尼古丁对Ca~（2+）/CaN-NFATc信号通路在哮喘发病机制中作用的影响
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摘要
第一部分Ca2+/CaN-NFATc信号通路在哮喘发病机制中作用的研究
第一节钙调神经磷酸酶在哮喘大鼠气道重塑中的作用
目的：通过测定哮喘大鼠肺组织中钙调神经磷酸酶(Calcineurin, CaN)蛋白、mRNA的表达及CaN活性,探讨CaN在哮喘大鼠气道重塑中的作用。
方法：将20只Wistar大鼠随机分为哮喘组和对照组,每组10只。用10%卵白蛋白(o valbumin，OVA)溶液致敏,2%OVA溶液雾化吸入激发,建立哮喘模型。HE染色观察气道炎症情况；图像分析观察支气管管壁厚度；实时定量(Real-time)PCR测定肺组织中CaN mRNA水平；免疫组织化学法检测肺组织中CaN蛋白的表达；生物化学法检测大鼠肺组织CaN活性；流式细胞仪测定外周血单个核细胞细胞周期时相分布；ELISA法(酶联免疫吸附试验)测定血浆中IL-4、TNF-α的含量。
结果：
1.哮喘组大鼠支气管管壁厚度[(20.1190±2.4045)μm2／μm]较对照组[(13.0476±1.4327)μm2／μm]明显增加,差异有统计学意义(P<0.01)。
2.哮喘组大鼠肺组织中CaNmRNA水平的相对表达(1.21-2.71)、CaN蛋白的相对表达量(0.3725±0.0586)及CaN的活性[(0.0588±0.0150)μmolPi/(mgprot·hour-1)]均较对照组[分别为(0.84-1.19)、(0.2407±0.0450)和(0.0379±0.0120)μmolPi/(mgprot·hour-1)]高,差异有统计学意义(分别P<0.05,P<0.01和P<0.01)。
3.哮喘组大鼠血浆中IL-4、TNF-α的含量[分别为(327.5443±39.8024)pg／ml和(228.0855±60.1975) pg/ml]较对照组[分别为(223.3017±40.9510)pg/ml和(174.1766±33.0778) pg/ml]增加,差异有统计学意义(分别P<0.01和P<0.05)。
4.哮喘组外周血单个核细胞G0／G1期百分率(90.0980±2.3468)%较对照组(95.1840±2.6970)%降低,S期、S+G2／M期百分率[分别为(7.3950±1.9028)%和(9.9020±2.3468)%]均较对照组[分别为(4.8160±2.6970)%和(3.2440±2.1460)%]增加,差异均有统计学意义(P均<0.01)。
5.(1)大鼠肺组织CaN活性与大鼠支气管管壁厚度呈正相关(P<0.01),与处于S期、S+G2／M期的外周血单个核细胞百分率呈正相关(P均<0.05),与血浆中IL-4的含量亦呈正相关(P<0.05)；
(2)血浆中IL-4含量与TNF-α含量及大鼠支气管管壁厚度亦呈正相关(P均<0.05)。
结论：哮喘大鼠肺组织CaN的活性增加,其可能通过促进气道管壁增厚、外周血单个核细胞的增殖、活化及L-4和TNF-α的产生而参与哮喘气道重塑的形成。
第二节哮喘大鼠肺组织Ca2+/CaN-NFATc活性及其与Th1／Th2失衡的关系
目的：探究哮喘大鼠肺组织Ca2+/CaN-NFATc的活性及其与Th1／Th2失衡的关系。
方法：20只Wistar大鼠随机分为哮喘组和对照组,每组10只。用10%OVA溶液致敏和2%OVA溶液雾化吸入激发,建立大鼠哮喘模型。HE染色观察气道炎症,图像分析测量气道管壁厚度；生物化学法检测肺组织钙含量、CaN活性；Western blotting法测定肺组织去磷酸化NFATc蛋白的表达及ELISA法测定IL-4、IL-2的含量。
结果：
1.哮喘组大鼠支气管管壁厚度[(20.1190±3.0795)μm2／μm]较对照组[(13.0476+2.0616)μm2/μm]支气管管壁厚度明显增加(P<0.01)。
2.哮喘组大鼠肺组织中IL-4含量[(4.7309±0.2356)pg/ml]、IL-4／IL-2比值(1.1255±0.1092)分别较对照组[(3.4635±0.4468)pg/ml和(0.6649±0.0875)]增高,而哮喘组IL-2含量[(4.2251±0.2751)pg/ml]较对照组[(5.2161±03093)pg/ml]降低,差异有统计学意义(P均<0.01)。
