Objectives: To explore the effect and mechanism of acupuncture at "Feishu" (BL 13) and "Dingchuan" (EX-B 1), and "Kongzui" (LU 6) and "Yuji" (LU 10) for relaxing the airway smooth muscle in the rats during acute asthma attack and compare the effect among the two pairs of acupoints and the acupoints combination.
Methods: Forty SD male rats with SPF grade were randomly divided into a blank group, a model group, a pair-point A group (acupuncture at "Feishu" [BL 13] and "Dingchuan" [EX-B 1]), a pair-point B group (acupuncture at "Kongzui" [LU 6] and "Yuji" [LU 10]) and a point combination group (acupuncture at "Feishu" [BL 13] , "Dingchuan" [EX-B 1], "Kongzui" [LU 6] and "Yuji" [LU 10]), with 8 rats in each group. Except the rats in the blank group, the model of acute asthma attack was induced by ovalbumin (OVA) combined with aluminum hydroxide gel in the rest groups. Started on the 15th day of modeling, except in the blank group and the model group, acupuncture was delivered in the other groups, 30 min in each intervention, once daily, for 14 days. In each group, the latent period of asthma inducing was measured; the lung resistance (LR) and dynamic lung compliance (Cdyn) were determined using lung function detector; the levels of endothelin-1 (ET-1), tumor necrosis factor-α (TNF-α), cyclic adenosine monophosphate (cAMP) and cyclic guanosine monophosphate (cGMP) in serum and bronchoalveolar lavage fluid (BALF) were measured by ELISA; with Masson staining and electron microscopy adopted, the morphology and ultrastructure of airway smooth muscle of the rats were observed; the mRNA and protein expressions of ET-1 and beta-2 adrenergic receptor (β2-AR) were detected by quantitative real-time fluorescence and Western blot, respectively.
Results: Compared with the blank group, the latent period of asthma inducing was shortened (P<0.05), RL increased and Cdyn decreased (P<0.05) with the different concentrations of methacholine (0.025 mg/kg, 0.05 mg/kg, 0.1 mg/kg, 0.2 mg/kg) in the model group. In the pair-point A group, the pair-point B group and the point combination group, the latent period of asthma inducing was prolonged (P<0.05), RL decreased and Cdyn increased (P<0.05) with different concentrations of methacholine when compared with those in the model group; and the latent period of asthma inducing in the point combination group was longer than that in the pair-point A group (P<0.05). Compared with the blank group, the levels of ET-1, TNF-α and cGMP in the serum and BALF were elevated (P<0.05), and those of cAMP reduced (P<0.05) in the model group. The levels of ET-1, TNF-α and cGMP in the serum and BALF were reduced (P<0.05), and those of cAMP elevated (P<0.05) in the pair-point A group, the pair-point B group and the point combination group when compared with those in the model group. In the blank group, the lung tissue was normal structurally. In the model group, the collagen fibers were proliferated increasingly, the smooth muscle was thickened, the mitochondria were swollen, and their cristae disrupted and reduced massively. In the pair-point B group, the collagen fibers were proliferated, the smooth muscle was thicker compared with that in the blank group, the mitochondria were mildly swollen and their cristae disrupted partially. In the pair-point A group and the point combination group, the lung tissue changes were obviously alleviated in comparison with the model group, the mitochondria were slightly swollen and their cristae disrupted occasionally. Compared with the blank group, the mRNA and protein expression of ET-1 increased and that of β2-AR decreased in the lung tissue of the model group (P<0.05). In the pair-point A group, the pair-point B group and the point combination group, the mRNA and protein expression of ET-1 was reduced and that of β2-AR elevated in the lung tissue when compared with those in the model group (P<0.05). In comparison with the pair-point A group, the mRNA expression of β2-AR was elevated in the point combination group (P<0.05). When compared with the pair-point B group, the mRNA expression of β2-AR increased, the protein expression of ET-1 decreased (P<0.05) in the point combination group.
Conclusions: Acupuncture at "Feishu" (BL 13) and "Dingchuan" (EX-B 1), "Kongzui" (LU 6) and "Yuji" (LU 10), two pairs of acupoints relieves the airway smooth muscle spasm in the rats during acute asthma attack, which may be related to inhibiting the mRNA and protein expression of ET-1 to reduce the excretion of ET-1 and TNF-α; while enhancing the mRNA and protein expression of β2-AR to balance the levels of cAMP and cGMP. The effect is optimal when acupuncture is delivered at two pairs of acupoints simultaneously.
