Influence Of Nicotine On The Role Of Ca²⁺/CaN-NFATc Signal Pathway In The Pathogenesis Of Asthma

Objective:To investigate the expreession and activity of calcineurin (CaN) in asthmatic rat lungs, and to explore the role of calcineurin in airway remodeling with asthma.Methods:Twenty Wistar rats were randomly divided into two groups:the control group and the asthma group, ten rats each group. The rats in asthma group were sensitized with 10% ovalbumin and challenged with 2% ovalbumin to establish the asthma model, while the rats in control group were sensitized and challenged with saline instead. The following parametes were measured:airway inflammation by HE staining, the bronchial wall thickness (WAt/Pi) by computer-assisted image analysis system; the expression of CaN mRNA by Real-time PCR, the protein expression of CaN by immunohistochemistry and the activity of CaN by biochemical method; the cell cycle distribution of monocyte in peripheral blood by flow cytometry and the level of IL-4 and TNF-a in plasma by ELISA.Results:1. The bronchial wall thickness of the asthma group [(20.1190±2.4045)μm2/μm]was significantly increased compared with that of the control group [(13.0476±1.4327)μm2/μm] (P＜0.01).2. The expression of CaN mRNA, the protein expression of CaN and the activity of CaN in the asthma group[(1.21-2.71),(0.3725±0.0586),(0.0588±0.0150)μmolPi/(mgprot·hour-1), respectively] were significantly higher than those of the control group [(0.84-1.19), (0.2407±0.0450), (0.0380±0.0120)μmolPi/(mgprot-hour-1)](P<0.05,P<0.01,P<0.01, respectively).3. The level of IL-4 and TNF-a in plasma of asthma group[(327.5443±39.8024) pg/ml and(228.0855±60.1975)pg/ml] was significantly higher than that in control group[(223.3017±40.9510)pg/ml and (174.1766±33.0778) pg/ml] (P＜0.01, P＜0.05, respectively).4. The percentage of monocyte in the G0/G1 phase in peripheral blood was lower in the asthma group (90.0980±2.3468)% than that in the control group(95.1840±2.6970)%; howover, the percentage of monocyte in the S phase and S+G2/M phase in the asthma group was [(7.3950±1.9028)% and (9.9020±2.3468)% respectively],which was significantly higher than that in the control group [(3.2440±2.1460)% and (4.8160±2.6970)%](all P<0.01)].5.(1)There was a positive correlation between the activity of calcineurin in rat lungs and the percentage of monocyte in the S phase and S+G2/M phase in peripheral blood (all P<0.05). The positive correlation existed between the activity of calcineurin and the level of IL-4 in plasma or the bronchial wall thickness too(P<0.05, P＜0.01, respectively).(2)There was a positive correlation between the level of IL-4 and the level of TNF-a in plasma(P<0.05). The positive correlation existed between the level of IL-4 in plasma and the bronchial wall thickness too(P<0.05).Conclusions:The activity of CaN in rat lungs with asthma was increased and it might play an important part in the development of airway remodeling in asthma by promoting the increment of bronchial wall thickness and causing the proliferation and activation of monocyte in peripheral blood,even causing the producing of IL-4 or TNF-α. Objective:To evaluate the activity of Ca2+/CaN-NFATc, and to studuy its association with the imbalance of Thl/Th2 in the rat lungs with asthma.Methods:Twenty Wistar rats were randomized to the asthma group and the control group, ten rats each group. The rats were sensitized with 10% ovalbumin and challenged with 2% ovalbumin to establish the asthma model. Airway inflammation was observed by HE staining. The thickness of bronchial wall (WAt/Pi) was measured by computer-assisted image analysis system. The quantity of Ca2+ and activity of CaN were evaluated by biochemical method. The protein expression of dephosphorylated NFATc was assayed by Western blotting. The level of IL-4 and IL-2 was measured by ELISA.Results:1. The thickness of bronchial