Cumulative antioxidant defense against oxidative challenge in galactose-induced cataractogenesis in Wistar rats

Natural dietary ingredients are known for their antioxidant activity. Of such, curcumin, the active principle of turmeric, at 0.01% in the diet proved as pro-oxidative in galactose-induced cataract in vivo. The purpose of this study was to investigate the effect of vitamin E (VE), a well-known antioxidant, in combination with curcumin on the onset and maturation of galactose induced cataract. Periodic slit-lamp microscope examination indicated that in combination with vitamin-E, 0.01% curcumin (G-IV) delayed the onset and maturation of galactose-induced cataract. Biochemical analyses revealed that combined treatment of 0.01% curcumin and vitamin-E diet exhibited an efficient antioxidant effect, as it inhibited lipid peroxidation and contributed to a distinct rise in reduced glutathione content. The results indicate that natural dietary ingredients are effective in combination rather than the individual administration as they are complementing each other in reducing the risk of galactose induced cataract.     

The cataract causing Cx50-S50P mutant inhibits Cx43 and intercellular communication in the lens epithelium

Mutations in Connexin50 (Cx50) cause cataracts in both humans and mice. The mechanism(s) behind how mutated connexins lead to a variety of cataracts have yet to be fully elucidated. Here, we tested whether the cataract inducing Cx50-S50P mutant interacts with wild-type Connexin43 (Cx43) to form mixed channels with attenuated function. Using dual whole-cell voltage clamp, immunofluorescent microscopy and in situ dye transfer analysis we identified a unique interaction between the mutant subunit and wild-type Cx43. In paired Xenopus oocytes, co-expression of Cx50-S50P with Cx43 reduced electrical coupling ≥ 90%, without a reduction in protein expression. In transfected cells, Cx50-S50P did not target to cell-cell interfaces by itself, but co-expression of Cx50-S50P with Cx43 resulted in its localization at areas of cell-cell contact. We used Cx43 conditional knockout, Cx50 knockout and Cx50-S50P mutant mice to examine this interaction in vivo. Mice expressing both Cx43 and Cx50-S50P in the lens epithelium revealed a unique expression pattern for Cx43 and a reduction in Cx43 protein. In situ dye transfer experiments showed that the Cx50-S50P mutant, but not the Cx50, or Cx43 conditional knockout, greatly inhibited epithelial cell gap junctional communication in a manner similar to a double knockout of Cx43 and Cx50. The inhibitory affects of Cx50-S50P lead to diminished electrical coupling in vitro, as well as a discernable reduction in epithelial cell dye permeation. These data suggest that dominant inhibition of Cx43 mediated epithelial cell coupling may play a role in the lens pathophysiology caused by the Cx50-S50P mutation.     

比较不同类型白内障形成过程中非蛋白质巯基与蛋白质巯基的动态变化及关系

本文报道了在亚硒酸钠、平阳霉素及半乳糖诱发大鼠产生白内障过程中晶状体中非蛋白质巯基及蛋白质巯基的动态变化,并探讨了其变化机理及相互关系。在亚硒酸钠诱发白内障过程中,给药24h后晶状体中非蛋白质巯基减少到正常的二分之一,以后又逐渐回升,但始终未达到正常水平,至第7天,非蛋白质巯基又再度减少。在平阳霉素及半乳糖诱发白内障过程中,晶状体中非蛋白质巯基分别在给药后的第7天及第3天开始大量减少,以后继续减少,至第15天时,其含量分别为正常的十分之一及五分之一。在体外,亚硒酸钠有促进还原型谷胱甘肽自氧化的作用,半乳糖对此作用无影响,而平阳霉素可阻止其进行,但能加强亚硒酸钠的促进作用。在三种白内障晶状体中,蛋白质巯基开始减少的时间均较非蛋白质巯基为晚,这表明只有非蛋白质巯基减少到一定程度后蛋白质巯基才会被大量氧化,同时也说明非蛋白质巯基具有保护蛋白质巯基免受氧化的作用。只有这种保护作用减弱后,才会使蛋白质巯基遭受氧化而导致白内障。     