目的: 探讨针刺“肺俞”“定喘”和“孔最”“鱼际”松弛哮喘急性发作大鼠气道平滑肌的作用机制，并比较两组穴位及联合配伍的疗效。方法: 将40只SPF级雄性SD大鼠随机分成空白组、模型组、肺俞定喘组（针刺“肺俞”“定喘”）、孔最鱼际组（针刺“孔最”“鱼际”）和联合组（针刺“肺俞”“定喘”“孔最”“鱼际”），每组8只。除空白组外，其余各组均以卵清蛋白（OVA）联合氢氧化铝凝胶的方法诱导大鼠哮喘急性发作模型。第15天起，除空白组和模型组外，其余各组分别予针刺干预，每次30 min，每日1次，连续14 d。比较各组大鼠的引喘时间，应用肺功能检测仪测定各组大鼠气道阻力（RL）和肺顺应性（Cdyn），ELISA法检测各组大鼠血清及支气管肺泡灌洗液（BALF）内皮素1（ET-1）、肿瘤坏死因子α（TNF-α）、环磷酸腺苷（cAMP）、环磷酸鸟苷（cGMP）含量，Masson染色及透射电镜观察各组大鼠气道平滑肌形态及超微结构，实时荧光定量PCR法检测各组大鼠肺组织ET-1、β2肾上腺素受体（β2-AR）mRNA表达，Western blot法检测各组大鼠肺组织 ET-1、β2-AR蛋白表达。结果: 与空白组比较，模型组大鼠引喘时间缩短（P<0.05），不同乙酰甲胆碱浓度（0.025 mg/kg、0.05 mg/kg、0.1 mg/kg、0.2 mg/kg）下RL均升高、Cdyn均降低（P<0.05）；与模型组比较，肺俞定喘组、孔最鱼际组及联合组大鼠引喘时间延长（P<0.05），不同乙酰甲胆碱浓度下RL均降低、Cdyn均升高（P<0.05）；与肺俞定喘组比较，联合组大鼠引喘时间延长（P<0.05）。与空白组比较，模型组大鼠血清及BALF ET-1、TNF-α、cGMP含量升高（P<0.05），cAMP含量降低（P<0.05）；与模型组比较，肺俞定喘组、孔最鱼际组、联合组大鼠血清及BALF ET-1、TNF-α、cGMP含量降低（P<0.05），cAMP含量升高（P<0.05）。空白组大鼠肺组织结构正常；模型组肺组织可见大量胶原纤维增生，平滑肌增厚，线粒体明显肿胀，嵴大量断裂、减少；孔最鱼际组大鼠肺组织大量胶原纤维增生，平滑肌较空白组增厚，线粒体轻度肿胀，嵴部分断裂；肺俞定喘组、联合组大鼠肺组织改变较模型组明显减轻，线粒体略微肿胀，嵴少量断裂。与空白组比较，模型组大鼠肺组织ET-1 mRNA及蛋白表达升高、β2-AR mRNA及蛋白表达降低（P<0.05）；与模型组比较，肺俞定喘组、孔最鱼际组、联合组大鼠肺组织ET-1 mRNA及蛋白表达降低、β2-AR mRNA及蛋白表达升高（P<0.05）；与肺俞定喘组比较，联合组大鼠肺组织β2-AR mRNA表达升高（P<0.05）；与孔最鱼际组比较，联合组大鼠肺组织β2-AR mRNA表达升高、ET-1蛋白表达降低（P<0.05）。结论: 针刺“肺俞”“定喘”、“孔最”“鱼际”及针刺两组穴位均可缓解哮喘急性发作大鼠的气道平滑肌痉挛，其作用机制可能是抑制ET-1 mRNA及蛋白表达，使ET-1、TNF-α分泌减少；同时升高β2-AR mRNA及蛋白表达，平衡cAMP和cGMP含量。针刺两组穴位疗效更佳。.
Keywords: acupuncture; asthma; beta-2 adrenergic receptor; endothelin-1; smooth muscle spasm.