射干麻黄汤及其辛、苦味药组拆方对寒哮大鼠气道炎症及肺组织TRPV1/TAS2R14表达的影响

Effect of Shegan Mahuangtang and Its Pungent and Bitter Chinese Herbs on Airway Inflammation and Expression of TRPV1/TAS2R14 in Lung Tissue of Rat Model of Cold Asthma

摘要:

目的 探讨射干麻黄汤辛、苦味药组对哮喘寒哮证大鼠肺组织中瞬时受体电位通道香草醛亚型1（TRPV1）、苦味受体14（TAS2R14）蛋白表达的影响。方法 70只SD大鼠随机分为正常组、模型组、射干麻黄汤组、辛味药组、苦味药组、地塞米松组、桂龙咳喘宁组，10%卵清蛋白（OVA）结合氢氧化铝腹腔及四肢皮下注射致敏，联合2% OVA雾化及寒冷（2~4 ℃）刺激，激发大鼠寒哮模型。射干麻黄汤、辛味药、苦味药（6.43 g·kg^-1）、地塞米松（0.5 g·kg^-1）、桂龙咳喘宁组（10 g·kg^-1）灌胃及雾化，空白组、模型组以等量生理盐水灌胃及雾化，持续干预3周。末次激发后，对大鼠肺组织进行苏木素-伊红（HE）、碘酸雪夫（PAS）、马松（Masson）染色观察气道炎症反应和细胞增殖情况。采用酶联免疫吸附测定法（ELISA）检测血清中白细胞介素-5（IL-5）、肿瘤坏死因子-α（TNF-α）、胸腺基质淋巴细胞生成素（TSLP）、转化生长因子-β₁（TGF-β₁）含量；免疫荧光法检测TRPV1、TAS2R14的表达；蛋白免疫印迹法（Western blot）检测大鼠肺组织TRPV1、TAS2R14、磷脂酶Cβ₂（PLCβ₂）、B细胞淋巴瘤-2（Bcl-2）、α-平滑肌肌动蛋白（α-SMA）表达。结果 与空白组比较，模型组大鼠饮水、饮食及体质量下降，气道炎性细胞浸润、杯状细胞增生、组织纤维化及胶原蛋白沉积情况增多，血清IL-5、TNF-α、TSLP和TGF-β₁含量升高（P<0.01），TRPV1、PLCβ₂、α-SMA的表达增加、TAS2R14、Bcl-2的表达降低（P<0.05，P<0.01）；与模型组比较，射干麻黄汤、辛味药及苦味药物组大鼠饮水、饮食及体质量改善，气道炎性细胞浸润、杯状细胞增生、组织纤维化改变及胶原蛋白沉积情况减少；血清中IL-5、TNF-α、TSLP和TGF-β₁含量降低（P<0.01），TRPV1、PLCβ₂、α-SMA的表达减少，TAS2R14、Bcl-2表达增加（P<0.05，P<0.01）。结论 射干麻黄汤及其辛、苦味药物组可以明显减轻OVA诱发的哮喘大鼠气道炎症，降低TRPV1、PLCβ₂、α-SMA蛋白表达，激活TAS2R14、Bcl-2蛋白表达，其中苦味药组较辛味药组佳，辛苦相合其疗效增强，达到配伍之效。提示射干麻黄汤及其辛、苦味药物组能够减轻OVA诱发的哮喘气道炎症可能与抑制辛味相关TRPV1蛋白及激活苦味相关TAS2R14蛋白相关。

Abstract:

Objective To investigate the effects of Shegan Mahuangtang and its pungent and bitter Chinese herbs on the expression of transient receptor potential vanilloid-1 （TRPV1） and bitter taste receptor 14 （TAS2R14） in the lung tissue of the rat model of cold asthma.Method Seventy SD rats were randomized into 7 groups： normal， model， Shegan Mahuangtang， pungent Chinese herbs， bitter Chinese herbs （6.43 g·kg^-1）， dexamethasone （0.5 g·kg^-1）， and Guilong Kechuanning （10 g·kg^-1）. The rat model of cold asthma was established by intraperitoneal injection and subcutaneous injection of 10% ovalbumin （OVA） and aluminium hydroxide in the limbs， combined with 2% OVA atomization and cold （2-4 ℃） stimulation. The rats were treated with corresponding drugs by gavage and atomization， and the normal and model groups were treated with the same amount of normal saline for 3 weeks. After the last excitation， airway inflammation and cell proliferation were observed by hematoxylin-eosin （HE）， periodic acid-Schiff （PAS）， and Masson staining of the lung tissue. The levels of interleukin-5 （IL-5）， tumor necrosis factor-α （TNF-α）， thymic stromal lymphopoietin （TSLP）， and transforming growth factor-β₁ （TGF-β₁） in the serum were measured by enzyme-linked immunosorbent assay （ELISA）. The expression of TRPV1 and TAS2R14 was detected by immunofluorescence. The expression of TRPV1， TAS2R14， phospholipase Cβ₂ （PLCβ₂）， B-cell lymphoma-2 （Bcl-2）， and α-smooth muscle actin （α-SMA） in the lung tissue was determined by Western blot.Result Compared with the normal group， the model group showed decreased water intake， food intake， and body weight， increased airway inflammatory cell infiltration， goblet cell proliferation， tissue fibrosis and collagen deposition， elevated levels of IL-5， TNF-α， TSLP， and TGF-β₁ in the serum （P<0.01）， upregulated expression of TRPV1， PLCβ₂， and α-SMA， and downregulated expression of TAS2R14 and Bcl-2 （P<0.05， P<0.01）. Compared with model group， Shecgan Mahuangtang， pungent Chinese herbs， and bitter Chinese herbs increased the water intake， food intake， and body weight， reduced the inflammatory cell infiltration and goblet cell proliferation， alleviated tissue fibrosis and collagen deposition， lowered the levels of IL-5， TNF-α， TSLP， and TGF-β₁ in the serum （P<0.01）， downregulated the expression of TRPV1， PLCβ₂， and α-SMA， and upregulated the expression of TAS2R14 and Bcl-2 （P<0.05， P<0.01）.Conclusion Shegan Mahuangtang and its pungent and bitter Chinese herbs can reduce OVA-induced airway inflammation， downregulate the expression of TRPV1， PLCβ₂， and α-SMA， and upregulate the expression of TAS2R14 and Bcl-2 in asthmatic rats. Moreover， bitter Chinese herbs outperformed pungent Chinese herbs， and the combination of them enhanced the therapeutic effect. It is suggested that Shegan Mahuangtang and its pungent and bitter Chinese herbs may ameliorate the OVA-induced airway inflammation by inhibiting TRPV1 and activating TAS2R14.