| Tetramethylpyrazine nicolate (TMPN) is a new compound with the potential effects on the treatment of cerebrocardiac vascular disease, derived from tetramethylpyrazine(TMP). Tetramethylpyrazine is one of the major efficient components abstracted from the Chinese traditional medicine herb Chuanxiong (Ligusticum wallichii Franchat). Since 1970s, TMP is widely used in China as an oxygen free radical scavenger, an agent of antithrombusa and antihypertension, and a new kind of calcium antagonist for the treatment of ischemic cerebrocardiac vascular disease. In recent years, more studies indicate that TMP can penetrate through the blood-brain barrier, consequently be enriched in the brain, especially in the brainstem. Because the methyls moiety in TMP molecule can be easily oxygenized and transformed to metabolin with high water solubility which can be eliminated quickly, the half-life of TMP is very short and its bioavailability is low in vivo. According to the structure-activity relationship of TMP, we synthesized TMPN by remaining its pharmacophore and linking nicotinoyl to 2-methyl. Nicotinic acid has potent action on decreasing lipid and anti-thrombois. By keeping glycolysis during periods of ischemia and improving subendocardial blood flow during reperfusion, nicotinic acid improved the function of myocardium suffered from ischemia-reperfusion. It has much possibility that the pharmacodynamic action and liposolubility of TMPN would be increased through the synergistic effect of nicotinic acid, accordingly with longer half-life and higher bioavailability. Thereby, we supposed TMPN have stronger effects in central nervous system (CNS). In this paper, we used cultured primarily cortical cells and humanneuroblastoma SH-SY5Y cells (SH-SY5Y), which were suffered from oxidative stress by hydrogen peroxide and oxygen and glucose deprivation, to observe the protection of tetramethylpyrazine nicolate on the injured nerve cells, consequently elucidated the possible mechanisms in vitro. 1 Protection of TMPN on SH-SY5Y cells and primarily cortical cells injured by H2O21.1 Effect of TMPN on viability of SH-SY5Y cells and primarily cortical cells injured by H2O2SH-SY5Y cells and primarily cortical cells cultured in 96-well plate were bathed in culture media containing 150umol'L"'and 75U.1T1O1-L"1 H^Oafor 12 hours respectively, at the same time nerve cells were treated with TMPN and TMP. Then the optical density of each well was determined at 570nm. Results indicated that the viability of nerve cells descended significantly after oxidative damage (PO.01), TMPN at the dosage of lOumol-L'1, lOOumol-L"1 and TMP at the dosage of lOumol-L"1 effectively inhibited this descent induced by H2O2 (PO.05).1.2 Effect of TMPN on LDH activity in extra-cellular media of SH-SY5 Y cells and primarily cortical cells injured by H2O2We collected the extra-cellular bathing media of each experimental condition and determined the activity of LDH by kit. Results showed that after oxidative damage, the permeability of cellular membrane became serious and LDH efflux increased, then activity of LDH in extra-cellular markedly ascended (P<0.01). After the treatment of TMPN at the dosage of lumol-L'1, lOu-molL'1, 100umolL"'and TMP at the dosage of lOujnol-L"1, the leaking of LDH induced by H2O2 attenuated and LDH level in extra-cellular decreased (PO.01).1.3 Effect of TMPN on MDA contents and SOD, GSH-Px activities of SH-SY5Y cells and primarily cortical cells injured by H2O2After being damaged by H2O2, SH-SY5Y cells and primarily cortical cells cultured in 24-well plate were pelleted by low-speed shake and homogenized in lysis buffer solution. The homogenate was centrifuged at 10000 rpm at 4°C for 30min and the supernatant was used for enzyme assays. The MDA, SOD and GSH-Px assays were performed using kit, at the same time protein content of the samples was measured using the method ofcoomassie brilliant blue. Results showed that after cultures were incubated with H2O2, the content of MDA increased and the activity of SOD and GSH-Px decreased markedly (P<0.01). lumol-L"1, lOumol-L'1, lOOumol-L"1 TMPN and 10umol-L"! TMPsignificantly inhibited the change of MDA (P<0.01)and SOD and GSH-Px (P<0.05).1.4 Effect of TMPN on the intracellular concentration of free calcium of SH-SY5Y cells injured by H2O2SH-SY5Y cells were cultured in 6-well plate and incubated with 150|xmol-L"' H2O2 and TMPN for 12 hours. After the culture medium was removed, cells were collected from dishes and washed with PBS containing 2% glucose of 4°C. Then cells were incubated for 45 min at 37°C with a solution containing O.lmmolL'1 Fluo-3AM. After the cells were washed with PBS containing 2% glucose, the fluorescent intensity was measured using Flow Cytometry and concentration of free calcium was reflected at the relative values. Results showed that after oxidative damage, the intracellular concentration of free calcium increased significantly (P<0.01), lOumol-L'1, lOOumol-L*1 TMPN and lOumol-L"1 TMP inhibited the overload of concentration of free calcium induced by H2O2 (P<0.05).1.5 Effect of TMPN on apoptosis of primarily cortical cells injured by H2O2 Approximately 2 X 106 primarily cortical cells were harvested and DNA wasextracted. The cultured cells were treated with lysis buffer and proteinase K, followed