wall of the asthma group [(20.1190±3.0795)μm2/μm] was significantly higher than that of the control group [(13.0476±2.0616)μm2/μm] (P<0.01).2. The level of IL-4[(4.7309±0.2356) pg/ml] and the IL-4/IL-2 ratio (1.1255±0.1092) in rat lungs of asthma group were significantly higher than those in control group [(3.4635±0.4468) pg/ml and (0.6649±0.0875)](P<0.01,respectively). However, the level of IL-2 was lower in asthma group [(4.2251±0.2751) pg/ml] than that in control group [(5.2161±03093) pg/ml] (P＜0.01).3. The activity of CaN [(0.0560±0.0202)μmolPi/(mgprot·hour-1)] and the protein expression of NFATc (1.0685±0.0497) in asthma group were higher than those in control group [(0.0362±0.0121)μmolPi/(mgprot·hour-1) and (0.6260±0.0377)] (P<0.01, respectively), but the quantity of Ca2+ was lower in asthma group[(0.0796±0.0284) mmol/gprot]than that in control group [(0.1497±0.0426) mmol/gprot] (P＜0.01).4. (1)There was a positive correlation between the activity of CaN and the protein expression of dephosphorylated NFATc(P<0.05).(2)There was a positive correlation between the protein expression of dephosphorylated NFATc and the IL-4/IL-2 ratio(P<0.01).(3)There was a positive correlation between the thickness of bronchial wall and the IL-4/IL-2 ratio(P<0.01).Conclusions:The activity of CaN-NFATc was increased in rat lungs of the asthma group and it might promote the IL-4/IL-2 ratio to increase. So the signal pathway of CaN-NFATc probably takes part in the imbalance of Th1/Th2 and airway remodeling in asthmatic rat lungs. Objective:To evaluate the effects of the activity Ca2+/CaN-NFATc on the activation and proliferation of Lymphocyte in sensitized rats.Methods:Thirty Wistar rats were randomly divided into the sensitized group, the CsA group and the control group, ten rats each group. The rats of the sensitized group and the CsA group were sensitized with 10% ovalbumin to inject intraperitoneally and challenged with 2% ovalbumin to aerosolize, forty minutes every day for two weeks in all. The control group was sensitized and challenged with saline instead. Lymphocyte was separated from spleen and cultured for 24 hours after the last challenge. PHA-p (5μg/ml) was added to the culture medium in every group and CsA (1.0μg/ml) was added to the CsA group. The other operation was the same among three groups. The activity of CaN in Lymphocyte was evaluated by biochemical method. The protein expression of dephosphorylated NFATc was assayed by immunocytochemistry. The level of IL-4 and IL-2 was measured by ELISA. The concentration of [Ca2+] and the cell cycle distribution of Lymphocyte were analyzed by flow cytometry. The protein expression of CyclinE was assayed by Western blotting.Results:1. The concentration of [Ca2+] in Lymphocyte of the sensitized group (145.37±2.17) was significantly higher than that of the control group (123.34±5.44) (P＜0.01), while the CsA group (119.41±3.01)was lower than the control group(P<0.05) and the sensitized group(P<0.01).2. The activity of CaN in Lymphocyte of the sensitized group [(0.0844±0.0338)μmolPi/(mgprot·hour-1)] was significantly higher than that of the control group [(0.0587±0.0304)μmolPi/(mgprot·hour-1)] (P＜0.05), while the CsA group [(0.0324±0.0134)μmolPi/(mgprot·hour-1)] was lower than the control group(P<0.05) and the sensitized group(P＜0.01).3. The protein expression of dephosphorylated NFATc in Lymphocyte of the sensitized group (81.2080±14.3910) was higher than that of the control group(63.6597±9.0225))(P<0.01), while the CsA group was lower than the control group(P<0.01) and the sensitized group (P＜0.01).4.(1)The level of IL-4[(1.5488±0.1884)×10-2pg/ml] and the IL-4/IL-2 ratio(0.8117±0.1153) in culture supernatants of the sensitized group were higher than those of the control group [(0.9931±0.1208)×10-2pg/ml and(0.4899±0.4900)](P<0.01, respectively). Though the level of IL-2 in culture supernatants of the sensitized group[(1.9165±0.1428)×10-2pg/ml] was lower than the control group[(2.0309±0.1803) pg/ml], it was no difference(P>0.05).