Lens Organ Culture Research Articles

Uric acid–driven NLRP3 inflammasome activation triggers lens epithelial cell senescence and cataract formation

Excessive uric acid (UA) is associated with age-related cataract. A previous study showed that a high UA level in the aqueous humor stimulated the senescence of lens epithelial cells (LECs), leading to cataract progression. To better understand the underlying mechanisms, we investigated UA-driven senescence in human lens tissue samples obtained during surgery, rat lens organ cultures, and in vivo experiments, using senescence-associated β-galactosidase (SA-β-gal) staining, electronic microscopy, Western blotting, and histological analyses. Initially, we identified markedly higher expressions of NLRP3 and caspase-1 in the lens capsules of hyper-uricemic patients compared to normo-uricemic patients. This increase was accompanied by a significant rise in the SA-β-gal positive rate. We next built a cataract model in which rat lenses in an organ culture system were treated with an increasing dosage of UA. Notably, opacification was apparent in the lenses treated with 800 μM of UA starting on the fifth day. Mechanistically, UA treatment not only significantly induced the expression of NLRP3, caspase-1, and IL-1β, but also upregulated the levels of SA-β-gal and the senescence regulators p53 and p21. These effects were fully reversed, and lens opacification was ameliorated by the addition of MCC950, a selective NLRP3 antagonist. Moreover, an in vivo model showed that intravitreal UA injection rapidly induced cataract phenotypes within 21 days, an effect significantly mitigated by co-injection with MCC950. Together, our findings suggest that targeting the UA-induced NLRP3 inflammasome with MCC950 could be a promising strategy for preventing cataract formation associated with inflammageing.

Investigation of Mitochondrial Homeostasis Changes in Lens Epithelium of High-Myopic Cataract

Purpose High myopia is demonstrated as a pathogenic factor for nuclear cataract. The main mechanism of high-myopia cataracts (HMC) is oxidative damage, which causes mitochondrial homeostasis imbalance. This study aimed to explore the mitochondrial homeostasis alterations in lens epithelial cells (LECs) of HMC. Methods The lens epithelium tissues of 20 patients with HMC and 20 control subjects with age-related cataracts (ARC) were collected. The real-time quantitative PCR and western blot assays were performed for gene expressions. Immunofluorescence (IF) assays were performed for mitochondrial marker TOM20, DNA damage marker 15A3, and autophagosome marker LC3. Transmission electron microscopy (TEM) was used to observe the changes in mitochondria morphology. Mitochondrial ROS, and mitochondrial membrane potential were detected by MitoSOX fluorescence, and JC-1 MitoMP staining, respectively. Rat lenses cultured in vitro were pretreated with CCCP and H2O2 (10 and 400 µM) for 24 h. Results The copy number of mtDNA was decreased in HMC patients compared to the ARC patients. Increased mitochondrial-oriented oxidative stress response was detected in LECs of HMC compared to that of ARC. Altered expressions of mitochondrial homeostasis and mitophagy markers, including TFAM, PGC1α, MFN1, MFN2, Drp1, PINK1, Parkin and LC3, were found in HMC patients. Reciprocally, no significant differences in the expression of BNIP3 and FUNDC1 were found between HMC and ARC patients. Importantly, TEM revealed that the obvious mitochondrial fission and mitophagy phenomena occur in the LECs of HMC patients compared to the ARC patients. Moreover, CCCP aggreated the mitoROS production and depolarized mitochondrial membrane potential in the H2O2-treated human lens epithelial cells line (SRA01/04); Most important, rat lens organ culture experiments indicated a significant increase in H2O2-induced lens opacity following mitochondrial uncoupling CCCP treatment. Conclusion This study has identified for the first time the abnormal mitochondrial homeostasis in HMC, and provide a new perspective on the potential mechanisms of HMC, which occurs earlier and at a higher incidence rate than ARC.

Hesperetin treatment attenuates glycation of lens proteins and advanced‑glycation end products generation.

