| Objective:cerebral ischemia is one of the most dangerous threats to human health, whose pathophysiological mechanism is very complicated. Recent studies have revealed that ASICs is closely related to acidosis and calcium overload during cerebral ischemia. Catechin, a major tea polyphenols derived from green tea, has been reported to have the protective effect of catechin on neuronal injury.However, the detailed mechanisms have still not yet been completely resolved.The aim of this study is to investigate the protective effect of CA on ischemia-reperfusion and acidosis and provide rationale for the research and development of brain-protective drugs using Whole-cell patch clamp to observe the effects of catechin on ASICs current.Methods:Primary cortical neurons from post-natal day 1 Wistar albino rats were cultured. Glial cell growth was suppressed by cytosine arabinoside.Under these conditions, neuronal purity is estimated by immunocytochemical. Oxygen-glucose deprivation(OGD) and acidosis were applied to simulate in vivo ischemia/reperfusion.Cortical neurons were randomly divided into 6groups:normal, model, (+)-catechin A(lmmol/L), (+)-catechin B(0.5mmol/L),(+)-catechin C(0.25mol/L) and (+)-catechin D groups.The survival rate was measured by MTT assay, the activity of NOS and LDH were assayed and thepathology was observed, so as to study the protection of Catechin on neurons.Whole-cell patch-clamp electrophysiology was used to record the ASICs current in cultured rat cortical neurons.Result:1 The protective effect of (+)-catechin on neuronal injury induced by acidosis.(1) The cellular survival rate of each group in cortical neurons after 4h treated under acid are as follow:normal group(100±0.00), model group(29.13±1.89), amiloridum group (48.07±4.27), (+)-catechin A group (37.30±6.26), (+)-catechin B group (42.66±1.41), (+)-catechin C group (43.24±2.99), (+)-catechin D group (41.94±2.45)。The cellular survival rate in mordel and (+)-catechin treatment groups are lower than that in normal group(P<0.01). In comparing the model groups, the cellular survival rate in the (+)-catechin treatment groups are significant highter (P< 0.05).The cellular survival rate in (+)-catechin A and (+)-catechin D groups are obviously lower than that in amiloridum group (P<0.05), There is no difference in (+)-catechin B, (+)-catechin C groups and amiloridum group about NOS activities(P <0.05).(2) The LDH concentration of each group in cortical neurons after 4h treated under acid are as follow:normal group (112.9±9.62),model group(463.7±13.85), amiloridum group(288.2±20.10),(+)-catechin Agroup (314.4±14.74), (+)-catechin B group (336.3±14.69), (+)-catechin C group (344.8±26.24), (+)-catechin D group (381.2±15.66)The LDH activities of extracellular fluid in mordel and (+)-catechin treatment groups are obviously lower than that in normal group(P<0.01). In comparing the model groups, the (+)-catechin treatment groups show significant decreased of the LDH activities.(P<0.05). The LDH activities in (+)-catechin C groups is and (+)-catechin D groups obviously higher than that in amiloridum group (P<0.05).There is no difference in (+)-catechin A, (+)-catechin B groups and amiloridum group about LDH activities(P<0.05). (3) The NOS activities of each group in cortical neurons after 4h treated under acid are as follow:normal group (0.67±0.098), model group(1.75±0.095)。amiloridum group(1.15±0.087), (+)-catechin A group (1.23±0.190), (+)-catechin B group (1.28±0.092), (+)-catechin C group (1.43±0.140), (+)-catechin D group (1.49±0.085)。The NOS activities of extracellular fluid in mordel and (+)-catechin treatment groups are obviously highter than that in normal group(P<0.01). In comparing the model groups, the (+)-catechin treatment groups show significant decreased of the NOS activities.(P<0.05) The NOS activities in (+)-catechin Cand (+)-catechin D groups are obviously higher than that in amiloridum group (P<0.05), There is no difference in (+)-catechin A, (+)-catechin B groups and amiloridum group about NOS activities(P<0.05).2 The protective effect of (+)-catechin on neuronal injury induced by OGD.(1) The cellular survival rate of each group in cortical neurons are as follow:normal group (100±0.00), model group (35.27±3.41), amiloridum group (48.50±1.94), (+)-catechin A group (52.53±3.79), (+)-catechin B group (57.22±2.99), (+)-catechin C group (58.68±1.80), (+)-catechin D group (50.75±2.29)。The cellular survival rate in mordel and (+)-catechin treatment groups are lower than that in normal group(P<0.01). In comparing the model groups, the cellular survival rate in the (+)-catechin treatment groups are significant highter (P< 0.05).The cellular survival rate in (+)-catechin Band (+)-catechin C groups are obviously highter than that in amiloridum group (P<0.05), There is no difference in (+)-catechin A, (+)-catechin D groups and amiloridum group about NOS activities(P <0.05).(2) The LDH activities of each group are as follow:normal group(151.6±8.50) model group(672.3±28.51), amiloridum group(398.5±23.82),(+)-catechin A group (385.7±29.08), (+)-catechin B group (406.3±41.88), (+)-catechin C group (434.8±33.27), (+)-catechin D group (457.9±19.36)The LDH activities of extracellular fluid in mordel and (+)-catechin treatment groups are obviously highter than that in normal group(P<0.01). In comparing the model groups, the (+)-catechin treatment groups show significant decreased of the LDH activities.(P<0.05). There is no difference in (+)-catechin A, (+)-catechin B groups and (+)-catechin C group about the NOS activities(P<0.05). The NOS activities in (+)-catechin D groups is obviously higher than that in amiloridum group (P<0.05).(3) The NOS activities of each group are as follow:normal group(1.04±0.125), model group(3.05±0.163), amiloridum group(2.25±0.111), (+)-catechin A group (1.84±0.147), (+)-catechin B group (1.87±0.201), (+)-catechin C group (2.26±0.266), (+)-catechin D group (2.42±0.092)。The NOS activities of extracellular fluid in mordel and (+)-catechin treatment groups are obviously highter than that in normal group(P<0.01). In comparing the model groups, the (+)-catechin treatment groups show significant decreased of the NOS activities.(P<0.05). There is no difference in (+)-catechin C, (+)-catechin D groups and amiloridum group about NOS activities(P<0.05). The NOS activities in (+)-catechin A group and (+)-catechin B group are obviously lower than that in amiloridum group (P<0.05).3 The effects of (+)-catechin on ASICs current(+)-catechin treatment group(500μmol/1) and amiloridum(10μmol/1) show significant decreased of the ASICs current amplitude than in the control group without (+)-catechin at pH6.0. The current amplitude in amiloridum(10μmol/1) group and (+)-catechin treatment group(500μmol/l) were reduced to 65.2±4.13% (n=8-12) and 81.7±3.74%(n=8-12). The current amplitude in (+)-catechin treatment group(500μmol/1) are obviously highter than that in amiloridum(10μmol/1) group (P<0.05)Conclusion1 The cellular survival rates in cortical neurons treated under acid and OGD are significant decreased. (+)-Catechin can markedly increase the survival rate and decrease the LDH and NOS activities.2 (+)-Catechin has protective effect on neuronal injury, and its mechanism may be related to improving the brain tissues energy metabolism, resisting neurotoxicity induced by NO and inhibiting the opening activities on ASICs channel. |
PDF Full Text Request