3.哮喘组CaN活性[(0.0560±0.0202)μmolPi/(mgprot·hour-1)]和去磷酸化NFATc蛋白的相对表达量(1.0685±0.0497)均较对照组[分别为(0.0362±0.0121)μmolPi/(mgprot·hour-1)和(0.6260±0.0377)]增加,差异有统计学意义(P均<0.01),哮喘组大鼠肺组织钙含量[(0.1497±0.0426)mmol/gprot]较对照组[(0.0796±0.0284)mmol/gprot]降低,差异有统计学意义(P<0.01)。
4.(1)肺组织中CaN活性与去磷酸化NFATc蛋白的相对表达量呈正相关(P<0.05)；
(2)肺组织中去磷酸化NFATc蛋白的相对表达量与IL-4／IL-2比值呈正相关(P<0.01)；
(3)肺组织中IL-4／IL-2比值与大鼠支气管管壁厚度成正相关(P<0.01)。
结论：哮喘大鼠肺组织中CaN-NFATc的活性增加,其活性的增加可促进IL-4／IL-2比值增加,推测CaN-NFATc信号通路可能参与了哮喘大鼠肺组织Th1/Th2失衡及气道重塑的形成。
第三节Ca2+/CaN-NFATc信号通路在致敏大鼠淋巴细胞增殖、活化中的作用
目的：探讨Ca2+/CaN-NFATc信号通路在致敏大鼠淋巴细胞增殖、活化中的作用。
方法：30只Wistar大鼠随机分为对照组、致敏组和环孢菌素A(cyclosporinA, CsA)干预组,每组10只。致敏组和CsA干预组大鼠以10%OVA溶液腹腔注射致敏及2%OVA溶液雾化吸入激发,每天40分钟,共2周,末次激发后分离大鼠的脾淋巴细胞,加PHA-p(终浓度为5μg/ml)培养24小时。CsA干预组同时加入CsA稀释液(CsA终浓度为1.0μg/ml)。对照组以生理盐水代替OVA溶液致敏,余相同。生化法检测淋巴细胞CaN的活性；免疫细胞化学染色法检测淋巴细胞中去磷酸化NFATc蛋白的表达量；ELISA法测定细胞上清液中IL-4和IL-2的水平；流式细胞仪测定淋巴细胞内[Ca2+]i的浓度及淋巴细胞细胞周期各时相分布；Western blotting法测定淋巴细胞CyclinE蛋白的表达。
结果：
1.致敏组淋巴细胞内[Ca2+]的浓度(145.37±2.17)较对照组(123.34±5.44)高,差异有统计学意义(P<0.01)；CsA干预组(119.41±3.01)低于对照组和致敏组,差异有统计学意义(分别P<0.05,P<0.01)。
2.致敏组淋巴细胞CaN的活性[(0.0844±0.0338)μmolPi/(mgprot·hour-1)]较对照组[(0.0587±0.0304)μmolPi/(mgprot-hour-1)]高,差异有统计学意义(P<0.05)；CsA干预组[(0.0324±0.0134)μmolPi/(mgprot·hour -1)]较对照组和致敏组均低,差异有统计学意义(P<0.05,P<0.01)。
3.致敏组淋巴细胞中去磷酸化NFATc蛋白的相对表达量(81.2080±14.3910)较对照组(63.6597±9.0225)高,差异有统计学意义(P<0.01)；CsA干预组(47.1932±7.1620)较对照组和致敏组均低,差异均有统计学意义(P均<0.01)。
4.(1)致敏组细胞上清液中IL-4水平[(1.5488±0.1884)×10-2pg/ml]和IL4／IL2比值(0.8117±0.1153)均较对照组[分别为(0.9931±0.1208)×10-2pg／ml和(0.4899±0.4900)]高,差异有统计学意义(P均<0.01),而IL-2水平[(1.9165±0.1428)×10-2pg/ml]低于对照组[(2.0309±0.1803)×10-2pg/ml]但差异无统计学意义(P>0.05)；(2)CsA干预组细胞上清液中IL-4水平[(0.4687±0.0847)×10-2pg/ml]和IL-2水平[(0.6171±0.7132)×10-2pg/ml]分别低于对照组和致敏组,差异有统计学意义(P均<0.01),IL4／IL2比值(0.7762±0.1957)较对照组高,差异有统计学意义(P<0.01),而较致敏组减低,但差异无统计学意义(P>0.05)。
