黃嘌呤衍生物減緩大鼠慢性神經壓迫損傷誘導之神經性疼痛經由調控脊髓Cav2.2, Nav1.3 及KATP通道

A xanthine derivative KMUP-1 alleviates chronic constriction injury-induced neuropathic pain via modulation of Cav2.2, Nav1.3 and KATP channels in rat spinal cord

王韵茿(Yun-Ju Wang)

指導教授 ： 吳炳男

高雄醫學大學/醫學院/醫學研究所碩士班/碩士(2016年)

摘要

神經性疼痛可被定義為在周邊或中樞神經系統受到慢性神經損傷後異常疼痛的感覺，且會導致自發性疼痛、痛覺過敏以及觸覺痛。近年來，電位依賴性鈉離子通道、電位依賴性鈣離子通道和ATP敏感性鉀離子通道已被證實對於改善神經性疼痛方面是具有潛力的角色。而在先前的研究顯示，周邊神經受損後，脊髓神經中的Nav1.3及Cav2.2離子通道會正向調控，而KATP離子通道則會負向調控。因此在本篇論文，我們想要去探討KMUP-1是否可以藉由抑制大鼠脊髓神經中的Nav1.3和Cav2.2離子通道表現以及活化KATP離子通道來減緩慢性神經壓迫損傷所誘導之神經性疼痛。
本篇實驗是使用Sprague-Dawley公鼠，將其隨機分成四個組別，分別為控制組(sham group)、控制組投予藥物KMUP-1(sham + KMUP -1 group)、兩側坐骨神經壓迫損傷(CCI group)以及神經壓迫損傷後投予藥物KMUP-1(CCI + KMUP-1 group)，KMUP-1以一天一次，濃度5mg/kg腹腔注射的方式給予。於手術模擬神經損傷後的第七天將大鼠犧牲並取下兩側腰椎(lumbar)第四節到第六節的脊髓神經(spinal cord)以進行西方點墨法、定量即時聚合酶鏈鎖反應以及免疫螢光染色法實驗，進一步去探討Nav1.3、Cav2.2與KATP離子通道的表現情形，最後再以脊髓鞘內注射藥物來反轉KMUP-1的作用，並藉由行為監測反應去了解KMUP-1的可能機轉。從結果得知，Nav1.3和Cav2.2離子通道基因表現在CCI組有明顯增加，而在給予KMUP-1治療後能夠有效的改善；相比之下，KATP通道基因表現在CCI組則會減少，而給予KMUP-1後有改善的趨勢。此外，西方點墨法結果也顯示出Nav1.3和Cav2.2離子通道在CCI組時會正向調控，而KATP離子通道則會負向調控，且以上三者在經由KMUP-1治療後皆有明顯改善的效果；利用免疫螢光染色進一步去證實CCI所誘導之神經損傷會去促使Nav1.3和Cav2.2離子通道表現以及減少KATP離子通道，而此作用亦會受到KMUP-1的影響而有所改善，最後從行為監測結果亦可證實KMUP-1能夠透過Nav1.3、Cav2.2及KATP離子通道來減緩坐骨神經慢性壓迫損傷所誘導之溫覺過敏以及機械性觸覺痛。
綜合以上結果，可得知KMUP-1可以透過Nav1.3和Cav2.2離子通道之抑制以及KATP離子通道之活化來減緩CCI所造成的神經性疼痛，因此，KMUP-1應該會是個很有潛力用於加強神經性疼痛治療效果的藥物。

關鍵字

黃嘌呤衍伸物 ； 神經性疼痛 ； 離子通道

並列摘要

Neuropathic pain is defined as an abnormal pain sensation in the peripheral or central nervous system after chronic nerve injury, and leads to spontaneous pain, hyperalgesia and allodynia. Recently, voltage-gated sodium (Nav) and calcium (Cav) channels, and ATP-sensitive potassium (KATP) channels were demonstrated to have a potential role for improving neuropathic pain. Previous reports showed that the expression of spinal Nav1.3 and Cav2.2 (N-type calcium channel) were upregulated after peripheral nerve injury, but KATP was downregulated. In this study, we attempt to investigate whether KMUP-1 could reduce chronic constriction injury (CCI)-induced neuropathic pain by inhibiting the expression of Nav1.3 and Cav2.2 channels, as well as activating KATP channels in rat spinal cord.
Adult male Sprague-Dawley rats were used for this study and divided into four groups: sham, sham+KMUP-1, CCI and CCI+KMUP-1 (5 mg/kg/day, i.p.). Each rat was sacrificed at day 7 and spinal cord (L4-L6) isolated for the following experiments. Western blotting, quantitative real-time polymerase chain reaction (qPCR), and immunofluorescent staining were used to detect the expression of Nav1.3, Cav2.2, and KATP channels, then spinal intrathecal injection of drugs to reverse the KMUP-1 effects as well as to investigate the mechanism by behavioral monitoring. The results suggest that mRNA levels of Nav1.3 and Cav2.2 channels were increased in CCI group and improved in KMUP-1-treated group. By contrast, KATP mRNA levels were reduced in CCI group and ameliorated in KMUP-1-treated group. Additionally, western blotting also revealed upregulation of Nav1.3 and Cav2.2 channels, as well as downregulation of KATP channels in CCI group and improved in KMUP-1-treated group. Immunofluorescent staining further demonstrated that CCI-induced nerve injury increased Nav1.3 and Cav2.2 and decreased KATP channels, and those effects also improved by treating KMUP-1.Finally, KMUP-1 could attenuate CCI-induced thermal hypersensitivity and mechanical allodynia by Nav1.3,Cav2.2 and KATP channels.
In summary, KMUP-1 prevents CCI-induced neuropathic pain by Nav1.3 and Cav2.2 channels inhibition and KATP channels activation in peripheral nerve injury model. Therefore, KMUP-1 could be a promising candidate for strengthening the therapy of neuropathic pain.

並列關鍵字

xanthine derivative ； neuropathic pain ； ion channels

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