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.

Theissenolactone C Exhibited Ocular Protection of Endotoxin-Induced Uveitis by Attenuating Ocular Inflammatory Responses and Glial Activation.

The aim of this study was to investigate the effects of a natural component, theissenolactone C (LC53), on the ocular inflammation of experimental endotoxin-induced uveitis (EIU) and its related mechanisms in microglia. Evaluation of the severity of anterior uveitis indicated that LC53 treatment significantly decreased iridal hyperemia and restored the clinical scores. Additionally, the deficient retina functions of electroretinography were improved by LC53. LC53 significantly reduced levels of tumor necrosis factor (TNF)-α, monocyte chemoattractant protein-1, protein leakage and activation of matrix metalloproteinases in the anterior section during EIU. Moreover, LC53 treatment decreased the oxidative stress as well as neuroinflammatory reactivities of GFAP and Iba-1 in the posterior section. Furthermore, LC53 decreased the phosphorylation of p65, expression of HSP90, Bax, and cleaved-caspase-3 in EIU. According to the microglia studies, LC53 significantly abrogated the productions of TNF-α, PGE2, NO and ROS, as well as inducible NO synthase and cyclooxygenase-2 expression in LPS-stimulated microglial BV2 cells. The microglial activation of IKKß, p65 phosphorylation and nuclear phosphorylated p65 translocation were strongly attenuated by LC53. On the other hand, LC53 exhibited the inhibitory effects on JNK and ERK MAPKs activation. Our findings indicated that LC53 exerted the ocular-protective effect through its inhibition on neuroinflammation, glial activation, and apoptosis in EIU, suggesting a therapeutic potential with down-regulation of the NF-κB signaling for uveitis and retinal inflammatory diseases.

Anti-inflammatory properties of shikonin contribute to improved early-stage diabetic retinopathy.

Diabetic retinopathy (DR), a major microvascular complication of diabetes, leads to retinal vascular leakage, neuronal dysfunction, and apoptosis within the retina. In this study, we combined STZ with whole-body hypoxia (10% O2) for quicker induction of early-stage retinopathy in C57BL/6 mice. We also compared the effects of a high glucose condition combined with hypoxia (1% O2) to a low glucose condition by using retinal pigment epithelial (RPE) cells, which are a crucial component of the outer blood-retinal barrier and the damage is related to retinopathy. In the retina of DM/hypoxic C57BL/6 mice, abnormal a-wave and b-wave activity, yellowish-white spots, hyperfluorescence, and reduced retinal thickness were found using electroretinography (ERG), fundus photography (FP), fundus fluorescein angiography (FFA), and optical coherence tomography (OCT). Shikonin dose-dependently (0.5-50 mg/kg, per os) prevented DM/hypoxia-induced lesions. In eye tissue, administration of shikonin also attenuated DM/hypoxia-induced pre-apoptotic protein BAX expression as well as the production of inflammatory proteins cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). We also demonstrated that shikonin administration rescues high glucose/hypoxia (1% O2)-induced inflammation, decreased junction protein expression, and permeability in RPE cells. These results indicate that shikonin treatment may prevent the loss of vision associated with DR.

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.

Effect of Guanabenz on Rat AMD Models and Rabbit Choroidal Blood Flow.