Advanced glycation end products (AGEs) in lens proteins increase with aging, thus inducing cataracts and/or presbyopia. Hesperetin (Hst), which is an abundant plant flavanone largely derived from citrus species, and its derivatives attenuate cataracts and presbyopia invivo and invitro; however, no reports have described its effects on AGE formation in lens proteins. The present study demonstrated that AGEs in lens proteins increase with age in mice. Additionally, it showed that Hst can prevent AGEs and N(ε)‑carboxymethyl‑lysine generation and modification of lens proteins using invitro in human lens epithelial cell lines and exvivo in mouse lens organ cultures. Furthermore, treatment with Hst prevented lens hardening and decreased chaperone activity in lens proteins. These results suggested that Hst and its derivatives are good candidates for the prevention of presbyopia and cataracts.

Repurposing a Cyclin-Dependent Kinase 1 (CDK1) Mitotic Regulatory Network to Complete Terminal Differentiation in Lens Fiber Cells.

During lens fiber cell differentiation, organelles are removed in an ordered manner to ensure lens clarity. A critical step in this process is removal of the cell nucleus, but the mechanisms by which this occurs are unclear. In this study, we investigate the role of a cyclin-dependent kinase 1 (CDK1) regulatory loop in controlling lens fiber cell denucleation (LFCD). We examined lens differentiation histologically in two different vertebrate models. An embryonic chick lens culture system was used to test the role of CDK1, cell division cycle 25 (CDC25), WEE1, and PP2A in LFCD. Additionally, we used three mouse models that express high levels of the CDK inhibitor p27 to test whether increased p27 levels affect LFCD. Using chick lens organ cultures, small-molecule inhibitors of CDK1 and CDC25 inhibit LFCD, while inhibiting the CDK1 inhibitory kinase WEE1 potentiates LFCD. Additionally, treatment with an inhibitor of PP2A, which indirectly inhibits CDK1 activity, also increased LFCD. Three different mouse models that express increased levels of p27 through different mechanisms show impaired LFCD. Here we define a conserved nonmitotic role for CDK1 and its upstream regulators in controlling LFCD. We find that CDK1 functionally interacts with WEE1, a nuclear kinase that inhibits CDK1 activity, and CDC25 activating phosphatases in cells where CDK1 activity must be exquisitely regulated to allow for LFCD. We also provide genetic evidence in multiple in vivo models that p27, a CDK1 inhibitor, inhibits lens growth and LFCD.

Protection of Human Lens Epithelial Cells from Oxidative Stress Damage and Cell Apoptosis by KGF-2 through the Akt/Nrf2/HO-1 Pathway.

Oxidative stress exerts a significant influence on the pathogenesis of various cataracts by inducing degradation and aggregation of lens proteins and apoptosis of lens epithelial cells. Keratinocyte growth factor−2 (KGF-2) exerts a favorable cytoprotective effect against oxidative stress in vivo and in vitro. In this work, we investigated the molecular mechanisms of KGF-2 against hydrogen peroxide- (H2O2-) induced oxidative stress and apoptosis in human lens epithelial cells (HLECs) and rat lenses. KGF-2 pretreatment could reduce H2O2-induced cytotoxicity as well as reactive oxygen species (ROS) accumulation. KGF-2 also increases B-cell lymphoma-2 (Bcl-2), quinine oxidoreductase-1 (NQO-1), superoxide dismutase (SOD2), and catalase (CAT) levels while decreasing the expression level of Bcl2-associated X (Bax) and cleaved caspase-3 in H2O2-stimulated HLECs. LY294002, the phosphatidylinositol-3-kinase (PI3K)/Akt inhibitor, abolished KGF-2's effect to some extent, demonstrating that KGF-2 protected HLECs via the PI3K/Akt pathway. On the other hand, KGF-2 activated the Nrf2/HO-1 pathway by regulating the PI3K/Akt pathway. Silencing nuclear factor erythroid 2-related factor 2 (Nrf2) by targeted-siRNA and inhibiting heme oxygenase-1 (HO-1) through zinc protoporphyrin IX (ZnPP) significantly decreased cytoprotection of KGF-2. Furthermore, as revealed by lens organ culture assays, KGF-2 treatment decreased H2O2-induced lens opacity in a concentration-dependent manner. As demonstrated by these data, KGF-2 resisted H2O2-mediated apoptosis and oxidative stress in HLECs through Nrf2/HO-1 and PI3K/Akt pathways, suggesting a potential protective effect against the formation of cataracts.

