| Objective: One study reported that neural psychological stress and signal transduction pathway of cyclic AMP response element binding protein(CREB)are closely related to occurrence and development of malignant tumor, but related researches on NPS and CREB with non-small cell lung cancer(NSCLC)research are rarely reported. The study is proposed to observe influence of NPS on tumor cell growth and apoptosis of mice bearing pulmonary adenocarcinoma through establishing model of nude mouse bearing pulmonary adenocarcinoma, meanwhile; detect some important neurotransmitter and receptor expression changes in serum and tumor tissue of mice bearing pulmonary adenocarcinoma under the action of NPS; study some key signal factor expression related to CREB signal transduction pathway under stimulation of NPS; primarily discuss influence of NPS on NSCLC tumor cell growth, apoptosis and important neurotransmitter in serum and tumor tissue,and mechanism of action of NPS for CREB signal transduction pathway in NCSLC, which provide new a strategy and theoretical basis for NSCLC treatment and prevention in the future.Method: Select 64 nude mice of males and 6 weeks age(Harlan Sprague Dawley Inc); cell strain is human pulmonary adenocarcinoma NCI- H441(concentration is 3×106/0.2 ml). Establishment of model of nude mouse bearing pulmonary adenocarcinoma: nude mouse armpit subcutaneous inoculation human pulmonary adenocarcinoma NCI-H441 cell strain with 0.2ml(concentration is 3×106/0.2ml). Establishment method of NPS exposure:regularly replace composition of nude mice in each cage once every two days to make nude mice in each cage after replaced come from different cages before replaced; avoid each mouse repeated in the same cage and cause nude mouse in NPS status. According to different experimental conditions, nude mouse bearing the tumor should be divided into four groups, 16 mice for each group; group without NPS stimulation(CON group); group with NPS stimulation(NPS group); group with NPS stimulation and simultaneously applying NPS inhibitor(GABA)(N+G Group); group without NPS stimulation but applying NPS inhibitor GABA(GABA Group). Feed NPS group and N+G group for four weeks by exposed to NPS conditions after establishing model of nude mouse bearing pulmonary adenocarcinoma; feed CON group and GABA Group for four weeks under normal conditions. All nude mice are fed with autoclaving dedicated fodder for rodent and sterile drinking water. Observe general state, volume and weight changes of tumor growth for each group of nude mouse bearing the tumor; measure tumor volume: tumor volume(mm3)=(length/2) ×(width2); execute experimental animals by taking off their necks after four weeks; remove eyeball and get serum; completely strip transplantation tumor and weigh; detect concentration(ng/ml) of epinephrine,noradrenaline, cortisol and cyclic adenosine monophosphate(c AMP) in serum by using ELISA method; determine tumor cell apoptosis of transplantation tumor by using Tunnel method; detect kit by apoptosis in situ and mark with fluorescein; criterion of apoptotic cell: volume of tumor cell below microscope is shrinking; nucleoplasm is concentrated; nuclear membranes and nucleolus are broken; apoptotic cell stain is mainly located in cell nucleus, showing tawny; a little visible cytoplasm is dispersed; apoptotic index(A1)=apoptotic cell population/( apoptotic cell population+ normal cell population). Detect expression of adrenoreceptor of β1, β2, nicotine acetylcholine receptor of 3, 5,7(n ACh R3, 5, 7) and signal transduction pathway of CREB, phosphorylation cyclic AMP response element binding protein(P-CAEB), protein kinase A(PKA) in tumor tissue; the result should be carried out gray-scale by using Phoretix ID software; the value is relative optical density obtained by dividing corresponding egg white by internal reference egg white(GAPDH). All values show by mean± standard deviation(±S); compare among four experimental groups by analysis of variance; compare between two groups by q detection;detect difference of percentage by x2; P<0.05 shows a statistically significant differences.Results:(1) Volume and weight of transplantation tumor under different experimental conditions: volumes of transplantation tumor in NPS group, CON group, N+G group and GABA group are 105.60±24.53mm3, 80.21±21.84mm3,65.02±17.96mm3 and 49.61±15.74 mm 3 respectively; there are significant differences between four groups(P=0.031). Weight of NPS group is obviously greater than CON group, N+G group and GABA group(P=0.046, P=0.039,P=0.021, P=0.008); weight of transplantation tumor in NPS group, CON group,N+G group and GABA group are 0.17±0.09 g, 0.121±0.10 g, 0.113±0.06 g and0.098±0.07 g respectively; weight in four groups is obviously different and differences are statistical significance(P=0.011): Weight of NPS group is obviously greater than CON group, N+G group and GABA group(P=0.027,P=0.023, P=0.003).