Effects Of α-Toxin Of Staphylococcus Aureus On Aquaporin-1 Expression In Cultured Mouse BALB/c3T3 Cells

PrefaceAquaporins ( AQPs) that were found in recent years are specific water transportation proteins located on cellular membrane of mammals. Some researches suggest that AQPs play an important role in the alveolar fluid transportation , among which AQPl is the crucial factor for the pulmonary vasopermeabili-ty.α— Toxin is the main pathogenic agent of Staphylococcus aureus (SA) , which can cause the imbalance of the cellular osmotality. This research is to explore effects of SA α - Toxin on the expression of AQPl in BALB/c mouse fibro-blasts, and approach the correlation between the pathologic mechanism of a -toxin and AQP1.Materials and methodsMaterials. Polyclonal , affinity - purified rabbit antibody for AQPl was purchased from Santa Cruz Biotechnology. Horseradish peroxidase - couple secondary antibody was purchased from Amersham Pharmacia. MEM was purchased from Sigma. Electrophoresis agents were from Bio - Rad, and the bicinchoninic acid (BCA) protein assay kit was from Pierce.Cell culture and Harvest. BALB/c mouse fibroblasts ( BALB/c3T3) were cultured in MEM supplemented with 10% (vlo/vol) FBS,2Mm L - gluta-mine ,40units/ml Penicillinn, and 40ug/ml streptomysin at 37℃ in a 5% CO₂ in air atmosphere until cells were 90% confluend and ready to be used into the ex-periments. 30jxg/ml a — Toxin was added into the experimental group and lasted for 4 hrs or 6 hrs. Observe the changes of the cells under the microscope and then harvest the cells. Cell lysate protein concentrations were determined by the BCA assay.SDS - PAGE and Western blotting. Cell lysates were subjected to SDS/ PAGE and transferred to poly membrane for protein immunoblotting as described. Blots were visualized by enhanced chemiluminescence and autoradio-graphy (Kodak).Statistic analysis. Densitometric analysis of protein immunoblots was expressed as mean ± SEM for each group . Unpaired t tests were performed to assess the effect of different intervention. Date were analyze by SPSS11.0 statistic software.ResultsThe morphology of BALB/c3T3 cells is obviously changed. Normal BALB/c mouse fibroblasts (BALB/c3T3) are mainly in fusiform shape, ceri-oid, cells in dividing phase are round like, and grow in monolayer and stick to the wall. After adding a — Toxin for 4 hours and 6 hours the cells become larger and larger and fragmentations , cytolysis and dead cells increase.AQP1 protein expression is increased by adding a - Toxin. Lighting band can be seen at the site of 28 KD. The expression of AQP1 in cells adding a - Toxin increase with the time.DiscussionAquaporin ( AQP) is the molecular family of a group of selective water channels. Recent researches have found that the distribution of AQP1 in tissues is correlated with the water permeability. Researches showed that mouse with AQP1 gene knock - out had diabetes insipidus, and it has been testified by ex vivo experiments that lack of AQP1 makes the water permeability and reabsorp-tion in proximal convoluted tubule decrease;The expression of AQP1, AQP4,AQP5 mRNA of rats increase in circumnatal period;When pregnancy, congestive heart failure, the expression of AQPl and AQP2 also increase;Adenovirus (ADV) infection can cause low expression of AQPl and AQP5;Lipopolysac-charide (LPS) , TNF - a and Interleukin - 1 (3 can trigger the depressing expression of AQPl in mouse pulmonary capillary endotheliocyte;Tumor growth is inhibited obviously in mouse lack of AQPl. It is clear by pathological analysis that tumor regenerated vessels decrease, while necrosis increase. Experiments in vitro found that the invasiveness of aortic endotheliocyte in mouse lack of AQPl has been weakened obviously. From which we can suppose that abnormal expression and dysfunction of AQPl are correlated with some water metabolic diseases.This experiment employs SA a - Toxin to trigger mouse BALB/c3T3 cells for 4 hours and 6 hours , After that, under microscope we could see cells became round gradually from fusiform shape, the cellular volume became large, cellular plasma was light staining , cells necrosis and ablate, which is correlated with the pore formation of a - Toxin on cellular membrane. We did assay after adding a — Toxin and found that the protein concentration at 4h time point increased compared to that in control group. But the protein concentration at 6h time point decreased compared to that of 4h, which is correlated with the decrease or even stop of protein synthesis in cells after the toxin acting on cells. But Western - blot suggested that the expression of AQPl raised as the time increased. Statistic analysis shows that the inter — group difference has significance. All of which suggest that after a -Toxin added on BALB/c3T3 cells, the expression of AQPl increased instead of decreasing. So we suppose that once cells have contact with a - Toxin, they will trigger the expression of AQPl in order to increase outflow of water which can relieve such damage or change the number and distribution of AQPl directly or indirectly which can result in the protein number increasing on cell membrane while decreasing in cell plasma.Conclusion1. After SA a - Toxin acted on mouse fibroblast, the cellular morphologyhas a significant change.2. SA a - Toxin can make the expression of AQPl in mouse BALB/c3T3 cells.