脊髓JAK2-STAT3信号通路参与大鼠电针耐受调节
发布时间：2018-08-12 12:04
【摘要】：针灸作为一种传统疗法在治疗疾病和缓解疼痛方面具有悠久的历史。电针(EA)衍生于传统手针并逐渐替代手针,因为电针的镇痛效果更佳,副作用少,并且其参数可被客观的量化和控制。但是,如同吗啡耐受一样,长时间或反复电针刺激会使镇痛效果降低甚至消失,学术上称之为“电针耐受”。临床上电针耐受现象普遍存在,引起学者们高度关注。研究发现电针能通过诱导内源性阿片肽类物质释放产生镇痛作用,如内啡肽和脑啡肽,同时还可以促进一些具有抗镇痛作用物质的释放,如孤啡肽、八肽胆囊收缩素和血管紧张素Ⅱ等。研究证实了吗啡耐受与电针耐受存在双向交叉耐受,提示二者可能存在共同的作用机制。研究发现在吗啡耐受中,大鼠脊髓的STAT3的磷酸化水平升高。另外大量研究表明JAK2-STAT3信号通路在神经病理性痛大鼠的脊髓背角被激活,对疼痛发展和维持有重要作用,抑制JAK2-STAT3信号通路可以缓解痛觉触敏和痛觉过敏,提示JAK2-STAT3信号通路在疼痛反应起到重要作用。然而JAK2-STAT3信号通路是否参与电针耐受还未有报道。本试验将单次电针后甩尾潜伏期升高超过50%的SD大鼠判定为有效鼠。将70只有效鼠(250±20g)随机分成2组:电针组(EA组)和假针组。通过反复电针刺激穴位建立大鼠耐受模型(镇痛效果显著降低或消失表明耐受形成)。EA组采用2Hz频率电针刺激大鼠双侧足三里和三阴交穴位,每天1次,持续30min,连续8d。假针组大鼠只穴位处扎针,不通电,其余处理与电针组相同。通过热辐射法测量大鼠的甩尾潜伏期即痛阈。每次电针前后分别测量痛阈,计算痛阈变化率,通过痛阈变化率评估电针的镇痛效果。为探索脊髓JAK2-STAT3信号通路在电针耐受中的作用,取电针0(电针前)、2、4、6和8d后的大鼠L4~6段脊髓样品,采用荧光定量PCR检测JAK2和STAT3的基因表达变化,Western Blot检测JAK2和STAT3蛋白的磷酸化水平,并用免疫组织化学确定pJAK2和pSTAT3的分布。通过鞘内注射WP1066(JAK2-STAT3信号通路抑制剂)进一步确定脊髓JAK2-STAT3信号通路对电针耐受的影响。另选取45只有效鼠,随机分成3组:EA组、EA+二甲基亚砜(DMSO)组和EA+WP1066组,每组15只。DMSO(溶剂)和WP1066分别于电针前注入腰膨大部。电针前后测量痛阈,计算痛阈变化率,并采用免疫组织化学检测电针后脊髓背角JAK2和STAT3磷酸化水平。反复电针结果显示,随着电针次数增加镇痛效果减弱。假针组大鼠的痛阈变化率各时间点均无显著变化(p0.05)。EA组大鼠痛阈变化率在1~5d显著高于假针组(p0.05),6~8d与假针组无异(p0.05),提示电针耐受形成。荧光定量PCR结果显示EA组与假针组JAK2和STAT3在mRNA水平上表达没有差异(p0.05),Western Blot结果显示,与假针组相比电针后2~6d pJAK2和pSTAT3表达显著升高(p0.05),第4d达到峰值,第8d与电针前无显著差异(p0.05)。免疫组织化学结果显示pJAK2和pSTAT3主要在脊髓背角Ⅱ区表达有差异,且变化趋势与Western Blot结果相符。鞘内注射试验结果显示EA组与EA+DMSO组无显著性变化(p0.05),且6~8d电针效果基本消失。EA+WP1066组镇痛效果虽然也随着电针次数增加而减弱,但4~8d的电针效果都显著高于EA组与EA+DMSO组(p0.05)。免疫组织化学结果显示EA组JAK2和STAT3蛋白磷酸表达趋势与反复电针实验一致,2~6d表达升高,第4d达到峰值。EA+DMSO组与EA组无显著差异(p0.05)。EA+DMSO组JAK2和STAT3蛋白磷酸变化水平逐渐升高。上述结果表明脊髓JAK2-STAT3信号通路参与了电针耐受调节,鞘内注射WP1066抑制JAK2-STAT3信号通路的激活能弱化电针耐受的形成。
[Abstract]:Acupuncture has a long history as a traditional treatment for diseases and pain relief. Electroacupuncture (EA) derives from traditional hand acupuncture and gradually replaces hand acupuncture, because EA has better analgesic effect, fewer side effects, and its parameters can be objectively quantified and controlled. However, as with morphine tolerance, long-term or repeated EA stimulation can occur. Electro-acupuncture tolerance is a common phenomenon in clinic, which has aroused great concern of scholars. It has been found that electro-acupuncture can induce the release of endogenous opioid peptides to produce analgesic effects, such as endorphins and enkephalin, and also can promote some anti-analgesic effects. Release of substances such as orphanin, cholecystokinin octapeptide and angiotensin II. Studies have confirmed that there is a bi-directional cross-tolerance between morphine tolerance and electroacupuncture tolerance, suggesting that both may have a common mechanism of action. Studies have found that in morphine tolerance, the phosphorylation level of STAT3 in the spinal cord of rats increases. In addition, a large number of studies have shown that JAK2-STAT3. Inhibition of JAK2-STAT3 signaling pathway can alleviate hyperalgesia and hyperalgesia, suggesting that JAK2-STAT3 signaling pathway plays an important role in pain response. However, whether JAK2-STAT3 signaling pathway is involved in electroacupuncture tolerance has not been reported. In this study, SD rats with tail-flick latency increased by more than 50% after single electroacupuncture were judged to be effective. Seventy effective rats (250 + 20g) were randomly divided into two groups: electroacupuncture group (EA group) and sham acupuncture group. Stimulation of Zusanli and Sanyinjiao acupoints once a day for 30 minutes for 8 consecutive days. Sham-acupuncture group rats were only acupoint-pricked, not electrified, the rest of the treatment was the same as electroacupuncture group. The tail flick latency of rats was measured by thermal radiation. The