二甲双胍对新冠病毒感染所致小鼠肺纤维化影响的研究

Effect of metformin on pulmonary fibrosis induced by COVID-19 infection in mice

目的探讨二甲双胍对新冠病毒感染所致小鼠肺纤维化的影响,为了解新冠病毒肺炎后遗症肺纤维化的发病机制以及干预措施积累实验数据,为新冠病毒肺炎的临床诊疗保健提供相关理论依据。方法取8-10周的CAG-hACE2-IRES-Luc转基因(hACE2)小鼠,给予50μL SARS-CoV-2病毒溶液滴鼻,连续监测4天后进行解剖取材,取肺组织经福尔马林固定后进行组织病理学检查,得到小鼠感染SARS-CoV-2动物模型。将感染SARS-CoV-2(hACE2)小鼠的动物模型随机分到实验组和对照组,实验组给予二甲双胍250mg/kg每天治疗,对照组给予5mL生理盐水口服,共治疗14天时间后进行取血获取血清、解剖取材获取肺泡灌洗液及肺组织,检测血清IL-6、IL-1β、TNF-α浓度;检测肺泡灌洗液TGF-β1、HYP浓度;肺组织经福尔马林固定后进行组织病理学HE染色并进行肺纤维化评分。结果(hACE2)小鼠吸入SARS-CoV-2病毒溶液4天后可感染新冠病毒形成间质性肺炎,后续形成肺纤维化。每天给药二甲双胍250mg/kg,持续14天治疗后能降低小鼠血清的IL-6[实验组(79.24±15.42)pg/mL,对照组(87.53±14.11)pg/mL];IL-1β[实验组:(54.42±11.23)pg/mL,对照组:(60.78±12.03)pg/mL];TNF-α[实验组:(334.71±25.54)pg/mL,对照组:(350.58±26.59)pg/mL]含量;降低肺泡灌洗液TGF-β1[实验组:(193.60±28.47)pg/mL,对照组:(209.08±24.57)pg/mL]、HYP[实验组:(896.83±50.11)ug/mL,对照组:(926.03±44.96)ug/mL]的浓度;降低肺纤维化的评分(实验组:1.89±1.08,对照组:2.64±1.18)。结论(hACE2)小鼠可感染SARS-CoV-2病毒患新冠肺炎并形成肺纤维化,二甲双胍能有效地减轻(hACE2)小鼠感染SARS-CoV-2的炎症反应并抑制肺纤维化的形成。

Objective To explore the effect of metformin on pulmonary fibrosis caused by COVID-19 infection in mice,accumulate experimental data for understanding the pathogenesis and intervention measures of pulmonary fibrosis in sequelae of COVID-19 pneumonia,and provide a relevant theoretical basis for clinical diagnosis,treatment and health care of COVID-19 pneumonia.Methods 8-10 weeks of CAG-HACE2-IRES-Luc transgenic mice(hACE2)were given 50μL SARS-CoV-2 virus solution nasal drip.After continuous monitoring for 4 days,anatomical samples were taken,and the lung tissues were fixed by formalin then histopathological examination was conducted to obtain an animal model of mice infected with SARS-CoV-2.The mouse animal model infected with SARS-CoV-2(hACE2)was randomly divided into the experimental group and the control group.The experimental group was given 250mg/kg metformin daily treatment,and the control group was given 5 mL oral normal saline.After a total of 14 days of treatment,blood samples were collected to obtain serum and anatomical samples were collected to obtain alveolar lavage fluid and lung tissue.Serum levels of IL-6,IL-1β,and TNF-αwere detected.The concentrations of TGF-β1 and HYP in alveolar lavage fluid were detected.After formalin fixation,the lung tissue was stained with histopathological HE,and the pulmonary fibrosis score was performed.Results hACE2 mice infected with SARS-CoV-2 virus for 4 days after inhalation of SARS-CoV-2 virus solution could develop interstitial pneumonia and subsequent pulmonary fibrosis.After 14 days of treatment,metformin 250 mg/kg per day could reduce the serum IL-6 of mice[experimental group(79.24±15.42)pg/mL,control group(87.53±14.11)pg/mL].IL-1β[experimental group:(54.42±11.23)pg/mL,control group:(60.78±12.03)pg/mL];TNF-αcontent[experimental group:(334.71±25.54)pg/mL,control group:(350.58±26.59)pg/mL];Decreased alveolar lavage fluid TGF-β1[experimental group:(193.60±28.47)pg/mL,control group:(209.08±24.57)pg/mL],HYP[experimental group:(896.83±50.11)ug/mL,control group:(926.03±44.96)ug/mL];Decreased the pulmonary fibrosis score(experimental group:1.89±1.08,control group:2.64±1.18).Conclusion hACE2 mice can be infected with the SARS-CoV-2 virus and develop COVID-19 with pulmonary fibrosis.Metformin can effectively reduce the inflammatory response of hACE2 mice infected with SARS-CoV-2 and inhibit the formation of pulmonary fibrosis.