Suppression of PI3K signaling is linked to autophagy activation and the spatiotemporal induction of the lens organelle free zone

The terminal steps of lens cell differentiation require elimination of all organelles to create a central Organelle Free Zone (OFZ) that is required for lens function of focusing images on the retina. Previous studies show that the spatiotemporal elimination of these organelles during development is autophagy-dependent. We now show that the inhibition of PI3K signaling in lens organ culture results in the premature induction of autophagy within 24 h, including a significant increase in LAMP1+ lysosomes, and the removal of lens organelles from the center of the lens. Specific inhibition of just the PI3K/Akt signaling axis was directly linked to the elimination of mitochondria and ER, while pan-PI3K inhibitors that block all PI3K downstream signaling removed all organelles, including nuclei. Therefore, blocking the PI3K/Akt pathway was alone insufficient to remove nuclei. RNAseq analysis revealed increased mRNA levels of the endogenous inhibitor of PI3K activation, PIK3IP1, in differentiating lens fiber cells preceding the induction of OFZ formation. Co-immunoprecipitation confirmed that PIK3IP1 associates with multiple PI3K p110 isoforms just prior to formation of the OFZ, providing a likely endogenous mechanism for blocking all PI3K signaling and activating the autophagy pathway required to form the OFZ during lens development.

The transcription factor CREB acts as an important regulator mediating oxidative stress-induced apoptosis by suppressing αB-crystallin expression.

The general transcription factor, CREB has been shown to play an essential role in promoting cell proliferation, neuronal survival and synaptic plasticity in the nervous system. However, its function in stress response remains to be elusive. In the present study, we demonstrated that CREB plays a major role in mediating stress response. In both rat lens organ culture and mouse lens epithelial cells (MLECs), CREB promotes oxidative stress-induced apoptosis. To confirm that CREB is a major player mediating the above stress response, we established stable lines of MLECs stably expressing CREB and found that they are also very sensitive to oxidative stress-induced apoptosis. To define the underlying mechanism, RNAseq analysis was conducted. It was found that CREB significantly suppressed expression of the αB-crystallin gene to sensitize CREB-expressing cells undergoing oxidative stress-induced apoptosis. CREB knockdown via CRISPR/CAS9 technology led to upregulation of αB-crystallin and enhanced resistance against oxidative stress-induced apoptosis. Moreover, overexpression of exogenous human αB-crystallin can restore the resistance against oxidative stress-induced apoptosis. Finally, we provided first evidence that CREB directly regulates αB-crystallin gene. Together, our results demonstrate that CREB is an important transcription factor mediating stress response, and it promotes oxidative stress-induced apoptosis by suppressing αB-crystallin expression.

Anticataract effects of S. cumini and A. marmelos on goat lenses in an experimental diabetic cataract model

