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National Chung Hsing University Institutional Repository - NCHUIR > 生命科學院 > 生物醫學研究所 > 依資料類型分類 > 碩博士論文 >  探討高血糖老鼠在坐骨神經手術後TLR/MYD88相關訊號之變化

Please use this identifier to cite or link to this item: http://nchuir.lib.nchu.edu.tw/handle/309270000/152675

標題: 探討高血糖老鼠在坐骨神經手術後TLR/MYD88相關訊號之變化
Study on TLR/MYD88-related signals in hyperglycemic rat after sciatic nerve injury
作者: 劉子瑄
Liu, Tzu-Hsuan
Contributors: 陳春榮
Chun-Jung Chen
生物醫學研究所
關鍵字: 高血糖;坐骨神經手術;類鐸受體;病理神經痛
Hyperglycemia;Spared nerve injury;TLR;Neuropathic pain
日期: 2013
Issue Date: 2013-11-18 11:04:43 (UTC+8)
Publisher: 生物醫學研究所
摘要: 累積的臨床觀察及動物實驗發現,當坐骨神經、背根神經節、脊髓背角等周邊神經損傷後,常會伴隨著複雜的免疫發炎反應,這些免疫發炎反應包括神經膠細胞的活化及發炎因子的釋放,進而影響周邊與中樞神經元細胞的運作,嚴重者甚至引發神經病理痛。糖尿病是周邊神經損傷的危害因子,通常糖尿病患者末期會出現以疼痛為主的神經病變併發症,稱之為糖尿病疼痛性神經病變。許多研究指出高血糖會導致類鐸受體表達增加,而類鐸受體的活化就足以誘發神經病理痛。類鐸受體活化後可啟動多種細胞內訊息傳遞路徑,調控細胞活性,發炎因子相關基因更是重要標的。因此我們認為,高血糖會進一步惡化神經損傷誘發的神經病理痛,其中類鐸受體應該是串聯高血糖及神經病理痛的關鍵分子,類鐸受體及下游發炎反應路徑更是重要媒介。本篇論文的主軸即透過高血糖鼠和坐骨神經損傷動物模式來驗證這個假說。
本實驗將Sprague-Dawley品系大鼠隨機分成四組:正常血糖對照組、高血糖對照組、正常血糖手術組、高血糖手術組。大鼠以鏈佐黴素(streptozotocin,65 mg/kg)腹腔注射一劑方式來誘發高血糖,當鏈佐黴素注射後三天空腹血糖超過200 mg/dl大鼠即選用為本實驗之高血糖鼠。坐骨神經損傷動物模式則透過手術截斷脛神經及腓總神經方式來建立。藉由Von Frey filament及Hot plate test的行為測試發現,當動物進行坐骨神經損傷手術後,正常血糖動物及高血糖動物的手術傷害側,都出現對機械性刺激及熱刺激產生痛覺敏感的反應。免疫組織化學染色的結果顯示,手術傷害側坐骨神經及脊髓背角有神經膠細胞活化現象。西方點漬分析(Western blot)發現,(TLR4、MYD88、TNFR1、TRAF2、p-IκΒ−α、IκΒ−β、ΝF-κΒ)等類鐸受體及下游發炎媒介相關路徑的蛋白質,在手術傷害側坐骨神經都有表現增加的情形。膠體電泳移動變動分析(Gel mobility shift assay, EMSA)也發現手術傷害側坐骨神經ΝF-κΒ活性增加。透過real time PCR分析也觀察到手術傷害側坐骨神經TNF-α、 IL-1β、 IL-6基因表現的增加。這些變化在高血糖手術組都明顯高於正常血糖手術組。
本實驗觀察到大鼠坐骨神經損傷能造成神經病理痛,過程中伴隨著坐骨神經類鐸受體活化及發炎相關基因表現。單純高血糖的狀態下,亦可輕度誘發類鐸受體活化。高血糖能惡化坐骨神經損傷所造成的痛覺敏感及類鐸受體調控的發炎反應。總合本實驗結果及相關研究發現顯示,類鐸受體及相關訊號參與高血糖惡化的神經病理痛。
Accumulating clinical observations and animal studies show that the degeneration of peripheral nerves is accompanied by inflammatory responses which includes glial activation and cytokine expression and has impact on neuronal activity and finally even results in neurpathic pain. Diabetes mellitus is viewed as a risk factor for all sympatomatic presentations of distal peripheral neuropathy. Generally, patients of diabetes mellitus develop neuropathic pain-related complications at the end stage, called diabetic neuropathy. There are several studies showing that hyperglycemia causes an elevated expression of toll-like receptors. The activation of toll-like receptor-mediated signal alone is enough to induce neuropathic pain. It is recognized that the activation of toll-like receptor can turn on several intracellular signaling pathways in regulating cellular activities including inflammatory mediator expression. Thus, we hypothesized that hyperglycemia could augment peripheral nerve injury-provoked neuropathic pain and toll-like receptor-associated downstream inflammatory cascades might play crucial roles. This study was designed to prove the possibility of our hypothesis through animal models of hyperglycemia and sciatic nerve injury.
Adult male Sprauge-Dawley rats were randomly allocated into four groups: nomoglycemic control, hyperglycemic control, nomoglycemic sciatic nerve injury, and hyperglycemic sciatic nerve injury. Hyperglycemic rats were produced by intraperitoneally injection of streptozotocin (65 mg/kg) once. Those rats with fasting glucose more than 200 mg/dl 3 days after streptozotocin injection were enrolled to this study. Spared nerve injury model was chosed to establish sciatic nerve injury by transecting tibial and peroneal nerves in the right side. The results of Von Frey filament test and hot plate test revealed that spared nerve injury induced mechanical allodynia and thermal hyperalgesia in nomoglycemic and hyperglycemic rats. Immunohistochemical studies showed that spared nerve injury caused glial activation in sciatic nerves and spinal cord dorsal horns. The results of Western blotting revealed that spared nerve injury increased toll-like receptor and associated downstream effector expression such as TLR4, MYD88, TNFR1, TRAF2, P-IκB-α, IκB-β, and NF-κB subunits. EMSA further demonstrated an increased NF-κB DNA binding activity after spared nerve injury. The elevated expression of TNF-α, IL-1β, and IL-6 caused by spared nerve injury was detected by quantitative real-time RT-PCR. Those nociceptive and inflammatory responses were further augmented in spared nerve injured-rats with hyperglycemia.
The results of this study showed that spared nerve injury in rats induced neuropathic pain and this process was accompanied by the activation of toll-like receptors and expression of inflammatory cytokines. Hyperglycemia alone caused a moderate activation of toll-like receptors. Hyperglycemia augmented spared nerve injury-induced activation of toll-like receptor-related signals and inflammation as well as the development of neuropathic pain. Together with other relevant studies, our findings provide supporting evidence showing that the toll-like receptor-related signals might be potential linkers between hyperglycemia and augmented neuropathy.
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