Cx43, ZO-1, alpha-catenin and beta-catenin in cataractous lens epithelial cells

Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n?=?52) undergoing cataract surgery. Specimens were divided into three groups based on the type of cataract: nuclear cataract, cortical cataract and posterior subcapsular cataract (PSC). Clear lenses (n?=?11) obtained from donor eyes were used as controls. Expression was studied by immunofluorescence, real-time PCR and Western blot. Statistical analysis was done using the student’s t-test. Immunofluorescence results showed punctate localization of Cx43 at the cell boundaries in controls, nuclear cataract and PSC groups. In the cortical cataract group, cytoplasmic pools of Cx43 without any localization at the cell boundaries were observed. Real-time PCR results showed significant up-regulation of Cx43 in nuclear and cortical cataract groups. Western blot results revealed significant increase in protein levels of Cx43 and significant decrease of ZO-1 in all three cataract groups. Protein levels of alpha-catenin were decreased significantly in nuclear and cortical cataract group. There was no significant change in expression of beta-catenin in the cataractous groups. Our findings suggest that ZO-1 and alpha-catenin are important for gap junctions containing Cx43 in the LECs. Alterations in cell junction proteins may play a role during formation of different types of cataract.     

The connexin 46 mutant (V44M) impairs gap junction function causing congenital cataract

Connexin 46 (Cx46) is important for gap junction channels formation which plays crucial role in the preservation of lens homeostasis and transparency. Previously, we have identified a missense mutation (p.V44M) of Cx46 in a congenital cataract family. This study aims at dissecting the potential pathogenesis of the causative mutant of cataract. Plasmids carrying wild-type (wt) and mutant (V44M) of Cx46 were constructed and expressed in Hela cells respectively. Western blotting and fluorescence microscopy were applied to analyse the expression and subcellular localization of recombinant proteins, respectively. Scrape loading dye transfer experiment was performed to detect the transfer capability of gap junction channels among cells expressed V44M mutant. The results demonstrated that in transfected Hela cells, both wt-Cx46 and Cx46 V44M were localized abundantly in the plasma membrane. No significant difference was found between the protein expressions of the two types of Cx46. The fluorescent localization assay revealed the plaque formation, significantly reduced in the cells expressing Cx46 V44M. Immunoblotting analysis demonstrated that formation of Triton X-100 insoluble complex decreased obviously in mutant Cx46. Additionally, the scrape-loading dye-transfer experiment showed a lower dye diffusion distance of Cx46 V44M cells, which indicates that the gap junction intercellular communication activity was aberrant. Human Cx46 V44M mutant causing cataracts result in abnormally decreased formation of gap junction plaques and impaired gap junction channel function.     

Connexin 37 profoundly slows cell cycle progression in rat insulinoma cells

In addition to providing a pathway for intercellular communication, the gap junction-forming proteins, connexins, can serve a growth-suppressive function that is both connexin and cell-type specific. To assess its potential growth-suppressive function, we stably introduced connexin 37 (Cx37) into connexin-deficient, tumorigenic rat insulinoma (Rin) cells under the control of an inducible promoter. Proliferation of these iRin37 cells, when induced to express Cx37, was profoundly slowed: cell cycle time increased from 2 to 9 days. Proliferation and cell cycle time of Rin cells expressing Cx40 or Cx43 did not differ from Cx-deficient Rin cells. Cx37 suppressed Rin cell proliferation irrespective of cell density at the time of induced expression and without causing apoptosis. All phases of the cell cycle were prolonged by Cx37 expression, and progression through the G(1)/S checkpoint was delayed, resulting in accumulation of cells at this point. Serum deprivation augmented the effect of Cx37 to accumulate cells in late G(1). Cx43 expression also affected cell cycle progression of Rin cells, but its effects were opposite to Cx37, with decreases in G(1) and increases in S-phase cells. These effects of Cx43 were also augmented by serum deprivation. Cx-deficient Rin cells were unaffected by serum deprivation. Our results indicate that Cx37 expression suppresses cell proliferation by significantly increasing cell cycle time by extending all phases of the cell cycle and accumulating cells at the G(1)/S checkpoint.     

Prevention of cataract development in severely galactosemic rats by the aldose reductase inhibitor, tolrestat

With a fixed time period of galactose feeding, the rate of appearance of lenticular opacities depended on the severity of galactosemia, while with a fixed amount of galactose fed, the rate was time dependent. The capacity of tolrestat, a structurally novel inhibitor of aldose reductase (AR), to control cataract development was assessed in rats fed 30-50% galactose with the diet for 7 to 277 days. In rats fed 30% galactose for 31 days, the controlling effect of tolrestat was dose dependent, and no cataracts were detected at a dose of 35 mg/kg/day. In rats given tolrestat with the diet for 14 days, then rendered severely galactosemic with a diet containing 50% galactose, and subjected to continued treatment with tolrestat at a dose of 43 mg/kg/day, no changes were detected by slit-lamp microscopy after 207 days. The preventive effect was also dose dependent. In view of the established similarity in the pathogenesis of galactosemic and diabetic cataracts, the results obtained with tolrestat support its potential for controlling cataract development in diabetics.     

