本文選題：IL-6 + 鈣超載��； 參考：《蘇州大學(xué)》2014年博士論文

【摘要】：白細(xì)胞介素-6（interleukin-6, IL-6）是目前發(fā)現(xiàn)的幾十種細(xì)胞因子中的一種。過(guò)去認(rèn)為IL-6主要與免疫系統(tǒng)和造血系統(tǒng)中細(xì)胞的誘導(dǎo)、生長(zhǎng)和分化有關(guān)。但現(xiàn)在的研究發(fā)現(xiàn)，IL-6也存在于中樞神經(jīng)系統(tǒng)（central nervous system, CNS）中，而且來(lái)源于腦細(xì)胞的內(nèi)源性生成。盡管正常腦內(nèi)IL-6水平較低，但在神經(jīng)系統(tǒng)功能紊亂時(shí)，例如：腦損傷、神經(jīng)變性、感染、缺血和多發(fā)性硬化等，IL-6濃度迅速及明顯地增加，這提示IL-6與神經(jīng)系統(tǒng)疾病關(guān)系密切。已有研究說(shuō)明IL-6具有神經(jīng)保護(hù)作用。我們實(shí)驗(yàn)室先前的研究也表明IL-6能夠抑制谷氨酸或N-甲基-D-天門冬氨酸（N-methyl-D-aspartate, NMDA）誘發(fā)的細(xì)胞內(nèi)鈣超載，因而具有神經(jīng)保護(hù)效應(yīng)。然而，IL-6抑制細(xì)胞內(nèi)鈣超載繼而產(chǎn)生神經(jīng)保護(hù)作用的機(jī)制仍不清楚。為此，本研究利用膜片鉗、鈣成像、形態(tài)學(xué)和分子生物學(xué)等實(shí)驗(yàn)技術(shù)，從神經(jīng)元膜上電壓門控性鈣通道（voltage-gated calcium channel, VGCC）和NMDA受體（NMDA receptor, NMDAR）通道的角度來(lái)研究IL-6抑制細(xì)胞內(nèi)鈣超載的機(jī)制，并進(jìn)一步以IL-6信號(hào)轉(zhuǎn)導(dǎo)途徑與NMDAR離子通道的聯(lián)系為切入點(diǎn)來(lái)探討IL-6神經(jīng)保護(hù)作用的機(jī)制。研究揭示了IL-6一方面經(jīng)抑制L-型鈣通道（L-type calcium channel, LCC）的活動(dòng)減少細(xì)胞內(nèi)鈣超載，另一方面通過(guò)Janus激酶（Janus Kinases，JAKs）-信號(hào)轉(zhuǎn)導(dǎo)和轉(zhuǎn)錄活化因子3（signaltransducer and activator of transcription3，STAT3）-鈣調(diào)神經(jīng)磷酸酶（calcineurin, CaN）的信號(hào)途徑抑制NMDAR亞基NR2B和NR2C的活動(dòng)減少了細(xì)胞內(nèi)鈣超載從而發(fā)揮神經(jīng)保護(hù)作用。本研究可為IL-6在神經(jīng)系統(tǒng)疾病中的作用及作用機(jī)制提供更多新的證據(jù)。 第一部分IL-6經(jīng)抑制L-型電壓門控性鈣通道的活動(dòng)減少神經(jīng)元內(nèi)鈣超載 目的： 說(shuō)明IL-6抑制神經(jīng)元內(nèi)鈣超載的細(xì)胞膜上VGCC的類型及機(jī)制。 方法： 1. IL-6（120ng/ml）預(yù)處理小腦顆粒神經(jīng)元（cerebellar granule neurons, CGNs）24h，然后應(yīng)用膜片鉗技術(shù)，在全細(xì)胞記錄模式下，觀察IL-6對(duì)培養(yǎng)的CGNs全細(xì)胞鈣通道電流和LCC電流的作用。 2.鈣離子熒光探針Fluo-3/AM標(biāo)記CGNs，應(yīng)用共聚焦激光掃描顯微鏡（confocallaser scanning microscope，CLSM）檢測(cè)LCC拮抗劑nifedipine和IL-6+預(yù)處理對(duì)高K誘發(fā)的細(xì)胞內(nèi)鈣超載的影響。 3. Western blot方法檢測(cè)IL-6對(duì)培養(yǎng)的CGNs表達(dá)LCC亞基Cav1.2水平的影響。 結(jié)果： 1.將細(xì)胞鉗制電壓設(shè)為-80mV，在去極化電壓至-20mV，-10mV，0mV和10mV四個(gè)階躍電壓下，IL-6預(yù)處理細(xì)胞的全細(xì)胞鈣通道電流密度要低于對(duì)照組（未經(jīng)IL-6預(yù)處理的細(xì)胞），表明IL-6可抑制全細(xì)胞鈣通道電流。 