5.致敏组处于G0／G1期淋巴细胞所占比例(89.3300±3.3850)%较对照组(94.1260±1.4389)%和CsA干预组(92.2100±1.9267)%减小(P均<0.01),而处于S期(7.8600±2.8241)%及S+G2／M期(10.6700±3.3850)%的淋巴细胞所占比例分别较对照组S期(1.7470±0.8545)%及S+G2／M期(5.8740±1.4389)%和CsA干预组S期(4.8600±1.959)%及S+G2／M期(7.9900±1.9405)%淋巴细胞所占比例增加(P均<0.01)；CsA干预组与对照组差别不大,差异无统计学意义(P＞0.05)。
6.致敏组淋巴细胞中CyclinE蛋白的相对表达量(0.9327±0.0370)较对照组(0.8374±0.0637)高,差异有统计学意义(P6＜0.01)；CsA干预组(0.6840±0.0485)较对照组和致敏组均低,差异有统计学意义(P均<0.01)。
7.(1)淋巴细胞内[Ca2+]i浓度与CaN的活性及CaN的活性与去磷酸化NFATc蛋白的相对表达量均呈正相关(分别P<0.01，P＜0.05)；(2)淋巴细胞中去磷酸化NFATc蛋白的相对表达量与上清液中IL-4的水平及淋巴细胞内CyclinE蛋白的相对表达量均呈正相关(分别P 结论：致敏大鼠淋巴细胞Ca2+/CaN-NFATc信号通路的活性增加；其活性的增加可通过促进IL-4的产生和分泌,导致Thl／Th2失衡,也可通过促进CyclinE蛋白的表达而引起淋巴细胞的增殖。
第二部分尼古丁对致敏大鼠淋巴细胞Ca2+/CaN-NFATc信号通路及淋巴细胞增殖和活化的影响
目的：探讨尼古丁对致敏大鼠淋巴细胞增殖、活化及Thl／Th2失衡的影响以及可能的影响机制。
方法：Wistar大鼠40只,用10%OVA溶液腹腔注射及2%OVA溶液雾化吸入激发使其致敏,每天40分钟,共2周,末次激发后分离大鼠的脾淋巴细胞,分组且分别予不同浓度的尼古丁刺激(终浓度分别为尼古丁0组：0μmol/L，尼古丁1组：1.0μmol/L,尼古丁2组：10μmol/L,尼古丁3组：100μmol/L),且每组分别加入PHA-p(终浓度为5μg／ml)培养24小时。ELISA法测定细胞上清液中IL-2、IL-4的水平,western blotting法测定淋巴细胞CyclinE蛋白及去磷酸化NFATc蛋白的表达,实时定量(Real-time)PCR检测淋巴细胞CyclinE mRNA的表达,流式细胞仪测定淋巴细胞的细胞周期分布及细胞内[Ca2+]1浓度,生物化学法检测淋巴细胞CaN活性。
结果：
1.(1)细胞上清液中IL-4的水平分别为尼古丁O组[(1.5527±0.2066)×10-2pg/ml]、尼古丁1组[(1.5287±0.0955)×10-2pg/ml]、尼古丁2组[(1.7903±0.1424)×10-2pg/ml]、尼古丁3组[(0.9234±0.2185)×10-2pg/ml],组间比较差异有统计学意义(P<0.01)；
(2)细胞上清液中IL-2水平分别为尼古丁0组[(1.9041±0.1545)×10-2pg/ml]、尼古丁1组[(0.9183±0.0848)×10-2pg/ml]、尼古丁2组[(0.7943±0.0642)×10-2pg／ml]、尼古丁3组[(0.6567±0.0790)×10-2 pg/ml],组间比较差异有统计学意义(P<0.01)；
(3)细胞上清液中IL-4／IL-2比值分别为尼古丁0组(0.8196±0.1254)、尼古丁1组(1.6843±0.2497)、尼古丁2组(2.2670±0.2631)、尼古丁3组(1.4149±0.3372),组间比较差异有统计学意义(P<0.01)。
2.(1)淋巴细胞中CyclinE mRNA的相对表达分别为尼古丁0组(1.0794±0.4809)、尼古丁1组(1.6287±0.3123)、尼古丁2组(2.3411±0.3802)、尼古丁3组(0.6182±0.2401),组间比较差异有统计学意义(P<0.01)；