(2)The level of IL-4[(0.4687±0.0847)×10-2pg/ml] and the level of IL-2 [(0.6171±0.7132)×10-2 pg/ml] in culture supernatants of the CsA group were lower than those of the sensitized group and the control group (allP<0.01). The IL-4/IL-2 ratio in culture supernatants of the CsA group (0.7762±0.1957) was higher than that of the control group(P<0.01), but it was no difference between the CsA group and the sensitized group(P>0.05).5. The percentage of Lymphocyte in the S phase (7.8600±2.8241)% and S+G2/M phase (10.6700±3.3850)% in sensitized group was increased than those in control group [(1.7470±0.8545)%,(5.8740±1.4389)%,respectively] and in CsA group [(4.8600±1.959)%,(7.9900±1.9405)%,respectively] (all P<0.01). The percentage of G0/G1 phase was lower in sensitized group(89.3300±3.3850)% than those in control group (94.1260±1.4389)% and the CsA group(92.2100±1.9267)%(allP<0.01). It was no significant difference between the control group and the CsA group (P>0.05).6. The protein expression of CyclinE in Lymphocyte of the sensitized group (0.9327±0.0370) was higher than that of the control group (0.8374±0.0637) (P＜0.01), while the CsA group (0.6840±0.0485) was lower than the control group and the sensitized group (P＜0.01, respectively).7. (1) There was a positive correlation between the concentration of [Ca2+]i and the activity of calcineurin in Lymphocyte. The positive correlation existed between the activity of calcineurin and the protein expression of dephosphorylated NFATc in Lymphocyte too (P<0.01,P<0.05, respectively). (2) There was a positive correlation between the protein expression of dephosphorylated NFATc in Lymphocyte and the level of IL-4 in culture supernatants. The positive correlation existed between the protein expression of NFATc and CyclinE in Lymphocyte too(all P＜0.01).Conclusions:The activity of Ca2+/CaN-NFATc was inereased in Lymphocyte of the sensitized rats. The rising of it might result in the imbalance of Thl/Th2 by promoting the expression of IL-4 and might lead to the proliferation of Lymphocyte by promoting the expression of CyclinE. Objective:To explore the influence of nicotine on the activation and proliferation of Lymphocyte in sensitized rats and its underlying mechanisms.Methods:Forty Wistar rats were sensitized with 10% ovalbumin to inject intraperitoneally and challenged with 2% ovalbumin to aerosolize, forty minutes every day for two weeks in all. Lymphocyte was separated from spleen, grouped and cultured for 24 hours after the last challenge. At the same time, PHA-p (5μg/ml) was added to the culture medium in every group and nicotine at a range of concentrations (0,1.0,10, 100μmol/L) was added to different groups(group0, group 1, group2 and group3). The level of IL-4 and IL-2 in culture supernatants was measured by ELISA. The expression of cyclinE mRNA in Lymphocyte was detected by Real-time PCR. The protein expression of CyclinE and dephosphorylated NFATc in Lymphocyte was assayed by Western blotting. The cell cycle distribution of Lymphocyte and the concentration of [Ca2+]i in Lymphocyte were analyzed by flow cytometry. The activity of CaN in Lymphocyte was evaluated by biochemical method.Results:1. (1) The difference of the level of IL-4 in culture supernatants was significant among the group0 [(1.5527±0.2066)×10-2pg/ml], group1[(1.5287±0.0955)×10-2pg/ml], group2[(1.7903±0.1424)×10-2pg/ml] and group3[(0.9234±0.2185)×10-2pg/ml] (P＜0.01);(2) The difference of the level of IL-2 in culture supernatants was significant among the group0 [(1.9041±0.1545)×10-2 pg/ml], group1[(0.9183±0.0848)×10-2 pg/ml], group2[(0.7943±0.0642)×10-2 pg/ml] and group3[(0.6567±0.0790)×10-2 pg/ml] (P<0.01);(3) The difference of the IL-4/IL-2 ratio in culture supernatants was significant