Efficacy of Biodegradable Curcumin Nanoparticles in Delaying Cataract in Diabetic Rat Model

Curcumin, the active principle present in the yellow spice turmeric, has been shown to exhibit various pharmacological actions such as antioxidant, anti-inflammatory, antimicrobial, and anti-carcinogenic activities. Previously we have reported that dietary curcumin delays diabetes-induced cataract in rats. However, low peroral bioavailability is a major limiting factor for the success of clinical utilization of curcumin. In this study, we have administered curcumin encapsulated nanoparticles in streptozotocin (STZ) induced diabetic cataract model. Oral administration of 2 mg/day nanocurcumin was significantly more effective than curcumin in delaying diabetic cataracts in rats. The significant delay in progression of diabetic cataract by nanocurcumin is attributed to its ability to intervene the biochemical pathways of disease progression such as protein insolubilization, polyol pathway, protein glycation, crystallin distribution and oxidative stress. The enhanced performance of nanocurcumin can be attributed probably to its improved oral bioavailability. Together, the results of the present study demonstrate the potential of nanocurcumin in managing diabetic cataract.     

Orally active multi-functional antioxidants delay cataract formation in streptozotocin (type 1) diabetic and gamma-irradiated rats

Background

Age-related cataract is a worldwide health care problem whose progression has been linked to oxidative stress and the accumulation of redox-active metals. Since there is no specific animal model for human age-related cataract, multiple animal models must be used to evaluate potential therapies that may delay and/or prevent cataract formation.

Methods/Principal Findings

Proof of concept studies were conducted to evaluate 4-(5-hydroxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 4) and 4-(5-hydroxy-4,6-dimethoxypyrimidin-2-yl)-N,N-dimethyl-3,5-dioxopiperazine-1-sulfonamide (compound 8), multi-functional antioxidants that can independently chelate redox metals and quench free radicals, on their ability to delay the progression of diabetic “sugar” cataracts and gamma radiation-induced cataracts. Prior to 15 Gy of whole head irradiation, select groups of Long Evans rats received either diet containing compound 4 or 8, or a single i.p. injection of panthethine, a radioprotective agent. Compared to untreated, irradiated rats, treatment with pantethine, 4 and 8 delayed initial lens changes by 4, 47, and 38 days, respectively, and the average formation of posterior subcapsular opacities by 23, 53 and 58 days, respectively. In the second study, select groups of diabetic Sprague Dawley rats were administered chow containing compounds 4, 8 or the aldose reductase inhibitor AL1576. As anticipated, treatment with AL1576 prevented cataract by inhibiting sorbitol formation in the lens. However, compared to untreated rats, compounds 4 and 8 delayed vacuole formation by 20 days and 12 days, respectively, and cortical cataract formation by 8 and 3 days, respectively, without reducing lenticular sorbitol. Using in vitro lens culture in 30 mM xylose to model diabetic “sugar” cataract formation, western blots confirmed that multi-functional antioxidants reduced endoplasmic reticulum stress.

Conclusions/Significance

Multi-functional antioxidants delayed cataract formation in two diverse rat models. These studies provide a proof of concept that a general cataract treatment focused on reducing oxidative stress instead of a specific mechanism of cataractogenesis can be developed.     

四种中草药对大鼠半乳糖性白内障相关酶活性的影响

本实验测定了中草药对大鼠半乳糖性白内障延缓及治疗中五种酶活性的影响。结果表明,在白内障晶状体中,醛糖还原酶的活性明显升高;多元醇脱氢酶,己糖激酶,6磷酸葡萄糖脱氢酶及过氧化氢酶的活性明显降低。在注射半乳糖的同时,分别用黄芩、石斛、菟絲子及玉蝴蝶四种中草药水煎剂灌胃,醛糖还原酶的活性没有明显升高,其余四种酶的活性均基本恢复到正常,表明这四种中草药对半乳糖所致的酶活性异常变化有抑制或纠正作用。     

