Ergosta-7,9(11),22-trien-3ß-ol Alleviates Intracerebral Hemorrhage-Induced Brain Injury and BV-2 Microglial Activation.

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-3ß-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.

A drug-tunable Flt23k gene therapy for controlled intervention in retinal 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.

The fungus-derived retinoprotectant theissenolactone C improves glaucoma-like injury mediated by MMP-9 inhibition.

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-1ß (IL-1ß) 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-1ß-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-1ß 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-1ß 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-1ß-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-1ß-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.

The natural retinoprotectant chrysophanol attenuated photoreceptor cell apoptosis in an N-methyl-N-nitrosourea-induced mouse model of retinal degenaration.

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.