AIM: The effects of Guanabenz, an agonist of α2-adrenergic receptors routinely used in human medicine as an antihypertensive drug, were studied on NaIO(3)-induced retinal pigment epithelium (RPE) degeneration, laser-induced choroidal neovascularization (CNV) and choroidal blood flow, in animal models. METHODS: The 35mg/kg NaIO(3)-induced RPE degeneration rat eyes were instilled with 1% Guanabenz eye drops 3 times a day for 7 days before NaIO(3) injection, and then 2 to 4 weeks thereafter. RPE function was measured with c-wave of electroretinogram (ERG). Male Brown Norway rats were anesthetized to receive Nd:YAG laser to break the Bruch's membrane. One percent Guanabenz eye drops were given likewise. The development of CNV was determined by fluorescein angiography performed on week 2 and week 4 using sodium fluorescein (FA) or fluorescein isothiocyanatedextran (FD70-FA). Colored microsphere technique was used for in vivo experiments to determine the choroidal blood flow in ocular hypertensive (40 mmHg) rabbit eyes. RESULTS: The RPE function was protected significantly by Guanabenz according to the c-wave of ERG. Four weeks after NaIO(3) injection, the amplitude of ERG c-wave was 0.422±0.092 millivolts in the control group, 0.103±0.04 millivolts in the NaIO(3) group, and 0.254±0.061 millivolts in the Guanabenz+NaIO(3) group. There was a significant protection of the ERG c-wave by Guanabenz as compared to NaIO(3) group (P<0.01). The angiograms of FD70-FA showed decreased lesion size in the Guanabenz group. Four weeks after laser treatment, the size of the CNV lesion was 2.99±0.18 mm(2) in the control group, and 1.24±0.16 mm(2) in the Guanabenz group (P<0.01). The choroidal blood flow was significantly increased at 30 and 60 minutes after Guanabenz instillation as compared to corresponding controls. CONCLUSIONS: Guanabenz significantly protected RPE from NaIO(3)-induced degeneration, inhibited the development of CNV in laser-induced rat AMD model and increased choroidal blood flow markedly in vivo.

Effect of quercetin on formation of choroidal neovascularization (CNV) in age-related macular degeneration(AMD).

PURPOSE: Age-related macular degeneration (AMD) as a disease entity is "dry" at early stage and made up of two main components at late stage: atrophic AMD and exudative AMD. Quercetin acts as an anti-oxidant to protect retinal pigment epithelial cells (RPE) from damaged by oxidative stress, but its effect on formation of choroidal neovascularization (CNV) in AMD is unclear. The aim of this study is to investigate the effect of quercetin on the formation of CNV in AMD. METHODS: The development of CNV induced by laser was detected by fluorescein angiography (FA). Colored microsphere technique was used to determine the choroidal blood flow in ocular hypertensive rabbit eyes. In in vitro studies, HUVECs were treated with NaIO3, H2O2 and NaN3 to induce oxidative cell damages. The effect of quercetin on various oxidations-induced injuries in HUVECs was measured by MTT assay. HUVECs migration was assessed using a wound healing assay. RESULTS: Quercetin significantly inhibited the formation of laser-induced CNV. The choroidal blood flow in rabbit eyes was significantly increased after quercetin instillation. In vitro results showed quercetin enhanced various oxidations-induced injuries in HUVECs and inhibited migration of HUVECs during wound healing. CONCLUSION: Quercetin inhibited the formation of CNV both in vivo and in vitro and increased choroidal blood flow. It could become a promising candidate for the treatment of AMD.

Effect of naringenin on NaIO(3)-induced retinal pigment epithelium degeneration and laser-induced choroidal neovascularization in rats.

AIM: To study the effects of naringenin eye drops on NaIO(3)-induced retinal pigment epithelium (RPE) degeneration and laser-induced choroidal neovascularization (CNV) in rat eyes. METHODS: The 35mg/kg NaIO(3)-induced RPE degeneration was prevented by 10g/L naringenin eye drops 3 times a day for 7 days in advance of NaIO(3) injection, and then 2 to 4 weeks thereafter, RPE function was measured with C-wave of electroretinogram (ERG). The laser-induced CNV rats were treated with laser to break the Bruch's membrane and the CNV formation was prevented by 10g/L naringenin eye drops instilled 3 times a day for 2 to 4 weeks. The CNV formation was measured with fluorescein angiography (FA) and flat mount. RESULTS: Two weeks after NaIO(3) injection, the amplitude of ERG C-wave fell markedly in NaIO(3) group to 53% of normal group (P<0.01). No apparent difference was observed in naringenin+NaIO(3) group. Four weeks later, the NaIO(3) group fell to 37% of normal group (P<0.01), while the naringenin+ NaIO(3) group fell to only 57% of normal group (P<0.01). There was a 52% reversal of the ERG C-wave by naringenin as compared to NaIO(3) treated group (P<0.05). Two weeks and four weeks after laser treatment, naringenin reduced the CNV formation to 53% and 49% of control group (100%) measured by FA (P<0.01). Four weeks after laser treatment, naringenin reduced the CNV formation by 47% as compared to control group measured with flat mount (P<0.01). CONCLUSION: Naringenin can significantly protect RPE from NaIO(3) induced degeneration and also prevent CNV formation.