BackgroundCataractogenesis in diabetes mellitus is mainly due to generation of free radicals causing oxidative stress. Antioxidants are known to delay cataractogenesis. Indigenous plants are potential promising sources of antioxidants. ObjectivesThe present study was done in goat lenses for exploring local antioxidant and anticataract potential of Syzygium cumini (Jamun) and Aegle marmelos (Bael) and comparing their activities. Material and methods“Lens organ culture technique” was employed using “tissue culture medium 199” (TC 199). Lenses were divided into four groups of 30 each. Group 1 was “Normal Control”. In remaining 3 groups, experimental diabetic cataract was produced using dextrose (110 mM). Group 2: “Toxic Control” (untreated experimental diabetic cataract lenses). Group 3: S. cumini seed extract (0.25%) treated lenses. Group 4: A. marmelos leaf extract (0.25%) treated lenses. Biochemical parameters measured in lens homogenates included total soluble lens proteins (index of cataractogenesis), malondialdehyde (index of lipid peroxidation), and superoxide dismutase, glutathione reductase, and glutathione peroxidase (indices of antioxidant enzyme activity). Lens morphology was compared in all groups. ResultsS. cumini and A. marmelos showed significantly increased activity of all three antioxidant enzymes, preserved total soluble proteins and decreased malondialdehyde (MDA). Lens morphology was well preserved with these extracts. S. cumini aqueous seed extract scored better over A. marmelos. ConclusionIn goat lenses with dextrose-induced experimental diabetic cataract, S. cumini and A. marmelos showed antioxidant and anticataract properties and preservation of lens morphology (p < 0.0001 to 0.05). S. cumini showed better anticataract activity than A. marmelos.

Effects of D-Limonene on aldose reductase and protein glycation in diabetic rats

Diabetes mellitus and hyperglycemia can lead to other health complications. The pathogenesis of diabetes mellitus can affect numerous tissues in the eye structure and cataracts are one of the major causes of visual impairment in diabetes mellitus patients. In this study, we aim to investigate the ability of D-limonene to inhibit aldose reductase hypoglycemic activity in lens organ culture and its anti-glycating properties in STZ-induced cataracts of rats with diabetes. D-limonene repressed aldose reductase in-vitro with IC50 ranges of ~13.9 ± 0.31 µg/mL in an uncompetitive manner. This compound also prevented hyperglycemic induced increases in aldose reductase activity, accumulation of sorbitol, altered crystalline proteins (α, β and γ), and opacification of the rat lens in ex-vivo lens organ culture. Supplementation of D-limonene in STZ-induced diabetic rats restored these changes and delayed the progression of cataracts. This study demonstrates that D-limonene is a pharmaceutically active component that inhibits rat-lens aldose reductase. Therefore, this study showed that D-limonene prevents advanced glycation end product formation contributing to the integrity of α-crystallin chaperone activity and delays cataract development. It has been concluded, D-limonene has a role in inhibiting aldose reductase activity and advanced glycation end products formation in vivo, and in delaying the development of diabetic cataracts. Further studies of D-limonene are needed to assess other complications in DM as they may provide additional evidence for establishing D-limonene as a potential pharmacological target therapy for diabetes mellitus complications.

Dietary flavonoids inhibit the glycation of lens proteins: implications in the management of diabetic cataract

The intervention of functional foods as complementary therapeutic approach for the amelioration of diabetes and sugar induced cataractogenesis is more appreciated over the present day chemotherapy agents owing to their nontoxic and increased bioavailability concerns. Dietary flavonoids, a class of bioactive phytochemicals is known to have wide range of biological activities against variety of human ailments. In the present study, we demonstrate anti-cataract effect of eight dietary flavonoids in sugar induced lens organ culture study. We present data on processes like inhibition of glycation-induced lens cloudiness, lens protein aggregation, glycation reaction and advanced glycation end products formation that can act as biochemical markers for this disease. The selected flavonoids were also tested for their aldose reductase (AR) inhibition (experimental and in silico). The molecular dynamics simulation results shed light on mechanistic details of flavonoid induced AR inhibition. The outcome of the present study clearly focuses the significance of kaempferol, taxifolin and quercetin as potential candidates for controlling diabetic cataract.

