Research On The Protective Effects Of Regulating Liver Formula For The Hippocampal Neurons Against The Stressed Injury

ObjectiveStress is a kind of non-specificity reaction of the organisim caused by excessive and adverse stimulus which break down the homeostasis of the internal environment. Moderate stress is important for maintaining the living of the organism, while excessive and prolonged stress would cause a series of pathology. The hippocampus, which is the centre of CNS for the accommodation of stress, would be impaired by excessive stress. In turn it fails to give negative feedback to the hyperactivity of HPA axis, inducing a infernal circle, finally caused a series of stress disorders. Our previous researches have demonstrate that Regulating Liver Formula-JWSNS could regulate the stress reaction, resist to the toxicity of EEA, possibly by down-regulating the NMDA receptors of hippocampal neurons. In this paper, we established two injury models of hippocampal cells in vitro to further explore the CNS effects and the effect mechanisms of JWSNS.MethodsSerum-free primary hippocampal cell culture was adopt to establish the injury models induced by excessive Glu and CORT. Serum pharmacology method of Chinese medicine was applied to further imply the protective effects of JWSNS on hippocampal cells. In the first part we used MTT method to assess the survival rates of neurons to explore the suitable concentration of Glu and CORT inducing the damage to hippocampal cells. In the second part, first we use MTT method to explore the best concentration of JWSNS serum on the protective effects against the injury caused by excessive Glu and CORT, then LDH was measured and apoptosis rates were detected using Flow cytometry to further interpret its protective effects. In the third part Fura-2 was applied to detected the [Ca²⁺]_i and immune fluorescence cell chemistry method was used to detect the expression of NMDA receptors.Results1.Compared with control group,100μmol/L, 200μmol/L and 500μmol/L Glu could decrease the cell survival rates (P<0.01) ,while 10_⁷mol/L CORT showed no significant difference (P>0.05), both 10^-5mol/L CORT and 10^-6mol/L CORT could decrease the cell survival rates (P<0.01). Based on the morphology changes, 200μmol/LGlu and 10^-6mol/L CORT is suggested to use as the moderate concentrations to establish the injury models for further research.2.The cell survival rates in 10% Low, Middle, High dose of JWSNS serum treated groups showed significant differences compared with Glu group (P<0.01). While compared with 10% control serum +Glu group, the Middle dose group had a higher cell survival rates (P<0.05), however the Low and High dose groups showed no significant differences (P>0.05).The cell survival rates in 10% Low, Middle, High doses of JWSNS serum treated groups showed significant differences compared with CORT group (P<0.01) and 10% control serum group (P<0.05). While compared with 10% control serum +CORT group, the Middle dose group had a higher cell survival rates (P<0.01).3.Compared to the control group, LDH increased in the Glu group, CORT group (P<0.01). JWSNS could decrease the LDH induced by excessive CORT, compared to the control serum group (P<0.01) and CORT group (P<0.01). Compared with the Glu group and control serum group, JWSNS could decrease the LDH (P<0.01,P<0.05)4.Compared to the control group, apoptosis rates increased in Glu group and CORT group (P<0.01). JWSNS could decrease the apoptosis rates induced by excessive CORT, compared to the control serum group (P<0.01) and CORT group (P<0.01). Compared with the Glu group and control serum group, JWSNS could decrease the apoptosis rates (P<0.01,P<0.05).5.With analysis and measured by the Tillvision system, it was observed that [Ca²⁺]_i of the model group increased significantly compared with control group (P<0.01); JWSNS could decrease the [Ca²⁺]_i compared with Glu group (P<0.01) and control serum group (P<0.05). The [Ca²⁺]_i in JWSNS group decreased compared with the CORT (P<0.01) group and the control Serum group (P<0.05).6.After treated with Glu and CORT for 24h, the intensity of green fluorescence of NR1 and NR2B increased in the control groups (P<0.05). While in JWSNS groups, this dropped down compared to model groups and Control serum group (P<0.05). However, it showed no significant differences during these groups on the intensity of green fluorescence of NR2A (P>0.05).Conclusions1.Regulating Liver Formula-JWSNS could resist to the damage caused by excessive Glu and CORT to the hippocampal cells by increasing the cell survival rates.2.Regulating Liver Formula-JWSNS could decrease the LDH induced by the excessive Glu,CORT to the hippocampal cells and decrease the apoptosis rates of them.3.Regulating Liver Formula-JWSNS could resist to the increase of [Ca²⁺]_i of hippocampal cells induced by excessive Glu and CORT, and it could regulate the [Ca²⁺]_i homeostasis of the hippocampal cells.4.Regulating Liver Formula-JWSNS could down-regulate the hyperactivity of NR1 and NR2B induced by the excessive Glu and CORT to the hippocampal cells. Its neuro-protective effects has some relationship to regulating the NMDA receptors' functions.