pain threshold was measured before and after each electroacupuncture, and the change rate of pain threshold was calculated. To explore the role of spinal JAK2-STAT3 signaling pathway in electroacupuncture tolerance, the L4-6 segments of the spinal cord of rats were taken from 0 (before electroacupuncture), 2, 4, 6 and 8 days after electroacupuncture. The gene expression of JAK2 and STAT3 was detected by fluorescence quantitative PCR, and the phosphorylation levels of JAK2 and STAT3 proteins were detected by Western Blot assay, and pJ was determined by immunohistochemistry. Distribution of AK2 and pSTAT 3. The effect of spinal cord JAK2-STAT3 signaling pathway on Electroacupuncture tolerance was further determined by intrathecal injection of WP1066 (inhibitor of JAK2-STAT3 signaling pathway). Another 45 rats were randomly divided into three groups: EA group, EA+DMSO group and EA+WP1066 group, 15 rats in each group. DMSO (solvent) and WP1066 were injected into the lumbar region before electroacupuncture respectively. The pain threshold was measured before and after electroacupuncture, and the change rate of pain threshold was calculated. The phosphorylation levels of JAK2 and STAT3 in spinal dorsal horn were detected by immunohistochemistry. The results of repeated electroacupuncture showed that the analgesic effect was weakened with the increase of electroacupuncture times. The expression of JAK 2 and STAT3 mRNA in EA group and sham needle group were not significantly different (p0.05). The Western Blot results showed that the expression of pJAK 2 and pSTAT3 was significantly higher (p0.05) at 2-6 days after EA than that in sham needle group (p0.05), and at 4 days after EA than that in sham needle group (p0.05). The results of immunohistochemistry showed that pJAK2 and pSTAT3 were mainly expressed in the spinal dorsal horn II region, and the change trend was consistent with that of Western Blot. The analgesic effect of EA group was significantly higher than that of EA group and EA+DMSO group (p0.05). Immunohistochemical results showed that the expression of JAK 2 and STAT3 protein phosphoric acid in EA group was consistent with that of EA group. The expression of JAK 2 and STAT3 protein phosphoric acid increased from 2 to 6 days and reached a peak at 4 days. There was no significant difference between EA+DMSO group and EA group (p0.05). The results showed that JAK2-STAT3 signaling pathway in spinal cord participated in the regulation of EA tolerance. Intrathecal injection of WP1066 inhibited the activation of JAK2-STAT3 signaling pathway and weakened the formation of EA tolerance.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S853.6
本文编号：2179004
[Abstract]:Acupuncture has a long history as a traditional treatment for diseases and pain relief. Electroacupuncture (EA) derives from traditional hand acupuncture and gradually replaces hand acupuncture, because EA has better analgesic effect, fewer side effects, and its parameters can be objectively quantified and controlled. However, as with morphine tolerance, long-term or repeated EA stimulation can occur. Electro-acupuncture tolerance is a common phenomenon in clinic, which has aroused great concern of scholars. It has been found that electro-acupuncture can induce the release of endogenous opioid peptides to produce analgesic effects, such as endorphins and enkephalin, and also can promote some anti-analgesic effects. Release of substances such as orphanin, cholecystokinin octapeptide and angiotensin II. Studies have confirmed that there is a bi-directional cross-tolerance between morphine tolerance and electroacupuncture tolerance, suggesting that both may have a common mechanism of action. Studies have found that in morphine