Guo-Tong Xu

The possible mechanism of naphthalene cataract in rat and its prevention by an aldose reductase inhibitor (ALØ1576)

The naphthalene-induced cataract in rats has been studied for many years as a possible model of human aging-related cataract. While the molecular mechanism of this cataract is unclear, it has recently been demonstrated that the aldose reductase inhibitor ALO1576 can prevent lens opacification in this system. The present study was undertaken to investigate the molecular basis for the effects of naphthalene on the lens and the role of pigmentation in the cataractogenic mechanism. Cataracts were induced in five strains of rats (two pigmented, three albino) by oral administration of naphthalene. Initial lens changes were observed after 1 week by slit-lamp; by 3 weeks a distinct shell-like opacity was present in the deep cortex. Little difference in the course of opacification was found between the pigmented and albino strains. Major biochemical effects were a decrease of 20-30% in glutathione (GSH) by 1 week of feeding, disulfide cross-linking of lens proteins present by 3 weeks, and a nearly 20-fold increase in the content of protein-GSH mixed disulfide. No effect was seen in the ability of the affected lenses to accumulate activity [3H]choline or 86Rb from the medium in organ culture nor in the activity of the Na+/K(+)-ATPase. ALO1576 (10 mg kg-1 day-1) completely prevented all morphological and biochemical changes in the lenses of the naphthalene-fed rats in both pigmented and non-pigmented strains. These results indicate that pigmentation is not required for induction of naphthalene cataract in rats. Naphthalene dihydrodiol was found in the aqueous humor and lens of naphthalene-fed rats. It is proposed that naphthalene dihydrodiol produced in the liver reaches the aqueous humor and penetrates the lens where it is further metabolized ultimately to form the toxic species, naphthoquinone.

Intravitreal Injection of Exendin-4 Analogue Protects Retinal Cells in Early Diabetic Rats

PURPOSE To evaluate the protective effect of intravitreal injection of exendin-4 analogue (E4a) in early diabetic retinopathy (DR) and to explore its possible mechanism. METHODS Forty Sprague-Dawley rats were divided into three groups: normal (N), diabetic (D), and E4a-treated diabetic rats (E4a). Diabetes was induced by streptozotocin. Rats in the E4a group were treated with E4a (0.1 μg/2μL/eye), whereas the N and D groups were treated with the equivalent volume of normal saline. Electroretinography was performed at 1 month and 3 months after diabetes onset. Thicknesses and cell counts in each layer of the retina were evaluated. The concentration of glutamate was measured by high-performance liquid chromatography (HPLC). Expressions of glucagon-like peptide-1 receptor (GLP-1R) and GLAST (excitatory amino acid transporter) were detected at mRNA and protein levels and verified by immunohistochemistry in vitro and in vivo. The rMc-1 cells were cultured under high-glucose medium (25 mM), which mimicked diabetic conditions. Effects of E4a (10 μg/mL) were also tested in the rMc-1 culture system. RESULTS E4a prevented the reduction in b-wave amplitude and oscillatory potential amplitude caused by diabetes. It also prevented the cell loss of outer nuclear layer and inner nuclear layer; the thickness and cell count in the outer nuclear layer were decreased in 1-month diabetic rats. The concentration of glutamate in the retina was higher in diabetic rats and was significantly reduced in the E4a-treated group. Consistent with such changes, retinal GLP-1R and GLAST expression were reduced in the diabetic retina but upregulated in E4a-treated rats. No improvement was found in the retina in both functional and morphologic parameters 3 months after treatment. CONCLUSIONS Intravitreal administration of E4a can prevent the retina, functionally and morphologically, from the insults of diabetes in rats. GLP-1R and GLAST were proved to exist in the rat retina, and their lowered expressions in the diabetic retina might be related to retinal damage by increasing the retinal glutamate. E4a might protect the retina by reducing the glutamate level through upregulating GLP-1R and GLAST, as observed in retinal Müller cells in this study, but this protective effect was transient. Thus, this could be a potential approach for the treatment of DR.

Effects of intravitreal erythropoietin therapy for patients with chronic and progressive diabetic macular edema.

