| Background:Clinically,pleurisy and lung injury can be caused by a variety of factors such as infection,tumor and immune diseases.Oxidative stress and inflammation are involved in the disease progression of pleurisy.Therefore,anti-inflammation and antioxidant can be used as effective treatment to alleviate pleurisy and lung injury.Carrageenan（Car）induces pleurisy and lung injury mainly by inducing oxidative stress and inflammatory response,which can be used to screen effective treatment drugs for inflammatory injury of respiratory system.Amentoflavone（AMF）is a natural ingredient with anti-inflammatory,antioxidant and anti-tumor activities,which extracted from a variety of plants.However,the role of AMF on Car-induced pleurisy and lung injury and its mechanism remain unclear.Purpose:In this study,we adopted the Car-induced pleurisy and lung injury model mice to investigate the protective effect and mechanism of AMF on acute pleurisy and lung injury.Method:The model of acute pleurisy and lung injury was induced by intrathoracic injection of Car（2%）in C57BL/6 WT（wild type）or Nrf2-/-（Nrf2-konckout）mice.After starved for 12 hours,the mice in the treatment group were given AMF by intragastric administration twice（at time intervals of 12 h）.The mice in the control group were treated normal saline by the same method.After the second intragastric administration for 1 hour,2%Car was injected into the right thoracic cavity of the mice in the model group and the treatment group.Simultaneously,the mice in control group were treated with normal saline in the same way.After 4 h,the mice were euthanized,then pleural effusion and lung tissue were collected.BCA kit was used to detect the protein content of pleural effusion,Giemsa staining was used to analyze the type and number of inflammatory cells in pleural effusion,ELISA was used to detect inflammatory factors in pleural effusion of mice,HE staining was used to analyze the pathological changes of lung tissue,the corresponding kits were used to detect oxidative stress levels in pleural effusion and lung tissue,and western blotting was used to analyze the expression of the proteins related to oxidative stress including NOX2,NOX4,Keap1,Nrf2,HO-1,NQO1,GCLC and GCLM,and the proteins related to inflammatory pathway proteins such as NF-κB,MAPK,STAT3 and i NOS and COX2.Results:1.AMF can significantly improve the increased pleural exudation,increased protein content of exudate,massive exudation of inflammatory cells and pathological injury of lung tissue induced by Car in mice.2.In the Car-induced mouse model of acute pleurisy,AMF significantly reduced the number of F4/80 positive cells in lung tissue,decreased the levels of IL-1βand TNF-αin pleural fluid,and increased the level of IL-10.3.In the Car-induced mouse model of acute pleurisy,AMF significantly reduced ROS production of leukocytes in the exudate,decreased MPO and MDA production as well as improved GSH and SOD depletion in lung tissue.4.In the Car-induced mouse acute pleurisy model,AMF can effectively inhibit the up-regulation of NOX2 and NOX4,promote the degradation of Keap1 and the entry of Nrf2 into the nucleus,and up-regulate the expressions of GCLC,GCLM,NQO1 and HO-1 downstream of Nrf2.5.AMF effectively blocked Car-induced phosphorylation of IκB and p65 subunits,which inhibited NF-κB activation,and consequently inhibited Car-induced i NOS and COX2 expression.6.AMF inhibited the phosphorylation of ERK,JNK and STAT3,and blocked the translocation into nucleus of p-STAT3 in lung tissues of Car-induced pleurisy mice.7.Compared with WT mice,AMF did not significantly improve the exudation of inflammatory cells in the pleural cavity of Car-induced Nrf2-/-mice,the pathological damage of lung tissue,and IL-1β,TNF-α,and IL-10 levels in the exudate.8.In the Car-induced Nrf2-/-mouse model of acute pleurisy,AMF did not effectively block NF-κB activation,inhibit i NOS and COX2 expression,nor significantly inhibit STAT3 phosphorylation and p-STAT3 nuclear translocation.9.In the Car-induced Nrf2-/-mouse pleurisy model,AMF did not significantly inhibit ROS production,the depletion of GSH and SOD,and the expression of NOX2and NOX4.Conclusion:1.AMF alleviated Car-induced oxidative stress and inflammatory response by inhibiting the activation of NF-κB,MAPK and STAT3 inflammatory pathways.2.The inhibitory effect of AMF on NF-κB and STAT3 was regulated by Nrf2.3.As an anti-inflammatory and antioxidant natural medicinal ingredient,AMF may have clinical potential to the treatment of acute pleurisy and lung injury. |
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