Inhibitory effects of α-crystallin on optic nerve astrocytes proliferation, activation and secretion stimulated by lipopolysaccharide / 中华实验眼科杂志

Background Glial scaring induced by the activation and proliferation of astrocytes after optical nerve damage is one of causes of neural axons difficult to regeneration.Researches showed that α-crystallin can promote the regeneration and pass through scaring zone of retinal ganglion cells (RGCs) axons,and we speculate α-crystallin protect optical nerve tissue against scaring process.Objective This study was to investigate the influence of α-crystallin for the activation and secretion of inflammatory factors of astrocytes.Methods Optical nerver tissue was isolated from 3-5 day-old SPF Long Evans rats to culture and purify astrocytes.The cells were identified by detecting the expression of glial fibrillary acidic protein (GFAP) with immunofluorescence technique.The cells were cultured with regular culture medium in the normal control group,and 5 μg/ml lipopolysaccharides (LPS) was added in the LPS group,while 5 μg/ml LPS and 1 ×10-4 g/L α-crystallin were added in the α-crystallin group,and the cells were consecutively cultured for 24 hours.The proliferation (absorbance,A) of the cells was assayed by cell counting kit-8 (CCK-8).The expression of GFAP in the cells was detected by immunofluorescence technique and quantitated by Western blot.The contents in the cell supernatants of tumor necrosis factor-α (TNF-α) and interleukin-1β (IL-1β) were detected by ELISA.Results The morphology and size were well-proportioned in 3-4 generation of cells with the GFAP positive rate over 95％.The A values were 1.335±0.070,1.643±0.069 and 1.390±0.004 in the normal control group,LPS group and α-crystallin group,and the A values in the LPS group were significantly higher than those in the normal control group and α-crystallin group (t =3.315,3.681,both at P＜0.05).Immunofluorescence examination showed that the fluorescence intensity was evidently enhanced in the LPS group compared with the normal control group and α-crystallin group and presented the largest cell bodies in the LPS group.The relative expressions of GFAP in the cells were 0.851 ±0.076 in the LPS group,which were higher than those in the normal control group and α-crystallin group (0.786±0.091,0.569±0.049).Compared between the LPS group and α-crystallin group,there is a significant difference between the two groups (t =3.115,P＜ 0.0l).In addition,compared with the LPS group,the contents of TNF-α and IL-1β in the suspensions were significantly reduced in the normal control group and α-crystallin group (all at P＜0.05).Conclusions α-Crystallin protein can inhibit the activation and secretion of optic nerve astrocytes stimulated by LPS.

Effects of α-crystallin on bioactivity of iNOS in the activated retinal microglial cells / 中华创伤杂志

Objective To investigate effects of α-crystallin on proliferation of lipopolysaccharide (LPS)-activated retinal microglia and bioactivity of iNOS.Methods The retinal microglial cells cultured in vitro were analyzed and their purity was identified by cell immunofluorescence and flow cytometry.After microglia cells being intervened using LPS and α-crystallin at various concentrations,influence of α-crystallin on activity of LPS-activated retinal microglia was detected by MTT method and level of NO was measured by RT-PCR to observe changes of iNOS expression in microglia.Results Purity of primary cultured microglial cells was 94.15％ by GSA-IB4 immunohistochemical identification and 93.34％ by CD11b flow cytometry.α-crystallin of 10-4g/L awakened activity of microglia induced by 10-6g/L LPS (P ＜ 0.01).Expressions of iNOS protein and mRNA showed significant decrease in combined treatment group (P ＜ 0.05).Conclusion In clinical condition,α-crystallin decreases the harm of microglial cells on retinal ganglial cells (RGCs) after optical nerve injury by inhibiting the microglia cells to produce NO and iNOS.

