Effects of bushenhuoxue on activities of glutamine synthetase and retinal Müller cells under TGF-β2-intervene hypoxic condition

Abstract

Background Our previous study determined that bushenhuoxue decoction can induce the reactive astrogliosis of retinal Muller cells in rat and further plays a protecting effect on retinal ganglion cells against the damage due to diabetic retinopathy（DR）. However,its mechanism is still being investigated. Objective The present study was to observe the change of activity of glutamine synthetase（GS） and retinal Muller cells in vitro under hypoxic conditions or transforming growth factor-β2 （TGF-β2）-intervened hypoxie conditions and investigate the effect of bushenhuoxue decoction. Methods Retinal Muller cells were isolated from clean 7-day-old SD rats and cultured in the DMEM containing 20% fetal bovine serum for 24 hours and then in free-serum medium for other 24 hours. The cells were grouped based on the next cultivating procedure as follows： normal group （DMEM ＋ 20% serum culture） , TGF-β2 group（ DMEM ＋ 20% serum and 150 ng/L TGF-β2 ） , hypoxia group（ DMEM ＋ 20% serum and 1.0 mmol/L sodium hydrosulfite） , TGF-β2 ＋ hypoxia group, serum with drug cultured group, serum with drug ＋ TGF-β2 ＋ hypoxia group. The uhrastructure of retinal Mailer cells was observed under the transmission electron microscope. The content of lactate dehydrogenase （LDH） in extracellular fluid was detected to evaluate the activity of retinal Mailer cells,and the activity of GS was assessed with 490-enzyme-labeled instrument. Results The uhrastructure of retinal Muller cells in hypoxia group showed obvious pathological damages in hypoxic conditions,including vacuolization of cytoplasm, deformation and agglutination of cell nucleus,destroy of cell organs and increase of glucogen in microvilli. However, these changes were mild. Compared with normal group,the release of LDH was significantly enhanced, and the activity of GS was obviously weakened in TGF-β2 group, hypoxic group and TGF-β2 ＋ hypoxic group （ P 〈 0.05 ）. Compared with hypoxic group, the LDH level was significantly increased, whereas the activity of GS was obviously decreased in TGF-β2 ＋ hypoxic group （ P 〈 0.05 ）. In comparison with TGF-β2 group or hypoxia group, the LDH content in TGF-β2 ＋ hypoxic group was considerably lower （P 〈 0. 05）. In all the groups with bushenhuoxue ＋ serum,the release of LDH was declined in comparison to without bushenhuoxue ＋ serum groups at 24 and 48 hours （P〈0.05）,and bushenhuoxue ＋ serum groups also significantly enhanced the activity of GS both in 12 hours of normal control condition and 24 - 48 hours of TGF-β2-intervened hypoxic conditions （P 〈 0.05 ）. Conclusion TGF-β2 can aggravate the activity of GS and retinal Mailer cells in hypoxic condition. Bushenhuoxue medicine-containing serum can strengthen the activity of retinal MUller cell and GS. Bushenhuoxue may be a new approach to the prevention and treatment of DR. Key words: bushenhuoxue, transforming growth factor-β2, hypoxia; retinal Muller cells; lactate dehydrogenase; glutamine synthetase