Diabetes-induced abnormalities of mitochondrial function in rat brain cortex: the effect of n-3 fatty acid diet

High expression of connexins was found in a variety of cancers, but their role is still controversial. We investigated whether connexin43 (Cx43) contributed to bladder carcinogenesis through MAPK activation. In this study, we found that Cx43 expression was significantly increased in bladder cancer tissues and cell line. Overexpression of Cx43 in bladder cancer 5637 cells increased cell proliferation, promoted cell cycle progression, and inhibited apoptosis. Western blot showed that JNK and ERK pathways were dramatically activated in Cx43-overexpressed cells. Conversely, knockdown of Cx43 inhibited cell proliferation by increasing apoptosis and causing cell cycle arrest, concomitant with inhibition of JNK and ERK signaling. In addition, JNK and ERK pathways were also activated in bladder cancer tissues. In conclusion, abnormal high expression and cytoplasmic localization of Cx43 contributed to bladder cancer. Inhibition of Cx43 activity could be a potential therapeutic strategy for preventing the progression of bladder cancer.     

Allopurinol promotes and butylated hydroxy toluene prevents sugar-induced cataractogenesis

Rats fed a high galactose (30% galactose) diet (w/w) or made diabetic by injecting streptozotocin developed mature cataracts in approximately 45 and 90 days, respectively. Addition of allopurinol, a commonly used drug in the therapy of gout, to the high galactose diet or to the normal diet fed to diabetic rats advanced cataractogenesis in both the groups by approximately 50%. Allopurinol fed to control rats did not cause cataract formation. Feeding butylated hydroxy toluene (BHT), an antioxidant, prevented the allopurinol-induced advancement of cataract formation in galactosemic and diabetic rats. Assuming that these results are applicable in human subjects, there is need for caution in using allopurinol for the therapy of gout in diabetic subjects.     

Sex differences in connexin-43 expression in left ventricles of aging rats

Cardiac gap junctions have been implicated in maintaining intercellular electrical and metabolic couplings. The abnormalities in connexin-43 (Cx43) lead to conduction defects and contractile dysfunction. We have evaluated the expression and phoshorylation status of Cx43 in the left ventricular myocardium of male and female 16-month-old rats submitted to 14-week L-thyroxine (T4) treatment. Western blot analysis revealed the presence of fully or intermediately phosphorylated and unphosphorylated forms of Cx43. We have found no significant differences in Cx43 expression and phosphorylation between T4-treated and control untreated animals. However, expression of Cx43 was significantly higher in female compared to male rats. We conclude that T4 administration has no effect on Cx43 expression, but there are sex-dependent differences in Cx43 expression in the left ventricles between aging male and female rats.     

Dose-dependent effects of a genistein-enriched diet in the heart of ovariectomized mice

The isoflavone genistein is used as a pharmacological compound and as a food supplement. The duration and the level of exposure of humans to genistein are considerable. However, the magnitude of genistein-supplemented dietary interventions necessary to induce any changes in the heart has not been studied so far. The aim of this study was to investigate the dose-dependent effects of dietary genistein in the disease- and stress-free mouse heart. Female C57BL/6J mice at the age of 2 months were ovariectomized and randomly assigned to feed on diets with seven different genistein doses (0.01, 0.03, 0.1, 0.3, 1, 3 and 10 g genistein/kg food) for 3 months. Mice with intact ovaries or ovariectomized fed on soy-free diets were used as controls. Ovariectomy led to an increase in body weight, while the two highest genistein doses prevented this increase. Absolute uterus weight was decreased in the ovariectomized group and all genistein groups except for the 10 g/kg food group compared with the intact ovaries/soy-free group. Considering cardiac mass, although the 3 and 10 g/kg food groups had significantly lower absolute heart weight than all other groups, heart-to-body-weight ratios did not differ between these two groups and the intact ovaries/soy-free group, while all remaining groups had smaller ratios. Next, we observed dose-dependent effects of genistein on cardiac gene expression. The present findings indicate that exposure of female mice to the soy isoflavone genistein influences body weight and cardiac mass and gene expression in a dose-dependent manner. Human exposure to dietary genistein supplements may influence cardiac function.     