Proliferation of retinal pigment epithelial cells induced by (R,R)-XY-10 and (S,S)-XY-10 and their action mechanisms.

AIM: To investigate the mechanism of proliferation effect induced by (R,R)-XY-10 and (S,S)-XY-10 on retinal pigmented epithelial cells (ARPE-19). METHODS: Human retinal pigmented epithelial cells (ARPE-19) and human umbilical vein endothelial cells (HUVECs) were used to investigate the effect of (R,R)-XY-10 and (S,S)-XY-10 on cell growth, and their mechanisms of proliferative action by using ERK, AKT, PI3K, Protein kinase C (PKC) and Nitric oxide synthase (NOS) inhibitors. RESULTS: (R,R)-XY-10 and (S,S)-XY-10 dose-dependently increased ARPE-19 cell proliferation, but not on HUVECs. When treated with proliferative inhibitors, H-7 (5µmol/L), hypericin (20µmol/L), PD98059 (2µmol/L), LY294002 (50µmol/L), SH-5 (10µmol/L) and L-NAME (100µmol/L), the proliferative effect was reduced by H-7, hypericin, PD98059 and LY294002, but not by SH-5 and L-NAME. CONCLUSION: (R,R)-XY-10 and (S,S)-XY-10 can induce cell proliferation through MAPK and PI3K dependent pathway.

Effect of flavone on the ocular blood flow and formation of choroidal neovascularization.

AIM: To investigate the effect of flavone on ocular blood flow in rabbit eyes and the formation of choroidal neovascularization (CNV) in rat model of age-related macular degeneration (AMD). METHODS: In in vivo studies, colored microsphere technique was used to determine the ocular blood flow in ocular hypertensive rabbit eyes. The rat eyes were treated with 0.5% flavone eye drops 3 times a day for 1 week before and 4 weeks after laser-induced injury of Bruch's membrane. The development of CNV was determined by fluorescein angiography (FA) performed on the 2nd and 4th after injury. In in vitro studies, the effect of flavone on the viability of human umbilical vein endothelial cells HUVECs was measured by MTT assay. RESULTS: The ocular blood flow in rabbit eyes was significantly increased after flavone instillation. Flavone significantly inhibited the formation of laser induced CNV. In vitro results showed that flavone inhibited the proliferation of HUVECs. CONCLUSION: Flavone could increase ocular blood flow and inhibit the formation of CNV.

Effects of flavone on the oxidation-induced injury of retinal pigment epithelium cells.

AIM: To investigate the effect of flavone on oxidation-induced injury in retinal pigment epithelium cells. METHODS: In in vivo studies, NaIO(3)-induced RPE degeneration in rat eyes was treated with 0.5% flavone eye drops 3 times a day for 1 week before and 4 weeks after NaIO(3) injection. At the end of 2 and 4 weeks, all rats were measured c-wave by electroretinogram (ERG). In in vitro studies, ARPE-19 cells were treated with hypoxia, H(2)O(2), NaN(3) and t-BHP to induce cell damages. MTT assay was used to measure the viable cells. RESULTS: The ERG c-wave results showed that flavone reversed NaIO(3)-induced injury at the end of 4 weeks. In vitro results showed flavone reversed the various oxidants-induced injuries in RPE cells. CONCLUSION: Flavone could prevent the RPE from oxidation-induced injury both in vivo and in vitro.

Effect of tetramethylpyrazine on rat experimental choroidal neovascularization in vivo and endothelial cell cultures in vitro.

