HCN通道在神經(jīng)病理性痛及焦慮抑郁中的作用和機(jī)制研究
發(fā)布時(shí)間：2019-04-03 07:26
【摘要】：慢性神經(jīng)病理性痛和其伴發(fā)的焦慮抑郁產(chǎn)生機(jī)制復(fù)雜.最新研究發(fā)現(xiàn)外周神經(jīng)損傷后,超極化激活的環(huán)核苷酸門控通道(HCN)表達(dá)明顯改變,同時(shí)與焦慮、抑郁關(guān)系密切,但HCN通道在神經(jīng)病理性痛中的作用目前尚不清楚.本研究擬探討三個(gè)問題：1.側(cè)腦室給予HCN通道阻斷劑ZD7288是否可以緩解大鼠坐骨神經(jīng)分支阻斷模型(SNI)術(shù)后的疼痛,并改善動(dòng)物焦慮、抑郁行為?2.SNI術(shù)后腦內(nèi)HCN通道的變化如何,HCN通道的輔助亞基含Rab8b的TPR結(jié)構(gòu)域相互作用蛋白(TRIP8b)是否參與調(diào)節(jié)?3.給藥后腦內(nèi)γ-氨基丁酸((ABA)的變化和其機(jī)制是否與谷氨酸脫羧酶(GAD)相關(guān)?方法： 128只Wistar Kyoto(WKY)大鼠(抑郁大鼠)分為三部分進(jìn)行實(shí)驗(yàn).首先選取40只WKY大鼠隨機(jī)分為Veh、ZD7288(2.5μg/kg)、ZD7288(5μg/kg)、 ZD7288(10μg/kg)四組,通過自發(fā)活動(dòng)實(shí)驗(yàn)觀察ZD7288側(cè)腦室給藥后大鼠的自身活動(dòng).其次28只WKY大鼠隨機(jī)分為Sham、ZD7288(5μg/kg)、ZD7288(10μg/kg)、 Saline四組,通過開場(chǎng)實(shí)驗(yàn)、高架十字迷宮實(shí)驗(yàn)、強(qiáng)迫游泳實(shí)驗(yàn)觀察側(cè)腦室單次給予ZD7288后抑郁大鼠的焦慮、抑郁情緒.最后60只WKY大鼠隨機(jī)分為Sham、SNI、 SNI+ZD7288(5μg/kg)、SNI+ZD7288(10μg/kg)、SNI+Saline五組,通過機(jī)械性縮足反射實(shí)驗(yàn)、熱輻射潛伏期實(shí)驗(yàn)觀察ZD7288側(cè)腦室連續(xù)給藥14天痛閾的變化,通過開場(chǎng)實(shí)驗(yàn)、高架十字迷宮實(shí)驗(yàn)、懸尾實(shí)驗(yàn)觀察大鼠神經(jīng)病理性痛伴隨的焦慮、抑郁情緒。行為檢測(cè)后取材,通過免疫熒光染色觀察HCN通道的表達(dá),同時(shí)觀察HCN與錐體神經(jīng)元、中間神經(jīng)元的共定位,通過western blot方法檢測(cè)丘腦和海馬內(nèi)HCN通道、輔助亞基TRIP8b和GAD的表達(dá),通過高效液相色譜檢測(cè)大鼠腦內(nèi)GABA的含量,并通過全細(xì)胞膜片鉗記錄ZD7288(10μM)灌流后微小抑制性突觸后電流(mIPSCs)。結(jié)果：側(cè)腦室給予2.5 μg/kg、5μg/kg、10μg/kg三種劑量ZD7288都不影響大鼠自發(fā)活動(dòng)；側(cè)腦室單次給予ZD7288 10μg/kg可以增加抑郁大鼠在高架十字迷宮開放臂的停留時(shí)間百分比,5μg/kg、10μg/kg兩組抑郁大鼠強(qiáng)迫游泳實(shí)驗(yàn)自主活動(dòng)時(shí)間延長(zhǎng)；抑郁大鼠SNI術(shù)后側(cè)腦室連續(xù)14天給予ZD7288 5μg/kg、10μg/kg可以不同程度提高機(jī)械痛閾,以10μg/kg作用更明顯.在懸尾實(shí)驗(yàn)中,10μg/kg組自主活動(dòng)明顯增力口。Western blot結(jié)果顯示丘腦內(nèi)HCN2的表達(dá)明顯增加,給予ZD7288后HCN2的表達(dá)有不同程度下降,但是輔助亞基TRIP8b變化不明顯.在海馬內(nèi),SNI術(shù)后HCN1表達(dá)上升,10μg/kg組HCN1表達(dá)明顯下降,并且TRIP8b的表達(dá)與HCN1呈相反趨勢(shì).同時(shí)5μg/kg、10μg/kg兩組丘腦和海馬中GAD的表達(dá)與SNI組相比顯著升高。高效液相色譜結(jié)果顯示10μg/kg組丘腦中GABA表達(dá)明顯升高,5μg/kg、10μg/kg兩組海馬中GAB A的表達(dá)顯著增加.全細(xì)胞膜片鉗結(jié)果表明海馬腦片在給予ZD7288灌流后對(duì)mIPSCs沒有增頻增幅作用.結(jié)論：側(cè)腦室給予ZD7288可以對(duì)WKY大鼠產(chǎn)生鎮(zhèn)痛、緩解焦慮抑郁的作用,這可能是通過調(diào)節(jié)HCN通道的表達(dá)和興奮性從而影響了GAD的表達(dá),進(jìn)而改變GABA的水平介導(dǎo)的.
