本文選題：嗎啡耐受 + HMGB1　； 參考：《鄭州大學(xué)》2017年碩士論文

【摘要】：研究背景阿片類藥物嗎啡由于其強(qiáng)大、可靠的鎮(zhèn)痛效能,常被作為臨床治療頑固性疼痛的金標(biāo)準(zhǔn)。但嗎啡等鎮(zhèn)痛藥在多次反復(fù)應(yīng)用后,往往會產(chǎn)生鎮(zhèn)痛作用的下降,即產(chǎn)生了所謂的鎮(zhèn)痛耐受。雖然加大劑量可一定程度恢復(fù)其鎮(zhèn)痛的效能,但其副作用常使病人難以忍受。因此,嗎啡耐受嚴(yán)重限制其在臨床上的應(yīng)用。目前認(rèn)為反復(fù)應(yīng)用嗎啡后引起的阿片受體功能的變化、以及中樞痛易化神經(jīng)環(huán)路的激活是嗎啡耐受產(chǎn)生的主要原因。大量實(shí)驗(yàn)證明嗎啡可以引起脊髓膠質(zhì)細(xì)胞的激活,進(jìn)而促使其表達(dá)趨化因子、細(xì)胞因子等致痛物質(zhì),這些致痛物質(zhì)在嗎啡耐受的形成、發(fā)展和維持過程中起著至關(guān)重要的作用。HMGB1高遷移率族蛋白1(High Mobility Group Box-1 protein HMGB1)屬于HMGB家族,是一種與細(xì)胞因子類似的炎癥介質(zhì)。近年來的研究發(fā)現(xiàn),脊髓中的HMGB1在神經(jīng)病理性疼痛和骨癌痛中發(fā)揮了重要作用,但HMGB1在嗎啡耐受形成過程中的作用及其機(jī)制目前仍無報(bào)道。根據(jù)HMGB1促進(jìn)細(xì)胞因子的釋放以及自身所具有的細(xì)胞因子樣作用,我們推測HMGB1可能參與嗎啡耐受的形成。據(jù)此,我們實(shí)驗(yàn)室開展了系列研究,初步證明了脊髓HMGB1上調(diào)參與了嗎啡耐受的形成和維持。但嗎啡引起的脊髓HMGB1表達(dá)分泌的機(jī)制以及HMGB1介導(dǎo)嗎啡耐受的細(xì)胞分子原理仍需進(jìn)一步研究。研究目的本實(shí)驗(yàn)觀察嗎啡耐受及嗎啡停用后,大鼠脊髓(Spinal Cord SC)和背根神經(jīng)節(jié)(Dorsal Root Ganglion DRG)中HMGB1的表達(dá)變化,并且觀察HMGB1參與嗎啡耐受及其引起的痛覺過敏可能的信號通路中相關(guān)分子的表達(dá)變化,以確定HMGB1在嗎啡耐受形成過程中的表達(dá)調(diào)節(jié),并確定HMGB1介導(dǎo)嗎啡耐受形成的信號途徑。確定HMGB1在嗎啡耐受的形成過程中分子機(jī)制。實(shí)驗(yàn)結(jié)果1.鞘內(nèi)微量注射嗎啡對脊髓HMGB1及其受體、磷酸化NF-κB p65、TNF-α和IL-1β表達(dá)的影響連續(xù)6天進(jìn)行大鼠鞘內(nèi)注射10μg/10μl的嗎啡,每天兩次,第7天給予誘導(dǎo)劑量。在給藥的第1、3天嗎啡的鎮(zhèn)痛效率在90%-100%,給藥的第5天嗎啡的鎮(zhèn)痛效率只有20%左右(***P0.001),第7天嗎啡的鎮(zhèn)痛效率則幾乎為0(***P0.001)。在嗎啡停藥后,引起了大鼠雙側(cè)后足機(jī)械痛覺超敏和熱痛覺過敏(*P0.05***P0.001是與第8天的control組相比,###P0.001是與自身baseline相比)。分別在給藥的第1、3、5、7天以及停藥后第3天進(jìn)行大鼠脊髓的取材,具體為大鼠L4-L5的脊髓,之后進(jìn)行western blot檢測。Western Blot結(jié)果顯示在嗎啡注射的第1、3、5、7天,脊髓HMGB1及其受體RAGE和TLR4、磷酸化NF-κB p65以及致炎因子TNF-α、IL-1β的表達(dá)量維持在較高水平(*P0.05**P0.01),第三天達(dá)到峰值,停藥后其水平逐漸恢復(fù),且在停藥后的第3天其表達(dá)量恢復(fù)至正常水平。由此說明鞘內(nèi)微量注射嗎啡可以引起脊髓HMGB1及其受體、磷酸化NF-κB p65、TNF-α和IL-1β表達(dá)量升高。2.嗎啡對離體培養(yǎng)大鼠胎鼠脊髓神經(jīng)元HMGB1及其下游致炎因子TNF-α和IL-1β的表達(dá)的影響離體的脊髓神經(jīng)元培養(yǎng)10天細(xì)胞生長狀態(tài)穩(wěn)定,向培養(yǎng)基中加入1 mg/ml的嗎啡30μl,使其終濃度為20μM/L,分別作用0 h、3 h、6 h、12 h、24 h和48 h后提取蛋白進(jìn)行western blot檢測,結(jié)果顯示與嗎啡作用0 h組相比其他組的HMGB1和TNF-α、IL-1β的表達(dá)量顯著升高(*P0.05**P0.01***P0.001),作用6 h時三者的表達(dá)量達(dá)到峰值,且三者的表達(dá)變化趨勢一致。與上述相同,在細(xì)胞生長穩(wěn)定后向培養(yǎng)基中分別加入saline、1 mg/ml的嗎啡15μl、30μl、60μl、120μl和240μl使其終濃度分別為0、10、20、40、80和160μM/L,待嗎啡作用6 h后提取蛋白進(jìn)行western blot檢測,結(jié)果顯示與saline組相比,嗎啡組的HMGB1的表達(dá)量明顯升高(***P0.001),且嗎啡濃度為20μM/L時達(dá)到峰值。由以上結(jié)果說明嗎啡可以對離體的大鼠脊髓神經(jīng)元產(chǎn)生作用,可以導(dǎo)致神經(jīng)元中HMGB1和TNF-α、IL-1β表達(dá)的增加,且對嗎啡具有一定的時間和劑量依賴性。3.TLR4和μ受體阻斷劑對離體培養(yǎng)大鼠胎鼠脊髓神經(jīng)元HMGB1和致炎因子TNF-α表達(dá)的影響離體的大鼠脊髓神經(jīng)元培養(yǎng)10 d后,首先分別向培養(yǎng)基中加入TLR4阻斷劑TAK-242(10μM/L)、μ-受體阻斷劑CTOP(10μM/L)預(yù)先作用30 min后,加入嗎啡,使其終濃度為20μM/L,作用6 h后收取細(xì)胞,提取蛋白進(jìn)行western blot檢測,結(jié)果顯示與單獨(dú)加入嗎啡相比,加入Mor+TAK-242組的HMGB1、TNF-α的表達(dá)量明顯降低(*P0.05),并且單獨(dú)加入TAK-242組兩者的表達(dá)量與溶劑組、嗎啡加TAK-242組的相比沒有統(tǒng)計(jì)學(xué)意義。而Mor+CTOP組與嗎啡加溶劑組相比,HMGB1的表達(dá)量沒有改變,且這兩組的HMGB1的表達(dá)量明顯高于溶劑組和CTOP組。