Absorbance and Light Scattering of Lenses Organ Cultured with Glucose

ABSTRACTPurpose/Aim: Diabetes is one of the major factors related to cataract. Our aim is to determine if the attenuation of light through glucose treated lenses was due to light scattering from structural changes or absorbance from metabolic changes.Materials and Methods: Human and rat lenses were cultured in a medium with and without 55 mM glucose for a period of 5 days. Absorbance and light scattering were measured using a ultraviolet spectrometer. Aldose reductase and catalase activity, RAGE, and glutathione were measured using classical assays.Results: Almost all of the glucose related attenuation of light through the human lens was due to light scattering from structural changes. Glucose treatment caused three absorbance band to appear at 484, 540 to 644 and 657 nm in both the rat and human lens. The optimum time point for equilibration of human lenses was found to be between 2 and 3 days in organ culture. Glucose caused a more significant effect on the opacity of human lenses compared with rat lenses. Since the levels of glutathione, catalase, and aldose reductase were reduced in glucose treated rat lenses compared with untreated lenses, glucose may have caused oxidative stress on the rat lens.Conclusions: The absorbance and light scattering of glucose treated lenses in organ culture were quantitated for the first time which could be important for future studies designed to test the efficacy of agents to ameliorate the opacity. Almost all of the glucose related attenuation of light through the human lens was due to light scattering from structural changes and not absorbance from metabolic changes. Glucose caused a more significant effect on the opacity of human lenses compared with rat lenses. The lens model employed could be used to study the efficacy of agents that potentially ameliorate lens opacity.

The optical properties of rat, porcine and human lenses in organ culture treated with dexamethasone

Organ culturing of lenses is useful for testing the direct effect of moieties such as cations, glucose, oxygen, antioxidants or drugs on the lens. Lens opacity changes in organ cultured lenses have never been measured quantitatively. Our aims were twofold: to establish the time course of lens opacity changes in organ cultured rat, porcine and human lenses and to test the utility of our system to measure opacity changes in lenses treated with dexamethasone. Human, porcine and rat lenses were incubated with or without 5 μM dexamethasone in minimum essential medium for 13 days. Lens absorbance and light scattering were measured using a spectrometer. Lens opacity was graded from lens photographs. Sixteen human lenses were obtained from 10 donors. Light scattering accounted for all of the absorbance of the human lens above 550 nm, 72% of the absorbance at 500 nm and 50% of the absorbance at 450 nm. Similarly, for the porcine lens, light scattering accounted for all of the absorbance above 400 nm and 40% of the absorbance at 350 nm. For the rat lens, scattering accounted for all of the absorbance above 325 nm, and 37% of the absorbance at 300 nm. In the rat lens spectrum, the broad absorbance peak near 600 nm was unique to the rat lens. After day five in organ culture, the level of light scattering, absorbance and optical grade of human lenses were higher in dexamethasone treated lenses compared with those without dexamethasone. Similar differences were observed for rat lenses after one day in organ culture. The opacity of porcine lenses did not change with dexamethasone treatment. In conclusion, this study showed that dexamethasone-induced opacity in human and rat lenses could be measured which is important to future studies to test the positive or negative efficacy of moieties that cause or ameliorate cataract.

Chlorogenic acid attenuates hydrogen peroxide‑induced oxidative stress in lens epithelial cells.

