捻转补泻手法对应激性高血压大鼠下丘脑NOS-1、CGRP、ET-1基因表达的影响
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摘要
目的
     本研究以应激性高血压(stress-induced hypertension, SIH)大鼠模型为研究对象,运用荧光定量聚合酶链式反应(Q-PCR)和蛋白印迹(WB)技术,观察针刺“太冲”穴,施以捻转补法和泻法对应激性高血压大鼠下丘脑中神经细胞型一氧化氮合酶(NOS-1)、降钙素基因相关肽(CGRP)、内皮素1型(ET-1)基因表达和蛋白表达的影响,探讨捻转补法与泻法对SIH大鼠血压及血管内皮相关因子表达的差异,从中枢系统调控血管内皮功能角度分析捻转补泻手法降压的途径,为针刺捻转补泻手法的有效性、差异性提供客观依据,为针刺治疗高血压的机制途径提供实验依据。
     方法
     清洁级雄性Wistar大鼠60只,随机分为5组:空白组、造模组、针刺不手法组、捻转补法组和捻转泻法组。空白组不做处理,其他各组采用电击加噪声刺激制成应激性高血压模型,并在造模前1d,造模第3、6、.9、12、15d的造模刺激后2h测量大鼠收缩压(SBP)。造模完成后,对针刺不手法组进行两侧“太冲”穴针刺治疗,不行补泻手法;对捻转补法组和捻转泻法组进行两侧“太冲”穴针刺治疗,并分别施以捻转补法和捻转泻法,空白组和造模组进行相同的捉抓。每次手法操作持续1 min,留针20 min,每天1次,共治疗7d。治疗结束后测量各组大鼠收缩压,随后断头处死,取下丘脑,用Q-PCR和WB方法分别检测下丘脑中NOS-1、CGRP、ET-1的基因和蛋白表达情况。
     结果
     1各组大鼠收缩压的变化
     造模前各组血压之间无显著差异(p>0.05)。造模15d结束后,与空白组比较,进行造模的四组收缩压明显升高,差异具有统计学意义(p<0.01)。进行造模的四组血压值之间,差异无统计学意义(p>0.05)。针刺7d结束后,空白组与造模前、造模后收缩压变化无显著差异(p>0.05);造模组的血压仍较高,与空白组比较有显著差异(p<0.01),与造模前有显著差异(p<0.01),与造模后无显著差异(p>0.05);针刺不手法组和捻转泻法组针刺后血压下降,与造模前无显著差异(p>0.05),与造模后有显著差异(p<0.01);针刺不手法组比捻转泻法组血压高,两组有显著差异(p<0.05);捻转补法组在针刺后血压下降,但血压高于造模前(p<0.01),低于造模后(p<0.01)。
     2各组大鼠下丘脑中NOS-1基因和蛋白表达情况
     造模组、针刺不手法组下丘脑中NOS-1 mRNA的表达偏低,与空白组比较有显著差异(p<0.05),捻转泻法组与空白组比较有极显著差异(p<0.01),与捻转补法组比较有显著差异(p<0.05)。而捻转补法组与空白组比较没有显著差异(p>0.05)。
     造模组、针刺不手法组、捻转补法组和捻转泻法组的大鼠下丘脑中NOS-1表达下调,与空白组有显著差异(p<0.01)。其中造模组、针刺不手法组的表达水平明显低于捻转补法组和捻转泻法组(p<0.01);捻转泻法组的表达水平明显低于捻转补法组(p<0.01)。
     3各组大鼠下丘脑中CGRP基因和蛋白表达情况
     各组间大鼠下丘脑中CGRP mRNA的表达无显著差异(p>0.05)。空白组的大鼠下丘脑中CGRP的表达明显低于造模组、针刺不手法组、捻转补法组和捻转泻法组,差异有统计学意义(p<0.01)。造模组的大鼠下丘脑中CGRP的表达明显低于针刺不手法组、捻转补法组和捻转泻法组,差异有统计学意义(p<0.01)。针刺不手法组的大鼠下丘脑中CGRP的表达明显低于捻转补法组和捻转泻法组,差异有统计学意义(p<0.01)。同时,捻转补法组与捻转泻法组也有显著差异(p<0.01)。
     4各组大鼠下丘脑中ET-1基因和蛋白表达情况
     各组间大鼠下丘脑中ET-1 mRNA的表达无显著差异(p>0.05)。造模组、针刺不手法组、捻转补法组和捻转泻法组的大鼠下丘脑中ET-1表达水平与空白组有显著差异(p<0.01);造模组和捻转补法组表达水平低于针刺不手法组和捻转泻法组,差异有统计学意义(p<0.01)。
     结论
     1电击足底结合噪音刺激可有效建立应激性高血压大鼠模型。应激性高血压大鼠下丘脑中,NOS-1基因和蛋白的表达下调;CGRP和ET-1的基因表达未见明显变化,不排除检测时间错过高拷贝时间的因素;CGRP表达增加,ET-1表达下降,可能发挥着代偿性降压作用。
     2针刺“太冲穴”可有效降低应激性高血压大鼠收缩压。降压效果以施于捻转泻法最佳,单纯针刺效果次之,施于捻转补法效果再次。3以不同手法针刺“太冲”穴,其降压机制可能不同。捻转泻法的降压途径可能与下丘脑中CGRP的关系较为密切,而与NOS-1和ET-1无直接关系;捻转补法的降压途径与下丘脑中NOS-1的关系较为密切,与CGRP和ET-1有一定关系。
     4以“左旋为补,右旋为泻”的捻转补泻手法确实存在效应上差异,表明以“左旋为补,右旋为泻”确定捻转补泻手法是有效的。
Objective
     The study aims at observing the effect on the gene and protein expression of NOS, CGRP, ET in hypothalamic of SIH rats treated by twirling reinforcing and reducing manipulation with Q-PCR and WB, investigating the difference on blood pressure and vascular endothelial factor expression of SIH rats between twirling reinforcing manipulation and reducing manipulation, and analyzing how twirling reinforcing-reducing manipulation bring high blood pressure down from vascular endothelial function controlled by central nervous system angle. It provides scientific bases for the validity and diversity on twirling reinforcing-reducing manipulation and experimental bases for the mechanism that acupuncture treats hypotension.