據(jù)此我們得出結(jié)論阻斷TLR4可以抑制脊髓神經(jīng)元HMGB1、TNF-α的表達(dá),而阻斷μ受體不能抑制兩者的表達(dá),由此說明TLR4參與了HMGB1介導(dǎo)的嗎啡耐受的形成過程,而μ受體則沒有參與該過程。4.鞘內(nèi)注射TLR4阻斷劑對脊髓和背根神經(jīng)節(jié)HMGB1、磷酸化NF-κB p65表達(dá)及嗎啡耐受形成的影響實(shí)驗(yàn)組大鼠嗎啡注射前30 min進(jìn)行鞘內(nèi)注射TLR4阻斷劑TAK-242(劑量為5,10,20μg/10μl),一天一次,嗎啡一天兩次,連續(xù)六天;對照組為嗎啡+溶劑鞘內(nèi)注射,一天兩次,連續(xù)六天。行為學(xué)結(jié)果顯示鞘內(nèi)預(yù)注射TAK-242組與鞘內(nèi)預(yù)注射溶劑組相比較在注射嗎啡第5天(***P0.001)和第7天(***P0.001*P0.05)嗎啡的最大鎮(zhèn)痛效率有明顯的升高。在嗎啡注射后第8天(即嗎啡停止注射后第1天),TAK-242組與溶劑組相比較PWMT和PWHL顯著增高(**P0.01),但與自身的BL相比較無顯著差異,顯著的緩解了連續(xù)使用嗎啡后引起的痛覺過敏現(xiàn)象,并且具有一定的藥物劑量依賴性。在停藥后的第二天進(jìn)行western blot的取材。Western Blot結(jié)果顯示鞘內(nèi)預(yù)注射嗎啡+TAK-242組的脊髓背角和DRG中HMGB1、p-p65的表達(dá)量與嗎啡+溶劑組比較明顯減少(**P0.01)。以上結(jié)果說明阻斷TLR4可以緩解嗎啡耐受的形成,抑制脊髓和背根神經(jīng)節(jié)HMGB1、p-p65的表達(dá)釋放。5.鞘內(nèi)注射HMGB1 siRNA對嗎啡耐受形成的細(xì)胞內(nèi)信號途徑的影響實(shí)驗(yàn)組嗎啡注射前30 min注射HMGB1 siRNA、Scramble si RNA和Transfection regent,一天一次,嗎啡一天兩次,連續(xù)六天;對照組單獨(dú)嗎啡注射,一天兩次,連續(xù)六天。在停藥后的第二天進(jìn)行western blot的取材。Western Blot結(jié)果顯示鞘內(nèi)預(yù)注射嗎啡+HMGB1 siRNA組脊髓背角和DRG中HMGB1、p-p65、p-JNK的表達(dá)量與其他三組相比明顯減少(*P0.05**P0.01***P0.001)。說明鞘內(nèi)預(yù)注射HMGB1 siRNA可以緩解嗎啡耐受的形成,抑制脊髓和背根神經(jīng)節(jié)HMGB1、p-p65和p-JNK的表達(dá)釋放。結(jié)果提示鞘內(nèi)嗎啡誘導(dǎo)的脊髓HMGB1上調(diào)可能是通過NF-κB和JNK信號通路的激活介導(dǎo)嗎啡耐受的形成。結(jié)論嗎啡通過TLR4-NF-κB信號通路的激活介導(dǎo)了SC和DRG中HMGB1的表達(dá);脊髓HMGB1通過激活NF-κB、p-JNK信號通路,調(diào)控細(xì)胞因子的表達(dá)介導(dǎo)了嗎啡耐受的形成。
[Abstract]:Background opioid morphine is often used as the gold standard for clinical treatment of intractable pain because of its strong and reliable analgesic efficacy. However, morphine and other analgesics often produce a decrease in analgesic effect after repeated use of morphine, which produces a so-called analgesic tolerance. But the side effects often make the patient unbearable. Therefore, morphine tolerance severely restricts its clinical application. The changes in the function of opioid receptor caused by repeated use of morphine, and the activation of the central pain prone neural loop are the main causes of morphine tolerance. A large number of experiments have shown that morphine can cause spinal glial cells. Activation, thus promoting the expression of chemokines, cytokines, and other pain causing substances, which play a vital role in the formation, development and maintenance of morphine tolerance,.HMGB1 high mobility group protein 1 (High Mobility Group Box-1 protein HMGB1) belongs to the HMGB family, which is an inflammatory mediator similar to the cytokine. Recent studies have found that HMGB1 plays an important role in neuropathic and bone cancer pain in the spinal cord, but the role and mechanism of HMGB1 in the formation of morphine tolerance are still not reported. Do we speculate that HMGB1 may be involved in promoting the release of cytokines and the cytokine like effects of HMGB1 