Oxidative stress has an important role in the degradation, oxidation, cross-linking and aggregation of lens proteins, and can trigger lens epithelial cell apoptosis. To investigate the protective effect of chlorogenic acid (CGA) against hydrogen peroxide (H2O2)-induced oxidative stress, human lens epithelial cells (hLECs) were exposed to various concentrations of H2O2 in the presence and absence of CGA. Using MTT assay, reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and ELISA techniques, cell viability, and protein/mRNA levels of BCL2 apoptosis regulator (Bcl-2) and BCL2 associated X apoptosis regulator (Bax) were investigated. Additionally, the levels of intracellular reactive oxygen species (ROS) and apoptosis within cells were measured using flow cytometry to determine the protective effect of CGA on H2O2-induced oxidative stress. Furthermore, the protective effect of CGA on H2O2-induced apoptosis was also examined using rabbit lenses ex vivo. The results indicated that CGA reduced H2O2-induced cytotoxicity in a dose-dependent manner. Flow cytometry analysis demonstrated that simultaneous exposure of hLECs to H2O2 and CGA significantly decreased apoptosis and the levels of ROS. RT-qPCR analysis revealed a decrease in Bcl-2 and an increase in Bax in hLECs following exposure to H2O2 for 24 h, regardless of CGA presence. Furthermore, ELISA results indicate that CGA increased Bcl-2 expression and decreased Bax expression following treatment with H2O2 for 24 h and the Bax/Bcl-2 ratio was significantly decreased by CGA treatment. Lens organ culture experiments indicated a dose-dependent decrease in H2O2-induced lens opacity following CGA treatment. These results suggest that CGA suppresses hLECs apoptosis and prevents lens opacity induced by H2O2 via Bax/Bcl-2 signaling pathway. CGA may provide effective defenses against oxidative stress and, thus, haσ potential as treatment for a variety of diseases in clinical practice.

Inhibition of glycation and aldose reductase activity using dietary flavonoids: A lens organ culture studies

On the eve of increasing incidence of diabetes mellitus and related complications, the search for novel, safe and alternatives therapeutic approaches are evolving. In the present investigation, a panel of ten dietary flavonoids such as 4′-methoxyflavanone, formononetin, hesperetin, hesperidin, naringenin, naringin, rutin, diadzin, silibinin and silymarin was evaluated as possible inhibitors of sugar induced cataractogenesis using bovine lens organ culture studies. The effect of selected flavonoids was observed on glycation induced lens opacity, AGE fluorescence, carbonyl group formation (a biomarker of glycation), protein aggregation and aldose reductase (AR) inhibition. The results obtained clearly demonstrate the efficacy of rutin and silibinin as promising leads for inhibition of glycation reaction and amelioration of sugar induced cataractogenesis. The findings of the present study may be useful for designing and development of the novel lead molecules for the management of diabetic cataract.

Ghrelin Protects Human Lens Epithelial Cells against Oxidative Stress-Induced Damage.

Oxidative stress has been recognized as an important mediator in the pathogenesis of age-related cataracts; using antioxidant supplements is one plausible strategy to protect the antioxidative defense system against oxidative stress. Ghrelin administration is expected to reduce ROS, preventing the onset of different diseases. The role of ghrelin, if any, in protecting against oxidative stress in HLECs has never been examined. In the present study, we investigated the effects of ghrelin against H2O2-induced oxidative stress and the associated molecular mechanisms in HLECs and rat lenses. The results showed that pretreatment with ghrelin reduced H2O2-induced cellular apoptosis and ROS accumulation, increased the expression levels of SOD and CAT, and decreased the expression level of MDA. The morphological examination showed that the ghrelin-treated lens organ culture maintained transparency. This is the first report to show that ghrelin can protect HLECs from H2O2-induced oxidative stress. Our findings suggest that ghrelin may prevent the progression of cataracts, which has treatment value for ophthalmologists.

Ku80 Counters Oxidative Stress-Induced DNA Damage and Cataract Formation in the Human Lens.