2.將細(xì)胞鉗制電壓設(shè)為-80mV，去極化電壓至-10mV時(shí)，IL-6預(yù)處理細(xì)胞的全細(xì)胞LCC的電流密度要低于對(duì)照組，而全細(xì)胞非LCC的電流密度與對(duì)照細(xì)胞無(wú)明顯差異。經(jīng)Boltzmann方程擬合，IL-6預(yù)處理細(xì)胞與對(duì)照組細(xì)胞比較，電壓激活曲線的半激活電壓和斜率因子無(wú)統(tǒng)計(jì)學(xué)差異。這些結(jié)果說(shuō)明IL-6抑制了LCC的鈣電流，但未影響其通道的激活特征。 3.+高K（KCl）誘發(fā)了明顯的神經(jīng)元內(nèi)Ca2+濃度升高。神經(jīng)元用LCC拮抗劑nifedipine++預(yù)處理后，高K激發(fā)的胞內(nèi)鈣超載被減弱，說(shuō)明高K促使LCC的開放，導(dǎo)致Ca2+內(nèi)流增加。 4.神經(jīng)元經(jīng)IL-6+預(yù)處理后高K誘發(fā)的細(xì)胞內(nèi)鈣超載被抑制。并且，IL-6也明顯2+抑制了神經(jīng)元經(jīng)LCC流入的Ca。 5. IL-6預(yù)處理明顯下調(diào)神經(jīng)元LCC亞基Cav1.2的蛋白表達(dá)。 結(jié)論： IL-6經(jīng)下調(diào)神經(jīng)元LCC亞基Cav1.2的水平抑制了LCC的活動(dòng)，從而減少了經(jīng)LCC2流入的Ca+，抑制了細(xì)胞內(nèi)鈣超載，這是IL-6發(fā)揮神經(jīng)保護(hù)作用的一種機(jī)制。 第二部分IL-6抑制NMDA誘導(dǎo)的神經(jīng)元鈣超載和凋亡的受體通道機(jī)制及信號(hào)轉(zhuǎn)導(dǎo)途徑 目的： 說(shuō)明IL-6經(jīng)神經(jīng)元內(nèi)JAK-STAT3-CaN的信號(hào)轉(zhuǎn)導(dǎo)途徑抑制了NMDAR亞基NR2B和NR2C的活動(dòng)，從而減少NMDA誘導(dǎo)的神經(jīng)元鈣超載和凋亡而發(fā)揮神經(jīng)保護(hù)作用。 方法： 1. IL-6（120ng/ml）預(yù)處理CGNs24h后，再分別用胞內(nèi)鈣庫(kù)耗竭劑毒胡蘿卜素（thapsigargin, TG），NMDAR亞基的拮抗劑NVP-AAM077、ifenprodil和PPDA，JAK-STAT3-CaN信號(hào)通路的抑制劑AG490和FK506處理神經(jīng)元。 2.用鈣離子熒光探針Fluo-3/AM標(biāo)記已用IL-6和上述各種藥物處理后的神經(jīng)元，利用CLSM觀察神經(jīng)元在NMDA（100μM）激發(fā)下，胞內(nèi)鈣熒光強(qiáng)度的動(dòng)態(tài)變化。 3.應(yīng)用膜片鉗技術(shù)，在全細(xì)胞記錄模式下，，觀察IL-6及結(jié)合使用上述各種藥物處理后的神經(jīng)元對(duì)NMDA誘發(fā)電流的影響。 4.利用JC-1試劑盒，檢測(cè)神經(jīng)元在用IL-6及上述各種藥物處理后對(duì)NMDA刺激下的線粒體膜電位的影響。 5.使用TUNEL熒光標(biāo)記法，觀察神經(jīng)元在用IL-6及上述各種藥物處理后對(duì)NMDA誘發(fā)的神經(jīng)元凋亡的影響。 6. Western blot方法分別檢測(cè)IL-6及上述各種藥物處理后，神經(jīng)元在NMDA刺激下的NMDAR必需亞基NR1和調(diào)節(jié)亞基NR2A、NR2B、NR2C的表達(dá)水平及其磷酸化水平。 7.通過(guò)比色法和Western blot方法分別檢測(cè)CaN的活性水平和表達(dá)水平，觀察神經(jīng)元用IL-6和AG490處理后，在NMDA刺激下的CaN的活性水平和表達(dá)水平的變化。 結(jié)果： 1. NMDA激發(fā)神經(jīng)元內(nèi)鈣熒光強(qiáng)度升高至基礎(chǔ)水平的1.84倍。神經(jīng)元用胞內(nèi)鈣+庫(kù)耗竭劑TG預(yù)處理后，NMDA2仍可激發(fā)細(xì)胞內(nèi)Ca水平升高，說(shuō)明NMDA激活細(xì)胞膜上NMDAR離子通道使Ca2+內(nèi)流增加。