammation using an in vitro lipopolysaccharide (LPS)-stimulated BV2 microglial cell system. METHODS: Expression of AK148321 was analyzed by qPCR. Inflammatory cytokine expression levels were determined by ELISA assay. The interaction between AK148321, microRNA (miRNA), and its target gene was validated by luciferase reporter assay and RNA immunoprecipitation (RIP). Cell apoptosis was analyzed by Annexin V/PI staining. RESULTS: LPS treatment suppressed AK148321 expression in BV2 cells. Overexpression of AK148321 inhibited LPS-induced BV2 microglial cell activation and decreased the expression of inflammatory cytokine TNF-α and IL-1ß. AK148321 function as a competing endogenous RNA (ceRNA) by sponging microRNA-1199-5p (MiR-1199-5p). In LPS-stimulated BV2 cells, AK148321 exerted its inhibitory function via negatively modulating miR-1199-5p expression. Moreover, we identified that Heat Shock Protein Family A Member 5 (HSPA5) was a direct target of miR-1199-5p. RIP assay using the anti-Ago2 antibody further validated the relationship among AK148321, miR-1199-5p and HSPA5. The AK148321/miR-1199-5p/HSPA5 axis regulated the neuroinflammation in LPS-induced BV2 microglial cells. Microglial cell culture supernatant from LPS-stimulated, AK148321-overexpressing BV2 cells suppressed the cell apoptosis of mouse hippocampal neuronal cell HT22, while HSPA5 knockdown abrogated the suppression effect. CONCLUSION: Our findings suggest that AK148321 alleviates neuroinflammation in LPS-stimulated BV2 microglial cells through miR-1199-5p/HSPA5 axis.

A nano-predator of pathological MDMX construct by clearable supramolecular gold(I)-thiol-peptide complexes achieves safe and potent anti-tumor activity.

As alternatives to small-molecular proteolysis-targeting chimeras (PROTAC), peptide-based molecular glues (MG) are a broad range of dual-functional ligands that simultaneously bind with targetable proteins and E3 ligases by mimicking proteinprotein interaction (PPI) partners. Methods: Herein, we design a peptide-derived MG to target a tumor-driving protein, MDMX, for degradation, and nanoengineered it into a supramolecular gold(I)-thiol-peptide complex (Nano-MP) to implement the proteolysis recalcitrance, cellular internalization, and glutathione-triggered release. To optimize the tumor targeting, a pH-responsive macromolecule termed polyacryl sulfydryl imidazole (PSI) was synthesized to coat Nano-MP. Results: As expected, Nano-MP@PSI induced the MDMX degradation by ubiquitination and subsequently restored the anti-cancer function of p53 and p73. Nano-MP@PSI revealed potent anti-cancer activities in an orthotopic xenograft mouse model of retinoblastoma by intraocular injection and a patient-derived xenograft model of malignant pancreatic cancer by systemic injection, while maintaining a favorable safety profile and showing a highly favorable clearable profile of excretion from the living body. Conclusion: Collectively, this work not only provided a clinically viable paradigm for the treatment of a wide variety of tumors by multiple administration types, but, more importantly, it bridged the chasm between peptides and PROTACs, and likely reinvigorated the development of peptide-derived proteolysis-targeting chimeras for a great variety of diseases.

Upregulation of heme oxygenase-1 by Brahma-related gene 1 through Nrf2 signaling confers protective effect against high glucose-induced oxidative damage of retinal ganglion cells.

High glucose (HG)-induced oxidative damage of retinal ganglion cells (RGCs) contributes to the pathogenesis of diabetic retinopathy, a severe complication of diabetes mellitus. Brahma-related gene 1 (Brg1) has currently emerged as a cytoprotective protein that alleviates oxidative damage induced by various stress. However, whether Brg1 is involved in the regulation of HG-induced oxidative damage of RGCs remains unknown. In this study, we aimed to investigate the potential role and underlying mechanism of Brg1 in regulating HG-induced damage of RGCs. We found that Brg1 expression was significantly downregulated in RGCs in response to HG treatment. Functional experiments showed that Brg1 knockdown enhanced HG-induced apoptosis and production of reactive oxygen species, while Brg1 overexpression suppressed HG-induced apoptosis and reactive oxygen species production, showing a protective effect. Moreover, Brg1 overexpression resulted in an increase in nuclear expression of nuclear factor-erythroid-2-related factor-2 (Nrf2) and the expression of heme oxygenase-1 (HO-1) in RGCs. Notably, inhibition of Nrf2 or HO-1 significantly blocked Brg1-mediated protection against HG-induced damage. Overall, these findings demonstrate that Brg1 protects RGCs from HG-induced oxidative damage through promotion of Nrf2/HO-1 signaling, indicating a potential role of Brg1 in the pathogenesis of diabetic retinopathy.

