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Background & objectives: Cytoskeletal proteins are deregulated during oxidative stress and cataract formation. However, estrogen which protects against cataract formation and harmful effects of oxidative stress has not been tested on the cytoskeleton of lens epithelial cells (LECs). The current study was undertaken to assess if the protection rendered to LECs by estrogen was mediated by preserving the cytoskeletal proteins. Methods: Oxidative stress was induced by 50 μM of H2O2 in cultured goat LECs (gLECs) and effect of 1 μM 17β-estradiol (E2) was tested. After treatment, morphological analysis of cells was carried out using haematoxylin-eosin staining and cell density was also quantified. Cell viability was determined using Hoechst (Ho), YO-Pro (YP) and propidium iodide (PI). F-actin and vimentin were localized using phalloidin and anti-vimentin antibody, respectively, and viewed under fluorescence microscopy. Vimentin was further analysed at protein level by Western blotting. Results: H2O2 led to increased condensation of nucleus, cell death and apoptosis but these were prevented with pre- and co-treatment of E2 with increase in cell viability (P<0.001). E2 also prevented H2O2 mediated depolymerization of cytoskeleton but was not able to reverse the changes when given after induction of oxidative stress. Interpretation & conclusions: Our findings showed that E2 helped in preventing deteriorating effect of H2O2, inhibited cell death, apoptosis and depolymerisation of cytoskeletal proteins in LECs. However, the exact mechanism by which estrogen renders this protection to cytoskeleton of lens epithelial cells remains to be determined.
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Specimens of the anterior lens capsule with an attached monolayer of lens epithelial cells (LECs) were obtained from patients (n052) 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 (n011) 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.
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Background: Intestinal permeability increases early in the course of acute pancreatitis and is associated with sepsis and organ failure. Aim: To evaluate the intestinal permeability (IP) and anti-endotoxin antibodies immunoglobulin G and A (AEA IgG and A) in severe acute pancreatitis (SAP) as compared to healthy controls and determine their significance in relation to various complications of SAP. Methods: IP was measured by urinary lactulose/mannitol (LM) excretion ratio and antiendotoxin antibodies by Endocab ELISA kit at days one and seven of admission (D1 and D7). Results: Thirty one patients of SAP [mean age (42.0±15.8) years, APACHE II scores (8.8±5.4) and CT severity index (6.4±2.0)] were included in this study. Infected pancreatic necrosis was detected in 13 (42%) patients of whom three died. Six died of persistent organ failure. Median values of LM ratio at D1 and D7 were similar to those in healthy controls. Patients experiencing complications [organ failure (4/9, 44%), infected pancreatic necrosis (5/10, 50%) and death (1/ 2, 50%)] manifested a substantial increase in their intestinal permeability at D7. Anti-endotoxin antibodies IgG were lower (p=0.003) in patients than the controls at admission. AEA IgG were lower (p=0.03) in non-survivors as compared to survivors at D7. Conclusion: Patients experiencing complications of severe acute pancreatitis showed an increase in intestinal permeability. Higher endotoxemia predicted poor outcome in severe acute pancreatitis.