参苓白术散对肺脾两虚型COPD大鼠十二指肠Ghrelin、Obestatin及其受体的影响

Effects of Shen Ling Baizhu Prescription on duodenal Ghrelin,Obestatin and Their Receptors in Rats with Lung and Spleen Deficiency Type COPD

目的观察参苓白术散对肺脾两虚型慢性阻塞性肺疾病(Chronic Obstructive Pulmonary Disease,COPD)模型大鼠十二指肠中生长激素释放肽(Ghrelin)、肥胖抑制素(Obestatin)及其受体(GHSR、GPR39)基因及蛋白表达的影响,基于Ghrelin-Obestatin信号通路探讨参苓白术散防治COPD的作用机制。方法将60只SPF级Wistar大鼠随机分为空白对照组、肺脾两虚观察组、药物干预组(根据药物干预时间不同,分为28 d、35 d、42 d组),每组12只。观察组及药物干预组采用烟熏+气管内滴注脂多糖+灌服冰冷番泻叶浸液建立肺脾两虚型COPD大鼠模型;药物干预组给予参苓白术散灌胃,分别于药物干预第28天、35天、42天测定各组大鼠肺功能并处死大鼠,采用RT-PCR法及免疫组化法测定大鼠十二指肠组织中Ghrelin,Obestatin及其受体GHSR、Obestatin m RNA及蛋白表达变化。结果与空白对照组相比,肺脾两虚观察组大鼠肺功能吸气气流高峰流速(PIF)、呼气气流高峰流速(PEF)频率(F)明显增高(P<0.05)、呼出50%气量时流速(EF50)、肺泡通气量(AV)明显下降(P<0.05);与肺脾两虚观察组相比,参苓白术散28 d组PIF、PEF、EF50、AV、F降低(P<0.05);参苓白术散35 d、42 d组PIF、PEF、EF50、F降低(P<0.05),AV升高(P<0.05)。与空白对照组相比,肺脾两虚观察组大鼠十二指肠组织中Ghrelin、GHSR、GPR39 m RNA表达减少(P<0.05),与肺脾两虚观察组相比,各药物干预组Ghrelin、GHSR、GPR39 m RNA表达水平升高(P<0.05)。与空白对照组相比,肺脾两虚观察组大鼠十二指肠组织中Ghrelin、GHSR、Obestatin、GPR39平均光密度明显降低(P<0.05),与肺脾两虚观察组相比,参苓白术散28 d组大鼠十二指肠中Ghrelin、GHSR、GPR39的变化无统计学意义(P>0.05);参苓白术散35 d组Ghrelin、GHSR、Obestatin的变化无统计学意义(P>0.05),参苓白术散42 d组Ghrelin、GHSR、Obestatin、GPR39平均光密度有所升高,差异有统计学意义(P<0.05)。结论参苓白术散可改善肺脾两虚型COPD大鼠一般状况及肺功能,其机制可能是通过改善十二指肠病理改变,调节肺脾两虚型COPD大鼠模型十二指肠Ghrelin、Obestatin及其受体m RNA及蛋白表达而实现的。

Objective The effect of Shen Ling Baizhu prescription on the genes related to growth hormone releasing peptide（ghrelin）,obestatin and its receptor（GHSR,GPR39）in the duodenum of the rat model of chronic obstructive pulmonary disease（COPD）with lung and spleen deficiency was observed;the expression levels of ghrelin and obestatin and their receptors in the duodenum were detected.The mechanism of the method of reinforcing earth to generate metal by Ghrelin-Obestatin signal transduction pathways was analyzed.Methods Sixty SPF Wistar rats were randomly divided into blank control group,lung and spleen deficiency observation group and drug intervention groups（28 d,35 d and 42 d groups according to different drug intervention time）,12 rats in each group.Rats in the observation group and drug intervention group were smoked＋intratracheal instillation of lipopolysaccharide＋perfusion of cold senna extract to establish lung and spleen deficiency COPD rat models;rats in drug intervention groups were given Shen Ling Baizhu prescription.The rats were sacrificed on the 28th day,35th day and 42nd day.Rats’lung function was determined.RT-PCR and immunohistochemistry were used to determine the mRNA and protein expression levels of ghrelin,obestatin and GHSR and GPR39 in rats’duodenal tissue.Results Compared with the blank control group,lung-spleen deficiency group rats’PIF,PEF and F lung function were significantly higher（P〈0.05）;EF50,AV were significantly decreased（P〈0.05）.Compared withthe lung-spleen deficiency group,PIF,PEF,EF50,AV and F were decreased in the 28 d group（P〈0.05）.PIF,PEF,EF50 and F were decreased（P〈0.05）and AV was increased（P〈0.05）in the groups of 35 d and 42 d.Compared with the blank control group,the mRNA expression levels of ghrelin,GHSR and GPR39 in the duodenum of the lung-spleen deficiency group were decreased（P0.05）.Compared with the lung-spleen deficiency group,the mRNA levels of ghrelin,GHSR and GPR39 in all intervention groups were significantly higher than those in the observation group（P〈0.05）;and the mRNA contents of ghrelin,GHSR and GPR39 increased significantly with the intervention time increasing（P〈0.05）.Compared with the blank control group,the average optical density of ghrelin,GHSR,obestatin and GPR39 in the duodenum of lung-spleen deficiency group was significantly decreased（P〈0.05）.Compared with the lung-spleen deficiency group,there was no statistical significance（P〈0.05）of ghrelin,GHSR and GPR39 in the duodenum of rats in the Shen Ling Baizhu prescription 28 d group.The mean optical density of ghrelin,GHSR,obestatin in 35 d groups of Shen Ling Baizhu prescription increased with no statistical significance（P〈0.05）.Conclusion Shen Ling Baizhu prescription can regulate the mRNA and protein expression of Ghrelin,Obetatin and their receptors in duodenal of COPD model with lung and spleen deficiency,improve pathological changes of duodenum in rats,and improve malnutrition in COPD rats with deficiency of lung and spleen.The condition may be achieved through the regulation of Ghrelin-Obestatin signaling pathway to improve body energy metabolism.