P物质在夹脊电针调控炎性痛大鼠神经—免疫网络中的作用研究
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摘要
目的:通过阐明中枢内源性下行镇痛系统中的重要神经肽SP及其受体NK-1R在夹脊电针抑制大鼠炎性痛中的作用,从神经-免疫网络的视角,揭示夹脊电针的作用机制。
方法:在弗氏完全佐剂建立的单侧足底慢性炎症痛大鼠模型上,从行为学到形态学等不同方面,从分子到蛋白等不同水平,采用Real time-PCR、免疫组织化学染色、ELISA等技术,(l)观察下丘脑、脊髓中SP及其受体NK1R在大鼠炎症痛发展的不同阶段以及夹脊电针镇痛中的变化;(2)观察下丘脑、脊髓中TNF-α在大鼠炎症痛发展的不同阶段以及夹脊电针镇痛中的变化;(3)观察外周血中CD4+/CD8+在大鼠炎症痛发展的不同阶段以及夹脊电针镇痛中的变化。
干预方法:(1)夹脊电针:于造模后3h取C3和L4双侧“夹脊穴”给予电针刺激,以后每日早8:00开始给予电针治疗,1次/ d,至造模第14d。将大鼠置于固定器内,待大鼠安静20min后,在C3和L4棘突两侧各旁开2mm处取穴, 0.25mm×13mm毫针垂直刺入3mm,使针尖触及椎板,采用KWD-808Ⅱ型脉冲电针仪,二组导线分别上下连接针柄,正极在上,负极在下,规律交流脉冲电波(60Hz /1.05s与2Hz/2.85s交替),强度1mA,持续25min。(2)普通电针:于造模后3h给予电针刺激,以后每日早8:00开始给予电针治疗,1次/ d,至造模第14d。将大鼠置于固定器内,两后肢暴露于外,待大鼠安静20min后,0.25mm×13mm毫针垂直刺入大鼠左侧“足三里”和“三阴交”穴约3mm,采用KWD-808Ⅱ型脉冲电针仪,正极连接“足三里”,负极连接“三阴交”穴,规律交流脉冲电波(60Hz /1.05s与2Hz/2.85s交替),强度1mA,持续25min。(3)灌饲塞来昔布(西乐葆):造模当天开始给药,20mg/kg/day,每天早8:30给药一次,至造模第14d。
结果:
(1)给予治疗后3个时间点上,3个电针组的PWL与关节炎组比较均明显提高(P<0.001);给予治疗的第10、14次夹脊组,夹脊+常规组的PWL均较常规电针组显著性延长(P<0.05),但夹脊组与夹脊+常规组3个时间点的PWL无显著性差异(P=0.627,P=0.731,P=0.473)。
(2)给予治疗后3个时间点上,3个治疗组的PWL与关节炎组比较均明显提高(P <0.001);给予治疗的第4、10、14次夹脊电针组和西乐葆组的PWL无显著性差异( P =0.874, P =0.779, P =0.892),但在第10天和第14天,与夹脊电针组、西乐葆组相比,电针+西乐葆组的PWL进一步提高(P =0.023 to P =0.044)。
(3)在大鼠炎症痛发展的不同阶段下丘脑中SP、NK1R mRNA及免疫阳性细胞数表达较假造模组明显增加(P<0.001);夹脊电针组较模型组增加更为显著(P<0.01),且电针合用西乐葆能进一步增加炎症痛大鼠下丘脑SP、NK1R mRNA及免疫阳性细胞的表达( P<0.01)。
(4)在大鼠炎症痛发展的不同阶段脊髓中SP、NK1R mRNA及免疫阳性细胞数表达较假造模组明显增加(P<0.001);夹脊电针组的表达较模型组显著性降低(P<0.01),且灌饲西乐葆能加强夹脊电针对炎症痛大鼠脊髓中SP、NK1R mRNA及免疫阳性细胞表达的抑制作用(P<0.01)。
(5)在大鼠炎症痛发展的不同阶段下丘脑、脊髓TNF-αmRNA及蛋白表达较假造模组明显增加(P<0.001);夹脊电针组的表达较模型组显著性降低(P<0.01),且灌饲西乐葆能加强夹脊电针对炎症痛大鼠下丘脑、脊髓中TNF-αmRNA及蛋白表达的抑制作用(P<0.01)。
(6)在大鼠炎症痛发展的不同阶段外周血中CD4+/CD8+较假造模组明显增加( P<0.001 ) ;夹脊电针组CD4+/CD8+较模型组显著性降低( P<0.01 ),且灌饲西乐葆能加强夹脊电针法对炎症痛大鼠外周血中CD4+/CD8+的抑制作用( P<0.01)。
结论:
(1)多次电针对大鼠炎性痛具有显著的累加镇痛效应,夹脊电针法的抗热痛敏效果与灌饲西乐葆相当,强于电针“足三里”+“三阴交”穴,且灌饲西乐葆能加强夹脊电针法对炎症痛大鼠的镇痛作用。
(2)夹脊电针能够调控炎性痛大鼠中枢内源性下行镇痛系统中的重要神经肽SP和其受体NK-1R的表达,诱发下丘脑、脊髓的神经细胞兴奋,引起其神经—免疫作用,进而下调促炎细胞因子TNF-α的表达,降低外周血中CD4+/CD8+淋巴细胞比值。
Objective: The aim of this study was to reveal the mechanism of involvement of central endogenous descending pain control system SP,NK-1R in Inflammatory pain and the regulaion of network of immune-neuroendocrine interactions by JIAJI electroacupuncture.
