右归丸调节Leptin/JAK2/STAT3信号通路抑制肾阳虚COPD大鼠气道炎症

Youguiwan Reduces Airway Inflammation in COPD Rats with Syndrome of Kidney-Yang Deficiency by Inhibiting Leptin/JAK2/STAT3 Signaling Pathway

目的:观察右归丸对肾阳虚慢性阻塞性肺疾病(COPD)大鼠肺组织瘦素(Leptin)/酪氨酸激酶2(JAK2)/信号转导与转录激活因子3(STAT3)信号通路的影响。方法:采用第1、14天时气管滴注脂多糖,并连续烟熏6周,期间隔3 d肌注氢化可的松,复制40只肾阳虚COPD模型SD大鼠,随机分为模型组、右归丸高剂量组(11.7 g·kg^(-1))、右归丸中剂量组(5.85 g·kg^(-1))、右归丸低剂量组(2.93 g·kg^(-1))和氨茶碱组(0.054 g·kg^(-1)),另设8只空白组SD大鼠。造模完成后,给予各组大鼠相应药物灌胃,连续给药28 d,末次给药后,取材。肺功能分析仪评估大鼠肺功能,酶联免疫法检测肺泡灌洗液白细胞介素-17A(IL-17A)、白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)含量,苏木素-伊红染色观察肺组织病理变化,马松三色染色观察肺组织支气管周围蓝色胶原纤维沉积并计算肺部炎症积分,免疫荧光观察支气管Ⅰ型胶原蛋白(ColⅠ)和α-平滑肌肌动蛋白(α-SMA)蛋白含量,蛋白免疫印迹法检测肺组织Leptin、IL-17A、JAK2和STAT3蛋白含量,实时荧光定量聚合酶链式反应(Real-time PCR)检测肺组织Leptin、IL-17A、JAK2、STAT3 mRNA含量。结果:与空白组比较,模型组肺功能显著降低(P<0.01),肺泡灌洗液中IL-6、IL-17A、TNF-α含量显著升高(P<0.01),肺组织炎症评分、气道上皮下胶原纤维沉积和ColⅠ、α-SMA蛋白显著升高(P<0.01),肺组织Leptin、IL-17A、JAK2和STAT3蛋白及mRNA水平均显著升高(P<0.01),JAK2、STAT3蛋白磷酸化程度显著增强(P<0.01)。与模型组比较,右归丸高、中剂量组大鼠肺功能明显升高,肺泡灌洗液中IL-6、IL-17A、TNF-α含量明显降低,炎症评分降低,胶原纤维沉积减轻,ColⅠ、α-SMA蛋白降低,Leptin、JAK2、STAT3、IL-17A蛋白降低,JAK2、STAT3磷酸化程度减弱,Leptin、JAK2、STAT3、IL-17A mRNA表达明显降低(P<0.05,P<0.01);与氨茶碱组比较,右归丸高剂量组IL-17A、TNF-α水平明显降低,右归丸中、低剂量组IL-17A水平明显升高,右归丸低剂量组TNF-α水平明显升高,右归丸高、中剂量组肺组织支气管周围胶原纤维沉积明显降低,右归丸高、中剂量组组织支气管周围ColⅠ、α-SMA蛋白明显降低(P<0.05,P<0.01),右归丸高、中剂量组炎症评分降低,肺组织Leptin、JAK2、STAT3、IL-17A蛋白及mRNA均降低,但差异无统计学意义。结论:右归丸能抑制IL-17A改善COPD大鼠炎性反应及胶原沉积来阻止气道重塑,其机制可能与抑制Leptin/JAK2/STAT3信号通路相关。

Objective:To observe the effect of Youguiwan on the leptin/Janus kinase 2(JAK2)/signal transducer and activator of transcription 3(STAT3)signaling pathway in the lung tissue of the rat model of chronic obstructive pulmonary disease(COPD)due to kidney-Yang deficiency.Method:Forty rats were modeled for COPD with the syndrome of kidney-Yang deficiency by intratracheal instillation of lipopolysaccharide on day 1 and day 14 and continuous fumigation for 6 weeks,during which hydrocortisone was injected intramuscularly at an interval of 3 days.The modeled rats were randomized into model,high-(11.7 g·kg^(-1)),medium-(5.85 g·kg^(-1)),and low-dose(2.93 g·kg^(-1))Youguiwan,and aminophylline(0.054 g·kg^(-1))group.In addition,8 SD rats were set as the blank group.After the completion of modeling,the rats in each group were administrated with the corresponding drug by gavage for 28 consecutive days.After the last administration,samples were collected.A lung function analyzer was used to evaluate the lung function of rats.Enzyme-linked immunosorbent assay was employed to measure the levels of interleukin-17A(IL-17A),interleukin-6(IL-6),and tumor necrosis factor-α(TNF-α)in the bronchoalveolar lavage fluid(BALF).Hematoxylin-eosin staining was employed to observe the pathological changes in the lung tissue,and Masson staining was employed to observe the deposition of blue collagen fibers around bronchi in the lung tissue and calculate the inflammation score.The immunofluorescence assay was employed to measure the protein content of collagen typeⅠ(ColⅠ)andα-smooth muscle actin(α-SMA)in the bronchi.The protein and mRNA levels of leptin,IL-17A,JAK2,and STAT3 in the lung tissue were determined by Western blot and real-time fluorescence quantitative polymerase chain reaction,respectively.Result:Compared with the blank group,the model group showed decreased lung function(P<0.01),elevated levels of IL-6,IL-17A,and TNF-αin the BALF(P<0.01),and increased lung inflammation score,deposition of subcutaneous collagen fibers in the airway,and ColⅠandα-SMA proteins(P<0.01).Furthermore,the modeling up-regulated the proteins and mRNA levels of leptin,IL-17A,JAK2,and STAT3 in the lung tissue(P<0.01)and enhanced the phosphorylation of JAK2 and STAT3(P<0.01).Compared with the model group,high-and medium-dose Youguiwan improved the lung function,decreased the inflammation score,reduced collagen fiber deposition and ColⅠandα-SMA proteins,lowered the levels of IL-6,IL-17A,and TNF-αin the BALF,down-regulated the mRNA and protein levels of leptin,JAK2,STAT3,and IL-17A,and weakened the phosphorylation of JAK2 and STAT3(P<0.05,P<0.01).The aminophylline group had higher IL-17A and TNF-αlevels than the high-dose Youguiwan group,lower IL-17A level than the medium and low-dose Youguiwan groups,and lower TNF-αlevel than the low-dose Youguiwan group.Compared with the aminophylline group,the high-and medium-dose Youguiwan groups showed reduced deposition of collagen fibers and protein levels of ColⅠandα-SMA around the bronchi in the lung tissue(P<0.05,P<0.01),decreased inflammation score,and down-regulated protein and mRNA levels of leptin,JAK2,STAT3,and IL-17A in the lung tissue.Conclusion:Youguiwan can prevent airway remodeling by inhibiting IL-17A to reduce inflammation and collagen deposition in COPD rats,which may be related to the inhibition of the leptin/JAK2/STAT3 signaling pathway.