| OBJECTIVE As the traditional biomedical model is gradually replaced by the bio-psychology-social medical model,the relationship between negative psychological factors such as stress and the occurrence and development of malignant tumors has gradually attracted people’s attention.The purpose of this study was to establish a transplanted tumor model of mouse cervical cancer under chronic stress,and to explore the effects of the combination of 5-fluorouracil and benzodiazepine midazolam on the immunity of mice and the development of transplanted tumor.METHODS Firstly,the mouse cervical cancer U14 cells were transfected with Luc-TdT plasmid.The U14-Luc-TdT cells were separated by FACS.The growth and proliferation in vitro were tested with CCK,and the tumor formation was checked by subcutaneous inoculation into KM mice and C57 mice.T cells in peripheral blood of healthy KM mice were isolated and cultured,then a co-culture system of U14-Luc-TdTcells and T cells was established.Chemotherapy drugs 5-FU and anti-anxiety drugs midazolam with different concentrations(5-FU 50,100,150 mg/L,midazolam 0.2,0.5,0.1 mg/L)alone and jointly were applied to U14-Luc-TdT cells,T cells and the co-culture system.CCK8 was used to test the proliferation of U14-Luc-TdT cells and T cells which had been affected the two drugs.The contents of IFN-y and TNF-α in the supernatant of the co-culture system were tested by ELISA.Six-week-old female KM mice were employed and divided into control group(only tumor transplantation),tumor transplantation+anxiety group,tumor transplantation+anxiety+5-FU group,tumor transplantation+anxiety+5-FU+midazolam group,and tumor transplantation+anxiety+midazolam group.Using irregularly cutting off water supply,keeping bright light all night,keeping dark all day,turning on and turning off the light quickly,pounding cages,violently shaking cages and damp bedding as stressors to establish chronic stress mice model.Corresponding treatments were applied accordingly.5-FU was intraperitoneally injected at 40mg/kg body weight every other day,midazolam was intraperitoneally injected 0.3 mg/kg body weight at 9 o ’clock every night.Every three days both longest and shortest diameter of the transplanted tumors were measured,then the volume of transplanted tumors calculated and growth curve drawn.At d48 all mice were sacrified,and tumor,thymus,spleen collected.Thymus and spleen indexes were calculated.To study the effects of 5-FU on the gastroentestinal barrier,6-week-old Balb/C mice were employed for the experiment and randomly divided into control group,5-FU group,5-FU+prebiotics group,5-FU+probiotics group,and 5-FU+prebiotics+probiotics group.Corresponding treatments were given in each group for 2 weeks.5-FU was intraperitoneally injected at 40mg/kg body weight every other day,prebiotics were orally gavaged at 0.5ml daily,and probiotics were also orally gavaged at 0.5mL(1×109 CFU/ml)everyday.At the end of the experiment,intestinal tissues were sampled for hematoxylin-eosin staining(HE)and immunohistochemistry(IHC)staining.The histopathological changes of colon,the expression of mechanical/immunal/chemical barrier related molecules(claudin-1,MUC2,CD45,Lysozyme)were observed.RESULTS The cell morphology of U14-Luc-TdT was same as U14,which is long spindle/round.The U14-Luc-TdT cells were anchorage-dependent,and showed red fluorescence under fluorescence inverted microscope.Luciferase activity was also detected after substrate additon for the bioluminescence by in vivo iamging.The cell morphology and fluorescence expression remained the same after successive passage for 15 times.In vitro proliferation of U14-Luc-TdT cells were in the logarithmic growth stage between day 2 and 4,then it reached the growth plateau between day 4 and 5,similar to its parent cell U14.The in vivo tumorigenicity of U14-Luc-TdT cells was 100%which was also the same as that of U14 cells.The inhibition of 5-fluorouracil on U14-Luc-TdT cells increased with time and concentration(inhibition rate 11.39%-53.22%).Midazolam slightly inhibited U14-Luc-TdT cells at the concentration of 0.5mg/L on 48h(inhibition rate 6.13%),and cell proliferation was not affected at concentrations of 0.1 mg/L or 0.2mg/L.On 72h,midazolam inhibited the proliferation of U14-Luc-TdT cells at all concentrations(inhibition rate 5.85%～7.68%).The inhibitory effect on the proliferation of U14-Luc-TdT cells was additive between some combination of 5-fluorouracil and midazolam(5-FU 100mg/L+midazolam 0.2mg/L,5-FU100mg/L+midazolam 0.5mg/L,5-FU150mg/L+midazolam 