(2) Expression of neurotransmitter in serum of mice bearing pulmonary adenocarcinoma under different experimental conditions:concentration of epinephrine in NPS group, CON group, N+G group and GABA group are 2.37±0.72ng/ml, 1.96±0.84ng/ml, 1.83±1.07ng/ml and1.20±0.58ng/ml respectively; there are no significant differences between four groups(P=0.344). Concentration of noradrenaline in NPS group, CON group,N+G group and GABA group are 15.61±3.94ng/ml, 9.98±1.76ng/ml, 10.28±2.41ng/ml and 8.00±1.21ng/ml respectively; concentration of NPS group rises compared four groups and differences are statistical significance(P=0.026);concentration of NPS group rises significantly compared with CON group,N+G group and GABA group(P=0.026, P=0.015, P=0.011). Concentration of cortisol in NPS group, CON group, N+G group and GABA group are5.71±0.94ng/ml, 4.03±1.20ng/ml, 3.20±0.83ng/ml and 2.63±0.58ng/ml respectively; values in four groups are obviously different and differences are statistical significance(P=0.000); concentration of NPS group rises significantly compared with CON group, N+G group and GABA group(P=0.001,P=0.000,P=0.000). Concentration of cyclic adenosine monophosphate in NPS group, CON group, N+G group and GABA group are29.56±3.90ng/ml, 4.75±1.06ng/ml, 14.31±2.50ng/ml and 10.71±2.74ng/ml respectively; differences among groups are statistically significant(P=0.003);concentration of NPS group rises significantly compared with CON group,N+G group and GABA group(P=0.000, P=0.02, P=0.009).(3) Tumor cell apoptosis of mice bearing pulmonary adenocarcinoma under different experimental conditions: apoptosis rate in transplanted tumor tissue of NPS group is 26.2%; apoptosis rate of N+G group is 52.4%; apoptosis rate of GABA group is 58.6%; apoptosis rate of CON group is 41.8%. Differences of apoptosis rate of the four groups are obvious and have statistical significance(P=0.028); apoptosis rate of NPS group is obviously lower than that of CON group, N+G group and GABA group(P=0.034, P=0.021, P=0.026).(4)Expression of adrenoreceptor of β1, β2, n ACh R3, 5, 7 and CREB, p-CREB and PKA of mice bearing pulmonary adenocarcinoma under different experimental conditions detected by Western blotting: expressions of adrenoreceptor of β1 in transplantation tumor tissue in NPS group, CON group, N+G group and GABA group are 0.54±0.28, 0.26±0.14, 0.27±0.11 and 0.20±0.14 respectively;differences of the four groups are obvious and have statistical significance(P=0.004); expression of adrenoreceptor of β1 in NPS group rises significantly compared with CON group, N+G group and GABA group(P=0.003, P=0.005,P=0.001). Expressions of adrenoreceptor of β2 in transplantation tumor tissue in NPS group, CON group, N+G group and GABA group are 0.83±0.33,0.378±0.17, 0.41±0.19 and 0.38±0.15 respectively; differences of the four groups are obvious(P=0.001); expression of adrenoreceptor of β2 in NPS group rises significantly compared with CON group, N+G group and GABA group(P=0.001, P=0.002, P=0.001). Expression of CREB egg white in NPS group, CON group, N+G group and GABA group are 0.39±0.09, 0.31±0.20,0.36±0.11 and 0.32±0.18 respectively; there are no significant differences between four groups(P=0.971). Expression of P-CREB egg white in NPS group,CON group, N+G group and GABA group are 0.39±0.17, 0.21±0.11, 0.16±0.08 and 0.09±0.06 respectively; there are no significant differences between four groups(P=0.971); differences of apoptosis rate of the four groups are obvious and have statistical significance(P=0.011); P-CREB level in NPS group rises significantly compared with CON group, N+G group and GABA group(P=0.043,P=0.013,P=0.001). Expression of PKA egg white in NPS group,CON group, N+G group and GABA group are 0.73±0.32, 0.17±0.06, 0.28±0.16 and 0.19±0.10 respectively; there are significant statistical difference(P=0.042). Expression in NPS group rises significantly compared with CON group,N+G group and GABA group(P=0.010, P=0.034, P=0.026). Expression of n ACh R3 egg white in NPS group, CON group, N+G group and GABA group are 0.27±0.11, 0.15±0.06, 0.17±0.05 and 0.10±0.03 respectively; there are no significant statistical difference(P=0.344). Expression of n ACh R5 in NPS group, CON group, N+G group and GABA group are 0.39±0.12, 0.17±0.07,0.17±0.06 and 0.08±0.05 respectively; there are significant statistical difference compared four groups(P=0.014); expression of NPS group rises significantly compared with CON group, N+G group and GABA group(P=0.026, P=0.024,P=0.002). Expression of n ACh R7 in NPS group, CON group, N+G group and GABA group are 0.32±0.11, 0.08±0.01, 0.03±0.02 and 0.12±0.10 respectively;there are significant statistical difference compared four groups(P=0.019);expression of NPS group rises significantly compared with CON group, N+G group and GABA group(P=0.013, P=0.003, P=0.035).Conclusion: Neural psychological stress can promote tumor growth of nude mice bearing pulmonary adenocarcinoma, and proliferate and inhibit apoptosis of tumor cell, meanwhile, cause release of noradrenaline and cortisol of neurotransmitter in serum of mice bearing pulmonary adenocarcinoma and obvious rise of part β-adrenergic receptor and acetylcholine receptors;influence of neural psychological stress on tumor of mice bearing pulmonary adenocarcinoma is related to release of neurotransmitter and activation of CREB signaling pathway; inhibitory neurotransmitter and aminobutyric acid can inhibit release of neurotransmitter(noradrenaline and cortisol), expression ofβadrenergic receptor and acetylcholine receptors and affect growth and proliferation of tumor of nude mice bearing pulmonary adenocarcinoma. |
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