The Neuroprotective Effects And Mechanisms Of LMWH

Low Molecular Weight Heparins (LMWH) are an important new class of antithrombotic agents with the mean molecular weight 4kDa⁶kDa. In particular, low molecular weight heparins provide pharmacokinetic advantages compared with unfractionated heparin (UFH): longer half-life, better bioavailability, and lower binding to plasma proteins. These properties allow LMWHs to be administered qd. or at most bid. and without laboratory monitoring. The effect of LMWH on Alzheimer's disease (AD) has been investigated from 90's last century. It was previously shown that intraneuronal injection of conventional heparin (MW 12,000) in vitro prevents glutamate-induced calcium release from intracellular stores through its blocking action on I_P3 (inositol-1,4,5-triphosphate) receptors. IP3 is an important second messenger which can bind to IP3 receptors locating in endoplasmic reticulum and cause Ca²⁺ release. Additional researches indicate that IP₃/Ca²⁺ pathway present significant roles in progress of AD.In order to observer the protective effect of LMWH provided by Institute of Biochemical and Biotechnological Drug, Shandong University, we measure the cell viability and the lactate dehydrogenases (LDH) of cultured cortical neurons injured by glutamate, KC1, H₂O₂ and NaN₃. We also study the effect of LMWH against apoptosis and elevation of intracellular calcium induced by glutamate in cultured rat neurons. We measure the fluorescence density of Rhodamine123 which reflected the level of mitochondrial membrane (MMP) in primary rat neurons. The objective of our study is to verify the neuroprotective effect of LMWH and elucidate the possible mechanism. 1. Effect of LMWH on the cellular viability in the cultured rat neurons damaged by glutamate, potassium chloride, hydrogen dioxide and sodium azideTo study the protective effect of LMWH on cultured rat neurons, we make the cultured neuron-damaged model by adding glutamate, potassium chloride, hydrogen dioxide and sodium azide in the culture media of cortical neurons. The viability of the neuron is determined using MTT assay. The lactate dehydrogenase (LDH) leakage induced by glutamate is measured. We find that LMWH increases the viability of the neurons injured by glutamate, potassium chloride, hydrogen dioxide and sodium azide. The cell viabilities at 10μg·ml^-1 of LMWH are 90.1%, 82.7%, 81.9% and 75.9% respectively when cultured neurons are injured by the four above-mentioned toxins. Compared with model groups, the viabilities increase 13.3%, 27.9%, 66.3% and 24.0%, respectively. The LDH leakage of cultured neurons increases significantly when damaged by glutamate. LMWH can prevent LDH leaking from cultured neurons.2. Effect of LMWH on the apoptosis of cultured rat neuronsGlutamate (Glu) can induce apoptosis of cultured rat neurons. To observe the effect of LMWH on apoptotic neurons, we measure the percentage of apoptotic cells by means of flow cytometry (FCM), and we investigate the expression of Bcl-2, Bax and Caspase 3 using Western blot. We find that glutamate can remarkably increase the percentage of apoptotic cells, which shows significant different compared with that of normal control (P<0.01). Glu inhibited the expression of Bcl-2, up-regulated the expressions of Bax and Caspase 3. LMWH can decrease the rate of apoptotic neurons, enhance the expression of Bcl-2 and restrain the expressions of Bax and Caspase 3.3. Effect of LMWH on the intracellular calcium concentration in cultured rat neuronsFura-2/AM, a Ca²⁺ sensitive fluorescent indicator was used to load the cultured neurons, and free intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured. 500μmol·L^-1 of Glu could markedly elevate the free intracellular Ca²⁺ concentration (P<0.01), while LMWH could significantly inhibit the increase of [Ca²⁺]_i induced by glutamate in cultured neurons. The inhibitory ratio of LMWH 0.1μg·ml^-1 on [Ca²⁺]_i is 52.57%.4. Effect of LMWH on Mitochondrial Membrane Potential (MMP) of cultured rat neuronsThe neurons damaged by glutamate can cause the decline of MMP. Rhodaminel23 (Rhl23) aggregates mainly in mitochondrial, so the fluorescent density of Rhl23 measured by flow cytometry (FCM) reflects the level of MMP. LMWH inhibits the decrease of MMP induced by glutamate. Therefore LMWH could influence intracellular metabolism, which is in accord with the results of LMWH against apoptosis. All of these effects could suggest that the potential neuroprotective properties of LMWH. The exact anti-AD mechanism of LMWH remains to do further reserches.