IL-6神经保护作用的钙离子通道机制及信号转导途径

发布时间：2018-06-26 18:17

本文选题：IL-6 + 钙超载　；参考：《苏州大学》2014年博士论文

【摘要】：白细胞介素-6（interleukin-6, IL-6）是目前发现的几十种细胞因子中的一种。过去认为IL-6主要与免疫系统和造血系统中细胞的诱导、生长和分化有关。但现在的研究发现，IL-6也存在于中枢神经系统（central nervous system, CNS）中，而且来源于脑细胞的内源性生成。尽管正常脑内IL-6水平较低，但在神经系统功能紊乱时，例如：脑损伤、神经变性、感染、缺血和多发性硬化等，IL-6浓度迅速及明显地增加，这提示IL-6与神经系统疾病关系密切。已有研究说明IL-6具有神经保护作用。我们实验室先前的研究也表明IL-6能够抑制谷氨酸或N-甲基-D-天门冬氨酸（N-methyl-D-aspartate, NMDA）诱发的细胞内钙超载，因而具有神经保护效应。然而，IL-6抑制细胞内钙超载继而产生神经保护作用的机制仍不清楚。为此，本研究利用膜片钳、钙成像、形态学和分子生物学等实验技术，从神经元膜上电压门控性钙通道（voltage-gated calcium channel, VGCC）和NMDA受体（NMDA receptor, NMDAR）通道的角度来研究IL-6抑制细胞内钙超载的机制，并进一步以IL-6信号转导途径与NMDAR离子通道的联系为切入点来探讨IL-6神经保护作用的机制。研究揭示了IL-6一方面经抑制L-型钙通道（L-type calcium channel, LCC）的活动减少细胞内钙超载，另一方面通过Janus激酶（Janus Kinases，JAKs）-信号转导和转录活化因子3（signaltransducer and activator of transcription3，STAT3）-钙调神经磷酸酶（calcineurin, CaN）的信号途径抑制NMDAR亚基NR2B和NR2C的活动减少了细胞内钙超载从而发挥神经保护作用。本研究可为IL-6在神经系统疾病中的作用及作用机制提供更多新的证据。第一部分IL-6经抑制L-型电压门控性钙通道的活动减少神经元内钙超载目的：说明IL-6抑制神经元内钙超载的细胞膜上VGCC的类型及机制。方法： 1. IL-6（120ng/ml）预处理小脑颗粒神经元（cerebellar granule neurons, CGNs）24h，然后应用膜片钳技术，在全细胞记录模式下，观察IL-6对培养的CGNs全细胞钙通道电流和LCC电流的作用。 2.钙离子荧光探针Fluo-3/AM标记CGNs，应用共聚焦激光扫描显微镜（confocallaser scanning microscope，CLSM）检测LCC拮抗剂nifedipine和IL-6+预处理对高K诱发的细胞内钙超载的影响。 3. Western blot方法检测IL-6对培养的CGNs表达LCC亚基Cav1.2水平的影响。结果： 1.将细胞钳制电压设为-80mV，在去极化电压至-20mV，-10mV，0mV和10mV四个阶跃电压下，IL-6预处理细胞的全细胞钙通道电流密度要低于对照组（未经IL-6预处理的细胞），表明IL-6可抑制全细胞钙通道电流。 2.将细胞钳制电压设为-80mV，去极化电压至-10mV时，IL-6预处理细胞的全细胞LCC的电流密度要低于对照组，而全细胞非LCC的电流密度与对照细胞无明显差异。经Boltzmann方程拟合，IL-6预处理细胞与对照组细胞比较，电压激活曲线的半激活电压和斜率因子无统计学差异。这些结果说明IL-6抑制了LCC的钙电流，但未影响其通道的激活特征。 3.+高K（KCl）诱发了明显的神经元内Ca2+浓度升高。神经元用LCC拮抗剂nifedipine++预处理后，高K激发的胞内钙超载被减弱，说明高K促使LCC的开放，导致Ca2+内流增加。 4.神经元经IL-6+预处理后高K诱发的细胞内钙超载被抑制。并且，IL-6也明显2+抑制了神经元经LCC流入的Ca。 5. IL-6预处理明显下调神经元LCC亚基Cav1.2的蛋白表达。结论： IL-6经下调神经元LCC亚基Cav1.2的水平抑制了LCC的活动，从而减少了经LCC2流入的Ca+，抑制了细胞内钙超载，这是IL-6发挥神经保护作用的一种机制。第二部分IL-6抑制NMDA诱导的神经元钙超载和凋亡的受体通道机制及信号转导途径目的：说明IL-6经神经元内JAK-STAT3-CaN的信号转导途径抑制了NMDAR亚基NR2B和NR2C的活动，从而减少NMDA诱导的神经元钙超载和凋亡而发挥神经保护作用。方法： 1. IL-6（120ng/ml）预处理CGNs24h后，再分别用胞内钙库耗竭剂毒胡萝卜素（thapsigargin, TG），NMDAR亚基的拮抗剂NVP-AAM077、ifenprodil和PPDA，JAK-STAT3-CaN信号通路的抑制剂AG490和FK506处理神经元。 