基于PI3K信号通路探讨肺“俞募配穴”艾炷灸对哮喘小鼠RORγt/Foxp3表达的影响

Intervention mechanism of moxa-cone moxibustion at Shu- and Mu-acupoints on the expression of RORγt/Foxp3 in asthma mice: an analysis based on PI3K signaling pathway

目的:观察肺"俞募配穴"艾炷灸对哮喘小鼠血清及肺泡灌洗液(BALF)中相关炎性因子含量及对肺组织中磷脂酰肌醇-3-激酶(PI3K)、维甲酸相关孤儿受体γt(RORγt)、叉头/翼状螺旋转录因子3(Foxp3)表达的影响,并探讨其机制。方法:将60只雄性Balb/c小鼠随机分为正常组、模型组、LY剂组、电针组、艾炷灸组,每组12只。采用卵蛋白(OVA)致敏法复制哮喘小鼠模型。LY剂组予尾静脉注射PI3K抑制剂LY294002;电针组、艾炷灸组分别予电针、艾炷灸"肺俞""中府",1次/d,连续2周。ELISA法检测小鼠血清和BALF中免疫球蛋白E(IgE)、白细胞介素-4(IL-4)、干扰素-γ(IFN-γ)含量,荧光定量PCR法及免疫组织化学法检测肺组织中PI3K、RORγt、Foxp3的mRNA及蛋白表达。结果:与正常组比较,模型组小鼠血清和BALF中IgE及IL-4含量、肺组织中PI3K及RORγt mRNA和蛋白表达明显升高(P<0.01),血清和BALF中IFN-γ含量、肺组织中Foxp3 mRNA和蛋白表达明显降低(P<0.01);与模型组比较,LY剂组、电针组及艾炷灸组血清和BALF中IgE及IL-4含量、肺组织中PI3K及RORγt mRNA和蛋白表达明显降低(P<0.01,P<0.05),血清和BALF中IFN-γ含量、肺组织中Foxp3 mRNA和蛋白表达明显升高(P<0.01)。结论:肺"俞募配穴"艾炷灸能通过PI3K信号通路调节RORγt和Foxp3表达,从而减轻气道炎性反应,控制哮喘发作。

Objective To explore the effect of moxa-cone moxibustion at "Feishu"(BL13) and "Zhongfu"(LU1) on the contents of related inflammatory factors in serum and bronchoalveolar lavage fluid(BALF), and the expression of phosphatidy-linositol-3-kinase(PI3 K), retinoic acid related orphan receptor γt(RORγt) and fork/wing helix transcription factor 3(Foxp3) in lung tissue in asthma mice. Methods Sixty male Balb/c mice were divided into normal, model, LY294002(an inhibitor of PI3 K, LY), electroacupuncture(EA) and moxa-cone moxibustion(moxibustion) groups(n=12 in each group). The asthma model was established by intraperitoneal injection of ovalbumin sensitization. The mice in the LY group were injected with LY 294002(7.5 mg/kg) via the tail vein. EA or moxa-cone moxibustion was applied at BL13 and LU1 in the EA or moxibustion group once daily for 2 weeks. The levels of immunoglobulin E(IgE), interleukin-4(IL-4) and interferon-γ(IFN-γ) in serum and BALF were detected by enzyme-linked immunosorbent assay. The expressions of PI3 K, RORγt and Foxp3 in lung tissue were detected by real-time fluorescent quantitative polymerase chain reaction(QRT PCR) and immunohistochemistry. Results Compared with the normal group, the levels of IgE and IL-4 in serum and BALF, the expressions of PI3 K and RORγt mRNA and protein in lung tissue were significantly increased(P<0.01), while the levels of IFN-γ, and the expressions of Foxp3 mRNA and protein were significantly reduced(P<0.01) in the model group. Compared with the model group, the levels of IgE and IL-4 in serum and BALF, the expressions of PI3 K and RORγt mRNA and protein in lung tissue were significantly decreased(P<0.01, P<0.05), and the levels of IFN-γ in serum and BALF, the expressions of Foxp3 mRNA and protein in lung tissue were significantly increased(P<0.01) in the LY, EA and moxibustion groups. Conclusion Moxa cone moxibustion at Shu-and Mu-acupoints of the lung meridian can reduce airway inflammatory reaction and control asthma attacks in asthma mice, which is closely related to its effects in regulating the expressions of RORγt and Foxp3 through PI3 K signaling pathway.