本文選題：川芎嗪 + 星形膠質(zhì)膠質(zhì)細(xì)胞　； 參考：《南京醫(yī)科大學(xué)》2017年碩士論文

【摘要】：背景:慢性疼痛是危害人類健康的嚴(yán)重問(wèn)題。數(shù)據(jù)顯示,全球成人慢性疼痛的平均發(fā)病率約為30%,由于其病因復(fù)雜,治療非常困難。目前臨床上現(xiàn)有治療神經(jīng)痛的藥物,只能暫時(shí)性緩解疼痛,并且有許多副作用,因此當(dāng)務(wù)之急是了解神經(jīng)病理性疼痛的發(fā)生發(fā)展機(jī)制,并且尋找安全有效副作用少的治療藥物。越來(lái)越多的研究表明,脊髓膠質(zhì)細(xì)胞在神經(jīng)病理性疼痛過(guò)程中,發(fā)揮著重要的作用。膠質(zhì)細(xì)胞的調(diào)控逐漸成為治療神經(jīng)病理疼痛的新靶點(diǎn)。本實(shí)驗(yàn)室的前期研究結(jié)果與神經(jīng)科學(xué)領(lǐng)域的前沿研究均提示:川芎嗪是一個(gè)具有較大鎮(zhèn)痛潛能的天然產(chǎn)物。川芎是我國(guó)的一味傳統(tǒng)生物堿類中藥,作為慢性痛的治療藥物已經(jīng)有超過(guò)一百多年的歷史,具有活血化瘀的作用,同時(shí)又可理氣、疏風(fēng)止痛。川芎嗪被認(rèn)為是川芎發(fā)揮生物活性的主要物質(zhì),本實(shí)驗(yàn)室以往實(shí)驗(yàn)結(jié)發(fā)現(xiàn)在CCI誘導(dǎo)的神經(jīng)病理性疼痛模型中,川芎嗪可以緩解疼痛。但川芎嗪緩解神經(jīng)病理性疼痛的具體機(jī)制并不是很清楚。因此本文擬深入探討川芎嗪對(duì)神經(jīng)病理性疼痛進(jìn)程中的星形膠質(zhì)細(xì)胞功能紊亂的影響,并進(jìn)一步探索它的鎮(zhèn)痛機(jī)制。方法:(1)建立大鼠坐骨神經(jīng)慢性壓迫損傷(CCI)模型,利用行為學(xué)考察單次、多次給予川芎嗪對(duì)大鼠機(jī)械性縮足反射閾值影響。(2)利用Western blot和免疫熒光方法檢測(cè)川芎嗪對(duì)神經(jīng)病理性疼痛發(fā)生后脊髓星形膠質(zhì)細(xì)胞活化的影響。(3)利用明膠酶譜評(píng)估體內(nèi)川芎嗪對(duì)CCI造模的大鼠脊髓水平基質(zhì)金屬蛋白酶-9和2的作用。(4)利用免疫印跡(Western blot)法檢測(cè)單次、多次給予川芎嗪對(duì)CCI造模的大鼠脊髓水平MAPK家族蛋白p-P38、p-JNK、p-ERK的影響。免疫印跡(Western blot)法檢測(cè)單次、多次給予川芎嗪對(duì)CCI造模大鼠脊髓水平 p-PKCγ、p-NR1 和 IL-1β的影響。(5)為了進(jìn)一步探索川芎嗪潛在的鎮(zhèn)痛機(jī)制,利用分子模擬技術(shù),對(duì)接JNK的上游分子TAK1。(6)利用CCI模型,檢測(cè)TAK1和JNK抑制劑對(duì)大鼠機(jī)械性縮足反射閾值影響;使用TAK1和JNK抑制劑考察其對(duì)MMP-9和2的影響;利用免疫印跡(Western blot)法,在整體和具有星形膠質(zhì)細(xì)胞特性的C8細(xì)胞中檢測(cè)川芎嗪對(duì)p-TAK1和p-JNK的影響。結(jié)果:(1)單次川芎嗪腹腔給藥能夠劑量依賴性地抑制CCI誘導(dǎo)的機(jī)械超敏;連續(xù)五天給予川芎嗪(30mg/kg)能夠發(fā)揮持續(xù)的鎮(zhèn)痛效果;川芎嗪(30mg/kg)多次給藥能夠顯著延緩神經(jīng)病理性疼痛的發(fā)生;川芎嗪腹腔給藥能夠劑量依賴性的選擇性抑制脊髓的JNK的磷酸化水平。(2)多次川芎嗪腹腔給藥顯著抑制CCI誘發(fā)的脊髓背角星形膠質(zhì)細(xì)胞標(biāo)記物GFAP表達(dá)上調(diào)。(3)體內(nèi)明膠酶譜實(shí)驗(yàn)結(jié)果表明單次或多次腹腔給予川芎嗪,能夠顯著抑制CCI引起的大鼠脊髓水平MMP-9/2的激活。(4)川芎嗪能顯著抑制CCI誘導(dǎo)的大鼠脊髓MAPK家族蛋白p-JNK水平的升高和IL-1β活化以及PKCγ和NR1的磷酸化。(5)CCI可引起p-TAK1水平顯著上升。給予川芎嗪后,脊髓p-TAK1水平顯著降低。(6)鞘內(nèi)注射TAK1抑制劑和JNK抑制劑可以抑制CCI誘導(dǎo)的大鼠疼痛超敏的發(fā)生發(fā)展。(7)給予川芎嗪可以顯著抑制IL-1β引起的p-TAK1,p-JNK和MMP-9和2的升高。同時(shí),體外培養(yǎng)星形膠質(zhì)細(xì)胞C8細(xì)胞,IL-1β刺激后誘導(dǎo)C8細(xì)胞中TAK1、JNK的磷酸化,以及MMP-9/2的升高,而給予川芎嗪可以明顯降低CaMKII、TAK1、JNK的磷酸化,以及MMP-9/2的升高。結(jié)論:川芎嗪能夠顯著抑制CCI誘導(dǎo)的脊髓星形膠質(zhì)細(xì)胞的激活,通過(guò)抑制神經(jīng)炎癥產(chǎn)生鎮(zhèn)痛作用,我們的結(jié)果進(jìn)一步暗示JNK-MMP-2/9作為神經(jīng)性疼痛減輕的新靶標(biāo)。
[Abstract]:Background: chronic pain is a serious problem that endangers human health. Data shows that the average incidence of chronic pain in adults around the world is about 30%, and the treatment is very difficult because of its complicated causes. Currently, the current clinical drugs for neuralgia can only temporarily relieve pain and have many side effects, so the urgent task is to understand neuropathy. More and more studies have shown that the spinal glial cells play an important role in the process of neuropathic pain. The regulation of glial cells has gradually become a new target for the treatment of neuropathic pain. The frontier research in the field of neuroscience has suggested that Ligustrazine is a natural product with greater analgesic potential. Ligusticum chuanxiong is a traditional Chinese traditional Chinese medicine. As a chronic pain treatment drug, it has more than more than 100 years history. It has the function of activating blood and removing stasis, and it can also be rational and relieving pain. Ligustrazine is considered to be Ligusticum chuanxiong plays a major bioactive substance. In our laboratory, ligustrazine can relieve pain in the current CCI induced neuropathic pain model, but the specific mechanism of Ligustrazine to relieve neuropathic pain is not very clear. Therefore, this article is to explore the star of ligustrazine