The Neuroprotective Effcts And Mechanism Of LMWH-SOD

LMWH-SOD conjugate is gained by employing LMWH (low molecular weight heparin, LMWH, M.W. 4kDa~6kDa) to chemically modify SOD (superoxide dismutase, SOD). Acting as a scavenger towards superoxide ion, the conjugate has an efficient antioxidant action. Oxygen free radicals or oxidants have been implicated in the development of many neurological disorders and brain dysfunctions, which arise from the result of the overproduction of oxygen radicals and inactivation of detoxification systems. The free radicals can induce lipid peroxidation, which plays a key role in pathophysiological injuries of cerebral ischemia/reperfusion. Hence, acting as a scavenger towards free radicals, LMWH-SOD has a broad prospect for application. In the present study, we cultured the cerebral neurons and cerebellar granule cells of neonate rats in vitro, stimulated the pathological process in cerebral ischemia /reperfusion, and observed the neuroprotive effects of LMWH-SOD. In addition, we also observed it's action on the membrane fluidity and apoptosis of the cells.1. Effect of LMWH-SOD on the cellular viability in the cultured cortical neurons damaged by oxygen glucose deprivationIn this study, we observed the protective effects of LMWH-SOD on the injury induced by oxygen and glucose deprivation (OGD) in the cultured cortical neurons. The injury model of cultured neuron was made by the administration of sodium dithionite and glucose-deprived Earle's solution. The sodium dithionite and glucose-deprived Earle's solution were administrated for 6 hours in this model. Theviability of the neuron was analyzed using colorimetric MTT assay. The content of malondialdehyde (MDA) was determined by TBA method. The levels of nititric oxide (NO) and the activity of lactate dehydrogenase (LDH) in the medium were measured by NO and LDH kits.After the injury induced by oxygen and glucose deprivation, the viability of the neurons and the content of MDA, LDH, NO are greatly decreased. LMWH-SOD could obviously increase the MTT value and decrease the LDH activity. In the same time, LMWH-SOD at the concentration of 200u -ml^ lOOu -ml"1 ^ 50u 'ml'1, could increase the level of NO at the concentration of 200u ? ml'^ lOOu ? ml"1 and decrease the level of MDA content elevated by OGD in cultured cortical neurons in vitro. Compared with SOD(100u ? ml"1), LMWH-SOD(100U ? ml"1) only have a superiority only on the data, but there isn't the difference in statistics. The neuron protection of LMWH-SOD from injuries induced by oxygen and glucose deprivation may be related to decreasing the level of NO and the content of MDA.2. Effect of LMWH-SOD on the changes of membrane fluidity in cultured cortical neurons in ratsSince the cell membrane requires good fluidity to maintain homeostasis and metabolism in the body, fluidity is a useful index of cellular injury. In this study, we observed the effect of LMWH-SOD on the changes of membrane fluidity in cultured cortical neurons after the injuries induced by oxygen and glucose deprivation. The quantitative measurement of membrane fluidity was conducted with the fluorescence polarization indicator DPH (1, 6-phenyl 1-1,3,5-hexatriene) as a fluorescence probe. Preparations were suspended in PBS mixed with DPH , and incubated at 37 "Cfor 30 min. Fluorescence polarization was determined using a fluorescence spectrophotometer equipped with rotating polarizing filters. Samples were excited at 360 nm, and emission intensity was read at 435 nm, and calculated the values for polarization (P) ^ fluorescence anisotropy (y) and mean microviscosity(/7) of the samples. After labelled by DPH, the P, y and rj of the membranes can reflect the fluidity. The higher the P, y and rj are, the lower the membrane fluidity is.The result showed that P, y and r\ of the membranes greatly decreased after the injury of OGD. Since LMWH-SOD at the concentration of 200u ? ml"1 n 1 OOu ? ml"1 â– > 50u ? ml"1 obviously increased the P, y and rj of the membranes in cultured cortical neurons, we could conclude that LMWH-SOD improved the membrane fluidity. Compared with SOD(100u ? ml"1), LMWH-SOD(100u ? ml"1) seemed to have a superiority only on the data, but there was no difference in statistics.3. Effect of LMWH-SOD on the apoptosis of cultured cerebella granule cells in neonate ratsAfter cerebella granule cells from 7-day-old Wistar rats were cultured for 6-8 days, administrated sodium dithionite and glucose-deprived Earle's solution for 2 hours, then replaced the medium of incubated cells with basal Eagle medium for 18 hours. Necrosis was distinguished by MTT assay and apoptosis was determined by three methods: determination of the DNA ladder with agarose gel electrophoresis, the numbers of apoptotic cell with flow cytometry, and the expression of Bcl-2 in cells by immuneocytochemistry. After the injury, the cells exhibited typical DNA ladder on agarose gel electrophoresis, the number of apoptosis cells increased and the expression of Bcl-2 decreased..LMWH-SOD could reduce the DNA fragments, the number of apoptotic cells and increase the expression of Bcl-2, exerted neuroprotective action against the apoptosis induced by oxygen-glucose deprivation.It is concluded that as an effective scavenger of free radicals, LMWH-SOD can protect the neurons from OGD injury, increase the membrane fluidity and exhibit it's effect against apoptosis.