(2)淋巴细胞中CyclinE蛋白的相对表达量分别为尼古丁0组(0.9408±0.0145)、尼古丁1组(0.9755±0.0082)、尼古丁2组(1.2757±0.0701)、尼古丁3组(0.9242±0.0123),组间比较差异有统计学意义(P<0.01)；
(3)淋巴细胞细胞周期G0／G1期百分率分别为尼古丁0组(89.3300±3.3850)%、尼古丁1组(83.8670±3.4977)%、尼古丁2组(77.9480±2.7949)%、尼古丁3组(89.8280±3.400)%,组间比较差异有统计学意义(P<0.01)；S期和S+G2／M期百分率分别为尼古丁0组[(7.8600±2.8241)%和(10.6700±3.3850)%]、尼古丁1组[(10.9170±3.1836)%和(15.9330±3.81453)%]、尼古丁2组[(14.2100±2.6690)%和(22.0520±2.7949)%]、尼古丁3组[(7.5510±2.3390)%和(10.1720±3.4004)%],组间比较差异均有统计学意义(P均<0.01)。
3.(1)淋巴细胞中[Ca2+]i浓度分别为尼古丁0组(145.3720±2.1747)、尼古丁1组(151.9470±3.2908)、尼古丁2组(157.1350±4.8537)、尼古丁3组(132.1750±3.8274),组间比较差异有统计学意义(P<0.01)；
(2)淋巴细胞中CaN的活性分别为尼古丁0组[(0.0844±0.0338)μmolPi／(mgprot·hour-1)]、尼古丁1组[(0.2493±0.0568)μmolPi／(mgprot·hour-1)].尼古丁2组[(0.3203±0.0924)μmolPi／(mgprot·hour-1)].尼古丁3组[(0.1788±0.0728)μmolPi／(mgprot-hour-1)],组间比较差异有统计学意义(P<0.01)；
(3)淋巴细胞中去磷酸化NFATc蛋白的相对表达量分别为尼古丁0组(0.8379±0.0132)、尼古丁1组(0.9655±0.0144)、尼古丁2组(1.1011±0.0306)、尼古丁3组(0.8973±0.0167),组间比较差异有统计学意义(P<0.01)。
4.(1)淋巴细胞中[Ca2+]浓度与CaN的活性及CaN的活性与去磷酸化NFATc蛋白的相对表达量均呈正相关(P均<0.01)；
(2)淋巴细胞中去磷酸化NFATc蛋白的相对表达量与细胞上清液中IL4／IL2的比值及细胞中CyclinE蛋白的相对表达量均呈正相关(P均<0.01)。
结论：
(1)尼古丁浓度在0-10μmol／L范围内时,致敏大鼠淋巴细胞上清液中IL-4的水平及IL-4／IL-2比值随尼古丁浓度增加而增加,而IL-2水平随尼古丁浓度增加而渐减少；而尼古丁浓度为100μmol／L时,细胞上清液中IL-4水平、IL-2水平及IL-4／IL-2比值均显著减少。
(2)尼古丁浓度在0-10μmol／L范围内时,可使致敏大鼠淋巴细胞中CyclinE mRNA和CyclinE蛋白水平的表达渐增加,使处于DNA合成期和细胞分裂前期的淋巴细胞比例显著增加,促进了淋巴细胞的增殖,且呈浓度依赖性；尼古丁浓度为100μmol／L时,反而显著抑制了致敏大鼠淋巴细胞中CyclinE mRNA和CyclinE蛋白水平的表达,抑制了其淋巴细胞的增殖。
(3)尼古丁浓度在0-10μmol/L范围内时,致敏大鼠淋巴细胞中[Ca2+]i浓度渐增加,CaN的活性渐增加,去磷酸化NFATc蛋白的相对表达量亦渐增加,即淋巴细胞Ca2+／CaN-NFATc的活性随尼古丁浓度的增加而增加；而100μmol／L尼古丁反而显著抑制了致敏大鼠淋巴细胞Ca2+／CaN-NFATc的活性。
(4)淋巴细胞Ca2+／CaN-NFATc活性的改变可影响淋巴细胞的增殖、活化及细胞因子的产生。
总之,此部分实验结果说明尼古丁可能通过影响致敏大鼠淋巴细胞Ca2+／CaN-NFATc的活性而影响其淋巴细胞的增殖、活化和Thl/Th2的失衡。
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.
