新冠康复1号方治疗小鼠肺纤维化的抗氧化保护机制

Mechanism of Xinguan Kangfu No.1 Prescription(新冠康复1号方)in Treatment of Pulmonary Fibrosis in Mice

目的探讨新冠康复1号方对小鼠肺纤维化(pulmonary fibrosis,PF)的干预作用及其作用机制。方法72只SPF级雄性C57BL/6J小鼠,随机分为6组:对照组(Con组)、博来霉素模型组(BLM组)、新冠康复1号方低剂量组(XL组)、新冠康复1号方中剂量组(XM组)、新冠康复1号方高剂量组(XH组)、醋酸泼尼松组(P组),每组12只。采用气管注射5 mg/kg博来霉素(bleomycin,BLM)诱导小鼠肺纤维化模型。收集各组小鼠血清和肺组织,采用HE染色和Masson染色观察肺组织病理学改变;检测活性氧族(reactive oxygen species,ROS),采用试剂盒测定血清氧化应激指标[丙二醛(malondialdehyde,MDA)、超氧化物歧化酶(superoxide dismutase,SOD)、谷胱甘肽过氧化物酶(glutathione peroxidase,GSH-Px)];用细胞凋亡染色(Tunel染色)观察肺组织细胞凋亡情况,用免疫荧光染色(SP-C染色)染色观察氧化应激对肺泡上皮细胞损伤情况,使用电镜进一步观察小鼠肺泡上皮细胞及线粒体的变化,采用Western blotting检测B淋巴细胞瘤-2基因(B-cell lymphoma-2,Bcl-2)、Bax基因、半胱氨酸蛋白酶-3(Caspase-3)蛋白活性。结果HE染色、Masson染色结果提示BLM诱导的小鼠肺纤维化模型构建成功,给予新冠康复1号方后肺纤维化表型明显改善;氧化应激结果显示:新冠康复1号方可抑制ROS和MDA生成,增强GSH-Px、SOD的活性;Tunel染色、SP-C染色和电镜学观察显示新冠康复1号方可减轻BLM对小鼠肺泡上皮细胞(alveolar epithelial cells,AECs)的损伤程度,保护线粒体的结构和功能;Western blotting检测结果显示:新冠康复1号方可下调凋亡相关蛋白Bax、Caspase-3的表达,上调Bcl-2蛋白表达。结论新冠康复1号方可通过纠正氧化/抗氧化失衡,改善肺泡上皮细胞损伤,保护线粒体结构从而改善肺纤维化,为开发肺纤维化的治疗药物提供一定的理论基础。

Objective To investigate the intervention effect and mechanism of Xinguan Kangfu No.1 Prescription(新冠康复1号方)on pulmonary fibrosis in mice.Methods Seventy-two SPF male C57BL/6J mice were randomly divided into 6 groups:Control group(Con group),bleomycin model group(BLM group),Xinguan Kangfu No.1 Prescription low-dose group(XL group),Xinguan Kangfu No.1 Prescription medium-dose group(XM group),Xinguan Kangfu No.1 Prescription high-dose group(XH group),prednisone acetate group(P group),with 12 mice in each group.The pulmonary fibrosis model was induced by intratracheal injection of 5 mg/kg bleomycin(BLM).The serum and lung tissues of mice in each group were collected,and the pathological changes of lung were observed by HE staining and Masson staining.Reactive oxygen species(ROS)was detected,and serum oxidative stress indexes[malondialdehyde(MDA),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px)]were determined by kit.The apoptosis of lung tissue cells was observed by Tunel staining,the damage of alveolar epithelial cells caused by oxidative stress was observed by SP-C staining,the changes of alveolar epithelial cells and mitochondria were further observed by electron microscopy,and the activities of B-cell lymphoma-2(Bcl-2),Bcl-2-associated X protein(Bax)and cysteine protease 3(Caspase-3)proteins were detected by Western blotting.Results The results of HE staining and Masson staining indicated that the BLM-induced pulmonary fibrosis model of mice was successfully constructed,and the pulmonary fibrosis phenotype was significantly improved after the administration of the Xinguan Kangfu No.1 Prescription.The results of oxidative stress showed that Xinguan Kangfu No.1 Prescription could inhibit the production of ROS and MDA,and enhance the activities of GSH-Px and SOD.Tunel staining,SP-C staining and electron microscopy showed that Xinguan Kangfu No.1 Prescription could reduce the damage degree of BLM on mouse alveolar epithelial cells and protect the structure and function of mitochondria.Western blotting test results showed that Xinguan Kangfu No.1 Prescription may up-regulate the expression of Bcl-2 protein by down-regulating the expressions of apoptosis-related proteins Caspase-3 and Bax.Conclusion By correcting oxidative/antioxidant imbalance,Xinguan Kangfu No.1 Prescription can improve the damage of alveolar epithelial cells,protect the mitochondrial structure and thus improve pulmonary fibrosis,providing a theoretical basis for the development of therapeutic drugs for pulmonary fibrosis.