Difference analysis of proteome between diabetic cataract and age related cataract / 中华实验眼科杂志

Background With the changes of diet and living style,the diabetes has become the major diseases affecting human health.Diabetic cataract is a common complication of diabetes. Objective The present study was to investigate the difference of lens proteomics between diabetic cataract and age related cataract using two dimensional electrophoresis (2-DE) and mass spectrometry in order to postpone happening of diabetic cataract and offer the effective approach to the prevention and therapy of diabetic cataract. Methods The lenses were obtained from 8 diabetic patients and 12 age-related cataract patients during the surgery to extract the protein by lysis and centrifugation.The lens proteins were separated using immobilized pH gradients 2-DE.Image analysis was carried out using PDQuest Advanced-8.0.1 software package.Significant difference of the crystallines was identified by matrixassisted laser adsorption/ionization time of-flight-mass spectrometry (MALDI-TOF-MS) and peptide mass fingerprint combined with protein database. Results The maps of 2-DE showed that lens proteins of diabetic cataract and age related cataract were in the section of pH 5-9 with the relative molecular weight 14000-97000;while relative molecular weight of more abundant crystalline was localized at 20000-31000.About 3 differential protein spots were detected by image analysis software.Two crystallines,αB and βB1 crystallin,were identified using MALDI-TOF-MS.Conclusions Proteomic analysis of lens can be accomplished and the proteins can be well separated,moreover,differential proteins can be analyzed using 2-DE and mass spectrometry between diabetic cataract and age related cataract.These results indicate that αB and βB1 crystallin proteins accelerate the development of diabetic cataract.This technique offers a new avenue for clarity of lens proteins of diabetic cataract other than age related cataract.

The genetic and molecular basis of congenital cataract / Base genética e molecular da catarata congênita

Congenital cataracts are one of the most treatable causes of visual impairment and blindness during infancy, with an estimated prevalence of 1 to 6 cases per 10,000 live births. Approximately fifty percent of all congenital cataract cases may have a genetic cause. All three types of Mendelian inheritance have been reported for cataract; however, autosomal dominant transmission seems to be the most frequent. The transparency and high refractive index of the lens are achieved by the precise architecture of the fiber cells and the homeostasis of the lens proteins in terms of their concentration, stability, and supramolecular organization. Research on hereditary congenital cataract led to the identification of several classes of candidate genes that encode proteins such crystallins, lens specific connexins, aquaporine, cytoskeletal structural proteins, and developmental regulators. The purpose of this study was to review the literature on the recent advances made in understanding the molecular genetic basis of congenital cataracts.

A catarata congênita é uma das principais causas tratáveis de cegueira na infância, com prevalência estimada em 1 a 6 casos por 10.000 nascidos vivos, sendo a causa hereditária responsável por até metade dos casos. Dentre os padrões de herança já descritos para a catarata, a transmissão autossômica dominante é a mais frequente. A transparência e o alto índice refrativo do cristalino são resultados da disposição regular das fibras lenticulares e do equilíbrio homeostático; além da estabilidade e da organização supramolecular das proteínas do cristalino. Pesquisas sobre catarata congênita hereditária têm levado à identificação de várias classes de genes responsáveis pela codificação das proteínas do cristalino, tais como: cristalinas, conexinas, aquaporinas, proteínas do citoesqueleto e reguladores do desenvolvimento. O objetivo deste estudo foi a revisão da literatura sobre os recentes avanços na compreensão da base genética e molecular da catarata congênita.

Effects of intravenous injection of α-crystallin on retinal ganglion cells and some important organs / 中华眼底病杂志

Objective To investigate the effects of intravenous injection of α-crystallin on retinal ganglion cells (RGC) and some important organs of the Long Evans rats. Methods RGC were retrogradelabeled by fluorogold through bilateral superior colliculus and lateral geniculate body for seven days before optic nerve crush injury. Twenty-three Long Evans rats were used for this study, including three rats of normal control group and 20 rats of experimental group. Twenty rats were randomly divided into saline control group and three α-crystallin injection groups, which received tail vein injection of 1.25 ml isotonic saline and three different concentrations (1 × 10-2 , 1 × 10-1 and 1 g/L) of α-crystallin respectively, once every two days and totally seven times. After two weeks, the labeled RGC were counted, and the pathological changes on liver, kidney, brain, spleen and the lungs were investigated. Results Compared with the normal control group, although the number of RGC markedly decreased after two weeks of optic nerve crush injury in every group, the number of RGC in α-crystallin-treated groups was more than those in the saline control group. There were 2074± 150 RGC per mm2 in normal control group, 85 ± 15 RGC per mm2 in saline control group, 124±26 RGC per mm2 in 1 × 10-2 g/L α-crystallin group, 128± 31 RGC per mm2 in 1 × 10-1 g/L α-crystallin group, 164 ± 20 RGC per mm2 in 1 g/L α-crystallin group (F= 18. 660,P＜0. 01). No congestion, swelling, inflammation and other pathological changes were found in liver,kidney, brain, spleen and lung. Conclusions Intravenous injection of α-crystallin protein has protective effects on RGC after the optic nerve crush injury, and no significant effects on important organs.

