The Research Of JNK/MAPK Signal Pathway Mediated Protection For Hippocampus Neurons Injury By Tetramethylpyrazine

Cerebrovascular disease is the clinical common disease. Fifty-seventy percent of urvival patients has left over different levels of disabilities, which have serious impact on the health and survival quality of the patients. In addition, with the development of the medical technology, the risk of the injury after brain ischemia/reperfusion during the perioperative period also has increased. Therefore, the injury after brain ischemia/reperfusion during the perioperative period is the concerned question of clinical anesthesiology and recovery in recent years. The injury of brain ischemia/reperfusion could cause different levels of neurological deficits, which could hardly recover. Lots of researches have already proved that brain ischemia/reperfusion injury is a malignant cascade of damage with multifactor and mechanism. How to prevent brain ischemia/reperfusion injury during the perioperative period became a urgent issue.Tetramethylpyrazine(TMP) is usually used in the treatment of ischemic cerebrovascular disease and its sequela in clinical, however, we are discord and controversy on the mechanism. Especially, there is no literature report on the relationship between JNK signal transduction pathway and protection mechanism of TMP to brain ischemia/reperfusion injury. In this research, we plan to culture rat hippocampal neurons in vitro, then to observe the effect of TMP and a blocker of JNK signal transduction pathway, sP600125, on rat hippocampal neurons damage induced by anoxia-reoxygenation. We also exam the expression of JNK signal transduction pathway associated proteins. From this research, we will clarify the mechanism of TMP in the JNK signal transduction pathway to protect anoxia-reoxygenationed hippocampal neurons. Also, we will provide new theory basis for the treatment of brain ischemia injury. Objective1Clarify the role of JNK signal transduction pathway in the neurons apoptosis induced by ischemia/reperfusion.2Clarify the role of TMP in the JNK signal transduction pathway of anoxia-reoxygenationed hippocampal neurons.Methods1Rat hippocampal neurons were cultured in vitro for seven days.2Neurons were divided into six groupds. L group(TMP60ug/ml), M group (TMP200ug/ml), H group (TMP800ug/ml), C group (TMP Oug/ml), S group (JNK inhibitor SP60012510μmol/L), N group (normal group). Meanwhile, two control groups and one normal group were set in each experiment. After1hour place in an incubator with90%N2+10%C02for2hour, then place in an incubator with5%CO2and37℃for24hours to established rat hippocampal neurons damage by anoxia-reoxygenation3After step2, we used Annexin V-FITC to test the apoptosis ratio in different groups. The expressions of c-jun、c-fos、P-JNK proteins, and the relationship between them were tested by mmuno fluorescence method. We also tested the content of MKK4mRNA、MKK7mRNA and the correlation with p-JNK by using RT-PCR. Results1Anoxia-reoxygenation could increase the apoptosis ratio of hippocampal neurons (P<0.05). Comparing with group C, the apoptosis ratio in group S, L, M, H have decreased (P<0.01). Comparing with group L, H, the apoptosis ratio in group M has significant difference (P<0.01), however, significant differenc with group S.2Comparing with group C, the expression of C-fos、c-jun、P-JNK decreased in group S, L, M, H (P<0.01). Comparing with group L, H, the expression of C-fos、c-jun、P-JNK in group M has significant difference (P<0.01).3Compared with normal group, model group were increased to varying degrees the expression level of MKK4mRNA, MKK7mRNA. Comparing with group C, the content of MKK4mRNA、MKK7mRNA has decreased in group S, L, M, H (P<0.01). Comparing with group L, H, the content of MKK4mRNA、MKK7mRNA has significant difference (P<0.01).ConclusionTMP could decrease the apoptosis of anoxia-reoxygenationed hippocampal neurons in different levels, the expression of C-fos、c-ju、P-JNK, and the content of MKK4mRNA、MKK7mRNA. TMP has obvious neural protective action. The protective function of TMP preconditioning is concentration dependent. Under the suitable density, the protective function of TMP preconditioning is strongest. TMP could protect the hippocampal neurons through JNK signal transduction pathway.