The Effects And Mechanisms Of Kappa Opioid Receptor Agonist On Acute Lung Injury

Objective:Acute lung injury（ALI）is a common cause of death in critically ill patients in the world,which is an excessive inflammatory response syndrome and characterized by progressive dyspnea and intractable hypoxemia followed by severe infection or trauma.ALI patients have uncontrolled inflammatory response and increased vascular permeability,which can eventually lead to multiple organ failure or even death.In recent years,with the improvement of the treatment of ALI,anti-inflammatory drugs were used in patients.However,as the half-life of the medications and their analogues that promote the regression of inflammatory mediators is short and unstable.There is no effective drug or clinical strategy for the treatment of ALI nowadays.Therefore,exploring new drugs and therapeutic targets for ALI has become an important problem to be solved.ALI is commonly induced by Gram-positive bacteria and Gram-negative bacteria.S.aureus is the main cause of ALI,especially the antibiotic resistant strains,such as methicillin-resistant S.aureus（MRSA）,which leads to high incidence rate and mortality of ALI worldwide.In addition,lipopolysaccharide（LPS）,an effective pathogenic component of Gram-negative bacteria,can induce ALI and produce excessive inflammatory mediators.,thus leading to the imbalance of inflammatory and anti-inflammatory mechanisms.Therefore,extensive exploration of ALI caused by different pathogenesis and the development of novel anti-inflammatory targets and mechanisms in the treatment of ALI are the frontier hot spots focused by scholars at home and abroad.Opioids are widely used in perioperative and critically ill patients.However,little is known about their roles in pulmonary immune function and inflammatory response.Recent studies have found that Kappa opioid receptor（KOR）not only mediates analgesia and sedation,but also plays a vital role in immune regulation,which has been widely considered as a potential therapeutic target for inflammation related diseases.However,how KOR affects the occurrence and development of pulmonary immune function and inflammatory response is still unclear.The main objective of this study is to observe the protective effect of a novel highly selective non opioid KOR agonist Salvinorin A（SA）on ALI induced by Gram-positive bacteria and Gram-negative bacteria,and to elucidate the molecular mechanism of the protective effect.In this study,we first established an animal model of ALI induced by Gram-positive bacteria MRSA to observe the lung protective effect of KOR agonist SA and its effect on nuclear factor E2 related factor 2（Nrf2）pathway.Nrf2 gene knockout mice were used to clarify the mechanism of Nrf2 pathway mediated lung protection of SA on MRSA induced ALI..In vitro macrophage model,we used lipoteichoic acid（LTA）,the main pathogenic component of MRSA,to investigate the m RNA and protein levels of Nrf2 and the expression of its downstream genes HO-1,GCLM,Txn,SOD1 and SOD2.Furthermore,LPS,a pathogenic component of Gram-negative bacteria,was used to induce the activation of pulmonary macrophages.Different KOR agonists SA and U50488 and the KOR antagonist norbinaltorphimine（Nor-Bin）were used to clarify the protective effect of KOR agonist in ALI and to explore the molecular mechanism of lung protection,and provide the novel idea for the clinical use of SA in the treatment of ALI patients.Materials and Methods:Chapter I:The effect of Kappa opioid receptor agonist SA on Methicillin-Resistant Staphylococcus Aureus（MRSA）induced ALI in mice.The protective effect and molecular mechanism of KOR agonist SA on ALI were observed by observing the lung tissue morphology,lung injury score,protein concentration and cell count in alveolar lavage fluid,MPO immunohistochemical staining and activity measurement of lung tissue,m RNA expression of lung tissue inflammatory factors（TNF-α,IL-1β,IL-6,Nrf2,HO-1 and SOD1）,MDA content and SOD activity in peripheral blood.Chapter II:The role of Nrf2 in Kappa opioid receptor agonist SA alleviating ALI induced by methicillin-resistant Staphylococcus aureus in mice.In this study,Nrf2 knockout mice were constructed and the animal model of ALI was induced by MRSA intratracheal instillation.The