摘要
目的:本实验通过研究脾虚对哮喘大鼠嗜酸性粒细胞凋亡、对Fas mRNA、Bcl-2 mRNA表达的影响及针刺足三里的调节作用,探讨脾虚对哮喘的影响及针刺在哮喘炎性细胞凋亡中发挥的作用,从而探索哮喘的有效防治方法,以充分发挥中医针灸整体多层面、多靶点调节的治疗优势,进一步探求本法治疗哮喘的作用机制,更好地指导临床,优化针灸取穴方案。为进一步深化脾虚本质的研究,并为哮喘的针灸临床研究拓展新的领域。
方法:通过利用中医脾虚动物模型和西医哮喘动物模型的复合造模方法,建立大鼠脾虚哮喘病证结合的模型。在中医基础理论的指导下,根据培土生金理论,以脾虚哮喘和哮喘大鼠为研究对象,采取针刺足三里的方法进行治疗。采用双抗体夹心酶联免疫法(ELISA)检测BALF液中TGF-β、GM-CSF的浓度。以TUNEL法进行肺组织细胞凋亡的检测,采用原位杂交法进行肺组织中Fas mRNA、Bcl-2 mRNA的检测,并应用光镜对肺组织形态学进行病理观测。
结果:与正常对照组比较,脾虚哮喘组和哮喘组,均表现为EOS计数值升高、EOS凋亡率明显下降(P﹤0.01);与哮喘组(B1)比较,脾虚哮喘组(A1)的EOS计数值显著升高(P<0.01);与相应的疾病组(A1、B1)比较,两针刺组(A2,B2)中EOS凋亡率明显升高(P﹤0.01)。病理结果显示,脾虚哮喘组和哮喘组均存在不同程度的支气管及肺组织炎性浸润,两针刺组的支气管及肺组织的炎症病变均有不同程度减轻。与正常对照组(C)比较,脾虚哮喘组(A1)和哮喘组(B1),均表现为BALF中GM-CSF的浓度明显升高和TGF-β浓度的显著下降(P﹤0.01),与哮喘组(B1)比较,脾虚哮喘组(A1)的GM-CSF的浓度升高有显著差异(P< 0.05 );与相应的疾病组(A1、B1)比较,两针刺组(A2,B2) GM-CSF的浓度显著降低(P﹤0.01),与哮喘组比较(B1),哮喘针刺组(B2)TGF-β浓度的显著升高(P﹤0.05)。与正常对照组比较,脾虚哮喘组和哮喘组,均表现为EOS的Fas mRNA表达的明显减少和Bcl-2mRNA表达增多(P﹤0.01或P < 0.05),与哮喘组(B1)比较,脾虚哮喘组(A1)的Fas mRNA表达的减少,Bcl-2mRNA表达的增多存在显著差异(P<0.01);与相应的疾病组(A1、B1)比较,两针刺组(A2,B2) Bcl-2mRNA表达显著减少(P﹤0.01),Fas mRNA表达明显增多(P﹤0.01)。各组大鼠BALF中的TGF-β浓度水平与EOS的凋亡率呈明显正相关(r=0.538,p<0.01),大鼠BALF中的GM-CSF浓度水平与EOS的凋亡率呈显著负相关(r=–0.694,p<0.01);各组大鼠肺组织中Fas mRNA的表达与EOS的凋亡间存在明显的正相关(r=0.776,p<0.01),Bcl-2mRNA的表达与EOS的凋亡间存在着明显的负相关(r=–0.804,p<0.01)。
结论:脾虚状态下哮喘大鼠血液中嗜酸细胞的浓度明显增高,且与BALF中以及肺组织中嗜酸细胞的水平相一致。脾虚加重气道炎症反应是通过影响炎症细胞凋亡调控因子的浓度与相关基因的表达来实现的。针刺足三里能促进哮喘大鼠BALF中低水平TGF-β浓度的增高,抑制GM-CSF浓度增高的作用;针刺足三里可减轻哮喘大鼠气道炎性细胞的浸润并促进哮喘大鼠EOS的凋亡;针刺足三里能使哮喘大鼠肺组织中低水平Fas mRNA的表达增加,并促使Bcl-2mRNA的表达减少;针刺足三里能减轻模型大鼠的支气管及肺组织的炎性浸润。
Objective: In this research acupuncture treatments were given to the rats with spleen deficency asthma, and the influences of acupuncture at Zusanli on the morphological eosinophil apoptosis and Fas mRNA, Bcl-2 the mRNA expression were observed. The aim of this experiment was to explore that acupuncture treatment can regulate the inflammatory cell apoptosis and acupuncture is an effective method of treating asthma. It should be proved that acupuncture can play an important role in multi-aspects and at multi-targets so as to confirm the validity for treatment of asthma. The effective mechanism was studied in order to instruct clinic practice and optimize the scheme of selecting points to open a new field for Traditional Chinese Medicine and acupuncuture clinic research regarding asthma.
