补阳还五汤治疗放射性肺炎模型小鼠疗效及机制研究

Study on Therapeutic Effect and Mecha⁃nism of Buyang Huanwu Decoction(补阳还五汤)in Treatment of Model Mice with Radiation Pneumonitis

目的:从磷脂酰肌醇3-激酶/蛋白激酶B/哺乳动物雷帕霉素靶蛋白(PI3K/Akt/mTOR)信号通路探讨补阳还五汤对放射性肺炎模型小鼠的影响,评价其疗效与潜在机制。方法:将60只C57BL/6J雌性小鼠随机分为空白组、模型组、强的松组及补阳还五汤低、中、高剂量组,每组10只。空白组不进行照射,其余5组使用X生物射线辐照仪以15 Gy剂量诱导建立放射性肺炎模型。造模成功后,强的松组(0.325g·kg^(-1)·d^(-1))与补阳还五汤低、中、高剂量组(18.6 g·kg^(-1)·d^(-1)、37.2 g·kg^(-1)·d^(-1)、74.4 g·kg^(-1)·d^(-1))分别根据体质量灌胃相应药物,空白组及模型组灌胃生理盐水(20 mL·kg^(-1)·d^(-1)),每日1次,连续给药28 d后取材。观察各组小鼠体质量和肺湿重指数。苏木精-伊红(HE)染色观察小鼠肺组织病理形态学变化,酶联免疫吸附测定(ELISA)法检测肺组织白细胞介素-17(IL-17)、羟脯氨酸(HYP)、Ⅰ型胶原蛋白(ColI)和血清转化生长因子-β1(TGF-β1)含量,蛋白质印迹(Westernblot)法检测肺组织PI3K、磷酸化磷脂酰肌醇3-激酶(p-PI3K)、Akt、磷酸化蛋白激酶B(p-Akt)、mTOR、磷酸化哺乳动物雷帕霉素靶蛋白(p-mTOR)蛋白表达水平。结果:与空白组相比,模型组小鼠体质量明显降低、肺湿重指数显著增加(P<0.01);与模型组相比,各给药组小鼠体质量均上升、肺湿重指数均降低(P<0.01)。模型组小鼠肺组织结构破坏严重,可见管腔大量炎性渗出,肺泡间隔增宽、充血;肺泡结构不完整,肺泡塌陷,肺泡破坏严重。补阳还五汤各剂量组肺组织结构相对完整,炎症细胞浸润程度较低,炎症程度与模型组相比有改善。与空白组相比,模型组小鼠肺组织中IL-17、HYP、ColI与血清TGF-β1含量均显著升高(P<0.01)。与模型组相比,补阳还五汤高、中剂量组小鼠肺组织中IL-17、HYP、ColI与血清TGF-β1含量均显著下降(P<0.05或P<0.01)。与空白组相比,模型组小鼠肺组织中p-Akt/Akt、p-PI3K/PI3K、p-mTOR/mTOR相对蛋白表达量均升高(P<0.01)。与模型组相比,补阳还五汤高、中剂量组小鼠肺组织中p-Akt/Akt、p-PI3K/PI3K、p-mTOR/mTOR相对蛋白表达量均降低(P<0.05或P<0.01)。结论:补阳还五汤对放射性肺炎模型小鼠具有较好疗效,可以改善肺组织结构、降低炎症程度等,其机制可能与抑制PI3K/Akt/mTOR通路有关。

Objective:To explore the effect of Buyang Huanwu Decoction(补阳还五汤)on model mice with radiation pneumonitis(RP),and to evaluate its efficacy and potential mechanism from phosphoinositide 3-kinase/protein kinase B/mammalian target of rapamycin(PI3K/Akt/mTOR)signaling pathway.Methods:A total of sixty C57BL/6J female mice were randomly divided into blank group,model group,prednisone group,low-dose,medium-dose and high-dose groups of Buyang Huanwu Decoction,with 10 mice in each group.The blank group was not irradiated,while the remaining 5 groups of mice were induced to establish the RP models using an X-ray biological radiation irradiator at a dose of 15 Gy.After modeling,the prednisone group(0.325 g·kg^(-1)·d^(-1))and the low-dose,medium-dose and high-dose groups of Buyang Huanwu Decoction(18.6 g·kg^(-1)·d^(-1),37.2 g·kg^(-1)·d^(-1),74.4 g·kg^(-1)·d^(-1))were given corresponding drugs by gavage according to body weight.The blank group and model group were given normal saline(20 mL·kg^(-1)·d^(-1))by gavage once a day for 28 consecutive days.The body weight and wet weigh index of lung of each group were observed.Hematoxylin-eosin(HE)staining was used to observe the pathomorphological changes of lung tissue.Enzymelinked immunosorbent assay(ELISA)was used to detect the levels of interleukin-17(IL-17),hydroxyproline(HYP),type I collagen(ColI)and transforming growth factor-β1(TGF-β1)in lung tissue.The protein expression differences of PI3K,phosphorylated(p)-PI3K,Akt,p-Akt,mTOR and p-mTOR in lung tissue were detected by Western blot.Results:Compared with the blank group,the body weight of the mice in the model group was significantly decreased,and the wet weigh index of lung was significantly increased(P<0.01).Compared with the model group,the body weight of the mice in each drug group was increased and the wet weigh index of lung was decreased(P<0.01).The lung tissue structure of the model group was severely damaged,with a large amount of inflammatory exudation in the lumen,widening of the alveolar septum,and congestion,the alveolar structure was incomplete,the alveoli collapsed,and the alveoli were severely destroyed.The lung tissue structure of each dose group of Buyang Huanwu Decoction was relatively complete,and the degree of inflammatory cell infiltration was lower,and the degree of inflammation was improved compared with the model group.Compared with the blank group,the levels of IL-17,HYP,ColI in lung tissue and TGF-β1 in serum of the model group were significantly increased(P<0.01).Compared with the model group,the levels of IL-17,HYP,ColI in lung tissue and TGF-β1 in serum of mice in the high-dose and medium-dose groups of Buyang Huanwu Decoction were significantly decreased(P<0.05 or P<0.01).Compared with the blank group,the expression levels of p-Akt/Akt,p-PI3K/PI3K and p-mTOR/mTOR in lung tissue of the model group were significantly increased(P<0.05 or P<0.01).Compared with the model group,the expression levels of p-Akt/Akt,p-PI3K/PI3K and p-mTOR/mTOR in lung tissue of the high-dose and medium-dose groups of Buyang Huanwu Decoction were significantly decreased(P<0.05or P<0.01).Conclusions:Buyang Huanwu Decoction had a good therapeutic effect on RP in model mice,which can improve lung tissue structure and reduce inflammation degree,and its mechanism may be related to the inhibition of PI3K/Akt/mTOR pathway.