Role Of MiR-144/451 On Lipopolysaccharide-induced Acute Lung Injury And The Intervention Effect Of Huangkui Capsule

Research Background:Acute lung injury（ALI）and its most severe form,acute respiratory distress syndrome（ARDS）is a critical illness of a high mortality,which is characterized by pulmonary edema,inflammation and progressive hypoxemia.Mortality increases to over 40.0%when patients progress to severe ARDS.MicroRNAs（miRNAs）,a class of evolutionarily conserved small non-coding RNAs of approximately 19-22 nucleotides in length,function as posttranscriptional modulators of gene expression by either promoting mRNA degradation or blocking protein translation.Recent studies have revealed that miR-144/451 are important regulators of gene expression in immune cells and contribute to the pathogenesis of inflammatory lung disease.Here,we report that miR-144/451 knockout mice exaggerate the inflammation and oxidative stress in LPS-induced ALI mice and LPS-induced macrophage.In addition,we explore the effects of Huangkui capsule（HKC）on LPS-induced ALI in mice and the expression of miR-451 in the lung tissues.Objective:（1）To explore the role of miR-144/451 in LPS-induced ALI in mice（2）To explore the mechanism of miR-451 inhibiting LPS-induced inflammatory response and oxidative stress in macrophages（3）To investigate the effect of HKC on inhibiting LPS-induced ALI in mice and the expression of miR-451 in the lung tissuesMethod:（1）The role of miR-144/451 on LPS-induced ALI in miceMice were divided randomly into four groups:control groups（WT-Ctl,KO-Ctl）and LPS groups（WT-LPS,KO-LPS）.Mice in the LPS groups were anaesthetised by i.p.pentobarbital sodium（50 mg/kg）,following intratracheal LPS（250 μg/kg）instillation.Mice in the control groups were administrated by equal volume of saline.At 24 h after LPS challenge,bronchoalveolar lavage fluid（BALF）were collected to determine the total cell number and the content of TNF-α and IL-6 by ELISA.The degree of inflammatory injury in the lung tissues was were observed by HE staining.The myeloperoxidase（MPO）,catalase（CAT）and glutathione peroxidase（GPx）activities in lung tissue homogenate were measured.The expression of miR-144 and miR-451 was measured in lung tissue of mice in WT-Ctl and WT-LPS group.（2）The mechanism of miR-451 inhibiting LPS-induced inflammatory response and oxidative stress in macrophagesThe miR-451 mimics or miR-NC was mixed with Lipo6000TM transfection reagent in Raw 264.7 cells.Rac-1 mRNA level was determined by qRT-PCR and the protein expression of Racl was determined by western blotting.After 24 h of stimulation by LPS,the supernatant was assayed for the concentrations of TNF-α and IL-6.Intracellular ROS assay was performed by DCFH-DA.Mouse primary peritoneal macrophages were collected from WT-Ctl or KO-Ctl mice 4 days after intraperitoneal injection of 1mL of 4%thioglycollate medium.The relative expression of miR-451 in LPS-stimulated peritoneal macrophages from WT-Ctl mice was determined.Rac-1 mRNA level and the protein expression of Racl of peritoneal macrophages from WT and miR-144/451 KO were determined.Primary alveolar macrophages from mice were isolated from mouse lungs in WT-Ctl and KO-Ctl groups by collecting BALF.Macrophages were incubated with 100 ng/mL LPS for 24 h with or without Racl inhibitor NSC23766.Levels of TNF-α and IL-6 in supernatants were measured by ELISA.Intracellular ROS assay was performed by DCFH-DA.