Methods: The rat models with spleen-deficiency in Traditional Chinese Medicine (TCM) and bronchial asthma in West Medicine were set up. Under the guideline of the theory of TCM, after the rat models were established successfully, they were given acupuncture at Zusanli basing on the theory of reinforcing earth to strengthen metal. The concentration of TGF-βand GM-CSF in BALF were detected by enzyme linked immuoabsorbent assay (ELISA) and Apoptosis was detected by TUNEL.Fas mRNA and the Bcl-2 mRNA expressions in the tissue of lung were detected by hybridization in situ and The morphology of the lung tissue was observed with the light microscop.
Results: Compared with control group (C), in both the combined disease with syndrome group (A1) and the asthma group (B1), the count of EOS increased and EOS apoptosis rates decreased (P<0.01), compared with the asthma group (B1), in group A1, the the count of EOS increased (P<0.01). The pathological result showed: there was inflammatory lesion of the bronchial tube and the lung tissue in different degrees, in both the combined disease with syndrome group and the asthma group; the inflammatory lesion of the bronchial tube and the lung tissue was reduced in both acupuncture groups. Compared with control group (C), in both the combined disease with syndrome group (A1) and the asthma group (B1), the levels of GM-CSF remarkably increased (P<0.01) and the levels of TGF-βremarkably decreased in BALF (P <0.01). Compared with the asthma group (B1), in group A1, the levels of GM-CSF remarkably increased (P<0.05). Compared with both the combined disease with syndrome group (A1) and the asthma group(B1), the levels of GM-CSF remarkably increased (P<0.01) in both acupuncture groups (A2,B2). Compared with the asthma group (B1), the levels of TGF-βremarkably increased in BALF (P <0.05).Compared with control group, in both the combined disease with syndrome group and the asthma group, the expressions of Fas mRNA remarkably decreased and the expressions of Bcl-2 mRNA remarkably increased (P<0.01 or P<0.05), compared with the asthma group (B1), in group A1, the expressions of Fas mRNA remarkably decreased and the expressions of Bcl-2 mRNA remarkably increased (P<0.01); At the same time, compared with the corresponding group A1 and B1, in both acupuncture groups (A2,B2), Bcl-2mRNA expression was remarkably reduced (P<0.01), Fas mRNA expression obviously increased (P<0.01). A positively significant correlation was found between the EOS apoptosis and the concentration of TGF-βin BALF in each group rats (r= 0.538,p<0.01). The results indicated a very significant negative correlation of the EOS apoptosis with the concentration of GM-CSF in BALF(r=–0.694,p<0.01); A positively significant correlation was found between the Fas mRNA expression and EOS apoptosis in each group (r=0.776,p<0.01); and a very significant negative correlation was found between the Bcl-2mRNA expression and EOS apoptosis(r=–0.804,p<0.01).
Conclusion: Spleen deficiency can aggravate phlegmasia cell aggregation in peripheral blood of asthmatic rats, which accord with inflammatory reaction of airway on asthmatic organism. Inflammatory reaction of airway aggravated by spleen deficiency is through regulating factor density of inflammatory cell and related gene expression. Acupuncture at Zusanli could promote EOS apoptosis of rat models with asthma and asthma with spleen-deficency, and increase EOS superficial Fas mRNA expression of asthmatic rats with spleen-deficency, suppress EOS superficial Bcl-2 the mRNA expression and reduce concentration of TGF-β,GM-CSF in BALF. Acupuncture could reduce the inflammation of airway and lung tissue.
引文
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