The formation of endorphin, according to this, our laboratory has conducted a series of studies that preliminarily demonstrated that the HMGB1 up regulation of the spinal cord participates in the formation and maintenance of morphine tolerance. However, the mechanism of HMGB1 induced secretion of morphine and the molecular principle of HMGB1 mediated morphine tolerance are still needed to be further studied. Changes in the expression of HMGB1 in the spinal cord (Spinal Cord SC) and the dorsal root ganglion (Dorsal Root Ganglion DRG) after morphine discontinuation, and to observe the changes in the expression of the related molecules in the signaling pathway of the morphine tolerance and its induced hyperalgesia in order to determine the expression of HMGB1 in the formation of morphine tolerance and to determine HMGB. 1 to guide the signaling pathway of morphine tolerance formation. Determine the molecular mechanism of HMGB1 in the formation of morphine tolerance. Results the effects of microinjection of morphine on the HMGB1 and its receptor in the spinal cord in the 1. sheath, the effects of the expression of phosphorylated NF- kappa B p65, TNF- A and IL-1 beta in the rat sheath for 6 days for 6 days in the rat sheath were injected with 10 u g/10 Mu morphine, two times a day, and seventh days of induction. The analgesic efficiency of morphine on day 1,3 of the administration was 90%-100%. The analgesic efficiency of morphine was only about 20% (***P0.001) on the fifth day of administration, and the analgesic efficiency of morphine was almost 0 (***P0.001) in seventh days. After the withdrawal of morphine, the mechanical pain hypersensitivity and hyperalgesia of bilateral hind feet of the rats were induced (*P0.05***P0.001 and eighth days cont). Compared with the rol group, ###P0.001 was compared with its own baseline). The spinal cord of rats was taken on day 1,3,5,7 of the drug delivery and third days after the withdrawal of the drug, specifically the spinal cord of the rat L4-L5. Then the Western blot detection of.Western Blot showed that the.Western Blot result was on the day of the morphine injection. The expression of p65 and inflammation factor TNF- a, IL-1 beta was maintained at a high level (*P0.05**P0.01) and reached a peak at third days. After stopping the drug, its level gradually resumed and its expression recovered to the normal level on the third day after withdrawal. Thus, the intrathecal microinjection of morphine could cause HMGB1 and its receptor, phosphorylated NF- kappa B p65, TNF- A and IL-. 