基于PI3K/AKT/mTOR信号通路激活自噬通路研究荜茇改善小鼠肺纤维化的机制  

Mechanisms of Piperlongum L.on improving pulmonary fibrosis in mice by activating the autophagy pathway based on the PI3K/AKT/mTOR signaling pathway

作　　者：菅若男 甄华[1] 郭晶晶 JIAN Ruonan;ZHEN Hua;GUO Jingjing(The First Hospital of Hohhot,Hohhot,Inner Mongolia 010030,China;Baotou Medical College,Baotou,Inner Mongolia 014040,China)

机构地区：[1]呼和浩特市第一医院药剂科,内蒙古呼和浩特010030 [2]内蒙古科技大学包头医学院药学院,内蒙古包头014040

出　　处：《临床肺科杂志》2024年第5期675-682,共8页Journal of Clinical Pulmonary Medicine

基　　金：包头市青年创新人才项目(No.202229);内蒙古高等教育厅基金项目(No.NJZY23091)。

摘　　要：目的 研究荜茇是否可通过调控磷脂酰肌醇3-激酶(PI3K)/蛋白激酶B(Akt)/哺乳动物雷帕霉素靶蛋白(mTOR)信号通路诱导自噬改善小鼠肺纤维化。方法 将50只小鼠随机分为5组即空白组、博来霉素模型组(BLM组)、低剂量组(LD组)、高剂量组(HD组)、雷帕霉素组(RAPA组)。空白组予等容积生理盐水,其余小鼠通过气管内滴注博来霉素(5mg/kg)诱导建立PF模型,造模2周后开始灌胃给药,LD和HD组给予荜茇(5g/kg、10 g/kg)灌胃,RAPA组予自噬诱导剂雷帕霉素(5 mg/kg),连续给药4周。使用Anires2005系统测量小鼠肺功能即用力肺活量(FVC)和动态顺应性(Cdyn),并检测羟脯氨酸(HYP)含量水平。苏木素-伊红(HE)和马松(Masson)染色观察小鼠肺组织纤维化,免疫组化法和RT-qPCR测定α-平滑肌肌动蛋白(α-SMA)和转化生长因子β1(TGF-β1)表达水平,蛋白免疫印迹法(Western Blot)检测巨噬效应蛋白(Beclin-1)、自噬相关蛋白1轻链3Ⅱ(LC3Ⅱ)蛋白表达水平,并检测通路相关蛋白p-PI3K、p-AKT、p-mTOR相对蛋白表达量。结果 与空白组比较,BLM组FVC和Cdyn水平显著降低,HYP含量升高(P<0.01),α-SMA、TGF-β1蛋白表达增加(P<0.01),自噬蛋白LC3Ⅱ蛋白表达增加(P<0.05),而Beclin-1蛋白含量表达无显著差异,通路蛋白p-PI3K、p-AKT、p-mTOR表达增加(P<0.01);肺组织重度异常,大量肺泡萎缩塌陷,上皮细胞数量增多,肺实质化,肺实质内可见炎症细胞浸润,及粉红色纤维组织增生。与模型组比较,荜茇组及RAPA组FVC和Cdyn水平增高,HYP含量下降(P<0.05)。α-SMA、TGF-β1蛋白表达减少(P<0.05),Beclin-1、LC3Ⅱ表达增加(P<0.05),p-PI3K、p-AKT、p-mTOR表达减少(P<0.05);肺泡结构较为清晰,少量区域上皮细胞增多,肺泡壁增厚,炎症细胞数量明显减少。结论 荜茇可减少胶原纤维沉积,缓解肺纤维化进程,其作用机制可能是通过抑制PI3K/AKT/mTOR信号通路激活细胞自噬,从而缓解肺纤维化。Objective To investigate whether Piperlongum L.can induce autophagy to improve pulmonary fibrosis(PF) in mice by regulating phosphatidylinositol 3-kinase(PI3K)/protein kinase B(AKT)/mammalian target of rapamycin(mTOR) signaling pathway.Methods Fifty mice were randomly divided into five groups:blank group,bleomycin model group(BLM group),low dose group(LD group),high dose group(HD group),and rapamycin group(RAPA group).PF model was induced by intratracheal instillation of bleomycin(5mg/kg).After 2weeks of modeling,the mice in LD and HD groups were given Piperlongum L.(5g/kg,10 g/kg) by gavage,and the RAPA group was given autophagy inducer rapamycin(5mg/kg).The drug was administered continuously for 4weeks.Forced vital capacity(FVC) and dynamic compliance(Cdyn) were measured by the Anires2005 system,and hydroxyproline(HYP) content was detected.The expression levels of α-smooth muscle actin(α-SMA) and transforming growth factor-β1(TGF-β1) were detected by immunohistochemistry and RT-qPCR.Western blot was used to detect the protein expression levels of macrophage effector protein(Beclin-1) and autophagy-related protein 1 light chain 3 Ⅱ(LC3 Ⅱ),and.the relative protein expression levels of pathway-related proteins p-PI3K,p-AKT,and.pmTOR were detected.Results Compared with the blank group,the levels of FVC and Cdyn in the BLM group were significantly decreased,the content of HYP was increased(P<0.01),the expressions of α-SMA and TGF-β1 proteins were increased(P <0.01),and the expressions of autophagy proteins Beclin-1 and LC3 Ⅱ were decreased(P<0.01).The expressions of p-PI3K,p-AKT,and p-mTOR were significantly increased(P <0.01).The lung tissue was severely abnormal,with a large number of alveolar atrophy and collapse,an increased number of epithelial cells,lung parenchyma,inflammatory cell infiltration,and pink fibrous tissue proliferation in the lung parenchyma.Compared with the model group,the Piperlongum L.and RAPA groups had significant increases in FVC and Cdyn levels and a significant reduction in H

关 键 词：荜茇 肺纤维化 自噬 

分 类 号：R285.5[医药卫生—中药学]