Oxidative stress in the human lens leads to a wide range of damage including DNA strand breaks, which are likely to contribute to cataract formation. The protein Ku80 is a fundamental component of the nonhomologous end-joining pathway that repairs DNA double strand breaks. This study investigates the putative impact of Ku80 in cataract prevention in the human lens. The present study used the human lens epithelial cell line FHL124 and whole human lens organ culture. Targeted siRNA was used to deplete Ku80, with Western blot and immunocytochemistry employed to assess Ku80 expression levels. Oxidative stress was induced with hydrogen peroxide and DNA strand breaks measured by alkaline comet assay and γH2AX foci counts. Visual quality of whole human lenses was measured with image analysis software. Expression of Ku80 was predominately found in the cell nucleus of both FHL124 cells and native human lens epithelium. Treatment of FHL124 cells and whole lens cultures with siRNA targeted against Ku80 resulted in a significant knockdown at the protein level. Application of oxidative stress (30 μM H2O2) created more DNA strand breaks when added to Ku80 knockdown cells than in scrambled siRNA control cells as determined by the alkaline comet assay and the number of γH2AX foci. In whole lens cultures, exposure to 1 mM H2O2 resulted in more lens opacity in Ku80 knockdown lenses than match-paired controls. Depletion of Ku80 in the lens through acute change or a consequence of aging is likely to increase levels of DNA strand breaks, which could negatively influence physiological function and promote lens opacity. It is therefore feasible that Ku80 plays a role in retarding cataract formation.

Antiapoptotic Effects of Anthocyanin from the Seed Coat of Black Soybean Against Oxidative Damage of Human Lens Epithelial Cell Induced by H2O2

Purpose: To describe the protective effect of anthocyanin from black soybean in human lens epithelial cell line (HLE-B3) under H2O2-induced oxidative stress.Methods: Cytotoxicity of anthocyanin and H2O2 were determined by Cell Counting Kit-8 test. Viability of HLE-B3 cells under various H2O2 concentration (0, 50 and 100 μM) with or without pretreatment of anthocyanin (0, 50, 100 and 200 μg/ml) was measured. After quantifying the percentage of the apoptosis by Annexin V assay and APO-BrdU TUNEL assay, we conducted western blot and immunostaining of apoptosis-related molecules; Bcl2, BAD, BAX, p53 and caspase-3. To confirm the effect of anthocyanin on an ex vivo model, its effect on cultures of the lenses of porcine were examined.Results: Anthocyanin reduced cell death of HLE-B3 under H2O2-induced oxidative stress in a dose-dependent manner. In Annexin V analysis, anthocyanin protected HLE-B3 cells from apoptosis. H2O2 increased the expression of BAX, BAD, p53 and caspase-3 in a time-dependent manner, those of which anthocyanin significantly decreased. On the other hand, Bcl2 was increased from anthocyanin-treated lens cells. And in anthocyanin-treated lens organ culture, transparency was maintained.Conclusions: This study showed that anthocyanin protects HLE-B3 cells under oxidative stress from apoptosis, and the mechanism of the effect is related to the intrinsic pathway of apoptosis. Anthocyanin has a potential in prevention of cataract.

Design of an Amide N-Glycoside Derivative of β-Glucogallin: A Stable, Potent, and Specific Inhibitor of Aldose Reductase

β-Glucogallin (BGG), a major component of the Emblica officinalis medicinal plant, is a potent and selective inhibitor of aldose reductase (AKR1B1). New linkages (ether/triazole/amide) were introduced via high yielding, efficient syntheses to replace the labile ester, and an original two-step (90%) preparation of BGG was developed. Inhibition of AKR1B1was assessed in vitro and using transgenic lens organ cultures, which identified the amide linked glucoside (BGA) as a stable, potent, and selective therapeutic lead toward the treatment of diabetic eye disease.

Inhibition of aldose reductase by Aegle marmelos and its protective role in diabetic cataract

