| Cerebral vascular disease is one of the nerve system diseases, which is caused by blood supply obstacles. It can be classified into ischemic cerebrovascular disease (ICVD) and hemorrhagic cerebral vascular diseases (HCVD). The former is also called stroke, with the characteristic of high morbidity and lethality and causing severe damages to human health. It is known that the pathological basis of stroke is neurons ischemic death due to thrombus blocking vessels. At present, thrombolytic therapy is a common treatment, as it can recover the blood supply of ischemic tissues. However, stroke often leads to ischemia-reperfusion injury (I/R) because blood reperfusion will cause injury. Neurons death caused by stroke is classified into acute ischemic necrosis and delayed apoptosis. The former type is unable to be cured while the latter is likely to be alleviated or even reversed if only being treated in time. Up to now, there is still no effective drugs clinically. Consequently, exploration of the molecular mechanism involved in the pathogenesis of stroke in order to find new treatment strategies and develop new medicines is of great necessary. SMND-309 is a new compound found in the brain and heart after the rat is being injected with salvianolic acid B. Chemical structure analysis shows that this compound is a degradation product of salvianolic acid B. Now, it is being developed by researchers of Shandong Engineering Research Center for Natural Drugs to a new medicine to treat IVCD with self-owned intellectual property right. In this study, we investigated the protection and treatment of SMND-309 against I/R injury and discussed the possible mechanism in vitro.1.Human neuroblastoma (SH-SY5Y) cells were cultured in vitro. To simulate I/R injury in vivo, the oxygen and glucose deprivation/reperfusion (OGD/R) model was made. Simultaneously, SH-SY5Y cells were incubated with SMND-309 at doses of 2.5μmol/L,5.0μmol/L and 10μmol/L. The proliferation of SH-SY5Y cells was evaluated by the SRB assay, the activity of LDH in medium was estimated by colorimetry to assess the injuried degree, the change in morphology was detected by Hoechst33258 fluorescence staining. Apoptotic executor caspase-3 and apoptosis-related protein bcl-2 and bax were assayed by colorimetry and Western blotting, respectively. The results showed that viability of SH-SY5Y cells was decreased significantly (p<0.01) and LDH leakage was obviously increased (p<0.01) in OGD/R environment. The SH-SY5Y cells in OGD/R showed morphological characteristics of apoptosis:chromosome condensation and margination. The activity of caspase-3 was heightened (p<0.01) and expression of bcl-2 was diminished (p<0.01). Intervention with SMND-309 at the doses of 2.5μmol/L,5.0μmol/L and 10.0μmol/L, decreased the leakage of LDH (p<0.01 or p<0.05), increased the viability of SH-SY5Y cells (p<0.01 or p<0.05). While ameliorated caspase-3 activity (p<0.01) and up-regulated the expression of anti-apoptotic protein bcl-2 (p<0.01 or p<0.05), but failed to down-regulate the level of bax (p>0.05).2. SH-SY5Y cells were differentiated into neuron-like phenotype with long axon by all-trans retinoic acid (ATRA), and then axons were insulted in vitro with maintaining the cells under mild OGD/R condition. The dose of 0.5μmol/L was selected on basis of cellular viability assay. The length of axons were measured at 12 hour,24 hour and 48 hour after administration with SMND-309. The apoptosis of differentiated SH-SY5Y cells were assessed by Hoechst33258 fluorescence staining and Annexin-FITC/PI staining. The mitochondrial membrane potential and intracellular free Ca2+ concentration were evaluated with fluorochrome probe of Rhodamine123 and Fluro-3/AM, respectively. The levels of intracellular BDNF, t-Akt, p-Akt, t-CREB, p-CREB were detected with by Western blotting. The results showed that SH-SY5Y cells induced by ATRA displayed neuron-like phenotype with long neurite. The axons were retracted and the length was significantly shorten (p<0.01) in mild OGD/R. There was no obvious cellular apoptotic feature appeared in morphology and quantitative inspection (p>0.05) with fluorescence staining subsequently. But the mitochondrial membrane potential and intracellular free Ca2+ concentration of differentiated SH-SY5Y cells occured significant change (p<0.05). Treated with 0.5μmol/L SMND-309, the length of axons were longer than that of cells in OGD/R (p<0.01), mitochondrial membrane potential and intracellular free Ca2+ concentration were significantly improved (p<0.05), however, there was no significant change in the neuronal apoptotic rate (p>0.05). In the condition of OGD/R, there was no significant change in the expression of intracellular t-Akt and t-CREB (p>0.05), and the p-Akt and p-CREB were also generous down-regulated versus the control (p>0.05), but the level of BDNF was markedly down-regulated(p<0.05). The dose of 0.5μmol/L SMND-309 could up-regulate significantly the level of p-Akt, p-CREB and BDNF (p<0.01), while the positive role of SMND-309 was inhibited by incubcation with LY294002 (p<0.01).In conclusion, SMND-309 could inhibit neurons apoptosis induced by OGD/R, which has relativity with down-regulation of caspase-3 activity and up-regulation of anti-apoptotic bcl-2 protein expression. Consequently, SMND-309 possesed an obvious function of neuroprotection. Furthermore, SMND-309 was able to increase intracellular BDNF and promote axonal plasticity after OGD/R. This function was possibly accomplished through PI3K/Akt/CREB signaling pathway. |
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