基于“肺肠同治”的清肺化痰逐瘀汤治疗COPD的作用机制

Mechanism of Qingfei Huatan Zhuyu Decoction in Treatment of COPD Based on"Lung and Intestinal Co-treatment"

目的:观察清肺化痰逐瘀汤对慢性阻塞性肺病(COPD)大鼠肺肠功能的影响并进一步探讨其体现肺肠同治的深层次机制。方法:将60只Wistar大鼠随机分为6组,每组10只,组别为正常组、模型组、急支糖浆组(10 g·kg^(-1)·d^(-1))、清肺化痰逐瘀汤低、中、高剂量组(10、15、20 g·kg^(-1)·d^(-1))。以脂多糖气管滴注合并烟雾吸入法建立COPD大鼠模型,急支糖浆组与清肺化痰逐瘀汤组分别以相应剂量浓度药液灌胃给药,其余组采用生理盐水灌胃对照,末次给药后监测大鼠肺功能和血气指标。镜下观察大鼠肺、肠组织病理学变化;酶联免疫吸附测定法(ELISA)测定大鼠血清白细胞介素-6(IL-6)、肿瘤坏死因子-α(TNF-α)与结肠组织分泌型免疫球蛋白A(IgA)表达;测定大鼠血清二胺氧化酶(DAO)、D-乳酸、丙二醛(MDA)等生化指标变化;免疫组化检测大鼠结肠组织紧密连接蛋白(Occludin)表达,采用免疫荧光测定大鼠肺组织F4/80阳性肺泡巨噬细胞、α-肌动蛋白(α-SMA)、结肠闭锁小带蛋白^(-1)(ZO-1)表达;蛋白免疫印迹法(Western blot)检测大鼠肺组织磷酸化核转录因子-κB p65(p-NF-κB p65)、NF-κB p65和磷酸化p38丝裂原活化蛋白激酶(p-p38 MAPK)、p38 MAPK蛋白表达,以及结肠组织Occludin和ZO-1表达。结果:与正常组比较,模型组大鼠肺功能障碍,用力肺活量(FVC)、动脉血氧分压(PaO_(2))、动脉血氧饱和度(SaO_(2))、肺动态顺应性(Cdyn)降低(P<0.01),肺、肠病理学改变明显,血清IL-6、TNF-α、DAO、D-乳酸、MDA表达增高(P<0.05,P<0.01),肺组织p-NF-κB p65/NF-κB p65和p-p38 MAPK/p38 MAPK蛋白表达比值升高,肺组织F4/80阳性巨噬细胞表达增强,结肠组织IgA、Occludin、ZO-1表达减少(P<0.05,P<0.01);与模型组比较,急支糖浆和清肺化痰逐瘀汤各剂量组大鼠肺功能明显改善,FVC、PaO_(2)、SaO_(2)、Cdyn升高(P<0.05,P<0.01),肺、肠组织病理学改善明显,血清IL-6、TNF-α、DAO、D-乳酸、MDA表达降低(P<0.05,P<0.01),肺组织F4/80阳性巨噬细胞表达减少,肺组织p-NF-κB p65/NF-κB p65和p-p38 MAPK/p38 MAPK蛋白表达比值降低(P<0.01),结肠组织IgA、Occludin、ZO-1表达增高(P<0.01)。结论:清肺化痰逐瘀汤可以有效减轻COPD大鼠症状,其作用机制与抑制肺组织炎症反应,改善肠黏膜屏障功能有关。

Objective:To observe the effect of Qingfei Huatan Zhuyu decoction on the lung and intestinal function of rats with chronic obstructive pulmonary diseases(COPD)and explore the deep-seated mechanism of its embodiment of lung and intestinal co-treatment.Method:A total of 60 Wistar rats were randomly divided into six groups,with 10 rats in each group,and the groups were control group,model group,acute syrup group(10 g·kg^(-1)·d^(-1)),and low,medium,and high-dose groups(10,15,20 g·kg^(-1)·d^(-1))of Qingfei Huatan Zhuyu decoction.The COPD rat model was established by lipopolysaccharide tracheal drip combined with the smoke inhalation method,and the acute syrup group and the Qingfei Huatan Zhuyu decoction group were administered by gavage with corresponding dose concentrations respectively,while the rest groups were controlled by saline gavage,and the lung function and blood gas indexes of rats were monitored after the last administration.The histopathological changes in the lung and intestine were observed microscopically.The expression of serum interleukin-6(IL-6),tumor necrosis factor-α(TNF-α),and secretory immunoglobulin A(IgA)in colon tissue were measured by enzyme-linked immunosorbent assay(ELISA).The biochemical indexes such as serum diamine oxidase(DAO),D-lactic acid,and malondialdehyde(MDA)were measured.Immunohistochemistry was used to detect the expression of tight junction protein(Occludin)in rat colon tissue.The expression of F4/80 positive alveolar macrophages in rat lung tissue,and the expression ofα-actin(α-SMA)and colonic atresia small band protein-1(ZO-1)were determined by immunofluorescence.The protein expression of p-NF-κB p65,NF-κB p65,p-p38 MAPK,and p-p38 MAPK and the expression of Occludin and ZO-1 in colon tissue were detected in rat lung tissue by Western blot.Result:Compared with the normal group,the model group had pulmonary dysfunction,reduced forced vital capacity(FVC),arterial partial oxygen pressure(PaO_(2)),arterial oxygen saturation(SaO_(2)),and dynamic lung compliance(Cdyn)(P<0.01),and the pathological changes in the lung and intestine were obvious.The expressions of IL-6,TNF-α,DAO,D-lactic acid,and MDA in serum were increased(P<0.05,P<0.01),and the protein expression ratio of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in lung tissue was increased.The expression of F4/80 positive macrophages in lung tissue was enhanced.The expression of IgA,Occludin,and ZO-1 in colon tissue decreased(P<0.05,P<0.01).Compared with the model group,the pulmonary function of the rats in the acute syrup group and groups of Qingfei Huatan Zhuyu decoction was significantly improved,and the FVC,PaO_(2),SaO_(2),and Cdyn were increased(P<0.05,P<0.01).The pathological changes in the lung and intestine were significant.The expressions of IL-6,TNF-α,DAO,D-lactic acid,and MDA in serum were decreased(P<0.05,P<0.01),and the expressions of F4/80 positive macrophages in lung tissue were decreased(P<0.01).The protein expression ratio of p-NF-κB p65/NF-κB p65 and p-p38 MAPK/p38 MAPK in lung tissue decreased(P<0.01),and the expression of IgA,Occludin,and ZO-1 in colon tissue increased(P<0.01).Conclusion:Qingfei Huatan Zhuyu decoction can effectively reduce the symptoms of COPD rats,and its mechanism of action is related to inhibiting the inflammatory response of lung tissue and improving the barrier function of the intestinal mucosa.