A Case of Crystalline Keratopathy in Monoclonal Gammopathy of Undetermined Significance (MGUS)

A 62-year-old female visited our clinic with progressively decreased vision in both eyes beginning 12 years prior. Idiopathic corneal opacity in all layers of the cornea was found in both eyes. One year later, we performed penetrating keratoplasty on the undiagnosed right eye. During post-surgical follow-up, corneal edema and stromal opacity recurred, and penetrating keratoplasty was performed two more times. The patient's total serum protein level, which had previously been normal, was elevated prior to the final surgery. She was diagnosed with monoclonal gammopathy of undetermined significance. We made a final diagnosis of monoclonal gammopathy-associated crystalline keratopathy after corneal biopsy. Monoclonal gammopathy-associated crystalline keratopathy is difficult to diagnose and may lead to severe visual loss. A systemic work-up, including serologic tests like serum protein or cholesterol levels, is needed in patients with unexplainable corneal opacity.

Expression of αA-and αB-crystallin protein in retina after blue-light exposure / 中华眼底病杂志

Objective To observe the expression of αA-and αB-crystallin in retina after blue-light exposure.Methods Forty female Wistar rats were divided randomly into 4 groups:control group,and blue-light exposure for 6,12,and 24 hours groups,with 10 rats in each group.The rats in the control group were not intervened.The other three groups of rats were exposed to blue fluorescent lights for 6,12,and 24 hours respcetively.Then the rats were kept in darkness for 12 hours.The globes were enucleated after anaesthesia.The immunohistochemistry and Western blot were performed to detect the expression of αA-and αB-crystallin in retina.Results The absorbance value(A value)of retina αA-crystallin was 1.40573±0.70748 in the control group,and were 4.317 51±0.412 97,7.397 08±1.947 90,9.634 32±2.377 61,respectively in the other 3 groups;the difference among the groups was significant(F=24.569,P<0.001).The A value of retina αB-crystallin is 0.129 36±0.033 93 in the control group,and were 0.507 17±0.117 55,7.345 43±2.292 97,4.042 26±3.890 23,respectively in the other 3 groups;the difference among the groups was significant(F=40.102,P<0.001).The results of Western blot showed that the expression of αA-and αB-crystallin in groups with blue-light exposure was obviously higher than that in the control group.Conclusions Blue light may up-regulate the expression of αA-and αB-crystallin in rats' retina.

Accumulation and Aberrant Modifications of alpha-Crystallins in Anterior Polar Cataracts

Crystallins are the major proteins found in the lens, and the localization of specific crystallins is well known. Overexpression and accumulation of alphaB-crystallin has been observed in response to stress conditions or in certain diseases, such as brain tumors and neurodegenerative diseases. The purpose of this study was to examine whether alpha-crystallins are modified during pathological myofibroblastic changes in lens epithelial cells. Lens epithelial cells attached to the anterior capsules of patients with nuclear or anterior polar cataracts were analyzed quantitatively for alpha-crystallin proteins and mRNAs using Western blot and RT-PCR analysis., respectively. The degree of modification of alpha-crystallins was determined by 2-dimensional gel electrophoresis followed by Western blotting. Higher molecular weight protein bands that were immunoreactive to anti-alphaA- and anti-alphaB-crystallin antibodies around 45 kDa accumulated more in the anterior polar cataract samples than in those with the nuclear type of cataracts. Also monomeric alphaB-crystallins accumulated more in lens epithelial cells of patients with anterior polar cataracts. By comparison, no significant changes were found in the levels of the mRNAs encoding alphaA- and alphaB-crystallins in the different types of cataracts. Both alphaA- and alphaB-crystallin proteins seemed to undergo more extensive modification in anterior polar cataracts. Conclusion. In addition to fibrotic changes, which accompany increased levels of extracellular matrix molecules, accumulation and abnormal modification of alpha-crystallins might be implicated in the pathogenic mechanism of this type of cataract.