第一节钙调神经磷酸酶在哮喘大鼠气道重塑中的作用
目的：通过测定哮喘大鼠肺组织中钙调神经磷酸酶(Calcineurin, CaN)蛋白、mRNA的表达及CaN活性,探讨CaN在哮喘大鼠气道重塑中的作用。
方法：将20只Wistar大鼠随机分为哮喘组和对照组,每组10只。用10%卵白蛋白(o valbumin，OVA)溶液致敏,2%OVA溶液雾化吸入激发,建立哮喘模型。HE染色观察气道炎症情况；图像分析观察支气管管壁厚度；实时定量(Real-time)PCR测定肺组织中CaN mRNA水平；免疫组织化学法检测肺组织中CaN蛋白的表达；生物化学法检测大鼠肺组织CaN活性；流式细胞仪测定外周血单个核细胞细胞周期时相分布；ELISA法(酶联免疫吸附试验)测定血浆中IL-4、TNF-α的含量。
结果：
1.哮喘组大鼠支气管管壁厚度[(20.1190±2.4045)μm2／μm]较对照组[(13.0476±1.4327)μm2／μm]明显增加,差异有统计学意义(P<0.01)。
2.哮喘组大鼠肺组织中CaNmRNA水平的相对表达(1.21-2.71)、CaN蛋白的相对表达量(0.3725±0.0586)及CaN的活性[(0.0588±0.0150)μmolPi/(mgprot·hour-1)]均较对照组[分别为(0.84-1.19)、(0.2407±0.0450)和(0.0379±0.0120)μmolPi/(mgprot·hour-1)]高,差异有统计学意义(分别P<0.05,P<0.01和P<0.01)。
3.哮喘组大鼠血浆中IL-4、TNF-α的含量[分别为(327.5443±39.8024)pg／ml和(228.0855±60.1975) pg/ml]较对照组[分别为(223.3017±40.9510)pg/ml和(174.1766±33.0778) pg/ml]增加,差异有统计学意义(分别P<0.01和P<0.05)。
4.哮喘组外周血单个核细胞G0／G1期百分率(90.0980±2.3468)%较对照组(95.1840±2.6970)%降低,S期、S+G2／M期百分率[分别为(7.3950±1.9028)%和(9.9020±2.3468)%]均较对照组[分别为(4.8160±2.6970)%和(3.2440±2.1460)%]增加,差异均有统计学意义(P均<0.01)。
5.(1)大鼠肺组织CaN活性与大鼠支气管管壁厚度呈正相关(P<0.01),与处于S期、S+G2／M期的外周血单个核细胞百分率呈正相关(P均<0.05),与血浆中IL-4的含量亦呈正相关(P<0.05)；
(2)血浆中IL-4含量与TNF-α含量及大鼠支气管管壁厚度亦呈正相关(P均<0.05)。
结论：哮喘大鼠肺组织CaN的活性增加,其可能通过促进气道管壁增厚、外周血单个核细胞的增殖、活化及L-4和TNF-α的产生而参与哮喘气道重塑的形成。
第二节哮喘大鼠肺组织Ca2+/CaN-NFATc活性及其与Th1／Th2失衡的关系
目的：探究哮喘大鼠肺组织Ca2+/CaN-NFATc的活性及其与Th1／Th2失衡的关系。
方法：20只Wistar大鼠随机分为哮喘组和对照组,每组10只。用10%OVA溶液致敏和2%OVA溶液雾化吸入激发,建立大鼠哮喘模型。HE染色观察气道炎症,图像分析测量气道管壁厚度；生物化学法检测肺组织钙含量、CaN活性；Western blotting法测定肺组织去磷酸化NFATc蛋白的表达及ELISA法测定IL-4、IL-2的含量。
结果：
1.哮喘组大鼠支气管管壁厚度[(20.1190±3.0795)μm2／μm]较对照组[(13.0476+2.0616)μm2/μm]支气管管壁厚度明显增加(P<0.01)。
2.哮喘组大鼠肺组织中IL-4含量[(4.7309±0.2356)pg/ml]、IL-4／IL-2比值(1.1255±0.1092)分别较对照组[(3.4635±0.4468)pg/ml和(0.6649±0.0875)]增高,而哮喘组IL-2含量[(4.2251±0.2751)pg/ml]较对照组[(5.2161±03093)pg/ml]降低,差异有统计学意义(P均<0.01)。