[Abstract]:The mechanism of chronic neuropathic pain and its associated anxiety and depression is complicated. Recent studies have found that hyperpolarization-activated cyclic nucleotide gated channel (HCN) expression changes significantly after peripheral nerve injury, and is closely related to anxiety and depression. However, the role of HCN channel in neuropathic pain is still unclear. This study aims to explore three issues: 1. Can the intracerebroventricular administration of ZD7288, a HCN channel blocker, relieve the pain and improve the anxiety and depression behavior of the rat model of sciatic nerve branch occlusion after (SNI)? how about the changes of HCN channel in the brain after 2.SNI? Does the Rab8b-containing TPR domain interacting protein (TRIP8b) of the auxiliary subunit of the HCN channel participate in the regulation? 3. Is the change of 緯-aminobutyric acid (ABA) and its mechanism related to glutamic acid decarboxylase (GAD) after administration? Methods: 128 Wistar Kyoto (WKY) rats (depression rats) were divided into three parts. At first, 40 WKY rats were randomly divided into four groups: Veh,ZD7288 (2.5 渭 g / kg), ZD7288) (5 渭 g / kg), ZD7288 (10 渭 g / kg). Secondly, 28 WKY rats were randomly divided into four groups: Sham,ZD7288 (5 渭 g / kg), ZD7288) (10 渭 g / kg), Saline) group. The anxiety and depression of depression rats were observed by open field experiment, elevated maze test and forced swimming test after single administration of ZD7288 in lateral ventricle. Finally, 60 WKY rats were randomly divided into five groups: Sham,SNI, SNI ZD7288 (5 渭 g / kg), SNI ZD7288) (10 渭 g / kg), SNI Saline) group. The changes of pain threshold of ZD7288 were observed by mechanical foot contraction reflex test and thermal radiation latency test for 14 days after continuous administration of ZD7288. The anxiety and depression associated with neuropathic pain in rats were observed by opening experiment, elevated maze test and tail suspension test. The expression of HCN channel was observed by immunofluorescence staining, and the co-localization of HCN with pyramidal neurons and intermediate neurons was observed. The expression of HCN channel, auxiliary subunit TRIP8b and GAD in thalamus and hippocampus were detected by western blot method. The content of GABA in rat brain was determined by high performance liquid chromatography (HPLC), and the minimal inhibitory postsynaptic current (mIPSCs).) was recorded by whole cell patch clamp after ZD7288 (10 渭 M) perfusion. Results: administration of 2.5 渭 g / kg, and 5 渭 g / kg, 10 渭 g / kg of ZD7288 in lateral ventricle did not affect the spontaneous activity of rats. Single administration of ZD7288 10 渭 g / kg in lateral ventricle increased the percentage of open arm of depression rats in elevated maze, and prolonged the autonomous activity time of forced swimming in 5 渭 g / kg, 10 渭 g / kg group. Administration of ZD7288 5 渭 g / kg, 10 渭 g / kg into lateral ventricles of depressed rats after SNI for 14 days could increase the mechanical pain threshold in varying degrees, and the effect of 10 渭 g / kg was more obvious than that of 10 渭 g / kg. In tail suspension test, the results of. Western blot showed that the expression of HCN2 in thalamus was significantly increased in 10 渭 g / kg group, and the expression of HCN2 was decreased in different degrees after ZD7288 administration, but the change of auxiliary subunit TRIP8b was not obvious. In hippocampus, the expression of HCN1 increased after SNI, and the expression of HCN1 decreased significantly in 10 渭 g / kg group, and the expression of TRIP8b was opposite to that of HCN1. At the same time, the expression of GAD in thalamus and hippocampus in 5 渭 g / kg, 10 渭 g / kg group was significantly higher than that in SNI group. The results of high performance liquid chromatography (HPLC) showed that the expression of GABA in thalamus and GABA in hippocampus of 10 渭 g / kg group and 5 渭 g / kg, 10 渭 g / kg group were significantly increased. Whole-cell patch clamp results showed that hippocampal slices did not increase the frequency of mIPSCs after perfusion with ZD7288. Conclusion: intracerebroventricular administration of ZD7288 can induce analgesia and relieve anxiety and depression in WKY rats, which may be mediated by regulating the expression and excitability of HCN channel, thus affecting the expression of GAD and then changing the level of GABA.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R614