Protective effects of mixed crystamn on injured retinal ganglion cells after optic nerve injury in Long-Evans rats / 中华创伤杂志

Objective To investigate the effect of intraocular injection of mixed crystalline in vivo on survival of retinal ganglion cells(RGCs)after optic nerve cut.Methods Twenty Long-Evens rats were divided into normal control,and 7 day,14 day,and 21 day groups after optic nerve was cut off.There were 5 eyes in each group.Mixed crystallin(1x10~(-4)g/L)and isotonic saline solution were injected into the vitreous of fight eye and left eye respectively.The number of RGCs was counted 7,14 and 21 days after optic nerve cut.Results The density of RGCs declined clearly 7 days after optic nerve cut.In the group with crystallin injection,the density of RGCs declined to 71%,32% and 15% of the control group repec- tively,much higher than that of the controls on days 7,14 and 21 after optic nerve was cut off(P

The effect of sodium selenite on heat induced aggregation of lens crystallin / 解放军医学杂志

Objective To study the effect of sodium selenite on heat aggregation of lens crystallin. Methods 12 fresh and intact bovine lenses were incubated with different concentrations of sodium selenite for 72 hours. The lenses of the control group were incubated without sodium selenite for the same length of time. After removing the capsule homogenate, the ?-, ?- and ?-crystallin from the lenses were separated by gel filtration chromatography. After the separation and purification, the crystallin were heated at 60℃ for 30 minutes. Then the absorption value at 360nm was measured again. The difference in the percentage of the absorption value after aggregation was analyzed. Results Comparing with the control group, the lenses incubated with sodium selenite showed earlier opacification in a concentration-dependent manner. After the separation by gel filtration chromatography, the ?-crystallin peak showed an antelocation on the chromatogram, suggesting that there was some high molecular aggregation formation. The aggregation of selenite-treated fractions showed a concentration-dependent increasing absorption compared with normal control portions (P

Effect of crystallines on survival and growth of rat retinal ganglion cells in vitro / 中华创伤杂志

Objective To study the effect of crystallins on survival and growth of rat retinal ganglion cells (RGCs) in vitro and study elementarily the exact substance of the injured lens promoting RGCs survival and axonal regeneration. Methods RGCs in Long Evans rats were cultured in DMEM, crystallins and crystallins activated macrophage-conditioned media. The growth regularity and survival time of RGCs in vitro were observed under phase-contrast microscope. The number of RGCs with processes, length of the longest processes and cells activity were measured when RGCs were cultured for 1, 3 and 5 days respectively. Results (1) RGCs could survive for 12-14 days in DMEM containing crystallins, and the survival time of RGCs was longer significantly than other two groups (P

Phasic change of water soluble major crystallins with aging in rats / 中国病理生理杂志

AIM: To study the correlation between phasic change of the relative quantity of major crystallins with aging in rats. METHODS: Six groups of SD ra ts (age 1 d, 8 d,2 weeks,8 weeks,8 months and 1.5 years) were raised routine ly. Water soluble crystallins were extracted and separated by two-dimensional polyacrylamide gel electrophoresis. After comassize blue staining,the crystallins patterns were sc anned and analyzed. RESULTS: (1) Out of the eighteen water solu ble major rat cr ystallins tested in each group, seven showed gradual phasic changes in relative quantity of crystallins, but there were no significant changes in total quantity of water soluble crystallins. (2) Phasic changes in these crystallins pres ented four different patterns: increasing (?B 4??B 2??A 2??A 1), decreasi ng (? 7?? 8?? 2,3 ?? 5,6 ),relatively stable(?A 3??B 5) , and irregular. (3) The ratio of ?B 4 /?A 2 increased gradually with the r at aging process. CONCLUSION: T he gradual phasic changes in relative quantity of crystallins reflect the aging status of rat crystalline.