BACKGROUND AND OBJECTIVE To determine the effects of intravitreal injections of erythropoietin in eyes with severe, chronic diabetic macular edema, 5 eyes of 5 patients underwent injections of rHuEPO alpha (EPO). PATIENTS AND METHODS All eyes had progressive vision loss and persistent or worsening edema with prior multi-modal treatment. EPO (5U/50 microL) was injected intravitreally every 6 weeks for three doses and followed for an additional 6 weeks with complete ocular examinations, fluorescein angiography, optical coherence tomography (OCT), and central field acuity perimetry. RESULTS Visual acuity of all patients was subjectively improved by 3 or more lines in 3 eyes and 1 line in 2 eyes. Visual acuity improved to a larger extent than anatomic improvement by OCT. Clearing of hard exudates but only minor improvement in leakage on fluorescein angiography was observed. Improvement in vision occurred within 1 week after the first injection and was maintained until the end point of the current case series (at 18 weeks after the first injection). CONCLUSION This case series seems to show a short-term positive response to EPO for a specific group of patients with chronic diabetic macular edema who were unresponsive to currently available therapies.

SB-431542 inhibition of scar formation after filtration surgery and its potential mechanism.

PURPOSE To explore the inhibitive effect of SB-431542 (an ALK5 inhibitor) on scar formation after glaucoma surgery and to identify the potential pharmacologic target(s). METHODS Twenty-four New Zealand rabbits underwent filtration surgery on the right eye and were divided into a control group and three experimental groups (n=6). Human Tenons fibroblast monolayer was scraped to generate a single gap, and then the control medium with SB-431542 only or containing 10 microg/L TGF-beta1 and SB-431542 (1-20 microM) was added. The cells were pretreated with SB-431542 or in control medium for 30 minutes before induction with 10 microg/L TGF-beta1 or 1 microg/L TGF-beta2. The expression of alpha-SM-actin, CTGF, and Col I, as well as changes in the Smad, ERK, P38, and AKT signaling pathways were detected. RESULTS In comparison with the control rabbits, the IOPs in the experimental groups remained at lower levels until day 25 (P<0.05) after the surgery. Histologic profiles showed that there was only a mild deposition of collagen in the subconjunctival space in the experimental groups. The cell growth and migration were inhibited effectively by SB-431542, regardless of whether TGF-beta was present in the culture system. SB-431542 abrogated TGF-beta-induced upregulation of alpha-SM-actin, CTGF, and Col I. It effectively inhibited the phosphorylation of Smad2 stimulated by TGF-beta but not that of the components of the MAPK pathways. CONCLUSIONS SB-431542 inhibits scar formation after glaucoma filtration surgery. The mechanism may be that SB-431542 interferes in the phosphorylation of Smad2, thus abrogating TGF-beta-induced fibroblast transdifferentiation and then decreasing Col I synthesis.

Further studies on the dynamic changes of glutathione and protein-thiol mixed disulfides in H2O2 induced cataract in rat lenses: distributions and effect of aging

To further investigate the role of protein-thiol mixed disulfides in cataractogenesis, an in vitro H2O2 cataract model was used with rat lenses to study the effect of aging, and the dynamic changes in the cortex, nucleus and the lens protein fractions. A group of lenses was exposed to H2O2-containing media (0.6 mM) for 1 to 3 days so that cortical cataract was induced gradually. Another group of lenses was first subjected to H2O2 exposure for one day and then recovered in the oxidant-free media for one or two days. These lenses were examined for the distribution of free glutathione and protein-thiol mixed disulfides (protein-glutathione and protein-cysteine) in the cortical and nuclear regions as well as in the water soluble and water insoluble fractions. Similar to the results reported earlier, the glutathione depletion in the whole lens occurred immediately and extensively during the 3-day H2O2 exposure. This loss was evenly distributed in the cortical and nuclear fractions. The level of protein-glutathione increased rapidly and continued throughout the 3 days. Most of the accumulation was found in the cortex and in both lens protein fractions. The protein-cysteine modification responded more slowly and less to oxidative stress. The delayed formation occurred mainly in the nucleus and in both lens protein fractions. In the recovery group, glutathione depletion was less drastic in the cortical and nuclear regions, but the elevated protein-glutathione in both regions and both protein fractions spontaneously decreased to its respective basal level within 1 day. Protein-cysteine on the other hand remained quite high, and in some cases it continued to rise in the absence of oxidation. Aging showed little effect on the response of rat lenses to oxidative stress. Similar patterns in glutathione and protein-thiol mixed disulfides occurred in both age groups (1, 23 months) and in both chronic oxidative stress and recovery conditions.