0.1 mg/L,5-FU 150mg/L+midazolam 0.2mg/L)(q value 0.85～0.88).With the designed concentations in this study the inhibition of 5-FU on T cell proliferation was obvious with the increase of time and concentration(inhibition rate 23%-40%).And Midazolam could slightly promote T cells proliferation with inhibiton rate varied between-4.15%--7.94%.Compared with the same concentration of 5-FU used alone,the inhibition rate(21.76%-25.67%)of T cells was significantly reduced when 5-FU was combined with midazolam(q value 0.6-0.7).The amounts of TNF-α and IFN-y in the co-culture system of U14-Luc-TdT cells and T cells decreased under the effect of different concentrations of 5-FU(OD0.09～0.27 vs 0.37;0.03～0.07 vs 0.21),but the contents of TNF-α were significantly increased when midazolam was 0.2mg/L and 0.5mg/L(OD0.56-0.58 vs 0.37),and the amounts of IFN-y were significantly increased when midazolam was 0.5mg/L(OD0.24-0.26 vs 0.21).The amounts of TNF-α in the combination of 5-FU150mg/L+midazola0.5mg/L recovered compared with that of 5-FU150mg/L used only(OD0.13 vs 0.09),while the amounts of IFN-y in all combinations were higher than that of 5-FU used alone(0.08～0.14 vs 0.03～0.07),so it clearly showed that midazolam could reduce the toxic effect of 5-FU on T cells and partially rescue their function by restoring the TNF-α IFN-γ secretion.Different stressors were used to induce chronic stress in mice,and the immune function and the growth of transplanted tumor in mice were observed.Quantified anxiety in chronic stress,and used O maze to test the chronic anxiety model.It was found that mice with chronic anxiety had shorter duration in open area than that of the control group(29sec vs 60sec),and the duration in open area was prolonged after using midazolam for 0.3mg/kg body for one week(89sec vs 58sec).The transplanted tumor in the anxious group grew fastest in all 5 groups,while the anxious+5-FU+midazolam group had the slowest growth pate.After dissection,it was found that the average tumor weight in the anxiety group was the heaviest in all groups(average tumor weight 14.6g vs 7.25g or 10.95g),while the anxiety+5-FU+midazolam group had the lightest(average tumor weight 4.37g vs 9.41g).The indexes of spleen and thymus in the anxiety group decreased significantly(7.80 vs 13.66;1.55 vs 4.79),and the indexes of thymus in the anxiety+5-FU group and the anxiety+5-FU+midazolam group also showed a slow decline(1.83,1.83 vs 4.97),while those in the anxiety+5-FU+midazolam group had no significant change(2.63 vs 4.79).That was to say,anxiety could damage the immune organs such as spleen and thymus in body,while 5-FU also had a severe side effect on the thymus of the body.About effects of 5-fluorouracil on intestinal barrier in mice and the protective effects of probiotics and prebiotics,histopathology showed that compared with the control group,5-FU caused serious focal defects and shorter microvilli in mice colon mucosa,while lymphoid nodules is smaller and lesser.Prebiotics or probiotics treatment alleviated the damage,but the microvilli lesion still existed.Combination of prebiotics and probiotic treatment synergized the alleviating effects with almost normal intestinal structure,lymphoid nodules.The mucus layer is destroyed by 5-FU.The expressions of claudin-1,MUC2,CD45 and lysozyme were decreased,while all the molecules in the prebiotics/probiotics group showed improvement.CONCLUSION In this study,luciferase and red fluorescence labelled murine cervical cancer cell line U14-Luc-TdT was established.Midazolam slightly inhibited the proliferation of U14-Luc-TdT cells.Combined with 5-FU at specific concentrations,they showed additive inhibition on the proliferation of U14-Luc-TdT cells.Midazolam could slightly stimulate T cells proliferation and 5-FU can inhibite it.The combination of midazolam and 5-FU showed antagonistic effect on T cells proliferation.In the co-culture system of U14-Luc-TdT cells and T cells,midazolam could significantly alleviate the toxic effect of 5-FU on T cells and increase the production of immune factors TNF-α and IFN-y.In vivo experiment had shown that chronic stress can promote the growth of tumors,which may be related to the damage of spleen and thymus that caused by anxiety and the decrease of immune function.Midazolam combined with 5-FU could effectively slow down tumor growth in mice.5-FU can damage the mechanical,immune and chemical barriers of the colon while probiotics and prebiotics can alleviated the adverse effects. |
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