Ethnopharmacological relevanceAegle marmelos (L.) Corr. Serr. (Aegle marmelos) leaves were extensively used in the Ayurvedic, Unani and Siddha systems of Indian medicine as an anti-diabetic agent, which serves as hypoglycemic agent. However, the significance of this plant on secondary complications of diabetes such as cataract remained unknown. The aim of the study was to investigate the possible anti-cataractous activity of Aegle marmelos against streptozotocin (STZ) induced diabetic cataract in rats. Materials and methodsAegle marmelos leaf extract was prepared using three different solvents (petroleum ether, ethyl acetate and methanol) and tested for inhibition against rat lens aldose reductase (AR), a key enzyme of polyol pathway. Furthermore, the pharmacological potential of Aegle marmelos extract was investigated against osmotic stress induced opacification of lens in ex vivo organ culture and streptozotocin (STZ) induced diabetic cataract in rats. ResultsEthyl acetate extract of Aegle marmelos inhibited rat lens AR in vitro with an IC50 value of ∼15µg/ml. This extract also prevented the hyperglycemia induced increase in AR activity, sorbitol accumulation and opacification of rat lens in ex vivo lens organ culture. Supplementation of ethyl acetate extract of Aegle marmelos to STZ-induced diabetic rats decreased the blood glucose levels due to hyperglycemia and inhibited the AR activity and delayed cataract progression in dose dependent manner. α-crystallin isolated from diabetic rats fed with Aegle marmelos showed improved chaperone activity than that of isolated from rats naïve to Aegle marmelos. ConclusionThis study indicates that ethyl acetate extract of Aegle marmelos has pharmacologically active components with a potential to inhibit rat lens AR and consequential decrease in osmotic stress. Besides this, the present study also demonstrates that the extract prevented loss of antioxidants contributing to the integrity of α-crystallin's chaperone activity and thereby delaying cataract.

Potential Protection by Antioxidants of the Action of Tobacco Smoke on the Metabolism of Cultured Bovine Lenses

Purpose: Smoke from cigarette smoking (CS) has been proposed to be a major environmental risk factor for a variety of human diseases and was implicated also in cataract, an eye lens pacification, which is a major cause of blindness. We have undertaken a study to investigate the effect of smoke on the physiological integrity and metabolism of organ cultured lenses. Lenses in organ culture are metabolically active and have functional defense systems, thus they provide an appropriate model for studying the effects of smoke. Also the possible protective action of N-acetylL-cysteine (NAC) which is a precursor of glutathione and the iron chelators Deferoxamine (DFO), was estimated as potential protective agents against cataract. Methods: Bovine lenses were incubated in organ culture conditions at 35°Cfor 6 days. Treated lenses were 4 day in the culturally environment daily sated with a cigarette smoke under various doses of pressure-250,500 psi. Two of the experimental groups were treated with NAC (1 mM) and DFO (2.5 µg/ml) as antioxidants. An automated scanning laser system was used for daily testing of both treated and control lenses. At the end of the culture period, lenses were analyzed by inverted microscopy. We have used preparations of the forward monolayer epithelium bovine lenses from all experiments. For this purpose the capsule opened and crystalline lens fibers were cleaned. On the object-plate them there was only a capsule and a cellular monolayer epithelium. Changes of morphology of cells and the contents of the nucleic acids was estimated using the Einarson - DNA-RNA staining method. Reactive Oxygen Species (ROS) were estimated with 5- (and 6-) chloromethyl-2’,7’-dichloro-dihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA, C6827) to measure the level of cellular oxidation in the cells of lens epithelium. The levels of ROS were measured by monitoring the fluorescent intensity relative to that of control cultures under fluorescent microscopy. Nucleic acids were stained with Propidium Iodide. Results: Exposure of cigarette smoke in cultured medium under negative pressure, optical quality of lenses and the structural changes were demonstrated by decreased light transmission, increase in focal length variability and a decrease in morphological integrity such as hyperplasia and hypertrophy of epithelial cells. The group exposed to NAC and to DFO, demonstrated reduced optical changes representing smaller lens injury. The lenses show almost no volume changes. However, the baseline fluorescence of controls varied between experiments. A dose-dependent increase in ROS generation in cultures was also evident. Conclusions: We have shown that increasing the amount CS exposure, for a relatively short time, causes a sharp increase in the damage to the lens. Smoking is an independent risk factor that has dose-response effects. It causes morphological and functional changes to the lens. Based on the independent effects of NAC and DFO, we propose to possibly use them as means of prevention and/or treatment of cataract in heavily smoking people.