Pharmacokinetic and toxicity study of intravitreal erythropoietin in rabbits

AbstractAim:To study the pharmacokinetics and toxicity of intravitreal erythropoietin (EPO) for potential clinical use.Methods:For toxicity study, 4 groups (60 rabbits) with intravitreal injection (IVit) of EPO were studied (10U, 100U, or 1 000U) per eye for single injection and 0.6 U/eye (the designed therapeutic level in rabbits) for monthly injections (6×). Eye examination, flash electroretinogram (ERG), and fluorescein angiography (FA) were carried out before and after injection. The rabbits were killed for histological study at different intervals. For the pharmacokinetic study, after IVit of 5 U EPO into left eyes, 44 rabbits were killed at different intervals, and the EPO levels in vitreous, aqueous, retina and serum were analyzed by enzyme-linked immunosorbent assay.Results:At all of the time points examined, the eyes were within normal limits. No significant ERG or FA change was observed. The histology of retina remained unchanged. The pharmacokinetic profile of EPO in ocular compartments was summarized as follows. The half-life times of EPO in vitreous, aqueous and serum were 2.84, 3.24 and 2.12 d, respectively; and Cmax were 4615.75, 294.31 and 1.60 U/L, respectively. EPO concentrations in the retina of the injected eye peaked at 1.36 U/g protein at 6 h following injection, with the half-life observed to be 3.42 d.Conclusions:IVit of EPO in a wide range is well tolerated and safe for rabbit eyes. At doses up to 10-fold higher than therapeutic levels, EPO has a pharmacokinetic profile with faster clearance, which is favorable for episodic IVit.

Fullerenol protects retinal pigment epithelial cells from oxidative stress-induced premature senescence via activating SIRT1.

PURPOSE Oxidative stress-induced retinal pigment epithelium (RPE) senescence is one of the important factors in the pathogenesis of age-related macular degeneration (AMD). This study aimed to develop a new antisenescence-based intervention and clarify its possible molecular mechanism. METHODS A cell premature senescence model was established in both primary RPE cells and ARPE-19 cells by exposure of the cells to pulsed H₂O₂ stress for 5 days, and confirmed with senescence-associated β-galactosidase (SA-β-gal) staining. The final concentration of fullerenol (Fol) in the cell culture system was 5 μg/mL. Cellular redox status was determined by the examination of cellular reactive oxygen species (ROS) staining, catalase activity, and the ratio of reduced to oxidized glutathione, respectively. Deoxyribonucleic acid double-strand breaks were determined by quantitative analysis of γH₂AX. Cell cycle analysis was performed with flow cytometry. SIRT1 activity was examined with SIRT1 Assay Kit. SIRT1 overexpression and knockdown in ARPE-19 cells were performed with lentiviral-mediated infection. RESULTS Pulsed H₂O₂ exposure triggered the acetylation of p53 at lysine 382 (K382) and subsequent increase in its target p21(Waf1/Cip1). It also increased the number of accumulated phospho-γH2AX foci and the level of phosphor-ATM in RPE cells. Fullerenol protected the RPE cells, as it reduced the number of positive SA-β-gal-staining cells, alleviated the depletion of cellular antioxidants, and reduced genomic DNA damage. Its mechanism might involve the activation of deacetylase SIRT1, resulting in decreased levels of acetyl-p53 and p21(Waf1/Cip1). The roles of SIRT1 in protecting cells in response to Fol were further confirmed by applications of SIRT1 activator (resveratrol) and inhibitors (nicotinamide and sirtinol), and through SIRT1 overexpression and knockdown. CONCLUSIONS Fullerenol could rescue RPE cells from oxidative stress-induced senescence through its antioxidation activity and the activation of SIRT1. The protective effect of Fol is useful for the development of new strategies to treat oxidative stress-related retinal diseases like AMD.