in the process of neuropathic pain. The effect of glial cell dysfunction and further explore its analgesic mechanism. Methods: (1) establish the rat sciatic nerve chronic compression injury (CCI) model, use the behavior study to examine the single time, and give the effect of Ligustrazine on the mechanical contraction reflex threshold of rats many times. (2) Western blot and immunofluorescence were used to detect the neuropathology of Ligustrazine. The effect of astrocyte activation after the occurrence of sexual pain. (3) the effect of Tetramethylpyrazine on the spinal cord level matrix metalloproteinase -9 and 2 in CCI model rats was evaluated using gelatinase spectrum. (4) using the immunoblotting (Western blot) method to detect the single time, and the MAPK family protein p-P38, p- of the rat spinal cord with Ligustrazine on CCI The influence of JNK, p-ERK. Immunoblotting (Western blot) method was used to detect the effects of Ligustrazine on the level of p-PKC gamma, p-NR1 and IL-1 beta in the spinal cord of the rat model of CCI. (5) in order to further explore the potential analgesic mechanism of Ligustrazine, using molecular simulation technology, the upstream molecule TAK1. (6) of JNK was used for CCI model to detect TAK1 and inhibition. Effect of the agent on the reflex threshold of mechanical contraction in rats; the effects of TAK1 and JNK inhibitors on MMP-9 and 2; the effects of Tetramethylpyrazine on p-TAK1 and p-JNK were detected in the whole and astrocyte specific C8 cells by Western blot (Western blot). Results: (1) the single tetramethylpyrazine intraperitoneal administration could be dose-dependent. The mechanical hypersensitivity induced by CCI was inhibited; tetramethylpyrazine (30mg/kg) was given a continuous analgesic effect for five days; Ligustrazine (30mg/kg) could significantly delay the occurrence of neuropathic pain; tetramethylpyrazine intraperitoneal administration could selectively inhibit the phosphorylation of JNK in the spinal cord. (2) multiple Ligustrazine intraperitoneal administration The drug significantly inhibited the up-regulated expression of the astrocyte marker GFAP in the dorsal horn of the spinal cord induced by CCI. (3) the results of the gelatinase test in vivo showed that a single or multiple intraperitoneal administration of Ligustrazine could significantly inhibit the activation of MMP-9/2 in the spinal cord of rats induced by CCI. (4) tetramethylpyrazine could significantly inhibit the p-JNK water of MAPK family protein in spinal cord induced by CCI. Level elevation and IL-1 beta activation, and phosphorylation of PKC gamma and NR1. (5) CCI could cause a significant increase in p-TAK1 levels. After Ligustrazine, the level of p-TAK1 in the spinal cord decreased significantly. (6) intrathecal TAK1 inhibitors and JNK inhibitors could inhibit the occurrence of CCI induced pain hypersensitivity in rats. (7) tetramethylpyrazine could significantly inhibit the IL-1 beta. P-TAK1, p-JNK and MMP-9 and 2 increased. At the same time, cultured astrocyte C8 cells in vitro, IL-1 beta stimulated TAK1, JNK phosphorylation and MMP-9/2 increase in C8 cells, and tetramethylpyrazine could significantly reduce CaMKII, TAK1, JNK phosphorylation and increase. Conclusion: Ligustrazine can significantly inhibit the induced spinal star. Activation of the glial cells produces analgesic effects by inhibiting neuritis. Our results further imply that JNK-MMP-2/9 is a new target for neuropathic pain relief.

【學(xué)位授予單位】：南京醫(yī)科大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2017
【分類號(hào)】：R285.5

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本文編號(hào)：1837376