| Tetramethylpyrazine (TMP) is one of the major efficient components abstracted from the Chinese traditional medicine herb Chuanxiong (Ligusticum wallichii Franchat). TMP is widely used in China as an oxygen free radical scavenger, an agent of antithrombusa and antihypertension, and as a weak 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 and its derivates, we synthesize TMPDP by keeping its pharmacophore and using the (4,4'-fluorine)diphenyl-methyl-l-piperazidine to take place of the second methyl on TMP. The Flunarizine is an important drug for cardia and cerebral vessels disease, the (4,4'-fluorine)diphenyl-methy-1-piperazidine-Methyl-lium is its main active group. It has much possibility that the halflife of TMPDP could be longer and the bioavailability could be higher. And pharmacodynamic action of TMPDP can be increased with the synergistic effect of Flunarizine. Thereby, we supposed TMPDP have stronger effects on cardia and cerebral vessels disease.In this paper, we used human neuroblastoma SH-SY5Y cells (SH-SY5Y), which were suffered from oxidative stress by hydrogen peroxide to observe the protection of TMPDP on the injured nerve cells, consequently elucidated the possible mechanisms in vitro.1. Effect of TMPDP on the viability of normal SH-SY5Y cells.The viability of normal SH-SY5Y cells was detected by MTT assay. The results indicated that the viability of SH-SY5Y cells did not effect by administration of TMPDP at 5,10,20,40μmol/L, while decreased by 80μmol/L significantly (P<0.01). 2. Effect of TMPDP on the viability of SH-SY5Y cells damaged by H2O2SH-SY5Y cells were treated with 200μmol/L H2O2 and 5,10,20,40,80μmol/L TMPDP at the same time. Flunarizine (FLU) with the consistent concentration were given to cells as control groups. The viability of cells was detected by MTT assay. The results indicated that SH-SY5Y cells viability descended significantly after oxidative damage (P<0.01), but administration of TMPDP at 5,10,20,40μmol/L effectively inhibited the decrease of cells viability induced by H2O2, stronger than FLU (P<0.01). But at 80μmol/L, TMPDP has weak inhibitory effect than FLU. So,5,10,2μumol/L were chosen for TMPDP, 10μmol/L for FLU.3. Effect of TMPDP on LDH release from SH-SY5Y cells injured by H2O2SH-SY5Y cells cultured in 96-well plate were bathed in culture media containing 200μmol·L-1 H2O2 for 12 hours,5,10,20μmol/L TMPDP and 10μmol/L FLU were administered at the same time. The extra-cellular culture supernatants of each experimental group and cell lytic liquid were collected and the activity of LDH was determined by commercial kit. Then the release ratio of LDH from the cells was calculated. Results showed that after oxidative damage, cellular membrane became intact and LDH efflux increased (P<0.01). After the treatment of TMPDP at the concentrntion of 5,10 and 20μmol·L-1, the leaking of LDH induced by H2O2 attenuated and extra-cellular LDH level decreased significantly (P<0.01).4. The free radical-scavenging activity of TMPDP by DPPH* assayThe antioxidative potential of TMPDP was studied against DPPH*. The absorbance of the DPPH* solution including TMPDP/TMP/VitE was detected at 516nm. 120μmol·L-1 TMPDP demonstrated the %RSA of 40% radical scavenging activity, lower than VE, higher than TMP. This result indicated that TMPDP had moderate antioxidative effect.5. Effect of TMPDP on SOD activities, GSH contents and MDA formation in SH-SY5Y cells injured by H2O2After being damaged by H2O2, SH-SY5Y cells cultured in 6-well plate were colleted by low-speed shake and homogenized in lysate buffer solution. The homogenate was centrifuged at 13000rpm at 4℃for 15min and the supernatant was used for enzyme assays. The content of GSH and MDA, the activity of SOD were determined using commercial kit, at the same time protein contents of the samples were measured using the method of coomassie brilliant blue. Results showed that after incubated with H2O2, the content of MDA increased and the activity of SOD and GSH level decreased markedly (P<0.01).10 and 20μmol·L-1 TMPDP significantly inhibited the change of SOD activity, MDA content and GSH level in cells (P<0.01).6. Effect of TMPDP on the apoptosis of SH-SY5Y cells induced by H2O26.1 Effect of TMPDP on the cell morphologyThe morphology of apoptotic cells was observed by Hoechst-33258 fluorescence staining assay. The typical apoptotic cells with nuclear condensation and fragmentation under fluorescent light microscope were showed and an increase of apoptotic cells was observed in H2O2-treated group.10μmol·L-1 TMPDP could inhibit the apoptotic change of cell morphology.6.2 Effect of TMPDP on activity of Caspase-3 in SH-SY5Y cellsThe lysate of SH-SY5Y cells was collected to determine the activity of Caspase-3 by commercial kit. In cells exposed to H2O2, the activity of caspase-3 was enhanced obviously. However, H2O2-exposed cells treated with TMPDP at concentrations of 5, 10 and 20μM, revealed a decrease of the activity of Caspase-3 (P<0.01). 7. Effect of TMPDP on [Ca2+]i in SH-SY5Y cells injured by H2O2The fluorescent intensities of Fura 3-AM-loaded suspended cells were measured at 37℃with emission at 490 nm and excitation at 526 nm by Multilabel Counter. The results showed that addition of 5,10 and 20μmol/L TMPDP reduced [Ca2+]i in response to H2O2 stimulation (P<0.01).8. Effect of TMPDP on the content of NO and activity of iNOS in SH-SY5Y cells injured by H2O2The lysate of SH-SY5Y cells injured by H2O2 was collected to determine the content of NO and activity of iNOS by kit. The content of NO was increased in cells after exposed to H2O2 for 12h.10,20μmol/L TMPDP and 10μmol/L FLU reduced the content of NO in response to H2O2. The activities of iNOS were increased after exposed to H2O2 for 12h.5,10,20μmol/L TMPDP and 10μmol/L FLU decreased the activity of iNOS in cells cultured with H2O2. In conclusion, the protective effects of TMPDP on the human neuroblastoma SH-SY5Y cells damaged by H2O2 were observed. Results showed that TMPDP has potent protection on the injured SH-SY5Y by increasing the viability and decreasing the LDH release from cells and the intracellular content of MDA. TMPDP could inhibit the decrease of the activity of intracellular SOD and GSH. The cells apoptosis were induced by H2O2. TMPDP improved the cell morphology. Moreover, TMPDP could decrease the [Ca2+]i and the level of NO and activity of iNOS in SH-SY5Y. These results indicated that TMPDP protected damaged SH-SY5Y cells induced by H2O2 due to its effects of antioxidation, antiapoptosis and calcium antagonism. |
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