（3）To investigate the effect of Huangkui Capsule on inhibiting lipopolysaccharideinduced acute lung injury in mice by up-regulating miR-451Mice were allowed to acclimate for one week and were divided randomly into six groups:control,LPS,LPS+HKC（150,300,and 600 mg/kg）,and LPS+DEX（5 mg/kg;antiinflammatory positive control）.HKC was administered intragastrically once per day for 5 consecutive days.DEX was administered intraperitoneally once before LPS instillation.Mice in the control and model groups received equal volume of sterile 0.9%saline.At 0.5 h after drug administration on day 5,mice in the model,DEX,and HKC groups were anaesthetised with intraperitoneal administration of pentobarbital sodium（50 mg/kg）and were subsequently administered intratracheal LPS（250 μg/kg）instillation.Mice in the control group were provided equal volume of saline instead of LPS.At 24 h after LPS challenge,plasma IL-6 level was determined.The BALF was collected to determine the total cell number and the content of TNF-α and IL-6 by ELISA.The degree of inflammatory injury in the lung tissues was were observed by HE staining.MPO,CAT and GPx activities in lung tissue homogenate were measured.The expression of miR-451 in lung tissue was measured.Raw 264.7 cells and mouse primary peritoneal macrophages were incubated with HKC（25,50,100,and 200 μg/mL）and LPS（100 ng/mL）.After 24 h of stimulation by LPS,the concentrations of NO,TNF-α and IL-6 were measured using ELISA.Intracellular ROS assay was performed using DCFH-DA.TNF-α and IL-6 mRNA levels in Raw 264.7 cells were measured by qRT-PCR.Results:（1）miR-144/451 inhibits LPS-induced ALI in miceLoss-of-function experiments performed on miR-144/451 knockout mice showed that miR-144/451 gene inactivation exaggerated LPS-induced lung inflammation and oxidant stress compared with wild-type mice,as manifested by increased the total BALF cell and neutrophil counts,elevated levels of TNF-α and IL-6 in BALF,enhanced MPO,reduced CAT and GPx activities in lung tissues.We demonstrated that LPS leads to a significant decrease of miR-451 in the lung tissues.（2）The mechanism of miR-144/451 inhibiting lipopolysaccharide-induced macrophage inflammatory response and oxidative stressLPS-treated macrophages demonstrated significantly lower level of miR-451 compared to untreated cells.miR-144/451 KO further enhanced upregulation of Rac-1 in macrophages both in protein levels.Conversely,miR-451 overexpression downregulated on mRNA and protein levels of Rac-1 in Raw 264.7 cells.MiR-451 overexpression in LPS-induced Raw 264.7 cells remarkably reduced TNF-α,IL-6,and ROS production.In ex vivo stimulation experiments performed in isolated alveolar macrophages from miR-144/451 KO mice confirmed that Racl inhibitor alleviated increased levels of TNF-α and ROS in response to LPS stimulation compared with wild-type controls.（3）Huangkui Capsule inhibits lipopolysaccharide-induced acute lung injury in miceTreatment with HKC dramatically reduced the total cell count in BALF,and inhibited myeloperoxidase activity in the lung tissues 24 h after LPS challenge.Histopathological analysis demonstrated that HKC attenuated LPS-induced tissue oedema and neutrophil infiltration in the lung tissues.Additionally,the concentrations of TNF-α and IL-6 in BALF and IL-6 in the plasma reduced after HKC administration.Moreover,HKC could enhance glutathione peroxidase and catalase activities and upregulate the expression of miR-451 in the lung tissues.In vitro experiments revealed that HKC inhibited the production of nitric oxide,TNF-α,and IL-6 in LPS-induced RAW 264.7 cells and mouse primary peritoneal macrophages.Additionally,HKC downregulated LPS-induced transcription of Tnf-a and Il6 in RAW 264.7 cells.Conclusion:（1）miR-144/451 knockout mice exaggerate LPS-induced ALI.（2）The mechanism underlying this could involve inhibiting macrophage-mediated inflammatory response and oxidative stress through downregulation of Racl expression.（3）HKC inhibited LPS-induced inflammation and oxidative stress in mice and macrophage,and upregulated lung miR-451 expression.