Methods: Monoarthritis (MA) was induced by an injection of complete Freund’s adjuvant (CFA). By using the molecular biological techniques,including Real-time PCR,immunochemistry,ELISA,the present study was designed to explore,(1) the changes of expression of SP、NK-1R in hypothalamus and spinal cord of MA rats and EA analgesia; (2) the changes of expression of TNF-αin hypothalamus and spinal cord of MA rats and EA analgesia; (3) the changes of expression of ratio of CD4+/CD8+ T cells in peripheral blood of MA rats and EA analgesia;
Results:
(1)The PWL were significantly prolonged in JIAJI EA group than routine EA group at 10th day and 14th day post-EA( P<0.05) ,but there was no significant difference between JIAJI EA group and JIAJI+routine EA group during the experiment( P=0.627, P=0.731, P=0.473) .
(2)There was no significant difference between JIAJI EA group and Celebre group during the experiment( P =0.874,P =0.779,P =0.892), but which was further significantly enhanced by JIAJI EA +Celebre group at 10th day and 14th day post-EA( P =0.023 to P =0.044) .
(3)The mRNA levels detected by Real-time PCR and the expression detected by immunohistochemistry of SP ,NK-1R in hypothalamus in JIAJI EA group were significantly increaced as compared to MA group(P<0.01),which was further significantly enhanced by JIAJI EA +Celebre group during the experiment( P<0.01) .
(4) The mRNA levels detected by Real-time PCR and the expression detected by immunohistochemistry of SP ,NK-1R in spinal cord in JIAJI EA group were significantly decreaced as compared to MA group(P<0.01);Combination of EA with Celebre significantly enhanced the inhibitory effects(P<0.01).
(5) The mRNA levels detected by Real-time PCR and the expression detected by ELISA in hypothalamus and spinal cord of TNF-αin JIAJI EA group were significantly decreaced as compared to MA group( P<0.01);Combination of EA with Celebre significantly enhanced the inhibitory effects(P<0.01).
(6)The expression of ratio of CD4+/CD8+ T cells in peripheral blood in JIAJI EA group were significantly decreaced as compared to MA group(P<0.01);Combination of EA with Celebre significantly enhanced the inhibitory effects( P<0.01) .
Conclusion:
(1)Comulative EA has potent analgesia effect on inflammatory pain and JIAJI EA is more effectively than routine EA. Combination of EA with Celebre significantly enhanced the inhibitory effects on hyperalgesia.
