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.

Impact of diabetes on alpha-crystallins and other heat shock proteins in the eye.

Diabetes and its related complications represent a major growing health concern and economic burden worldwide. Ocular manifestations of diabetes include cataractogenesis and retinopathy, the latter being the leading cause of blindness in the working-age population. Despite numerous studies and recent progress, the exact pathophysiology of the disease remains to be fully elucidated and development of new and improved therapeutic strategies for this chronic condition are greatly needed. Heat shock proteins (Hsps) are highly conserved families of proteins, which are generally regarded as protective molecules that play a wide variety of roles and can be expressed in response to different types of cellular stresses. In recent years, numerous studies have reported their implication in various ocular diseases including diabetic retinopathy. The present review focuses on the potential implication of Hsps in ocular diabetic complications and discusses their specific mechanisms of regulation with respect to their expression, functions and alteration during diabetes. The review will conclude by examining the potential of Hsps as therapeutic agents or targets for the treatment of diabetic retinopathy.

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.

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.