1 beta expression increased the expression of.2. morphine on the HMGB1 of spinal cord and the expression of TNF- A and IL-1 beta in the spinal cord of the rat fetal rat in vitro. The growth state of the cells in the cultured spinal neurons was stable for 10 days, and 1 mg/ml of morphine 30 u l were added to the medium, and the final concentration was 20 mu M/L, which acted as 0 h, 3 h, 6 h, 12 h, 24 h and 48. After h, the extracted protein was detected by Western blot. The results showed that the expression of IL-1 beta was significantly increased (*P0.05**P0.01***P0.001) and the expression amount of the three in the other group was significantly higher than that of the other groups, compared with the 0 h group of morphine, and the expression of the three was at the peak value at 6 h, and the trend of expression of the three were the same. After adding saline, 1 mg/ml morphine 15 l, 30 mu L, 60 mu L, 120 mu L and 240 Mu L, the final concentration was 0,10,20,40,80 and 160 micron respectively. The protein extracted from morphine 6 h was detected by Western blot. The results showed that the expression of morphine was significantly higher than that of the morphine group, and the concentration of morphine was 20 mu. The results showed that morphine could produce the effect of morphine on the isolated rat spinal cord neurons, which could lead to the increase of the expression of HMGB1 and TNF- a, IL-1 beta in the neurons, and the expression of HMGB1 and the expression of inflammation factor TNF- alpha in the cultured rat spinal cord neurons in vitro by a certain dose dependent.3.TLR4 and the dose dependent.3.TLR4 receptor blocker. After 10 d of the isolated rat spinal cord neurons were cultured, the TLR4 blocker TAK-242 (10 M/L) was first added to the culture medium, and the micron receptor blocker CTOP (10 mu M/L) was pre acted on 30 min, and the final concentration was 20 u M/L, and the cells were collected after the action of 6 h, and the extracted protein was detected for Western blot. The results showed that the morphine was added to the morphine alone. The results showed that morphine was added separately to the morphine. The result showed that morphine was added to the morphine alone. The expression of HMGB1, TNF- alpha in the Mor+TAK-242 group was significantly lower (*P0.05), and the expression of the two groups in the TAK-242 group was not statistically significant compared with the solvent group and the morphine plus TAK-242 group. The expression of HMGB1 in the Mor+CTOP group was not changed compared with the morphine plus solvent group, and the expression of HMGB1 in these two groups was significantly higher. In the solvent group and the CTOP group, we concluded that blocking TLR4 could inhibit the expression of HMGB1 and TNF- alpha in the spinal cord neurons, while blocking the micron receptor did not inhibit the expression of the two. Thus, TLR4 was involved in the formation process of HMGB1 mediated morphine tolerance, while the micron receptor did not participate in the.4. sheath of the TLR4 blocker to the spinal cord and dorsal root. The effect of HMGB1, phosphorylated