小青龙汤及其方元对寒饮蕴肺证模型大鼠血清Ghrelin、Obestatin及骨骼肌COX4的调节作用

Regulatory Effects of Xiaoqinglong Decoction and Its Formula Elements on Serum Ghrelin,Obestatin,and Skeletal Muscle COX4 in Rats with Cold Fluid Accumulating Lung Syndrome Model

目的观察小青龙汤及其方元干预后寒饮蕴肺证大鼠血清生长激素释放肽(growth hormone releasing peptide,Ghrelin)、肥胖抑制素(Obestatin)及骨骼肌细胞色素C氧化酶调节亚基4(cytochrome c oxidase subunit 4,COX4)表达的变化。方法56只Wistar大鼠随机分为对照组、模型组、小青龙汤组、桂枝甘草组、芍药甘草组、姜辛味组、半夏麻黄组,每组8只。“形寒+饮冷+冷水浴”5周建立寒饮蕴肺证大鼠模型,小青龙汤及其方元药物干预2周。每周监测大鼠体质量;肺功能分析系统测定大鼠用力肺活量(forced vital capacity,FVC)、功能残气量(functional residual capacity,FRC)、平均呼气中期流量(mean mid expiratory flow,MMEF)、吸气时间(inspiratory time,tI)、呼气时间(expiratoty time,tE)等参数;HE染色观察大鼠肺组织病理形态学改变;ELISA检测血清Ghrelin、Obestatin含量;RT-qPCR和Western blot检测骨骼肌COX4表达。结果与对照组比较,模型组大鼠体质量明显减轻(P<0.01),肺功能FVC、FRC、MMEF下降(P<0.01),tI与tE延长(P<0.05),肺泡腔内组织水肿,炎性细胞浸润,支气管黏膜上皮粘连,血清中Ghrelin和Obestatin水平、Ghrelin/Obestatin比值下降(P<0.01),骨骼肌中COX4 mRNA及蛋白表达减少(P<0.01);与模型组比较,各给药组大鼠体质量上升(小青龙汤组>姜辛味组>桂枝甘草组>半夏麻黄组>芍药甘草组),肺功能好转,肺组织水肿状态改善,炎性细胞浸润减少,支气管黏膜粘连程度减轻,血清中Ghrelin/Obestatin上调(姜辛味组>小青龙汤组>桂枝甘草组>半夏麻黄组>芍药甘草组),骨骼肌中COX4 mRNA及蛋白表达增加。结论小青龙汤及其方元药物干预对寒饮蕴肺证大鼠具有保护作用,机制可能与上调血清Ghrelin/Obestatin比值及骨骼肌COX4含量有关,为小青龙汤通过补益肺脾之气发挥蠲饮作用提供了实验依据。

Objective To observe the changes of serum Ghrelin,Obestatin,and skeletal muscle COX4 expression in rats with the cold fluid accumulating lung syndrome under the intervention of Xiaoqinglong decoction and its formula elements.Methods 56 Wistar rats were randomly divided into control group,model group,Xiaoqinglong decoction group,Guizhi Gancao group,Shaoyao Gancao group,Jiangxinwei group,and Banxia Mahuang group,with 8 rats in each group.The rat model of cold fluid accumulating lung syndrome was established by the"coldness of body+drinking cold+cold bath"method for 5 weeks,and Xiaoqinglong decoction and its formula elements were administrated to treat the model rats for 2 weeks.The body weight of rats was monitored weekly.The parameters of FVC,FRC,MMEF,tI,and tE were measured by the lung function analysis system.The pathomorphological changes in lung tissue were observed by HE staining.Serum Ghrelin and Obestatin levels were detected by ELISA.The expression of COX4 in skeletal muscle was detected by RT-qPCR and Western blot.Results Compared with the control group,the weight of the model rats was significantly reduced(P<0.01),and the lung function parameter FVC,FRC,and MMEF were decreased(P<0.01)while the time of tI and tE prolonged(P<0.05).The alveolar cavity of model rat was filled with edema fluid,and inflammatory cell infiltration,with bronchial mucosal epithelial adhesion.The expression of Ghrelin,Obestatin,and Ghrelin/Obestatin were decreased(P<0.01),and the expression of COX4 mRNA and protein in skeletal muscle decreased(P<0.01)in the model rats.Compared with the model group,the body weight of rats in each treatment group increased(Xiaoqinglong decoction group>Jiangxinwei group>Guizhi Gancao group>Banxia Mahuang group>Shaoyao Gancao group),and lung function improved,lung tissue edema status improved,inflammatory cell infiltration decreased,bronchial mucosal adhesion degree reduced,serum Ghrelin/Obestatin upregulated(Jiangxinwei group>Xiaoqinglong decoction group>Guizhi Gancao group>Banxia Mahuang group>Shaoyao Gancao group),and COX4 mRNA and protein expression increased in skeletal muscle.Conclusion The intervention of Xiaoqinglong decoction and its formula elements has a protective effect on rats with cold fluid accumulating lung syndrome.The mechanism may be related to the up-regulation of serum Ghrelin/Obestatin ratio and skeletal muscle COX4 content,and may exert its relieving effect by nourishing lung and spleen Qi.