| Objective:Ischemic cerebrovascular disease(ICVD) is one of the three major diseases threating to human health. Among the many theories of cerebral ischemia injury mechanism, the theory of calcium overload has occupyed the central position, and that calcium overload is the final common pathway of neuron degeneration and death. Therefore, the research on inhibitors of calcium overload is of great significance for the prevention and treatment of ICVD. Our previous researches showed that phenolic compounds extracted from Gastrodia elata Blume. had significant brain protection on cerebral ischemia injury in rats, and further study found that they also had significant anti-platelet aggregation and relaxed vascular smooth muscle, which may be related to inhibition of calcium ion flowing into the cells. This project intended to duplicate the rat primary cortical neurons oxygen-glucose deprivation/ reintroduction(OGD/R) injury model, and study the effect of the phenolic compounds C# and D# on this model and calcium channels. The correlation between cerebral protective effect of phenolic compounds and inhibition of calcium ion channels was discussed preliminarily.Methods:1. Different concentrations of sugar-free containing Na2S2O4 Earle ’s fluid were used to cause oxygen-glucose deprivation and normal culture medium to cause reoxygenation. On the base of cell survival rate as the evaluation index, the best concentration of sugar-free containing Na2S2O4 Earle’ s fluid was screened to duplicate the rat primary cortical neurons oxygen-glucose deprivation/ reintroduction(OGD/R)injury model. 2. We set gradient concentrations of the phenolic compounds extracted from Gastrodia elata Blume. C# and D# and detected whether C# or D# had a protective effect on the primary cortical neurons which were affected by OGD/R injury by MTT. 3. The release quantity of NO from clear supernatant liquid of culture medium was detected by the kits of NO to see whether C# or D# had resistance to the release quantity of NO. 4. Intracellular Ca2+ concentration([Ca2+]i) change of the primary cortical neurons was detected by flow cytometry to see whether C# or D# had resistance to the intracellular calcium overload. 5. Western Blot and RT-PCR were used to study on whether C# or D# had influence on the protein and m RNA expressions of L-type calcium channel, NMDA receptor – type calcium channel and TRPM7 channel.Results:1. Condition on rat primary cortical neurons OGD/R injury model: compared with control group, 16 m M, 32 m M, 40 m M Na2S2O4 were carried out on the primary cortical neurons OGD/R injury significantly(P < 0.001). Na2S2O4 concentration in 32 m M induced cell survival rate at 55.10±9.09%, so 32 m M Na2S2O4 was used to establish model for subsequent experiments. 2. Research on the effect on the primary cortical neurons which were affected by OGD/R injury of the phenolic compounds extracted from Gastrodia elata Blume.: compared with model group, the phenolic compound C# 80 and 40μg/m L group and D# 100 and 50μg/m L group could elevate survival rate of the primary cortical neuron which were affected by OGD/R injury(P < 0.01, P < 0.05). 3. Research on the effect on the primary cortical neurons NO release which were affected by OGD/R injury of the phenolic compounds extracted from Gastrodia elata Blume.: compared with control group, model group of NO release increased(P < 0.05). Compared with model group, the phenolic compound C# 40 and 20μg/m L group and D# 50μg/m L group could decrease NO release of the primary cortical neurons(P < 0.05); MK- 801, L-type channel inhibitor Nim OGD/R injury could obviously reduce NOrelease of the primary cortical neurons(P < 0.001, P < 0.01). 4. Research on the effect on the primary cortical neurons intracellular Ca2+ concentration which were affected by OGD/R injury of the phenolic compounds extracted from Gastrodia elata Blume.: compared with control group, model group of intracellular calcium iron concentation increased significantly(P < 0.001). Compared with model group, the phenolic compound C# 40 and 20μg/m L group could reduce intracellular calcium iron concentration of the primary cortical neurons(P < 0.05, P < 0.01), and the phenolic compound D# 100μg/m L group could also reduce intracellular calcium iron concentration of the primary cortical neurons(P < 0.05); NMDA receptor calcium channel inhibitor MK- 801, TRPM7 channel inhibitor 2- APB could also reduce intracellular calcium iron concentration of the primary cortical neurons(P < 0.001, P < 0.05),and L-type calcium channel inhibitor Nim and TRPM7 channel inhibitor Gd had a trend of decreasing the intracellular calcium iron concentration(P > 0.05). 5. Research on the protein expressions of calcium channel of the primary cortical neurons which were affected by OGD/R injury of the phenolic compounds extracted from Gastrodia elata Blume.: compared with control group, model group NMDA receptor – type calcium channel subunit NR1 protein expression did not change(P > 0.05), L-type calcium channel and TRPM7 channel protein expression were significantly higher(P < 0.01, P < 0.05). Compared with model group, NMDA receptor calcium channel inhibitor MK- 801 and the phenolic compound C# and D# each dose group had no effect on protein expression of NR1(P > 0.05), the phenolic compound D# 100μg/m L group could decrease L-type calcium channel protein expression of the primary cortical neurons(P < 0.05), TRPM7 channel inhibitor Gd could obviously reduce the TRPM7 protein expression(P < 0.01), and the phenolic compound C# 80μg/m L group had a trend of decreasing the TRPM7 protein expression(P > 0.05). 6. Research on the gene expressions of calcium channel of the primary cortical neurons which were affected by OGD/R injury of the phenolic compounds extracted from Gastrodia elata Blume.: compared with control group, model group NMDA receptor – type calcium channel subunit NR1 m RNA expression was reduced(P < 0.05), NR2 A and NR2 B, L-type calcium channel m RNA expression did not change, TRPM7 m RNAexpression increased(P < 0.001). Compared with model group, the calcium channel inhibitors groups and the phenolic compound C# and D# each dose group of NR1, NR2 A, NR2 B and L-type calcium channel m RNA expression did not change(P > 0.05), the phenolic compound C# 40μg/m L group could decrease the TRPM7 m RNA expression(P < 0.01).Conclusion:1. The phenolic compounds extracted from Gastrodia elata Blume. C# could elevate survival rate of the primary cortical neurons which were affected by OGD/R injury, and the mechanism was associated with decreasing the primary cortical neurons NO release, intervention TRPM7 m RNA expression, and then reducing the primary cortical neurons intracellular calcium iron concentration. 2. The phenolic compound extracted from Gastrodia elata Blume. D# could elevate survival rate of the primary cortical neurons which were affected by OGD/R injury, and the mechanism was associated with decreasing the primary cortical neurons NO release, lower L-type calcium channel protein expression, and then reducing the primary cortical neurons intracellular calcium iron concentration. |
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