A dominant negative Cx43 mutant differentially affects tumorigenic and invasive properties in human metastatic melanoma cells

Previous reports have implicated connexin 43 (Cx43) as a tumor suppressor in early stages of tumorigenesis and in some cases as an enhancer of cell migration in later stages. To address the role of Cx43 in melanoma tumor progression, we utilized two melanoma cell lines derived from the same patient in pre‐metastasis (WM793B) and following isolation from a lung metastasis in nude mice (1205Lu). Our results demonstrate a strikingly increased expression of Cx43 in both the pre‐metastatic and metastatic melanoma cell lines that were actively migrating compared to non‐migrating cells. To further investigate the role of Cx43 in these melanoma cells, we overexpressed wild type (wt) Cx43 as well as a mutant dominant negative Cx43 mutant that causes closed channels (T154A). The metastatic 1205Lu cells expressing Cx43‐T154A showed a twofold decrease in colony formation on soft agar while the nonmetastatic WM793B cells showed no significant change. In invasion assays through a collagen matrix, the same Cx43‐T154A 1205Lu cells demonstrated a three‐ to fourfold increase in the invasion index compared to either wt Cx43 or vector control cells. The increase in invasiveness was eliminated by migration towards media with charcoal‐stripped serum, suggesting that migration may be directed towards a lipophilic compound(s). Our findings demonstrate that a dominant negative Cx43 mutant deficient in channel formation exhibits a dual pattern of regulation in metastatic melanoma cells with a decrease in anchorage‐independent growth and an increase in invasive potential. J. Cell. Physiol. 228: 853–859, 2013. © 2012 Wiley Periodicals, Inc.     

Dual benefit of reduced Cx43 on atherosclerosis in LDL receptor-deficient mice

Paracrine cell-to-cell interactions are crucial events during atherogenesis, however, little is known on the role of gap junctional communication during this process. We recently demonstrated increased expression of Cx43 in intimal smooth muscle cells and in a subset of endothelial cells covering the shoulder of atherosclerotic plaques. The purpose of this study was to examine the role of Cx43 in the development of atherosclerosis in vivo. Atherosclerosis-susceptible LDL receptor-deficient (LDLR(-/-)) mice were intercrossed with mice heterozygous for Cx43 (Cx43(+/-) mice). Male mice with normal (Cx43(+/+)LDLR(-/-)) or reduced (Cx43(+/-)LDLR(-/-)) Cx43 level of 10 weeks old were fed a cholesterol-rich diet (1.25%) for 14 weeks. Both groups of mice showed similar increases in serum lipids and body weight. Interestingly, the progression of atherosclerosis was reduced by 50% (P < 0.01) in the thoraco-abdominal aorta and in the aortic roots of Cx43(+/-)LDLR(-/-) mice compared with Cx43(+/+)LDLR(-/-) littermate controls. In addition, atheroma in Cx43(+/-)LDLR(-/-) mice contained fewer inflammatory cells and exhibited thicker fibrous caps with more collagen and smooth muscle cells, important features associated, in human, with stable atherosclerotic lesions. Thus, reducing Cx43 expression in mice provides beneficial effects on both the progression and composition of the atherosclerotic lesions.     

Moderate caloric restriction delays cataract formation in the Emory mouse

Eye lens senile cataract is a major cause of blindness, affecting the elderly in particular. The etiology of the disorder has been elusive, and attempts to delay the onset of senile cataracts have been unsuccessful. The need for more information is underscored by epidemiologists who estimate that the ability to delay cataract formation in humans by only 10 years would eliminate the need for 50% of the cataract extractions performed annually in the United States. The Emory mouse provides the best model for human senile cataracts. Feeding Emory mice a diet that was restricted in calories by approximately 21% delayed the onset of cataracts. This is the first study that demonstrates in vivo the delay of senile-type cataracts. In these animals, aging and cataracts are associated with diverse changes in the proportion of various proteins (particularly 21, 22, 31-34 kDa) and with transformation of proteins from a soluble to an insoluble state. In advanced cataracts, there is a loss of total protein. Within a cataract grade, there is no difference between restricted and nonrestricted animals in relative proportion of specific lens proteins or in amounts of total or soluble proteins. The transition from a clear to cataractous lens appears when the soluble-to-total protein ratio falls below about 0.58. The exclusive use of gamma-crystallin as an indicator of lens viability is questioned. To the extent that cataract formation is due to lens protein oxidation and/or an inability to proteolytically remove damaged protein, it would appear that caloric restriction results in enhanced protection against lens oxidative stress or in prolonged proteolytic function.