PURPOSE: To evaluate the effect of tetramethylpyrazine (TMP) on laser-induced experimental choroidal neovascularization (CNV) in rat model in vivo and on endothelial cell proliferation in vitro. METHODS: Male Brown Norway rats were anesthetized to receive Nd:YAG laser to break the Bruch membrane. TMP was given once daily through intraperitoneal injection after laser treatment for 4 weeks. The development of CNV was determined by angiography performed on week 2 and week 4 using sodium fluorescein (FA) or fluorescein isothiocyanate-dextran (FD70-FA). Human umbilical vein endothelial cells (HUVECs) were tested with proliferation assay with TMP at different concentrations. RESULTS: According to the angiograms of FA, intensity of fluorescein leakage from the photocoagulated lesions decreased significantly after TMP treatment. The number of rats with less leaky points (

Effect of interleukin-1 blockers, CK112, and CK116 on rat experimental choroidal neovascularization in vivo and endothelial cell cultures in vitro.

PURPOSE: The aim of this study was to investigate the effect of interleukin-1 blockers, CK112 and CK116, on laser-induced experimental choroidal neovascularization (CNV) in rat models in vivo and endothelial cell proliferation in vitro. METHODS: Male Brown Norway rats were anesthetized to receive Nd:YAG laser to break the Bruch's membrane. CK112, CK116, and prednisolone were given once-daily through intraperitoneal (i.p.) injection after laser treatment for 4 weeks. The development of CNV was determined by fluorescein angiography performed on weeks 2 and 4. Human umbilical vein endothelial cells (HUVEC) were tested with proliferation assay with CK112, CK116, and prednisolone at different concentrations. RESULTS: The intensity of fluorescein leakage from the photocoagulated lesions decreased significantly, compared to the control group (treated with dimethyl sulfoxide [DMSO] only), following CK112, CK116, and prednisolone treatment. Four (4) weeks after administration, CK112, at 10 mg/kg and 30 mg/kg, inhibited CNV development to 75% and 77% of the control group, respectively (P < 0.01). Both CK116, 10 mg/kg, and prednisolone, 5 mg/kg, inhibited the CNV development to 85% of the control group (P < 0.05). All three compounds interfered with the endothelial cell proliferation significantly. The reduction of the endothelial cells was 50.5% (P < 0.01), 28.5% (P < 0.05), and 23.1% (P < 0.05), respectively, in 500 microg/mL, 300 microg/mL, and 100 microg/mL of the CK112-treated group. CK116 inhibited the cell proliferation significantly to 77.2% of the control group at 500 microg/mL (P < 0.05). CONCLUSIONS: CK112 and CK116 inhibited the development of CNV in the rat model and interfered with vascular endothelial cell proliferation in vitro. Our results suggest that CK112 and CK116 may be good candidates to inhibit ocular neovascularization related to age-related macular degeneration (ARMD).

Apigenin inhibits laser-induced choroidal neovascularization and regulates endothelial cell function.

PURPOSE: The aim of this study is to evaluate the effect of apigenin on laser-induced experimental choroidal neovascularization (CNV) rat model and endothelial cell proliferation and migration. METHODS: Male Brown Norway rats were anesthetized to receive Nd:YAG laser to break the Bruch's membrane. Apigenin at 5, 15, or 30 mg/kg was given once-daily through intraperitoneal injection after laser treatment for 4 weeks. The development of CNV was determined by fluorescein angiography performed on weeks 2 and 4. Endothelial cell function was evaluated with proliferation assay and migration assay. RESULTS: The intensity of fluorescein leakage from the photocoagulated lesions decreased significantly, compared to the control group, following apigenin treatment. Four (4) weeks after administration, apigenin, at 15 and 30 mg/kg, inhibited CNV development to 84.5% and 83.6% of the control group, respectively (P<0.05). Apigenin also interfered with the endothelial cells' proliferation and migration. At 3 and 10 microg/ml, apigenin inhibited the growth of human umbilical vein endothelial cells (HUVEC) to 54% and 46% of the control group, respectively (P<0.01); inhibited the growth of choroidal endothelial cells to 47% and 8% of the control group, respectively (P<0.01); and inhibited HUVEC migration over 50%, compared with the control (P<0.01). CONCLUSIONS: Apigenin exerts an inhibitory effect on choroidal angiogenesis in vitro and in vivo. It should be further evaluated for its potential as a novel therapy for CNV.

Effects of dihydrogenation of flavones and number of hydroxy groups in the molecules on ocular blood flow in rabbits and retinal function recovery in rats.