m RNA levels of inflammatory factors in lung tissue,the content of MDA and the activity of SOD in peripheral blood were measured to explore whether SA can alleviate the oxidative stress induced by MRSA by activating Nrf2 pathway.Chapter III:Effects of Kappa opioid receptor agonist SA on Nrf2 pathway,apoptosis,inflammation and oxidative stress in LTA stimulating mice RAW264.7 cells.We used LTA stimulating RAW264.7 cells to construct ALI cell model.Flow cytometry was used to detect apoptosis.DHE probe was used to detect the expression of ROS.RT-q PCR was used to detect the m RNA expression of inflammatory factors Nrf2,HO-1,Keap-1,and Nrf2 downstream genes SOD1,SOD2,Gclm and Txn.Western blot was used to detect the protein expression of Nrf2 pathway.Chapter IV:The effect of kappa opioid receptor agonist on LPS induced inflammatory response of rat alveolar macrophage NR8383.Rat alveolar macrophages NR8383 were stimulated with LPS（100 ng/ml）to induce an inflammatory cell model and to investigate the effects of KOR agonists SA(10^-6～10^-15M)and U50488(10^-9～10^-11M)on inflammatory factors,such as nitrite,TNF-α,IL-1β,i NOS and COX-2.Norbinaltorphimine（Nor-Bin）,an antagonist of KOR,was used to further verify the lung protective effect of KOR agonist on the production of inflammatory cytokines by alveolar macrophages.Results:Chapter I:SA ameliorated MRSA-induced ALI in mice.SA markedly ameliorated the pathological changes of lung tissues.The protein concentration and total cell count in BALF was also significantly decreased in MRSA challenged mice after SA treatment.The results of MPO immumohistochemical staining and activity assay revealed that SA significantly decreased the concentration and activities of MPO in MRSA-induced ALI.Using RT-q PCR analysis,we also found that the m RNA levels of TNF-α,IL-1β,IL-6,Nrf2,HO-1 and SOD1 significantly reduced after SA treatment.SA reversed the increased MDA content and the decreased SOD activity after MRSA infection.Chapter II:SA did not inhibit the accumulation of neutrophils and the destruction of alveolar structure after intratracheal instillation of MRSA in Nrf2 KO mice.Meanwhile,the protein content and the total number of cells in BALF did not decrease significantly.In addition,SA could not decrease the activity of MPO and the expression of TNF-αand IL-1βm RNA in Nrf2 KO mice.Chapter III:SA significantly reduced the apoptosis and ROS content of mice RAW264.7cells stimulating by LTA.SA inhibited the m RNA level of TNF-α,IL-1βand IL-6induced by LTA in RAW264.7 cells.SA significantly increased the m RNA level of Nrf2 and HO-1,but had no significant effect on their protein content.In addition,SA upregulated the m RNA expression of Nrf2 downstream genes（SOD1,SOD2,Gclm and Txn）.This part of the study shows that SA can inhibit the inflammatory response,apoptosis and oxidative stress level in RAW264.7 cells induced by LTA by activating Nrf2 pathway.Chapter IV:The level of TNF-αin alveolar macrophages was significantly increased at 1h,2h and 6h after LPS（100ng/ml）stimulation.SA treatment reduced the increase of TNF-αand IL-1βlevels induced by LPS stimulation,thereby reducing the inflammatory response.In addition,SA treatment could reduce the LPS-induced increase of nitrite level and reduced the over-expression of i NOS and COX-2 within2h after LPS activation,which was partially blocked by Nor-Bin,a KOR antagonist.U50488,another nitrogenous KOR agonist,obtained the similar results like SA.Conclusion:1.KOR agonist SA attenuates MRSA-induced ALI through the Nrf2 signaling pathway.（1）KOR agonist SA significantly reduced ALI induced by MRSA in mice.（2）KOR agonist SA alleviated MRSA induced ALI by activating Nrf2signaling pathway in mice.（3）Nrf2 knockout abolished the protective role of SA on MRSA-induced ALI in mice.（4）KOR agonist SA activated the Nrf2 pathway and inhibited apoptosis,inflammation,and oxidative stress of RAW264.7 cells stimulated by LTA,the pathogenic component of Gram-positive bacteria.2.KOR agonist SA and U50488 has anti-inflammatory effect on pulmonary macrophages inflammation induced by LPS,the pathogenic component of Gram-negative bacteria.In conclusion,KOR agonists play a protective role in ALI and the mechanism is related to the activation of Nrf2 signaling pathway.