3.哮喘组CaN活性[(0.0560±0.0202)μmolPi/(mgprot·hour-1)]和去磷酸化NFATc蛋白的相对表达量(1.0685±0.0497)均较对照组[分别为(0.0362±0.0121)μmolPi/(mgprot·hour-1)和(0.6260±0.0377)]增加,差异有统计学意义(P均<0.01),哮喘组大鼠肺组织钙含量[(0.1497±0.0426)mmol/gprot]较对照组[(0.0796±0.0284)mmol/gprot]降低,差异有统计学意义(P<0.01)。
4.(1)肺组织中CaN活性与去磷酸化NFATc蛋白的相对表达量呈正相关(P<0.05)；
(2)肺组织中去磷酸化NFATc蛋白的相对表达量与IL-4／IL-2比值呈正相关(P<0.01)；
(3)肺组织中IL-4／IL-2比值与大鼠支气管管壁厚度成正相关(P<0.01)。
结论：哮喘大鼠肺组织中CaN-NFATc的活性增加,其活性的增加可促进IL-4／IL-2比值增加,推测CaN-NFATc信号通路可能参与了哮喘大鼠肺组织Th1/Th2失衡及气道重塑的形成。
第三节Ca2+/CaN-NFATc信号通路在致敏大鼠淋巴细胞增殖、活化中的作用
目的：探讨Ca2+/CaN-NFATc信号通路在致敏大鼠淋巴细胞增殖、活化中的作用。
方法：30只Wistar大鼠随机分为对照组、致敏组和环孢菌素A(cyclosporinA, CsA)干预组,每组10只。致敏组和CsA干预组大鼠以10%OVA溶液腹腔注射致敏及2%OVA溶液雾化吸入激发,每天40分钟,共2周,末次激发后分离大鼠的脾淋巴细胞,加PHA-p(终浓度为5μg/ml)培养24小时。CsA干预组同时加入CsA稀释液(CsA终浓度为1.0μg/ml)。对照组以生理盐水代替OVA溶液致敏,余相同。生化法检测淋巴细胞CaN的活性；免疫细胞化学染色法检测淋巴细胞中去磷酸化NFATc蛋白的表达量；ELISA法测定细胞上清液中IL-4和IL-2的水平；流式细胞仪测定淋巴细胞内[Ca2+]i的浓度及淋巴细胞细胞周期各时相分布；Western blotting法测定淋巴细胞CyclinE蛋白的表达。
结果：
1.致敏组淋巴细胞内[Ca2+]的浓度(145.37±2.17)较对照组(123.34±5.44)高,差异有统计学意义(P<0.01)；CsA干预组(119.41±3.01)低于对照组和致敏组,差异有统计学意义(分别P<0.05,P<0.01)。
2.致敏组淋巴细胞CaN的活性[(0.0844±0.0338)μmolPi/(mgprot·hour-1)]较对照组[(0.0587±0.0304)μmolPi/(mgprot-hour-1)]高,差异有统计学意义(P<0.05)；CsA干预组[(0.0324±0.0134)μmolPi/(mgprot·hour -1)]较对照组和致敏组均低,差异有统计学意义(P<0.05,P<0.01)。
3.致敏组淋巴细胞中去磷酸化NFATc蛋白的相对表达量(81.2080±14.3910)较对照组(63.6597±9.0225)高,差异有统计学意义(P<0.01)；CsA干预组(47.1932±7.1620)较对照组和致敏组均低,差异均有统计学意义(P均<0.01)。
4.(1)致敏组细胞上清液中IL-4水平[(1.5488±0.1884)×10-2pg/ml]和IL4／IL2比值(0.8117±0.1153)均较对照组[分别为(0.9931±0.1208)×10-2pg／ml和(0.4899±0.4900)]高,差异有统计学意义(P均<0.01),而IL-2水平[(1.9165±0.1428)×10-2pg/ml]低于对照组[(2.0309±0.1803)×10-2pg/ml]但差异无统计学意义(P>0.05)；(2)CsA干预组细胞上清液中IL-4水平[(0.4687±0.0847)×10-2pg/ml]和IL-2水平[(0.6171±0.7132)×10-2pg/ml]分别低于对照组和致敏组,差异有统计学意义(P均<0.01),IL4／IL2比值(0.7762±0.1957)较对照组高,差异有统计学意义(P<0.01),而较致敏组减低,但差异无统计学意义(P>0.05)。
