益气养阴活血方对肺纤维化大鼠TGF-β1、Smad3、MMP9及TIMP1表达的影响

Effect of Yiqi Yangyin Huoxue Prescription on Expressions of TGF-β1,Smad3,MMP9 and TIMP1 in Rats with Pulmonary Fibrosis

目的观察益气养阴活血方对肺纤维化大鼠的影响,探讨其改善肺纤维化的作用机制。方法采用博来霉素气管滴注制备肺纤维化大鼠模型,将大鼠随机分为模型组、醋酸泼尼松组和益气养阴活血方高、中、低剂量组(中药高、中、低剂量组),每组8只,另取8只正常大鼠作为空白组,给药组予相应药物灌胃28 d,空白组和模型组予等体积生理盐水灌胃。末次给药后,行肺功能检测,HE和Masson染色观察肺组织病理改变,免疫组化染色检测肺组织纤连蛋白(FN)和层粘连蛋白(LN)表达,RT-PCR检测肺组织转化生长因子-β1(TGF-β1)、Smad3、基质金属蛋白酶9(MMP9)和基质金属蛋白酶抑制剂1(TIMP1)mRNA表达,Western blot检测肺组织TGF-β1、Smad3、p-Smad3、MMP9和TIMP1蛋白表达。结果与空白组比较,模型组大鼠吸气时间和呼气时间显著缩短,每分钟通气量和呼吸频率显著增加(P<0.01),肺组织结构损伤明显,肺泡塌陷、萎缩、间隔变宽,有大量胶原纤维沉积,肺组织FN和LN表达显著升高(P<0.01),TGF-β1、Smad3、MMP9 mRNA和TGF-β1、p-Smad3、MMP9蛋白表达显著升高,TIMP1 mRNA和蛋白表达显著降低(P<0.01);与模型组比较,中药高剂量组大鼠吸气时间和呼气时间显著延长,每分钟通气量和呼吸频率显著减少(P<0.01),肺泡形态较完整、间隔变窄,炎性细胞浸润减少,胶原纤维沉积减少,肺组织FN和LN表达显著降低(P<0.05,P<0.01),TGF-β1、Smad3、MMP9 mRNA和TGF-β1、p-Smad3、MMP9蛋白表达显著降低(P<0.01,P<0.05),TIMP1 mRNA和蛋白表达显著升高(P<0.05,P<0.01)。结论益气养阴活血方可能通过调控TGF-β1、Smad3、p-Smad3表达,调节MMP-9/TIMP-1平衡,降解细胞外基质,延缓肺纤维化发展。

Objective To investigate the effects of Yiqi Yangyin Huoxue Prescription on pulmonary fibrosis(PF)rats;To explore its mechanism on improving PF.Methods The rat model of PF was prepared by intratracheal instillation of bleomycin,the rats were randomly divided into model group,prednisone acetate group,and Yiqi Yangyin Huoxue Prescription high-,medium-and low-dosage groups(TCM high-,medium-and low-dosage groups),with 8 rats in each group.Another 8 normal rats were set as the blank group.The rats in medication groups were given corresponding medicine for gavage for 28 d,while the blank group and model group were given equal volume of normal saline by gavage.The lung function detection was performed;pathological changes of lung tissue were observed by HE and Masson staining;the expression of FN and LN in lung tissue were detected by immunohistochemical staining;the expression of TGF-β1,Smad3,MMP9 and TIMP1 mRNA in lung tissue were detected by RT-PCR;the protein expressions of TGF-β1,Smad3,p-Smad3,MMP9 and TIMP1 were detected by Western blot.Results Compared with the blank group,the inspiratory time and expiratory time of the model group were significantly shortened,and the ventilation volume per minute and respiratory rate significantly increased(P<0.01),the structure of lung tissue was significantly damaged,the alveoli collapsed,atrophied,the septa widened,and a large number of collagen fibers were deposited,the expressions of FN and LN in lung tissue significantly increased(P<0.01),the expression of TGF-β1,Smad3 and MMP9 mRNA,and TGF-β1,p-Smad3 and MMP9 protein increased significantly,while the expression of TIMP1 mRNA and protein decreased significantly(P<0.01).Compared with the model group,the inspiratory time and expiratory time of rats in TCM high-dosage group were significantly prolonged,and the ventilation volume per minute and respiratory rate significantly decreased(P<0.01),the alveolar morphology was relatively complete,the interval was narrowed,the infiltration of inflammatory cells was reduced,and the deposition of collagen fibers was reduced,the expressions of FN and LN in lung tissue decreased significantly(P<0.05,P<0.01),the expressions of TGF-β1,Smad3,and MMP9 mRNA and TGF-β1,p-Smad3 and MMP9 protein decreased significantly(P<0.01,P<0.05),while the expression of TIMP1 mRNA and protein increased significantly(P<0.05,P<0.01).Conclusion Yiqi Yangyin Huoxue Prescription can delay the development of pulmonary fibrosis through regulating the expression levels of TGF-β1,Smad3 and p-Smad3,and regulate the balance of MMP-9/TIMP-1 to degrade extracellular matrix.