NF- kappa B p65 expression and morphine tolerance formation in the experimental group was 30 min before injection of morphine TLR4 blocker TAK-242 (dose 5,10,20 u g/10 micron L), once a day, two times a day for six days, and the control group was injected with morphine + Solution for six days, two times a day for six days. Compared with the preintrathecal PREINJECTED TAK-242 group and the intrathecal PREINJECTED solvent group, the maximum analgesic efficiency of morphine was significantly higher than that of morphine fifth days (***P0.001) and seventh days (***P0.001*P0.05). Eighth days after morphine injection (first days after morphine stopped injection), group TAK-242 was significantly higher than that of PWMT and PWHL (**P0.01) compared with that of the solvent group (**P0.01). There was no significant difference in the BL phase of its own, significantly relieving the hyperalgesia caused by the continuous use of morphine, and having a dose dependence. The.Western Blot results of Western blot in the second day after the withdrawal showed the spinal dorsal horn of the morphine +TAK-242 group and the HMGB1 in DRG, and the expression of p-p65. Compared with the morphine + solvent group (**P0.01), the results showed that blocking the TLR4 could alleviate the formation of morphine tolerance, inhibit the HMGB1 of the spinal cord and dorsal root ganglia, and the expression of p-p65 released the.5. intrathecal HMGB1 siRNA for the intracellular signaling pathway of morphine tolerance in the experimental group, the 30 Min injection HMGB1 siRNA, Scr before morphine injection. Amble Si RNA and Transfection regent, once a day, morphine two times a day for six days. The control group was injected with morphine alone, two times a day for six days. The.Western Blot results of Western blot in the second day after the withdrawal of the drug showed the intrathecal preinjection of morphine +HMGB1 siRNA group of spinal dorsal horn and DRG. The other three groups were significantly reduced (*P0.05**P0.01***P0.001). It was suggested that intrathecal pre injection of HMGB1 siRNA could alleviate the formation of morphine tolerance and inhibit the release of HMGB1, p-p65 and p-JNK in the spinal and dorsal root ganglia. The results suggest that intrathecal morphine induced up-regulated spinal HMGB1 may be mediated by the activation of NF- kappa B and JNK signaling pathway to mediate morphine tolerance. It is concluded that the activation of morphine through the TLR4-NF- kappa B signaling pathway mediates the expression of HMGB1 in SC and DRG, and spinal HMGB1 mediates the formation of morphine tolerance by activating NF- kappa B, p-JNK signaling pathway, and regulating the expression of cytokines.
【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R614

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