(2) These results show that JIAJI EA could regulate levels of SP,NK-1R and TNF-αin hypothalamus and spinal cord,suggesting that central endogenous descending pain control system SP,NK-1R and TNF-αplayed an important role in JIAJI electroacupuncture regulaing network of immune-neuroendocrine interactions.
方法:在弗氏完全佐剂建立的单侧足底慢性炎症痛大鼠模型上,从行为学到形态学等不同方面,从分子到蛋白等不同水平,采用Real time-PCR、免疫组织化学染色、ELISA等技术,(l)观察下丘脑、脊髓中SP及其受体NK1R在大鼠炎症痛发展的不同阶段以及夹脊电针镇痛中的变化;(2)观察下丘脑、脊髓中TNF-α在大鼠炎症痛发展的不同阶段以及夹脊电针镇痛中的变化;(3)观察外周血中CD4+/CD8+在大鼠炎症痛发展的不同阶段以及夹脊电针镇痛中的变化。
干预方法:(1)夹脊电针:于造模后3h取C3和L4双侧“夹脊穴”给予电针刺激,以后每日早8:00开始给予电针治疗,1次/ d,至造模第14d。将大鼠置于固定器内,待大鼠安静20min后,在C3和L4棘突两侧各旁开2mm处取穴, 0.25mm×13mm毫针垂直刺入3mm,使针尖触及椎板,采用KWD-808Ⅱ型脉冲电针仪,二组导线分别上下连接针柄,正极在上,负极在下,规律交流脉冲电波(60Hz /1.05s与2Hz/2.85s交替),强度1mA,持续25min。(2)普通电针:于造模后3h给予电针刺激,以后每日早8:00开始给予电针治疗,1次/ d,至造模第14d。将大鼠置于固定器内,两后肢暴露于外,待大鼠安静20min后,0.25mm×13mm毫针垂直刺入大鼠左侧“足三里”和“三阴交”穴约3mm,采用KWD-808Ⅱ型脉冲电针仪,正极连接“足三里”,负极连接“三阴交”穴,规律交流脉冲电波(60Hz /1.05s与2Hz/2.85s交替),强度1mA,持续25min。(3)灌饲塞来昔布(西乐葆):造模当天开始给药,20mg/kg/day,每天早8:30给药一次,至造模第14d。
结果:
(1)给予治疗后3个时间点上,3个电针组的PWL与关节炎组比较均明显提高(P<0.001);给予治疗的第10、14次夹脊组,夹脊+常规组的PWL均较常规电针组显著性延长(P<0.05),但夹脊组与夹脊+常规组3个时间点的PWL无显著性差异(P=0.627,P=0.731,P=0.473)。
(2)给予治疗后3个时间点上,3个治疗组的PWL与关节炎组比较均明显提高(P <0.001);给予治疗的第4、10、14次夹脊电针组和西乐葆组的PWL无显著性差异( P =0.874, P =0.779, P =0.892),但在第10天和第14天,与夹脊电针组、西乐葆组相比,电针+西乐葆组的PWL进一步提高(P =0.023 to P =0.044)。
(3)在大鼠炎症痛发展的不同阶段下丘脑中SP、NK1R mRNA及免疫阳性细胞数表达较假造模组明显增加(P<0.001);夹脊电针组较模型组增加更为显著(P<0.01),且电针合用西乐葆能进一步增加炎症痛大鼠下丘脑SP、NK1R mRNA及免疫阳性细胞的表达( P<0.01)。
(4)在大鼠炎症痛发展的不同阶段脊髓中SP、NK1R mRNA及免疫阳性细胞数表达较假造模组明显增加(P<0.001);夹脊电针组的表达较模型组显著性降低(P<0.01),且灌饲西乐葆能加强夹脊电针对炎症痛大鼠脊髓中SP、NK1R mRNA及免疫阳性细胞表达的抑制作用(P<0.01)。
(5)在大鼠炎症痛发展的不同阶段下丘脑、脊髓TNF-αmRNA及蛋白表达较假造模组明显增加(P<0.001);夹脊电针组的表达较模型组显著性降低(P<0.01),且灌饲西乐葆能加强夹脊电针对炎症痛大鼠下丘脑、脊髓中TNF-αmRNA及蛋白表达的抑制作用(P<0.01)。
(6)在大鼠炎症痛发展的不同阶段外周血中CD4+/CD8+较假造模组明显增加( P<0.001 ) ;夹脊电针组CD4+/CD8+较模型组显著性降低( P<0.01 ),且灌饲西乐葆能加强夹脊电针法对炎症痛大鼠外周血中CD4+/CD8+的抑制作用( P<0.01)。
结论:
(1)多次电针对大鼠炎性痛具有显著的累加镇痛效应,夹脊电针法的抗热痛敏效果与灌饲西乐葆相当,强于电针“足三里”+“三阴交”穴,且灌饲西乐葆能加强夹脊电针法对炎症痛大鼠的镇痛作用。
(2)夹脊电针能够调控炎性痛大鼠中枢内源性下行镇痛系统中的重要神经肽SP和其受体NK-1R的表达,诱发下丘脑、脊髓的神经细胞兴奋,引起其神经—免疫作用,进而下调促炎细胞因子TNF-α的表达,降低外周血中CD4+/CD8+淋巴细胞比值。
Objective: The aim of this study was to reveal the mechanism of involvement of central endogenous descending pain control system SP,NK-1R in Inflammatory pain and the regulaion of network of immune-neuroendocrine interactions by JIAJI electroacupuncture.