5.致敏组处于G0／G1期淋巴细胞所占比例(89.3300±3.3850)%较对照组(94.1260±1.4389)%和CsA干预组(92.2100±1.9267)%减小(P均<0.01),而处于S期(7.8600±2.8241)%及S+G2／M期(10.6700±3.3850)%的淋巴细胞所占比例分别较对照组S期(1.7470±0.8545)%及S+G2／M期(5.8740±1.4389)%和CsA干预组S期(4.8600±1.959)%及S+G2／M期(7.9900±1.9405)%淋巴细胞所占比例增加(P均<0.01)；CsA干预组与对照组差别不大,差异无统计学意义(P＞0.05)。
6.致敏组淋巴细胞中CyclinE蛋白的相对表达量(0.9327±0.0370)较对照组(0.8374±0.0637)高,差异有统计学意义(P6＜0.01)；CsA干预组(0.6840±0.0485)较对照组和致敏组均低,差异有统计学意义(P均<0.01)。
7.(1)淋巴细胞内[Ca2+]i浓度与CaN的活性及CaN的活性与去磷酸化NFATc蛋白的相对表达量均呈正相关(分别P<0.01，P＜0.05)；(2)淋巴细胞中去磷酸化NFATc蛋白的相对表达量与上清液中IL-4的水平及淋巴细胞内CyclinE蛋白的相对表达量均呈正相关(分别P
第二部分尼古丁对致敏大鼠淋巴细胞Ca2+/CaN-NFATc信号通路及淋巴细胞增殖和活化的影响
目的：探讨尼古丁对致敏大鼠淋巴细胞增殖、活化及Thl／Th2失衡的影响以及可能的影响机制。
方法：Wistar大鼠40只,用10%OVA溶液腹腔注射及2%OVA溶液雾化吸入激发使其致敏,每天40分钟,共2周,末次激发后分离大鼠的脾淋巴细胞,分组且分别予不同浓度的尼古丁刺激(终浓度分别为尼古丁0组：0μmol/L，尼古丁1组：1.0μmol/L,尼古丁2组：10μmol/L,尼古丁3组：100μmol/L),且每组分别加入PHA-p(终浓度为5μg／ml)培养24小时。ELISA法测定细胞上清液中IL-2、IL-4的水平,western blotting法测定淋巴细胞CyclinE蛋白及去磷酸化NFATc蛋白的表达,实时定量(Real-time)PCR检测淋巴细胞CyclinE mRNA的表达,流式细胞仪测定淋巴细胞的细胞周期分布及细胞内[Ca2+]1浓度,生物化学法检测淋巴细胞CaN活性。
结果：
1.(1)细胞上清液中IL-4的水平分别为尼古丁O组[(1.5527±0.2066)×10-2pg/ml]、尼古丁1组[(1.5287±0.0955)×10-2pg/ml]、尼古丁2组[(1.7903±0.1424)×10-2pg/ml]、尼古丁3组[(0.9234±0.2185)×10-2pg/ml],组间比较差异有统计学意义(P<0.01)；
(2)细胞上清液中IL-2水平分别为尼古丁0组[(1.9041±0.1545)×10-2pg/ml]、尼古丁1组[(0.9183±0.0848)×10-2pg/ml]、尼古丁2组[(0.7943±0.0642)×10-2pg／ml]、尼古丁3组[(0.6567±0.0790)×10-2 pg/ml],组间比较差异有统计学意义(P<0.01)；
(3)细胞上清液中IL-4／IL-2比值分别为尼古丁0组(0.8196±0.1254)、尼古丁1组(1.6843±0.2497)、尼古丁2组(2.2670±0.2631)、尼古丁3组(1.4149±0.3372),组间比较差异有统计学意义(P<0.01)。
2.(1)淋巴细胞中CyclinE mRNA的相对表达分别为尼古丁0组(1.0794±0.4809)、尼古丁1组(1.6287±0.3123)、尼古丁2组(2.3411±0.3802)、尼古丁3组(0.6182±0.2401),组间比较差异有统计学意义(P<0.01)；
(2)淋巴细胞中CyclinE蛋白的相对表达量分别为尼古丁0组(0.9408±0.0145)、尼古丁1组(0.9755±0.0082)、尼古丁2组(1.2757±0.0701)、尼古丁3组(0.9242±0.0123),组间比较差异有统计学意义(P<0.01)；