IL-6可減少TG處理后NMDA激發(fā)的細(xì)2+2+胞內(nèi)Ca升高，表明IL-6可抑制Ca經(jīng)NMDAR離子通道的流入。 2. NMDA的誘發(fā)電流可以被NMDAR拮抗劑MK-801所阻斷，表明NMDA的誘發(fā)電流經(jīng)NMDAR介導(dǎo)。經(jīng)IL-6預(yù)處理的神經(jīng)元，NMDA誘發(fā)電流的密度要明顯低于未經(jīng)IL-6處理的細(xì)胞，說(shuō)明IL-6可抑制NMDAR介導(dǎo)的內(nèi)向電流。 3. NMDAR亞基NR2B的拮抗劑ifenprodil和NR2C的拮抗劑PPDA均顯著抑制了NMDA誘發(fā)的細(xì)胞內(nèi)鈣超載，但NR2A的拮抗劑NVP-AAM077不能顯著抑制NMDA誘發(fā)的細(xì)胞內(nèi)鈣超載，說(shuō)明在NMDAR離子通道介導(dǎo)的Ca2+內(nèi)流中，NR2B和NR2C起關(guān)鍵作用。IL-6預(yù)處理的神經(jīng)元，ifenprodil和PPDA對(duì)NMDA誘發(fā)的胞+內(nèi)Ca2升高的抑制率低于未用IL-6預(yù)處理的神經(jīng)元，而NVP-AAM077并未表現(xiàn)出這種差異，表明IL-6主要抑制了NR2B和NR2C亞基的功能。 4. IL-6預(yù)處理的神經(jīng)元，ifenprodil和PPDA對(duì)NMDA誘發(fā)的內(nèi)向電流的抑制率明顯低于未用IL-6預(yù)處理的神經(jīng)元，而NVP-AAM077并未表現(xiàn)出這種差異，進(jìn)一步說(shuō)明IL-6主要通過(guò)抑制NR2B和NR2C亞基的功能從而減少NMDA誘發(fā)的內(nèi)向電流。 5. IL-6預(yù)處理的神經(jīng)元，ifenprodil和PPDA對(duì)NMDA誘發(fā)細(xì)胞凋亡的抑制率要明顯低于未用IL-6預(yù)處理的神經(jīng)元，表明IL-6經(jīng)抑制NR2B和NR2C亞基的活動(dòng)發(fā)揮抗凋亡作用。 6. IL-6下調(diào)神經(jīng)元的NR2B和NR2C的蛋白表達(dá)，也下調(diào)NR2C的磷酸化水平，但對(duì)NR2A的表達(dá)和磷酸化水平無(wú)明顯作用。 7. JAK-STAT3信號(hào)通路的抑制劑AG490和CaN的抑制劑FK506均可以阻斷IL-6抑制NMDA誘發(fā)的神經(jīng)元內(nèi)鈣超載。并且，AG490和FK506也阻斷IL-6抑制NMDA誘發(fā)的內(nèi)向電流。 8. IL-6預(yù)處理神經(jīng)元抑制了NMDA導(dǎo)致的線粒體膜電位下降，而AG490和FK506均阻斷了IL-6的這種作用。 9. IL-6預(yù)處理神經(jīng)元顯著抑制了NMDA誘發(fā)的細(xì)胞凋亡，AG490和FK506均阻斷了IL-6的抗凋亡作用。 10. IL-6預(yù)處理神經(jīng)元上調(diào)了CaN β亞基蛋白的表達(dá)，同時(shí)拮抗了由NMDA誘導(dǎo)的CaN活性的降低。AG490阻斷了IL-6的這種作用。 結(jié)論： 1. IL-6抑制神經(jīng)元膜上NMDAR通道的活動(dòng)，因此減少了由NMDA激發(fā)的胞2外Ca+經(jīng)NMDAR通道流入細(xì)胞內(nèi)。 2. IL-6通過(guò)抑制神經(jīng)元內(nèi)NMDAR通道亞基NR2B和NR2C的表達(dá)及活動(dòng)，從而減少NMDA誘發(fā)的胞內(nèi)鈣超載和神經(jīng)元凋亡。。 3. IL-6激活神經(jīng)元內(nèi)JAK-STAT3-CaN的信號(hào)通路，繼而抑制神經(jīng)元膜上NMDAR通道的活動(dòng)，發(fā)揮神經(jīng)保護(hù)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.
【學(xué)位授予單位】：蘇州大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2014
【分類號(hào)】：R741

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