with RnaseA. Then DNA was separated by electrophoresis in 1.0% agarose gel and stained with ethidium bromide. DNA was visualized using gel imaging system. After oxidative damage, apoptosis in primarily cortical cells was induced. Meanwhile, the typical apoptosis characteristic—a ladder pattern of DNA fragmentation appeared. lOumol-L'1, lOOnmol-I/'TMPN and lOumol-L"1 TMP partly blocked cell apoptosis and DNA fragmentatation.1.6 Effect of TMPN on the expression of Bcl-2 in SH-SY5Y cells and primarily cortical cells injured by H2O2After treatment with H2O2 and TMPN, SH-SY5Y cells and primarily cortical cells cultured on cover slips were washed and fixed. Then the expression of Bcl-2 protein was detected using SABC immunohistochemistry. The results showed that Bcl-2 leveldecreased markedly in nerve cells after oxidative damage (P<0.01), lOjimol-L"1, lOOumol-L"1 TMPN and lOumol-L'1 TMP prevented the down-regulation of Bcl-2 level induced by H2O2 (P<0.01).2 Protective effect of TMPN on SH-SY5Y cells and primarily cortical cells injured by oxygen and glucose deprivation2.1 Effect of TMPN on viability of SH-SY5Y cells and primarily cortical cells injured by oxygen and glucose deprivationSH-SY5Y cells and primarily cortical cells cultured in 96-well plate were bathed in low glucose DMEM containing 0.5mmol-L"1and 1.5mmol-L"1sodium dithionite for 12 hours respectively, at the same time nerve cells were treated with TMPN and TMP. Then numbers of surviving nerve cells were measured using MTT method. The results indicated that hypoxia and hypoglycemia impairment caused viability decrease of nerve cells significantly (PO.05), but TMPN at the dosage of 10u.mol-lA lOOumol-L^and TMP at the dosage of lOumol-L'1 inhibited the decrease of cells viability (P<0.05).2.2 Effect of TMPN on LDH in extra-cellular media of SH-SY5Y cells and primarily cortical cells injured by oxygen and glucose deprivationWe collected the extra-cellular bathing media of each experimental condition and determined the activity of LDH using kit. Results showed that after oxygen and glucose deprivation, the permeability of cellular membrane became serious and LDH efflux increased, so activity of LDH in extra-cellular media markedly elevated (P<0.05). TMPN at the dosage of lumol-L"', lOumol-L'1, lOOumol-L"1 and TMP at the dosage of lOumol-L"1 prevented the leaking of LDH induced by hypoxia and hypoglycemia damage (P<0.05).2.3 Effect of TMPN on MDA contents and SOD, GSH~Px levels in SH-SY5Y cells and primarily cortical cells injured by oxygen and glucose deprivationAfter being treated with every experimental condition, SH-SY5Y cells and primarily cortical cells cultured in 24-well plate were homogenized in lysis buffer solution. The homogenate were centrifuged at 10000 rpm at 4"C for 30min and the supernatant was used for enzyme assays. The MDA, SOD and GSH-Px assays were performed using kit, at the same time protein content of the samples was measured usingthe method of coomassie brilliant blue. Results showed that after cultures were incubated with sodium dithionite, the content of MDA increased and the activity of SOD and GSH-Px decreased markedly (PO.01). While lumol-L"1, lOumol-i;1, lOOpmol-L'1 TMPN and lOujnol-L"1 TMP significantly inhibited the generation of MDA (P<0.05) and the reduction of SOD and GSH-Px activity (P<0.05).2.4 Effect of TMPN on the intracellular concentration of free calcium of SH-SY5Y cells injured by oxygen and glucose deprivationSH-SY5Y cells were cultured in 6-well plate and incubated with 1.50m mol-L"1 sodium dithionite and TMPN for 12 hours. After the culture medium was removed, cells were collected from dishes and washed. Then cells were incubated for 45 min at 37°C with Fluo-3AM solution. The fluorescent intensity of cslls was measured by Flow Cytometry. Results showed that after oxygen and glucose deprivation, the intracellular concentration of free calcium in SH-SY5Y cells increased significantly (PO.01), lOumol-L'1, lOOumol-L"1 TMPN and lOumoM/1 TMP inhibited the overload of intracellular concentration of free calcium induced by hypoxia and hypoglycemia injury (PO.01).2.5 Effect of TMPN on apoptosis in primarily cortical cells injured by oxygen and glucose deprivationAfter primarily cortical cells from each experimental condition were harvested the DNA in cells was extracted and separated by electrophoresis in 1.0% agarose gel and stained with ethidium bromide. DNA was visualized by gel imaging system. The results indicated that apoptosis in primarily cortical cells was induced by hypoxia and hypoglycemia impairment. Meanwhile, a ladder pattern of DNA fragmentation occurred. After treatment with lOumoM/1, lOOumolL"' TMPN and lOumol-U1 TMP cell apoptosis and DNA fragmentation were partly blocked.2.6 Effect of TMPN on the expression of Bcl-2 in SH-SY5Y cells and primarily cortical cells injured by oxygen and glucose deprivationThe expression of Bcl-2 protein in SH-SY5Y cells and primarily cortical cells treated with sodium dithionite was detected using SABC immunohistochemistry. Results showed that Bcl-2 level decreased markedly in nerve cells after hypoxia and hypoglycemia injury(P<0.01), lOjjmol-L'1, lOOnmol-L'1 TMPN and lOumol-L"1 TMP prevented the down-regulation of Bcl-2 level (PO.05). |
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