Transplanted human embryonic neural stem cells survive, migrate, differentiate and increase endogenous nestin expression in adult rat cortical peri-infarction zone.

Transplantation of stem cells is a potential therapeutic strategy for stroke damage. The survival, migration, and differentiation of transplanted human embryonic neural stem cells in the acute post-ischemic environment were characterized and endogenous nestin expression after transplantation was investigated. Human embryonic neural stem cells obtained from the temporal lobe cortex were cultured and labeled with fluorescent 1,1'-dioctadecy-6,6'-di (4-sulfopheyl)-3,3,3',3'-tetramethylindocarbocyanin (DiI) in vitro. Labeled cells were transplanted into cortical peri-infarction zones of adult rats 24 h after permanent middle cerebral artery occlusion. Survival, migration, and differentiation of grafted cells were quantified in immunofluorescence-stained sections from rats sacrificed at 7, 14, and 28 days after transplantation. Endogenous nestin-positive cells in the cortical peri-infarction zone were counted at serial time points. The cells transplanted into the cortical peri-infarction zone displayed the morphology of living cells and became widely located around the ischemic area. Moreover, some of the transplanted cells expressed nestin, GFAP, or NeuN in the peri-infarction zone. Furthermore, compared with the control group, endogenous nestin-positive cells in the peri-infarction zone had increased significantly 7 days after cell transplantation. These results confirm the survival, migration, and differentiation of transplanted cells in the acute post-ischemic environment and enhanced endogenous nestin expression within a brief time window. These findings indicate that transplantation of neural stem cells into the peri-infarction zone may be performed as early as 24 h after ischemia.

SIRT6 protects retinal ganglion cells against hydrogen peroxide-induced apoptosis and oxidative stress by promoting Nrf2/ARE signaling via inhibition of Bach1.

Oxidative stress-induced damage of retinal ganglion cells (RGCs) is a major contributor to retinal degenerative diseases, such as glaucoma. Sirtuin 6 (SIRT6) has emerged as a cytoprotective protein against various insults. However, whether SIRT6 exerts a protective effect against oxidative stress-damaged RGCs remains unknown. In this study, we aimed to investigate the potential role and regulatory mechanism of SIRT6 in hydrogen peroxide (H2O2)-induced oxidative damage of RGCs in vitro. We found that SIRT6 expression was significantly downregulated in RGCs with H2O2 treatment. Functional experiments showed that overexpression of SIRT6 improved survival and reduced apoptosis and the production of reactive oxygen species (ROS) in H2O2-treated RGCs. In contrast, SIRT6 knockdown had the opposite effect. Moreover, we found that SIRT6 overexpression promoted the nuclear accumulation of nuclear factor erythroid 2-related factor 2 (Nrf2) and increased the activity of antioxidant response element (ARE). In addition, we found that the promotional effect of SIRT6 on Nrf2/ARE signaling was associated with inhibition of BTB and CNC homology 1 (Bach1), an inhibitor of Nrf2. However, overexpression of Bach1 or inhibition of Nrf2/ARE signaling partially reversed the SIRT6-mediated protective effect. Taken together, these results demonstrate that SIRT6 protects RGCs from oxidative stress-induced damage by promoting the activation of Nrf2/ARE signaling via inhibition of Bach1, suggesting a potential role of SIRT6 in retinal degenerative diseases.

Down-regulation of microRNA-27b promotes retinal pigment epithelial cell proliferation and migration by targeting Nox2.

Aberrant proliferation and migration of retinal pigment epithelium (RPE) cells contributes to the pathology of various ocular diseases. miR-27b has been reported to be crucial in the regulation of cell differentiation, proliferation, apoptosis, and migration. However, the role of miR-27b on RPE proliferation and migration remains largely unknown. Here the effect of miR-27b on ARPE-19 cells under platelet-derived growth factor (PDGF)-BB stimulation was explored. In this study, we found that the expression level of miR-27b was significantly reduced in ARPE-19 cells under PDGF-BB stimulation. Ectopic expression of miR-27b remarkably inhibited PDGF-BB-induced proliferation and migration in ARPE-19 cells. Furthermore, bioinformatic analysis and luciferase reporter assay showed that NADPH oxidase 2 (Nox2) was a direct target for miR-27b, and that knockdown of Nox2 expression mimicked the inhibitory effect of miR-27b on PDGF-BB -induced proliferation and migration in ARPE-19 cells, whereas, restoration of Nox2 expression showed an opposite effect. In addition, the ROS production and the activation of P13K/AKT/mTOR signaling induced by PDGF-BB were also suppressed by miR-27b overexpression or Nox2 silencing. Thus, these findings indicated that miR-27b exerted its protective role in RPE cells under PDGF-BB stimulation was partially through regulation of Nox2 and its downstream P13K/AKT/mTOR signaling, which might be a potential therapeutic approach for treatment of diseases caused by RPE proliferation, and migration.