(3)淋巴细胞细胞周期G0／G1期百分率分别为尼古丁0组(89.3300±3.3850)%、尼古丁1组(83.8670±3.4977)%、尼古丁2组(77.9480±2.7949)%、尼古丁3组(89.8280±3.400)%,组间比较差异有统计学意义(P<0.01)；S期和S+G2／M期百分率分别为尼古丁0组[(7.8600±2.8241)%和(10.6700±3.3850)%]、尼古丁1组[(10.9170±3.1836)%和(15.9330±3.81453)%]、尼古丁2组[(14.2100±2.6690)%和(22.0520±2.7949)%]、尼古丁3组[(7.5510±2.3390)%和(10.1720±3.4004)%],组间比较差异均有统计学意义(P均<0.01)。
3.(1)淋巴细胞中[Ca2+]i浓度分别为尼古丁0组(145.3720±2.1747)、尼古丁1组(151.9470±3.2908)、尼古丁2组(157.1350±4.8537)、尼古丁3组(132.1750±3.8274),组间比较差异有统计学意义(P<0.01)；
(2)淋巴细胞中CaN的活性分别为尼古丁0组[(0.0844±0.0338)μmolPi／(mgprot·hour-1)]、尼古丁1组[(0.2493±0.0568)μmolPi／(mgprot·hour-1)].尼古丁2组[(0.3203±0.0924)μmolPi／(mgprot·hour-1)].尼古丁3组[(0.1788±0.0728)μmolPi／(mgprot-hour-1)],组间比较差异有统计学意义(P<0.01)；
(3)淋巴细胞中去磷酸化NFATc蛋白的相对表达量分别为尼古丁0组(0.8379±0.0132)、尼古丁1组(0.9655±0.0144)、尼古丁2组(1.1011±0.0306)、尼古丁3组(0.8973±0.0167),组间比较差异有统计学意义(P<0.01)。
4.(1)淋巴细胞中[Ca2+]浓度与CaN的活性及CaN的活性与去磷酸化NFATc蛋白的相对表达量均呈正相关(P均<0.01)；
(2)淋巴细胞中去磷酸化NFATc蛋白的相对表达量与细胞上清液中IL4／IL2的比值及细胞中CyclinE蛋白的相对表达量均呈正相关(P均<0.01)。
结论：
(1)尼古丁浓度在0-10μmol／L范围内时,致敏大鼠淋巴细胞上清液中IL-4的水平及IL-4／IL-2比值随尼古丁浓度增加而增加,而IL-2水平随尼古丁浓度增加而渐减少；而尼古丁浓度为100μmol／L时,细胞上清液中IL-4水平、IL-2水平及IL-4／IL-2比值均显著减少。
(2)尼古丁浓度在0-10μmol／L范围内时,可使致敏大鼠淋巴细胞中CyclinE mRNA和CyclinE蛋白水平的表达渐增加,使处于DNA合成期和细胞分裂前期的淋巴细胞比例显著增加,促进了淋巴细胞的增殖,且呈浓度依赖性；尼古丁浓度为100μmol／L时,反而显著抑制了致敏大鼠淋巴细胞中CyclinE mRNA和CyclinE蛋白水平的表达,抑制了其淋巴细胞的增殖。
(3)尼古丁浓度在0-10μmol/L范围内时,致敏大鼠淋巴细胞中[Ca2+]i浓度渐增加,CaN的活性渐增加,去磷酸化NFATc蛋白的相对表达量亦渐增加,即淋巴细胞Ca2+／CaN-NFATc的活性随尼古丁浓度的增加而增加；而100μmol／L尼古丁反而显著抑制了致敏大鼠淋巴细胞Ca2+／CaN-NFATc的活性。
(4)淋巴细胞Ca2+／CaN-NFATc活性的改变可影响淋巴细胞的增殖、活化及细胞因子的产生。
总之,此部分实验结果说明尼古丁可能通过影响致敏大鼠淋巴细胞Ca2+／CaN-NFATc的活性而影响其淋巴细胞的增殖、活化和Thl/Th2的失衡。
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.
引文
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