Inhibition of naphthalene cataract in rats by aldose reductase inhibitors

Naphthalene-induced cataract in rat lenses can be completely prevented by AL01576, an aldose reductase inhibitor (ARI). In an attempt to understand the mechanism of this inhibition, several ARIs were examined to compare their efficacies in preventing naphthalene cataract, using both in vitro and in vivo models. Two classes of ARIs were tested: One group including AL01576, AL04114 (a AL01576 analog) and Sorbinil contained the spirohydantoin group, while Tolrestat contained a carboxylic acid group. Furthermore, to clarify if aldose reductase played a role in naphthalene-induced cataractogenesis in addition to its role in sugar cataract formation, a new dual cataract model was established for ARI evaluations. This was achieved by feeding rats simultaneously with high galactose and naphthalene or incubating rat lenses in culture media containing high galactose and naphthalene dihydrodiol. Under these conditions, both cortical cataract and perinuclear cataract developed in the same lens. It was found that at the same dosage of 10 mg/kg/day, both AL01576 and AL04114 completely prevented all morphological and biochemical changes in the lenses of naphthalene-fed rats. Sorbinil was less efficacious, while Tolrestat was inactive. AL01576 showed a dose-response effect in preventing naphthalene cataract and at 10 mg/kg/day, it was also effective as an intervention agent after cataractogenesis had begun. With the dual cataract model, Tolrestat prevented the high galactose-induced cortical cataract but showed no protection against the naphthalene-induced perinuclear cataract. AL01576, on the other hand, prevented both cataract formations. Results for dulcitol and glutathione levels were in good agreement with the morphological findings. AL04114, and ARI as potent as AL01576 but without its property for cytochrome P-450 inhibition, displayed similar efficacy in preventing naphthalene cataract. Based on these results, it was concluded that the prevention of the naphthalene cataract probably results from inhibition of the conversion of naphthalene dihydrodiol to 1,2-dihydroxynaphthalene and that the effect of the ARIs cannot be explained by their inhibition of the dihydrodiol dehydrogenase activity of aldose reductase.

Differential gene expression pattern of diabetic rat retinas after intravitreal injection of erythropoietin.

Background:  To profile the pattern of gene expression in diabetic rat retinas with or without intravitreal injection of erythropoietin.

BubR1 deficiency results in enhanced activation of MEK and ERKs upon microtubule stresses

Abstract.  Disruption of microtubules activates the spindle checkpoint, of which BubR1 is a major component. Our early studies show that BubR1 haplo‐insufficiency results in enhanced mitotic slippage in vitro and tumorigenesis in vivo. Objective: Given that both MAPKs/ERKs and MEK play an important role during mitosis, we investigated whether there existed regulatory relationship between the MAPK signalling pathway and BubR1. Method and Results: Here, we have demonstrated that BubR1 deficiency is correlated with enhanced activation of MEK and ERKs after disruption of microtubule dynamics. Specifically, treatment with nocodazole and paclitaxel resulted in hyper‐activation of ERKs and MEK in BubR1+/– murine embryonic fibroblasts (MEF) compared to that of wild‐type MEFs. This enhanced activation of ERKs and MEK was at least partly responsible for more successful proliferation completion when cells were treated with nocodazole. BubR1 knockdown via RNAi resulted in enhanced activation of ERKs and MEK in HeLa cells, correlating with inhibition of PP1, a negative regulator of MEK. Moreover, when BubR1 was partially inactivated due to premature missegregation of chromosomes after Sgo1 depletion, phosphorylation of ERKs and MEK was enhanced in mitotic cells; in contrast, little, if any activated ERKs and MEK were detected in mitotic cells induced by nocodazole. Furthermore, BubR1, activated ERKs and activated MEK all localized to spindle poles during mitosis, and also, the proteins physically interacted with each other. Conclusion: Our studies suggest that there exists a cross‐talk between spindle checkpoint components and ERKs and MEK and that BubR1 may play an important role in mediating the cross‐talk.