脊髓JAK2-STAT3信號通路參與大鼠電針耐受調(diào)節(jié)
發(fā)布時間：2018-08-12 12:04
【摘要】：針灸作為一種傳統(tǒng)療法在治療疾病和緩解疼痛方面具有悠久的歷史。電針(EA)衍生于傳統(tǒng)手針并逐漸替代手針,因為電針的鎮(zhèn)痛效果更佳,副作用少,并且其參數(shù)可被客觀的量化和控制。但是,如同嗎啡耐受一樣,長時間或反復電針刺激會使鎮(zhèn)痛效果降低甚至消失,學術上稱之為“電針耐受”。臨床上電針耐受現(xiàn)象普遍存在,引起學者們高度關注。研究發(fā)現(xiàn)電針能通過誘導內(nèi)源性阿片肽類物質(zhì)釋放產(chǎn)生鎮(zhèn)痛作用,如內(nèi)啡肽和腦啡肽,同時還可以促進一些具有抗鎮(zhèn)痛作用物質(zhì)的釋放,如孤啡肽、八肽膽囊收縮素和血管緊張素Ⅱ等。研究證實了嗎啡耐受與電針耐受存在雙向交叉耐受,提示二者可能存在共同的作用機制。研究發(fā)現(xiàn)在嗎啡耐受中,大鼠脊髓的STAT3的磷酸化水平升高。另外大量研究表明JAK2-STAT3信號通路在神經(jīng)病理性痛大鼠的脊髓背角被激活,對疼痛發(fā)展和維持有重要作用,抑制JAK2-STAT3信號通路可以緩解痛覺觸敏和痛覺過敏,提示JAK2-STAT3信號通路在疼痛反應起到重要作用。然而JAK2-STAT3信號通路是否參與電針耐受還未有報道。本試驗將單次電針后甩尾潛伏期升高超過50%的SD大鼠判定為有效鼠。將70只有效鼠(250±20g)隨機分成2組:電針組(EA組)和假針組。通過反復電針刺激穴位建立大鼠耐受模型(鎮(zhèn)痛效果顯著降低或消失表明耐受形成)。EA組采用2Hz頻率電針刺激大鼠雙側(cè)足三里和三陰交穴位,每天1次,持續(xù)30min,連續(xù)8d。假針組大鼠只穴位處扎針,不通電,其余處理與電針組相同。通過熱輻射法測量大鼠的甩尾潛伏期即痛閾。每次電針前后分別測量痛閾,計算痛閾變化率,通過痛閾變化率評估電針的鎮(zhèn)痛效果。為探索脊髓JAK2-STAT3信號通路在電針耐受中的作用,取電針0(電針前)、2、4、6和8d后的大鼠L4~6段脊髓樣品,采用熒光定量PCR檢測JAK2和STAT3的基因表達變化,Western Blot檢測JAK2和STAT3蛋白的磷酸化水平,并用免疫組織化學確定pJAK2和pSTAT3的分布。通過鞘內(nèi)注射WP1066(JAK2-STAT3信號通路抑制劑)進一步確定脊髓JAK2-STAT3信號通路對電針耐受的影響。另選取45只有效鼠,隨機分成3組:EA組、EA+二甲基亞砜(DMSO)組和EA+WP1066組,每組15只。DMSO(溶劑)和WP1066分別于電針前注入腰膨大部。電針前后測量痛閾,計算痛閾變化率,并采用免疫組織化學檢測電針后脊髓背角JAK2和STAT3磷酸化水平。反復電針結(jié)果顯示,隨著電針次數(shù)增加鎮(zhèn)痛效果減弱。假針組大鼠的痛閾變化率各時間點均無顯著變化(p0.05)。EA組大鼠痛閾變化率在1~5d顯著高于假針組(p0.05),6~8d與假針組無異(p0.05),提示電針耐受形成。熒光定量PCR結(jié)果顯示EA組與假針組JAK2和STAT3在mRNA水平上表達沒有差異(p0.05),Western Blot結(jié)果顯示,與假針組相比電針后2~6d pJAK2和pSTAT3表達顯著升高(p0.05),第4d達到峰值,第8d與電針前無顯著差異(p0.05)。免疫組織化學結(jié)果顯示pJAK2和pSTAT3主要在脊髓背角Ⅱ區(qū)表達有差異,且變化趨勢與Western Blot結(jié)果相符。鞘內(nèi)注射試驗結(jié)果顯示EA組與EA+DMSO組無顯著性變化(p0.05),且6~8d電針效果基本消失。EA+WP1066組鎮(zhèn)痛效果雖然也隨著電針次數(shù)增加而減弱,但4~8d的電針效果都顯著高于EA組與EA+DMSO組(p0.05)。免疫組織化學結(jié)果顯示EA組JAK2和STAT3蛋白磷酸表達趨勢與反復電針實驗一致,2~6d表達升高,第4d達到峰值。EA+DMSO組與EA組無顯著差異(p0.05)。EA+DMSO組JAK2和STAT3蛋白磷酸變化水平逐漸升高。上述結(jié)果表明脊髓JAK2-STAT3信號通路參與了電針耐受調(diào)節(jié),鞘內(nèi)注射WP1066抑制JAK2-STAT3信號通路的激活能弱化電針耐受的形成。
[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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S853.6
本文編號：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.
【學位授予單位】：華中農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S853.6
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