2.用钙离子荧光探针Fluo-3/AM标记已用IL-6和上述各种药物处理后的神经元，利用CLSM观察神经元在NMDA（100μM）激发下，胞内钙荧光强度的动态变化。 3.应用膜片钳技术，在全细胞记录模式下，，观察IL-6及结合使用上述各种药物处理后的神经元对NMDA诱发电流的影响。 4.利用JC-1试剂盒，检测神经元在用IL-6及上述各种药物处理后对NMDA刺激下的线粒体膜电位的影响。 5.使用TUNEL荧光标记法，观察神经元在用IL-6及上述各种药物处理后对NMDA诱发的神经元凋亡的影响。 6. Western blot方法分别检测IL-6及上述各种药物处理后，神经元在NMDA刺激下的NMDAR必需亚基NR1和调节亚基NR2A、NR2B、NR2C的表达水平及其磷酸化水平。 7.通过比色法和Western blot方法分别检测CaN的活性水平和表达水平，观察神经元用IL-6和AG490处理后，在NMDA刺激下的CaN的活性水平和表达水平的变化。结果： 1. NMDA激发神经元内钙荧光强度升高至基础水平的1.84倍。神经元用胞内钙+库耗竭剂TG预处理后，NMDA2仍可激发细胞内Ca水平升高，说明NMDA激活细胞膜上NMDAR离子通道使Ca2+内流增加。IL-6可减少TG处理后NMDA激发的细2+2+胞内Ca升高，表明IL-6可抑制Ca经NMDAR离子通道的流入。 2. NMDA的诱发电流可以被NMDAR拮抗剂MK-801所阻断，表明NMDA的诱发电流经NMDAR介导。经IL-6预处理的神经元，NMDA诱发电流的密度要明显低于未经IL-6处理的细胞，说明IL-6可抑制NMDAR介导的内向电流。 3. NMDAR亚基NR2B的拮抗剂ifenprodil和NR2C的拮抗剂PPDA均显著抑制了NMDA诱发的细胞内钙超载，但NR2A的拮抗剂NVP-AAM077不能显著抑制NMDA诱发的细胞内钙超载，说明在NMDAR离子通道介导的Ca2+内流中，NR2B和NR2C起关键作用。IL-6预处理的神经元，ifenprodil和PPDA对NMDA诱发的胞+内Ca2升高的抑制率低于未用IL-6预处理的神经元，而NVP-AAM077并未表现出这种差异，表明IL-6主要抑制了NR2B和NR2C亚基的功能。 4. IL-6预处理的神经元，ifenprodil和PPDA对NMDA诱发的内向电流的抑制率明显低于未用IL-6预处理的神经元，而NVP-AAM077并未表现出这种差异，进一步说明IL-6主要通过抑制NR2B和NR2C亚基的功能从而减少NMDA诱发的内向电流。 5. IL-6预处理的神经元，ifenprodil和PPDA对NMDA诱发细胞凋亡的抑制率要明显低于未用IL-6预处理的神经元，表明IL-6经抑制NR2B和NR2C亚基的活动发挥抗凋亡作用。 6. IL-6下调神经元的NR2B和NR2C的蛋白表达，也下调NR2C的磷酸化水平，但对NR2A的表达和磷酸化水平无明显作用。 7. JAK-STAT3信号通路的抑制剂AG490和CaN的抑制剂FK506均可以阻断IL-6抑制NMDA诱发的神经元内钙超载。并且，AG490和FK506也阻断IL-6抑制NMDA诱发的内向电流。 8. IL-6预处理神经元抑制了NMDA导致的线粒体膜电位下降，而AG490和FK506均阻断了IL-6的这种作用。 9. IL-6预处理神经元显著抑制了NMDA诱发的细胞凋亡，AG490和FK506均阻断了IL-6的抗凋亡作用。 10. IL-6预处理神经元上调了CaN β亚基蛋白的表达，同时拮抗了由NMDA诱导的CaN活性的降低。AG490阻断了IL-6的这种作用。结论： 1. IL-6抑制神经元膜上NMDAR通道的活动，因此减少了由NMDA激发的胞2外Ca+经NMDAR通道流入细胞内。 2. IL-6通过抑制神经元内NMDAR通道亚基NR2B和NR2C的表达及活动，从而减少NMDA诱发的胞内钙超载和神经元凋亡。。 3. IL-6激活神经元内JAK-STAT3-CaN的信号通路，继而抑制神经元膜上NMDAR通道的活动，发挥神经保护作用。
[Abstract]:-6 (interleukin-6, IL-6) is one of the dozens of cytokines found at present. In the past, it was thought that IL-6 was mainly related to the induction, growth and differentiation of cells in the immune system and the hematopoietic system. But the present study found that IL-6 also exists in the central nervous