PURPOSE: It has been found that the number of hydroxy groups in the molecule of flavones and flavanones affect the ocular blood flow significantly. However, the effects of dihydrogenation of flavones into flavanones on ocular blood flow and retinal function recovery have not been studied and required investigation. METHODS: The colored microsphere technique and electroretinography method were used for the study of ocular blood flow and retinal function, respectively. RESULTS: Maximum effects on ocular blood flow were obtained when there were 3 hydroxy groups in the molecule of flavones and flavanones. Dihydrogenation of flavones to flavanones increased the ocular blood flow further. The same is true for retinal function recovery after ischemic insult. CONCLUSION: These results indicate that hydrogenation is an excellent way to convert natural flavones into more potent compounds of flavanones.

Structural requirements of flavonoids for increment of ocular blood flow in the rabbit and retinal function recovery in rat eyes.

PURPOSE: We have recently reported that the effect of a flavonoid on ocular blood flow depends upon the number of hydroxy (OH) groups in its backbone structure. To elucidate the structural features on the number and type of functional groups present in the flavonoid molecule plus the number of OH groups, flavonoids with four to five OH groups, with or without methoxy groups, were studied on their effects to affect the ocular blood flow and the retinal function recovery. METHODS: A colored microsphere technique was used to determine the ocular blood flow in albino rabbit eyes and electroretinography was used to measure the retinal function recovery. RESULTS: Flavonols with four free OH groups produced no effects on the ocular blood flow (fisetin, kaempferol), whereas flavanone and flavones with four free OH groups and without the C2-C3 double bond produced the rapid increment on ocular blood flow (dihydrofisetin and luteolin, respectively). Similarly, flavonols with five free OH groups produced no effects on the ocular blood flow (morin, quercetin). Yet, flavanone with five free OH groups and without the C2-C3 double bond produced the rapid increment on ocular blood flow (dihydroquercetin). Flavanols with five free OH groups and without the C2-C3 double bond and the carbonyl group produced no effects on the ocular blood flow (catechin). Flavonols with four free OH groups and a methoxy group on the 7 position produced no effects on the ocular blood flow (Rhamnetin). Flavonols with four free OH groups and a methoxy group at the 5 (5-methylquercetin) or 3' position (isorhamnetin) produced positive effects on the ocular blood flow also. Flavonol with five methoxy groups but no OH group produced positive effects on the ocular blood flow (pentamethylquercetin). Flavonols with an excessive number of OH groups, having both a catechol-like structure in the C ring and a catechol at the B ring, produced no effect on the ocular blood flow (rhamnetin, quercetin). Parallel results were obtained on retinal function recovery after ischemic insult. CONCLUSION: The presence of OH groups at certain positions and the double bond at C2-C3 in the flavonoid molecules, which produces lipophilic action, can affect the increment on ocular blood flow and retinal function recovery. O-methylation can increase ocular blood flow and retinal function recovery as well.

Effects of some natural flavonoids on retinal function recovery after ischemic insult in the rat.

PURPOSE: There are six natural flavonoids studied recently and their effects on ocular blood flow measured with colored microsphere technique. It was found that three out of six compounds showed strong positive effects in increasing the ocular blood flow. In this study, we tried to find out whether these results can be translated on their effects to improve retinal function recovery after ischemic insult. METHODS: Electroretinography was used to measure the b-wave recovery as an indication of retinal function recovery. RESULTS: Naringenin, hesperetin, and rutin were found to produce marked positive effects on b-wave recovery, whereas naringin, hesperidin, and quercetin showed poor recovery of b-wave after ischemic insult of the retina. CONCLUSION: It was found that the compounds that showed strong increase of ocular blood flow also showed marked increase of retinal function recovery, whereas those that showed poor increase of ocular blood flow also showed poor effects on the retinal function recovery.

Structure-activity relationship (SAR) between some natural flavonoids and ocular blood flow in the rabbit.

