Effects Of JNK Signaling Pathway And The Changes Of NF-κBp65 Protein Expression During The Apoptosis Process Of Hippocampal Neurons Induced By Paclitaxel

Objective: Taxol(Paclitaxel) [1] is now widely used in clinical anti-cancer drugs, mainly used in ovarian cancer and breast cancer, and also have a certain effect in lung cancer and colorectal cancer. However, it shows up a dose-dependent toxicity in the course of clinical application[2], including neurotoxicity[3]and bone marrow suppression[4]. Neurotoxicity which includes central and peripheral neurotoxicity, but its pathogenesis and treatment is not clear. Therefore, this study provides a theoretical basis by exploring the molecular mechanism of central neurotoxicity of paclitaxel for clinical prevention and treatment. Studies have shown that cytology and molecular mechanisms of paclitaxel-induced neuralgia is mainly the activation of microglia mitogen-activated protein kinases(MAPKs) pathway [5] caused by apoptosis. JNK(C-Jun N-terminal kinase, JNK) signaling pathway is one of MAPKs pathways, and its physiological role is mainly showed on mediating the inflammation and apoptosis, and its downstream transcription factor NF-κB is rich in hippocampus region. Another study found that NF-κB can be activated by inflammatory mediators via the feedback pathway, thereby the inflammatory response is amplifying, the express of NF-κB is increased, and apoptosis is induced. After giving the inhibitor of NF-κB, the release of its downstream factor is reduced, such as IL-1β and TNF-α, and it could inhibit the damage and apoptosis of the brain neurons. But the role of JNK signaling pathway and the function of its downstream factor NF-κBp65 during paclitaxel-induced neuronal apoptosis in the hippocampus is unclear. In this study new-born Sprague Dawley rats within 24 hours were sacrificed. The hippocampuses of the rats were separated, and immunofluorescence staining with β-tublin III was used to identify whether hippocampal neuron cells were successful acquired. After the success of neurons cultured, they were treated with different concentrations of paclitaxel during different action time, using MTT assay(n=3,x±s) to determine the optimal concentration of paclitaxel of paclitaxel-induced apoptosis of hippocampal neurons, then we produced paclitaxel-induced hippocampal neurons apoptosis models. After the models were successful, paclitaxel and JNK inhibitor SP600125 were given. Cells were divided into 4 groups: control group(Group C), JNK pathway inhibitor group(Group S), paclitaxel group(Group N), JNK pathway inhibitor plus paclitaxel group(Group S+N). The apoptosis rates of hippocampal neurons induced by paclitaxel were measured by flow cytometry, and the expression of NF-κBp65 protein was determined by Western-blot method. Based on the results, we investigate the regulating effects of JNK pathway during the apoptosis process of the hippocampal neuron cells in new-born SD rats triggered by paclitaxel,and the changes of the expression of NF-κBp65 protein.Method: Healthy and clean newborn SD rats within 24 h, provided by the Experimental Animal Center of Hebei Medical University, were decapitated; then the hippocampal neurons of the rats were separated and seeded in culture plates for 5 days. Immunofluorescence staining with β-tublin III was used to identify whether hippocampal neurons were successful acquired. The concentration is about 1×106 /ml. Cell suspension were seeded in polylysine-coated 96 well culture plates with 200μl per well, culture 5d and then divided into five groups randomly: normal control group and four groups with different concentrations of paclitaxel(Final concentrations: 0.01μmol/L, 0.1μmol/L, 1μmol/L, 10μmol/L), each group had 6 wells. After the administration of 12,24,48,72 hours each well was added 20μl MTT of 5mg/ml. After cultured for 4h, each well was added 150μl DMSO, mixing by shaking in the water bath at 37 ℃ for 10 min. Then they were determined by comparing the absorbance values at 490 nm wavelength. Then we observed the effects of different concentrations and different action time of paclitaxel, in order to determine the optimal concentration of paclitaxel for producing paclitaxel-induced hippocampal neurons apoptosis models. Cells were divided into 4 groups randomly: control group(Group C), JNK pathway inhibitor group(Group S), paclitaxel group(Group N), JNK pathway inhibitor plus paclitaxel group(Group S+N). The apoptosis rates of hippocampal neurons induced by paclitaxel were measured by flow cytometry, and the expression of NF-κBp65 protein was determined by Western-blot method.Results: 1 The cell body of hippocampal neuron is subuliform, triangular or fusiformis, with a long axon, 1-2 dendritics which interweaving a net. After culturing for 5d, purity of cells is 90%, indicating that the hippocampus neurons are cultured successfully. 2 The inhibition of paclitaxel of on the survival rate hippocampal neurons has relationship with action time and concentration(F = 6.005, P<0.05): In the same concentration of paclitaxel, survival rate decreased with time extending(F = 6.151, P<0.05); in the same action time of paclitaxel, survival rate decreased with concentration increasing(F = 11.825, P<0.05). Hippocampal neurons are inhibited by 1μmol/L paclitaxel for 24 h, closing to the effective lethal dose, IC50=1. So we choose 1μmol/L as the optimum concentration of paclitaxel, the action time is 24 h. This condition can be successfully prepared for the apoptosis models for further experimental studies. 3 Compared with group C, the apoptosis rate of N group is increased(P<0.05). Group S compared with group C, the apoptosis rate is decreased(P<0.05). Group S+ N compared with group P, the apoptosis rate is decreased(P<0.05).Conclusion: Paclitaxel can induce apoptosis of primary cultured hippocampal neurons, and this process has relationship with survival time and concentration. JNK pathway plays the regulatory role in the apoptosis process of hippocampal neuron cells triggered by paclitaxel. Blocking JNK signaling pathway can inhibit paclitaxel-induced apoptosis of hippocampal neurons by down-regulate the expression of NF-κBp65 protein.