system (central nervous system, CNS), and is derived from brain cells. Endogenous generation. Although the level of IL-6 in normal brain is low, when nervous system dysfunction, such as brain injury, neurodegeneration, infection, ischemia and multiple sclerosis, the concentration of IL-6 increases rapidly and obviously, which suggests that the IL-6 is closely related to the nervous system disease. There has been a study that IL-6 has neuroprotective effect. Our laboratory has been shown to have a neuroprotective effect. Previous studies have also shown that IL-6 can inhibit intracellular calcium overload induced by glutamic acid or N- methyl -D- aspartic acid (N-methyl-D-aspartate, NMDA), thus having neuroprotective effects. However, the mechanism of IL-6 inhibition of intracellular calcium overload to produce neuroprotective effects is still unclear. Therefore, this study uses patch clamp, calcium imaging, and form. Experimental techniques such as state and molecular biology, from the angle of the voltage-gated calcium channel (VGCC) and the NMDA receptor (NMDA receptor, NMDAR) channel on the neuronal membrane, are used to study the mechanism of calcium overload in the IL-6 cells, and further the link between the IL-6 signal transduction pathway and the NMDAR ion channel is the breakthrough point. To explore the mechanism of IL-6 neuroprotective effect, the study revealed that IL-6, on the one hand, reduces intracellular calcium overload by inhibiting the activity of L-type calcium channel (LCC), and on the other hand, through the Janus kinase (Janus Kinases, JAKs) signal transduction and transcription activator 3 (signaltransducer) - calcium modulation. The signal pathway of calcineurin (CaN) inhibits the activity of NMDAR subunit NR2B and NR2C and reduces intracellular calcium overload and thus plays a neuroprotective role. This study can provide more new evidence for the role and mechanism of IL-6 in nervous system diseases.
Part 1 IL-6 inhibits calcium overload in neurons by suppressing the activity of L- voltage-gated calcium channels.
