Role Of Nur77/NR4A1 In Regulating Lung Macrophage Polarization In Lung Tissue Injury In Mice With Lipopolysaccharide-induced Acute Respiratory Distress Syndrome

Objective: To investigate the role of Nur77/NR4A1 modulation of lung macrophage polarization in lipopolysaccharide(LPS)-induced lung injury in ARDS mice.Methods:(1)One hundred and forty-four SPF-grade C57BL/6 wild-type mice(male and female)were randomly grouped into: saline group(NS group),NS+LPS intervention group(LPS group),LPS+Csn B group(Csn B group),and LPS+Csn B control group(Csn B control group).Twelve mice in each group.(2)The mouse ARDS model was established by intratracheal intubation method with intra-airway drops of LPS,and the mice were executed at 4h,12 h and 24 h after modeling,and the lung tissues and BALF were collected for subsequent experiments.(3)Lung tissue damage assessment was performed using hematoxylin-eosin(HE)staining of lung tissue sections,wet-dry specific gravity(W/D)of lung tissue,total protein concentration of bronchoalveolar lavage fluid(BALF),and enzyme-linked immunosorbent assay ELISA).(4)The expression levels of M1/M2 type lung macrophages were measured by immunohistochemistry(IHC),immunofluorescence(IF)and western blot to observe the effects of Nur77/NR4A1 and exogenous Nur77/NR4A1 agonists on lung macrophage polarization and lung tissue injury in mice with ARDS caused by LPS.Results:(1)After 4h,12h and 24h of establishing the mouse ARDS model,compared with the control group,the lung injury in the LPS group and LPS+NS group was significantly aggravated and time-dependent,the lung tissue W/D and BALF total protein concentrations were significantly increased,and the lung tissue pro-inflammatory factor TNF-α was increased,and the degree of lung tissue injury was significantly reduced after Csn B intervention,and the lung tissue W/D and BALF total protein concentrations at the corresponding time points were reduced,and the pro-inflammatory factor TNF-α was decreased,The total protein concentration of BALF decreased and the pro-inflammatory factor TNF-α decreased,suggesting that the mouse ARDS model was successfully established and could be used for subsequent experiments.(2)After 4h,12h and 24h of ARDS model establishment,the expression of i NOS and F4/80 immunofluorescence co-localization increased in the LPS and LPS+NS groups compared with the control group,and the expression of i NOS and F4/80 immunofluorescence co-localization decreased significantly at the corresponding time points after Csn B intervention.After 4h of LPS titration,Arg-1 and F4/80 immunofluorescence co-localization expression increased slightly in the LPS,LPS+Csn B and LPS+NS groups compared with the control group,but there was no significant difference between the observation groups.The expression of Arg-1 and F4/80 immunofluorescence co-localization in the LPS group,LPS+Csn B group and LPS+NS group were significantly increased compared with the control group after 24 hours of LPS titration.(3)After establishing the mouse ARDS model for 4h,12h and 24h,the expression of IL-1β in mouse lung tissues was significantly increased compared with the control group in a time-dependent manner,and the expression of IL-1β in mouse lung tissues was significantly decreased at the corresponding time points after Csn B intervention.After 4h of LPS drip,there was no significant expression of Arg-1 in lung tissues of mice in all groups,and the peak expression of Arg-1 in lung tissues of mice after 12h of LPS drip,while the rest of the control,LPS and LPS+NS groups showed no significant expression;after 24h of LPS drip,the LPS,LPS+Csn B and LPS+NS groups showed high expression compared with the control group.Conclusion:Nur77/NR4A1 can exert protective effects against LPS-induced ARDS in mice through its mediated anti-inflammatory effects and can regulate the early polarization of macrophages into M2-type macrophages,reversing the inflammatory microenvironment and exerting anti-inflammatory repair effects.