among the group0 (0.8196±0.1254), groupl(1.6843±0.2497), group2(2.2670±0.2631) and group3 (1.4149±0.3372) (P＜0.01).2. (1) The difference of the expression of cyclinE mRNA in Lymphocyte was significant among the group0 (1.0794±0.4809), groupl(1.6287±0.3123),group2(2.3411±0.3802) and group3(0.6182±0.2401) (P＜0.01);(2) The difference of the protein expression of cyclinE in Lymphocyte was significant among the group0(0.9408±0.0145), group1(0.9755±0.0082), group2(1.2757±0.0701) and group3(0.9242±0.0123) (P＜0.01);(3) The difference of the percentage of Lymphocyte in G0/G1 phase was significant among the group0 (89.3300±3.3850)%, groupl(83.8670±3.4977)%, group2(77.9480±2.7949)%, group3(89.8280±3.400)%(P<0.01, respectively). So did the difference of the percentage of S and S+G2/M phase among the group0[(7.8600±2.8241)% and (10.6700±3.3850)%], group1[(10.9170±3.1836)% and (15.9330±3.81453)%], group2[(14.2100±2.6690)% and (22.0520±2.7949)%] and group3[(7.5510±2.3390)% and (10.1720±3.4004)%](allP<0.01).3. (1) The difference of the concentration of [Ca2+]i in Lymphocyte was significant among the group0(145.3720±2.1747), groupl(151.9470±3.2908), group2(157.1350±4.8537) and group3(132.1750±3.8274) (P<0.01);(2) The difference of the activity of CaN in Lymphocyte was significant among the group0 [(0.0844±0.0338)μmolPi/(mgprot-hour-1)], group1[(0.2493±0.0568)μmolPi/(mgprot-hour-1)], group2[(0.3203±0.0924)μmolPi/(mgprot-hour-1)] and group3[(0.1788±0.0728)μmolPi/(mgprot·hour-1)] (P＜0.01);(3) The difference of the protein expression of dephosphorylated NFATc in Lymphocyte was significant among the group0(0.8379±0.0132), groupl(0.9655±0.0144), group2(1.1011±0.0306) and group3(0.8973±0.0167) (P<0.01).4. (1) There was a positive correlation between the concentration of [Ca2+]i and the activity of calcineurin in Lymphocyte. The positive correlation existed between the activity of calcineurin and the protein expression of dephosphorylated NFATc in Lymphocyte too (all P＜0.01).(2)There was a positive correlation between the protein expression of dephosphorylated NFATc in Lymphocyte and the IL-4/IL-2 ratio in culture supernatants. The positive correlation existed between the protein expression of NFATc and CyclinE in Lymphocyte too (all P＜0.01).Conclusions:(1) Exposure of Lymphocytes of the sensitized rats to nicotine at a concentration (0,1.0, 10μmol/L) led to decrease of the level of IL-2, increase the level of IL-4 and the IL-4/IL-2 ratio dose-dependently. In contrast, nicotine at concentration of 100μmol/L was also found to inhibit the production of IL-2 and IL-4 and reduce the IL-4/IL-2 ratio significantly.(2) Exposure of Lymphocytes of the sensitized rats to nicotine at a concentration (0,1.0, 10μmol/L) induced a dose-dependent increase of the expression of cyclinE mRNA and the protein expression of cyclinE and activated cell cycle progression by promoting the G0/G1-to-S+G2/M phase transition. While nicotine at concentration of 100μmol/L was found to inhibit the expression of cyclinE mRNA and the protein expression of cyclinE and the cell cycle entry.(3) Exposure of Lymphocytes of the sensitized rats to nicotine at a concentration (0,1.0, 10μmol/L) led to a dose-dependent increase of the concentration of [Ca2+]i, the activity of CaN and the protein expression of dephosphorylated NFATc in Lymphocyte, namly nicotine at a concentration (0,1.0, 10μmol/L) caused the activity of Ca2+/CaN-NFATc to rise; but nicotine at concentration of 100μmol/L was also found to inhibit the activity of Ca2+/CaN-NFATc in Lymphocyte.(4) The activity of Ca2+/CaN-NFATc in Lymphocyte might affect the proliferation and activation of Lymphocyte and the production of cytokines.In summary, nicotine might affect the proliferation and activation of Lymphocyte and might affect the imbalance of Th1/Th2 by regulating the activity of Ca2+/CaN-NFATc in Lymphocyte of sensentized rats.