Objective:
Explain the type and mechanism of VGCC in cell membrane that IL-6 inhibits calcium overload in neurons.
Method:
1. IL-6 (120ng/ml) pretreated cerebellar granule neurons (cerebellar granule neurons, CGNs) 24h, and then patch clamp technique was applied to observe the effect of IL-6 on the calcium channel current and LCC current of the cultured CGNs whole cell under the whole cell recording mode.
2. calcium ion fluorescence probe Fluo-3/AM was labeled CGNs, and the effect of LCC antagonist nifedipine and IL-6+ preconditioning on intracellular calcium overload induced by high K was detected by confocal laser scanning microscope (confocallaser scanning microscope, CLSM).
3. Western blot method was used to detect the effect of IL-6 on the expression of CGNs subunit Cav1.2 in cultured CGNs.
Result:
1. the cell clamp voltage was set to -80mV. Under depolarizing voltage to -20mV, -10mV, 0mV and 10mV, the total cell calcium channel current density of IL-6 pretreated cells was lower than that of the control group (without IL-6 pretreated cells), indicating that IL-6 could inhibit the whole cell calcium channel electric current.
2. when the cell clamp voltage was set to -80mV and the depolarized voltage to -10mV, the current density of the whole cell LCC in the IL-6 pretreated cells was lower than that of the control group, while the non LCC current density of the whole cell was not significantly different from that of the control cell. The IL-6 pretreated cells were compared with the control group by the Boltzmann equation, and the voltage activation curve was half activated by the IL-6 pretreated cells. The results showed that IL-6 inhibited the calcium current of LCC, but did not affect the activation characteristics of the channel.
3.+ high K (KCl) induces a significant increase in intracellular Ca2+ concentration. After preconditioning with LCC antagonist nifedipine++, intracellular calcium overload induced by high K is weakened, indicating that high K promotes LCC opening and leads to an increase in Ca2+ internal flow.
4. neurons were pretreated by IL-6+ and the intracellular calcium overload induced by high K was inhibited. Moreover, IL-6 also showed that 2+ inhibited the Ca. inflow of neurons through LCC.
5. pretreatment with IL-6 significantly reduced the protein expression of LCC subunit Cav1.2.
Conclusion:
IL-6 inhibits the activity of LCC by lowering the level of the neuron LCC subunit Cav1.2, which reduces the Ca+ flow through LCC2 and inhibits intracellular calcium overload, which is a mechanism for IL-6 to play a neuroprotective role.
The second part is the mechanism and signal transduction pathway of IL-6 inhibiting NMDA induced calcium overload and apoptosis in neurons.
Objective:
It is suggested that IL-6 inhibits the activity of NMDAR subunit NR2B and NR2C through the signal transduction pathway of JAK-STAT3-CaN in the neuron, thus reducing the calcium overload and apoptosis induced by NMDA and exerts a neuroprotective effect.
Method:
1. IL-6 (120ng/ml) pretreated CGNs24h, and then used the intracellular calcium library depleting agent carotene (thapsigargin, TG), NMDAR subunit antagonist NVP-AAM077, ifenprodil and PPDA, JAK-STAT3-CaN signaling pathway inhibitor AG490 and FK506 treated neurons.
2. the neurons treated with IL-6 and all kinds of drugs were labeled with calcium ion fluorescence probe Fluo-3/AM, and the dynamic changes of intracellular calcium fluorescence intensity were observed under the excitation of NMDA (100 mu) by CLSM.
3. patch clamp technique was used to observe the effects of IL-6 and the neurons treated with these drugs on NMDA induced currents in whole cell recording mode.
4. the JC-1 kit was used to detect the effects of IL-6 and the above drugs on mitochondrial membrane potential stimulated by NMDA.
5. TUNEL fluorescence labeling was used to observe the effects of IL-6 and the above drugs on NMDA induced neuronal apoptosis.
