Effects Of P38 Mitogen-activated Protein Kinase(p38MARKs ) Pathway And Its Downsteam Factors NF-κB On Changes Of GABA_B Receptors Expression In Paclitaxel-induced Apoptosis

Objective:To investigate the effects and molecular mechanism of p38 MAPK pathway on changes of GABAB receptors expression in paclitaxel-induced apoptosis by giving p38 MAPK inhibitor(SB203580) and NF-κB inhibitor(SN50);Methods:New-born SD rats in 24 h were sacrificed by decollation,carefully removed the brains;transferred the hippocampal cells into the fluid of DMEM with papain;put the neurons into incubator for 30 minutes;Mechanical percussion were seeded on polylysine coated culture plate.The serum containing plating medium were replaced by a serum-free Neurobasal medium supplemented with B27 and N2 after 6-8 hours.Change the half of the culture medium every 2 days;and used phase-contrast microscope to observe the morphological characteristic of hippocampal cells;used the neurons for experiment on the fifth day.The primary cultured hippocampal cells had been cultivated in vitro for five days were selected randomly,the concentration is about 1×106/ml.These neurons were separately given different concentrations of paclitaxel(0、0.01μmol/L、0.1μmol/L、1μmol/L、10μmol/L).The changes of hippocampal neuronral inhibitory rate and apoptosis rate were detected through MTT method and flow cytometry,as a result,the optimal concentration of taxol to induce apoptosis were determined. According to the test results determined by MTT colorimetric method,cell inhibition rate was calculated =(1- AD value/controls AD value) x 100%,and then the neurons half inhibitory concentration was calculated using improved karber formula(lg IC50 = Xm- I(P-(3- Pm- Pn) / 4).Using IC50 as the optimal concentration of taxol in further experiments.And this experiment was repeated four times.Observed the affections of neural inhibition ratewith different drug concentrations in different time,and calculated the mean.According to the test results of the flow cytometry method,taking Annexin V as horizontal axis and PI for the longitudinal axis.The upper left quadrant for mechanical damage cells,the upper right for late apoptotic cells or dead cells,the lower left for normal cells; the lower right for the early apoptosis of cells.The correctness of the optimum concentration of paclitaxel apoptosis by MTT colorimetric method was verified by flow cytometry instrument to detect the apoptosis rate.Another 5 days of primarily cultured hippocampal cells were selected,and randomly divided into six groups: those were control group(group C), 10μmol/L SB203580 group(K1 group),53μg/ml SN50 group(K2 group), the optimal concentration of paclitaxel group(N), 10μmol/L SB203580 + the optimal concentration of paclitaxel group(K1+N group).53μg/ml SN50 + the optimal concentration of paclitaxel group(K2+N group).The GABAB receptors and NF-k B expression of hippocampal neurons were detected by Western blot(n=5,x_±s).Results:At first,the primarily cultivated hippocampal cells were tiny and round,translucent,and in a signal suspension uniform distribution.After twenty-four hours,most of cells had changed;their morphologies were with various kinds of lengths of slender protrusions,and they were fusiform.On the third day,cells were full and the prominences were elongated and enhanced. The fifth day was the exponential growth phase.The cell body became larger and prominences were enlarged and elongated.Effect of paclitaxel on the hippocampal neurons viability had duration and concentration interaction(F=9.127, P<0.05).Due to the upper right was for late apoptotic cells or dead cells,The early apoptosis rate was mainly basis in the right lower quadrant in this experiment of the analysis on the apoptosis rate.The early apoptosis rate induced by 1μmol / L paclitaxel for 24 h was(48.63 ± 5.76)%.This is the experimental conditions for the determination of NF-k B and GABAB receptor proteins.Compared with the group C,the expression of GABAB receptor and NF-k B protein was significantly up-regulated in both group N、group K1+N and group K2+N(P<0.05),while the expression of GABAB receptor and NF-k B protein was down-regulated in group K1 and group K2(P<0.05);compared with the group K1,the expression of GABAB receptor and NF-k B protein was up-regulated in both group N and group K1+N(P<0.05);compared with the group K2,the expression of GABAB receptor and NF-k B protein was up-regulated in both group N and group K2+N(P<0.05);compared with the group N,the expression of GABAB receptor and NF-k B protein was down-regulated in group K1+N and group K2+N(P<0.05).Conclusion:The inhibition of p38 MAPK pathway can up-regulate the expression of GABAB receptors in the process of paclitaxel-induced apoptosis,while its downstream factor NF-k B may be the key target of regulating the expression of GABAB receptors. NF- k B can inhibit the neurons apoptosis induced by taxol by down-regulating the expression of GABAB receptor protein.