tolerance, the phosphorylation level of STAT3 in the spinal cord of rats increases. In addition, a large number of studies have shown that JAK2-STAT3. Inhibition of JAK2-STAT3 signaling pathway can alleviate hyperalgesia and hyperalgesia, suggesting that JAK2-STAT3 signaling pathway plays an important role in pain response. However, whether JAK2-STAT3 signaling pathway is involved in electroacupuncture tolerance has not been reported. In this study, SD rats with tail-flick latency increased by more than 50% after single electroacupuncture were judged to be effective. Seventy effective rats (250 + 20g) were randomly divided into two groups: electroacupuncture group (EA group) and sham acupuncture group. Stimulation of Zusanli and Sanyinjiao acupoints once a day for 30 minutes for 8 consecutive days. Sham-acupuncture group rats were only acupoint-pricked, not electrified, the rest of the treatment was the same as electroacupuncture group. The tail flick latency of rats was measured by thermal radiation. The pain threshold was measured before and after each electroacupuncture, and the change rate of pain threshold was calculated. To explore the role of spinal JAK2-STAT3 signaling pathway in electroacupuncture tolerance, the L4-6 segments of the spinal cord of rats were taken from 0 (before electroacupuncture), 2, 4, 6 and 8 days after electroacupuncture. The gene expression of JAK2 and STAT3 was detected by fluorescence quantitative PCR, and the phosphorylation levels of JAK2 and STAT3 proteins were detected by Western Blot assay, and pJ was determined by immunohistochemistry. Distribution of AK2 and pSTAT 3. The effect of spinal cord JAK2-STAT3 signaling pathway on Electroacupuncture tolerance was further determined by intrathecal injection of WP1066 (inhibitor of JAK2-STAT3 signaling pathway). Another 45 rats were randomly divided into three groups: EA group, EA+DMSO group and EA+WP1066 group, 15 rats in each group. DMSO (solvent) and WP1066 were injected into the lumbar region before electroacupuncture respectively. The pain threshold was measured before and after electroacupuncture, and the change rate of pain threshold was calculated. The phosphorylation levels of JAK2 and STAT3 in spinal dorsal horn were detected by immunohistochemistry. The results of repeated electroacupuncture showed that the analgesic effect was weakened with the increase of electroacupuncture times. The expression of JAK 2 and STAT3 mRNA in EA group and sham needle group were not significantly different (p0.05). The Western Blot results showed that the expression of pJAK 2 and pSTAT3 was significantly higher (p0.05) at 2-6 days after EA than that in sham needle group (p0.05), and at 4 days after EA than that in sham needle group (p0.05). The results of immunohistochemistry showed that pJAK2 and pSTAT3 were mainly expressed in the spinal dorsal horn II region, and the change trend was consistent with that of Western Blot. The analgesic effect of EA group was significantly higher than that of EA group and EA+DMSO group (p0.05). Immunohistochemical results showed that the expression of JAK 2 and STAT3 protein phosphoric acid in EA group was consistent with that of EA group. The expression of JAK 2 and STAT3 protein phosphoric acid increased from 2 to 6 days and reached a peak at 4 days. There was no significant difference between EA+DMSO group and EA group (p0.05). The results showed that JAK2-STAT3 signaling pathway in spinal cord participated in the regulation of EA tolerance. Intrathecal injection of WP1066 inhibited the activation of JAK2-STAT3 signaling pathway and weakened the formation of EA tolerance.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S853.6
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