6. Western blot method was used to detect the NMDAR essential subunit NR1 and the expression level of NR2A, NR2B, NR2C and phosphorylation level of the NMDAR essential subunits of the neurons under the NMDA stimulation after IL-6 and the various drugs were treated respectively.
7. the activity level and expression level of CaN were detected by colorimetric method and Western blot method, and the changes of activity level and expression level of CaN under NMDA stimulation were observed after IL-6 and AG490 were treated with NMDA.
Result:
The intracellular calcium fluorescence intensity of 1. NMDA excited neurons increased to 1.84 times as much as the basic level. After the neuron was pretreated with intracellular calcium + depletion agent TG, NMDA2 could still stimulate the increase of intracellular Ca level. It indicated that NMDA activation on the NMDAR ion channel on the membrane of the cell activated the Ca2+ inflow.IL-6 to reduce the NMDA excitation in the thin 2+2+ intracellular Ca. The inflow of Ca via the NMDAR ion channel is inhibited.
The induced current of 2. NMDA can be blocked by the NMDAR antagonist MK-801, indicating that the induced current of NMDA is mediated by NMDAR. The density of NMDA induced current by IL-6 pretreated neurons is significantly lower than that of the cells without IL-6 treated, indicating that IL-6 can inhibit the introversion current mediated by NMDAR.
3. NMDAR subunit NR2B antagonist ifenprodil and NR2C antagonist PPDA all significantly inhibited the intracellular calcium overload induced by NMDA, but NR2A antagonist NVP-AAM077 did not significantly inhibit the intracellular calcium overload induced by NMDA. The inhibitory rate of rodil and PPDA on the increase of intracellular Ca2 induced by NMDA was lower than that of non IL-6 pretreated neurons, but NVP-AAM077 did not show this difference, suggesting that IL-6 mainly inhibited the function of NR2B and NR2C subunits.
4. IL-6 pretreated neurons, ifenprodil and PPDA inhibited NMDA induced inward currents significantly lower than those without IL-6 pretreated neurons, and NVP-AAM077 did not show this difference, which further indicated that IL-6 is mainly by inhibiting the function of NR2B and NR2C subunits to reduce NMDA induced introversion.
5. IL-6 pretreated neurons, ifenprodil and PPDA inhibited NMDA induced apoptosis significantly lower than those without IL-6 pretreated neurons, indicating that IL-6 could inhibit apoptosis by inhibiting the activity of NR2B and NR2C subunits.
6. IL-6 down regulated the expression of NR2B and NR2C in neurons, and down regulated the phosphorylation level of NR2C, but had no significant effect on the expression and phosphorylation level of NR2A.
The inhibitor AG490 of 7. JAK-STAT3 signaling pathway and the inhibitor FK506 of CaN can block the intracellular calcium overload induced by IL-6, and AG490 and FK506 also block the introverted current induced by IL-6 inhibition NMDA.
8. IL-6 preconditioning inhibited the decrease of mitochondrial membrane potential induced by NMDA, while AG490 and FK506 blocked the effect of IL-6.
9. IL-6 preconditioning significantly inhibited NMDA induced apoptosis, while AG490 and FK506 blocked the anti apoptotic effect of IL-6.
10. IL-6 preconditioning neurons up-regulated the expression of CaN beta subunit protein, and antagonized the decrease of CaN activity induced by NMDA, and.AG490 blocked the effect of IL-6.
Conclusion:
1. IL-6 inhibited the activity of NMDAR channels on the membrane of neurons, thus reducing the extracellular Ca+ stimulated by NMDA and entering the cells through NMDAR channels in 2 of the cells.
2. IL-6 inhibits intracellular calcium overload and neuronal apoptosis induced by NMDA by inhibiting the expression and activity of NMDAR channel subunits NR2B and NR2C in neurons.
3. IL-6 activates the JAK-STAT3-CaN signaling pathway in neurons, which inhibits the activity of NMDAR channels on neurons and exerts neuroprotective effects.
【学位授予单位】：苏州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R741

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