     Method
     60 clean male Wistar rats were divided into 5 groups such as controling group, modeling group, the acupuncture group, reinforcing manipulation group and reducing manipulation group. Electric shock and noise were applied to stimulate the rats into stress-induced hypertension model except controlling group. After modeling, the acupuncture group was treated by needling "tai chong" point without manipulation. Reinforcing manipulation group was treated by needling "tai chong" point with twirling reinforcing manipulation and reducing group was treated by needling "tai chong" point with twirling reducing manipulation. The treatments were operated once per day with a 7-day period. The systolic blood pressure (SBP) was measured in all the groups 1 day before modeling, the third, sixth, ninth, twelfth, fifteenth during modeling and the day after treatment. Then, the gene and protein contents of NOS, CGRP, ET in hypothalamic were detected by means of Q-PCR and WB.
     Result
     1 comparison of the SBP in groups
     Before modeling, there was no significant difference among groups (p>0.05). After modeling for 15 days, compared with the controlling group, the SBP of the other four groups was significantly higher (p<0.01). And there was no significant difference between the four modeled groups (p>0.05). After acupuncture treatment, the SBP of the acupuncture group and reducing manipulation group was lower than the one after modeling (p<0.01), which is not significantly difference from the one when before modeling (p>0.05). The SBP of the reinforcing manipulation group was lower than the one after modeling (p<0.01) and higher than the one before modeling (p<0.01).
     2 comparisons of the NOS-1 gene and protein expression in the hypothalamic in groups
     Compared with controlling group, the NOS-1 mRNA expression of the modeling group, acupuncture group and reducing manipulation group was lower (p<0.05 or p<0.01). There was significant difference between reinforcing manipulation group and reducing manipulation group.
     Compared with controlling group, the NOS-1 expression in the hypothalamic of the modeling group, acupuncture group, reinforcing manipulation and reducing manipulation group was lower (p<0.01). And the one of the modeling group and acupuncture group was lower than reinforcing manipulation group and reducing manipulation group (p<0.01). There was significant difference between reinforcing manipulation group and reducing manipulation group (p<0.01).
     3 comparisons of the CGRP gene and protein expression in the hypothalamic in groups
     The CGRP mRNA expression in the hypothalamic was no significant difference among the groups (p>0.05).
     Compared with controlling group, the CGRP expression in the hypothalamic of the modeling group, acupuncture group, reinforcing manipulation and reducing manipulation group was higher (p<0.01). Compared with modeling group, the CGRP expression in the hypothalamic of acupuncture group, reinforcing manipulation and reducing manipulation group was higher (p<0.01). And there was significant difference among acupuncture group, reinforcing manipulation and reducing manipulation group (p<0.01).
     4 comparisons of the ET-1 gene and protein expression in the hypothalamic in groups
     The ET-1 mRNA expression in the hypothalamic was no significant difference among the groups (p>0.05).
     Compared with controlling group, the ET-1 expression in the hypothalamic of the modeling group, acupuncture group, reinforcing manipulation group and reducing manipulation group was lower (p<0.01). And the one of the modeling group and reinforcing manipulation group was lower than acupuncture group and reducing manipulation group (p<0.01).
     Conclusion
     1. The method can effectively establish the stress-induced hypertension model that Wistar rats are shocked by electric on foot and stimulated by noise. In the hypothalamic of SIH rats, the NOS-1 gene and protein expression could be reduced or restrained. There are no significant changes in CGRP and ET-1 mRNA. It may be related with the detection time. The expression of CGRP and ET-1 is decreased. They may play a compensatory effect to bring the SBP down.
     2. Needling "tai chong" point can effectively reduce the SBP of the SIH rats. In three treatments, the effect of twirling reducing manipulation is best, and twirling reinforcing manipulation is worst.
     3. The mechanism that the two manipulations bring the SBP down may be different. The approach that the twirling reducing manipulation bring the SBP down may be closely related to CGRP and there is no direct relationship with NOS-1 and ET-1. The approach that the twirling reinforcing manipulation brings the SBP down may be closely related to NOS, and there is a certain relationship with CGRP and ET-1.
     4. There are differences in the effects between the twirling reinforcing-reducing manipulations whose concept is reinforcing when turning left and reducing when turning right. And it is effective that turning left or right makes a distinction between twirling reinforcing manipulation and reducing manipulation.
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