[Abstract]:The mechanism of chronic neuropathic pain and its associated anxiety and depression is complicated. Recent studies have found that hyperpolarization-activated cyclic nucleotide gated channel (HCN) expression changes significantly after peripheral nerve injury, and is closely related to anxiety and depression. However, the role of HCN channel in neuropathic pain is still unclear. This study aims to explore three issues: 1. Can the intracerebroventricular administration of ZD7288, a HCN channel blocker, relieve the pain and improve the anxiety and depression behavior of the rat model of sciatic nerve branch occlusion after (SNI)? how about the changes of HCN channel in the brain after 2.SNI? Does the Rab8b-containing TPR domain interacting protein (TRIP8b) of the auxiliary subunit of the HCN channel participate in the regulation? 3. Is the change of 緯-aminobutyric acid (ABA) and its mechanism related to glutamic acid decarboxylase (GAD) after administration? Methods: 128 Wistar Kyoto (WKY) rats (depression rats) were divided into three parts. At first, 40 WKY rats were randomly divided into four groups: Veh,ZD7288 (2.5 渭 g / kg), ZD7288) (5 渭 g / kg), ZD7288 (10 渭 g / kg). Secondly, 28 WKY rats were randomly divided into four groups: Sham,ZD7288 (5 渭 g / kg), ZD7288) (10 渭 g / kg), Saline) group. The anxiety and depression of depression rats were observed by open field experiment, elevated maze test and forced swimming test after single administration of ZD7288 in lateral ventricle. Finally, 60 WKY rats were randomly divided into five groups: Sham,SNI, SNI ZD7288 (5 渭 g / kg), SNI ZD7288) (10 渭 g / kg), SNI Saline) group. The changes of pain threshold of ZD7288 were observed by mechanical foot contraction reflex test and thermal radiation latency test for 14 days after continuous administration of ZD7288. The anxiety and depression associated with neuropathic pain in rats were observed by opening experiment, elevated maze test and tail suspension test. The expression of HCN channel was observed by immunofluorescence staining, and the co-localization of HCN with pyramidal neurons and intermediate neurons was observed. The expression of HCN channel, auxiliary subunit TRIP8b and GAD in thalamus and hippocampus were detected by western blot method. The content of GABA in rat brain was determined by high performance liquid chromatography (HPLC), and the minimal inhibitory postsynaptic current (mIPSCs).) was recorded by whole cell patch clamp after ZD7288 (10 渭 M) perfusion. Results: administration of 2.5 渭 g / kg, and 5 渭 g / kg, 10 渭 g / kg of ZD7288 in lateral ventricle did not affect the spontaneous activity of rats. Single administration of ZD7288 10 渭 g / kg in lateral ventricle increased the percentage of open arm of depression rats in elevated maze, and prolonged the autonomous activity time of forced swimming in 5 渭 g / kg, 10 渭 g / kg group. Administration of ZD7288 5 渭 g / kg, 10 渭 g / kg into lateral ventricles of depressed rats after SNI for 14 days could increase the mechanical pain threshold in varying degrees, and the effect of 10 渭 g / kg was more obvious than that of 10 渭 g / kg. In tail suspension test, the results of. Western blot showed that the expression of HCN2 in thalamus was significantly increased in 10 渭 g / kg group, and the expression of HCN2 was decreased in different degrees after ZD7288 administration, but the change of auxiliary subunit TRIP8b was not obvious. In hippocampus, the expression of HCN1 increased after SNI, and the expression of HCN1 decreased significantly in 10 渭 g / kg group, and the expression of TRIP8b was opposite to that of HCN1. At the same time, the expression of GAD in thalamus and hippocampus in 5 渭 g / kg, 10 渭 g / kg group was significantly higher than that in SNI group. The results of high performance liquid chromatography (HPLC) showed that the expression of GABA in thalamus and GABA in hippocampus of 10 渭 g / kg group and 5 渭 g / kg, 10 渭 g / kg group were significantly increased. Whole-cell patch clamp results showed that hippocampal slices did not increase the frequency of mIPSCs after perfusion with ZD7288. Conclusion: intracerebroventricular administration of ZD7288 can induce analgesia and relieve anxiety and depression in WKY rats, which may be mediated by regulating the expression and excitability of HCN channel, thus affecting the expression of GAD and then changing the level of GABA.
【學(xué)位授予單位】：中國(guó)人民解放軍醫(yī)學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2015
【分類號(hào)】：R614
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