PURPOSE: Flavonoids with two to five hydroxy groups, with or without sugar, and/or methoxy groups were studied on their effects to affect ocular blood flow. METHODS: Colored microsphere technique was used to determine the ocular blood flow in rabbit eyes. RESULTS: Flavonoids with three free hydroxy (OH) groups seemed to produce the optimal effects in increasing ocular blood flow (naringenin and hesperitin, Pfalts and Bauer, Waterbury, CT). Whether the OH groups are below three (naringenin, hesperitin, Pfalts and Bauer, Waterbury, CT) or above four (Quercetin, Pfalts and Bauer, Waterbury, CT), they produced no effects on the ocular blood flow. When OH groups are four (rutin, Aldrich, Milwaukee, WI), it produced mixed effects on ocular blood flow. The attachment of rutinose and/or methoxy group in the structure did not affect the ocular blood flow one way or the other. CONCLUSION: The ocular blood flow is increased significantly by the number of OH group in the molecule, with three the best to increase the ocular blood flow.

Induction of nitric oxide synthase and over-production of nitric oxide by interleukin-1beta in cultured lacrimal gland acinar cells.

PURPOSE: Inflammation of the lacrimal gland is one of the major causative factors in aqueous tear-deficient dry eye syndrome. Pro-inflammatory cytokine production is upregulated in lacrimal gland autoimmune disease (i.e. Sjögren's syndrome) and is associated with cell death. The expression of inducible nitric oxide synthase (iNOS/NOS-2) is known to be induced in the presence of pro-inflammatory cytokines in several secretory epithelial cell types. We hypothesize that pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta), cause a marked increase in nitric oxide (NO) production via induction of iNOS in lacrimal gland epithelial cells and that this may be a significant pathophysiological pathway of dry eye syndrome. METHODS: Cultured immortalized rabbit lacrimal gland acinar cells were incubated with IL-1beta, iNOS inhibitor, or IL-1 receptor antagonist (IL-1ra). Colorimetric detection of NO(2)(-) and NO(3)(-) in the media, measured by the Griess reaction, was used as an index of NO production. Expression of iNOS was determined by SDS-PAGE and Western blot. RESULTS: IL-1beta stimulated a concentration-dependent and time-dependent increase in NO production. IL-1beta-induced NO production was significantly antagonized by co-incubation with IL-1ra or the iNOS-specific inhibitor, 1400W. Expression of iNOS protein was greatest at 4hr after addition of IL-1beta, and was nearly undetectable at 12hr. IL-1ra greatly reduced IL-1beta-induced iNOS expression. CONCLUSIONS: Lacrimal gland acinar cells are able to produce iNOS in response to the pro-inflammatory cytokine IL-1beta. The amount of iNOS expressed and the subsequent levels of NO that are produced by lacrimal cells are far lower than those seen in macrophages, but are consistent with those reported for other cell types in the literature. This pathway of iNOS induction and overproduction of NO may be a factor in lacrimal gland cell death in dry eye syndrome. Inhibitors of iNOS or IL-1 receptor may be beneficial for controlling lacrimal gland inflammation.

Relationship between nitric oxide production and choroidal blood flow.

PURPOSE: To invent a drug which can specifically facilitate choroid blood flow via increase of nitric oxide (NO). METHOD: Cell culture was used for in vitro experiments to determine the production of NO by NO donors and colored microsphere technique was used for in vivo experiments to determine the blood flow in various tissues of rabbit eyes. RESULTS: ZX-5 and ZX-4 are two geographic isomers with ZX-5 as trans-form and ZX-4 as cis-form. (1-phenyl-3-[3-methoxy-2-propoxy-5-[4-(3,4,5-trimethoxy-phenyl)-1,3-d lane-2-yl]phenyl]thiourea). It was found that ZX-5 released significant amount of NO at 3, 10, 30 microg/ml concentrations and increased choroid blood flow at 1%, 50 microl instillation into eyes. It was not effective on the blood flow of iris or ciliary body. The corresponding ZX-4 was not effective on ocular blood flow nor it released NO. CONCLUSION: ZX-5 can specifically increase the choroidal blood flow which could be useful to suppress the choroidal neovascularization in age-related macular degeneration (AMD). It is hoped that ZX-5 type of compounds could be used for the treatment/prevention of AMD in the elderly.