Methods: Monoarthritis (MA) was induced by an injection of complete Freund’s adjuvant (CFA). By using the molecular biological techniques,including Real-time PCR,immunochemistry,ELISA,the present study was designed to explore,(1) the changes of expression of SP、NK-1R in hypothalamus and spinal cord of MA rats and EA analgesia; (2) the changes of expression of TNF-αin hypothalamus and spinal cord of MA rats and EA analgesia; (3) the changes of expression of ratio of CD4+/CD8+ T cells in peripheral blood of MA rats and EA analgesia;
Results:
(1)The PWL were significantly prolonged in JIAJI EA group than routine EA group at 10th day and 14th day post-EA( P<0.05) ,but there was no significant difference between JIAJI EA group and JIAJI+routine EA group during the experiment( P=0.627, P=0.731, P=0.473) .
(2)There was no significant difference between JIAJI EA group and Celebre group during the experiment( P =0.874,P =0.779,P =0.892), but which was further significantly enhanced by JIAJI EA +Celebre group at 10th day and 14th day post-EA( P =0.023 to P =0.044) .
(3)The mRNA levels detected by Real-time PCR and the expression detected by immunohistochemistry of SP ,NK-1R in hypothalamus in JIAJI EA group were significantly increaced as compared to MA group(P<0.01),which was further significantly enhanced by JIAJI EA +Celebre group during the experiment( P<0.01) .
(4) The mRNA levels detected by Real-time PCR and the expression detected by immunohistochemistry of SP ,NK-1R in spinal cord in JIAJI EA group were significantly decreaced as compared to MA group(P<0.01);Combination of EA with Celebre significantly enhanced the inhibitory effects(P<0.01).
(5) The mRNA levels detected by Real-time PCR and the expression detected by ELISA in hypothalamus and spinal cord of TNF-αin JIAJI EA group were significantly decreaced as compared to MA group( P<0.01);Combination of EA with Celebre significantly enhanced the inhibitory effects(P<0.01).
(6)The expression of ratio of CD4+/CD8+ T cells in peripheral blood in JIAJI EA group were significantly decreaced as compared to MA group(P<0.01);Combination of EA with Celebre significantly enhanced the inhibitory effects( P<0.01) .
Conclusion:
(1)Comulative EA has potent analgesia effect on inflammatory pain and JIAJI EA is more effectively than routine EA. Combination of EA with Celebre significantly enhanced the inhibitory effects on hyperalgesia.
(2) These results show that JIAJI EA could regulate levels of SP,NK-1R and TNF-αin hypothalamus and spinal cord,suggesting that central endogenous descending pain control system SP,NK-1R and TNF-αplayed an important role in JIAJI electroacupuncture regulaing network of immune-neuroendocrine interactions.
引文
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