Regulatory Role And Mechanism Of NLRC5 In Controlling Inflammatory Cytokines Release In Alveolar Macrophages Through NF-κB And P38 MAPK Signal Pathway

Acute lung injury(acute lung injury,ALI)is a kind of the acute inflammatory diseases leading by non-cardiac disease factors,can step in to severely development stage known as acute respiratory distress syndrome(ARDS).Its progress is rapid,and late period is multiple organ dysfunction syndrome.Acute lung injury is still a major concern disease because of its high mortality rate of about 40%.There are many researches on the pathogenesis of acute lung injury at home and abroad,but it has not fully understood its pathogenesis,especially in cell and molecular gene level.For the treatment of acute lung injury,there is still lack of effective and specific means in clinical,mainly by symptomatic support therapy.Therefore,to study the pathogenesis of acute lung injury furtherly and to seek new targets for gene therapy,can provide new ideas for the target drugs of acute lung injury treatment.The pathogenesis of acute lung injury is complex.It is difficult to explain comprehensively in a single cellular pathway.Nor can it be limited to the simple sense of the lung itself,but the manifestation of systemic inflammatory response syndrome in the lungs.The basis of the disease is inflammation.Inflammatory response plays a central role in the development of acute lung injury.It releases a large number of inflammatory cytokines through inflammatory cells activated by signal transduction pathway.The activation of alveolar macrophages which is one of the inflammatory cells is crucial in the development of acute lung injury.Alveolar macrophages(AM),as a phagocytic cell in the lung,are mainly distributed in the alveolar cavity.It accounts for 80% of alveolar resident cells.AM is the key regulatory cell that starts the pulmonary inflammatory response and mediates the cellular immune response.It plays a key role to maintain the balance of organism,increase the rapid response of the internal and external stimulation and participate in the repair of tissue damages.The active ingredient of endotoxin is bacterial lipopolysaccharide(LPS),which is the main component of the outer membrane of gram-negative bacteria.LPS attacks the lungs first and activates inflammatory cells such as macrophages,which are the strongest stimulants of inflammatory cytokines such as TNF-α、IL-1β and IL-6.Inflammatory cytokines include both proinflammatory and anti-inflammatory cytokines.It can maintain the stability of the body’s environment by down-regulating the production of pro-inflammatory cytokines or up-regulating the production of anti-inflammatory cytokines.In recent years,studies have shown that both nuclear transcription factor kappa B(nuclear transcription factor kappa B,NF-κB)and p38 MAPK which is an important member of mitogen-activated protein kinase(mitogen-activated protein kinase,MAPK)are cell signal transduction pathways.They can participate in the expression of various inflammatory factors,synthesize and release inflammatory cytokines such as TNF-α、IL-1、IL-6、IL-8.They also can regulate the development of acute lung injury and affect the prognosis.NOD like receptor(NOD like receptors,NLRs)family is a class of pathogenic molecular recognition receptors located in the cytoplasm.Most of the family members promote the release of pro-inflammatory cytokines and participate in anti-infective immune response mainly through NF-κB、MAPK pathway and IL-1β related to inflammatory small body pathways.However,NLRC5(NLR family,CARD domain containing 5),one of the NLR family members discovered recently,has the opposite anti-inflammatory effect and participates in negative regulation of natural immunity.Cui et al.reported that NLRC5 was expressed in various tissues of human and mouse.NLRC5 m RNA expressions of human and mouse were high in the lung,spleen and thymus.Our previous studies have found that the expressions of NLRC5 were significantly increased in both mice peritoneal macrophages of acute liver injury model and RAW264.7 cells induced by LPS,and NLRC5 negative regulated the secretion of cytokines such as IL-6、TNF-α in RAW264.7 cells.In the previous study,NLRC5 was also found to regulate the expression of IL-6 and IL-lβ in TNF-α induced LX-2 cells by regulating NF-κB signaling pathway.The above results indicated that NLRC5 was involved in the regulation of the secretion of inflammatory factors,but it played different roles in different diseases.At present,there is no research report on the role and its mechanism of NLRC5 in regulating the secretion of inflammatory cytokines of alveolar macrophages in the development of acute lung injury at home and abroad.Combine with in vitro study(in MH-S cells)and in vivo study(acute lung injury model in mice),we will investigate the effect of NLRC5 on the secretion of inflammatory cytokines in alveolar macrophages.We will further clarify the molecular mechanism of NLRC5 regulating the secretion of inflammatory cytokines in alveolar macrophages.This study will provide new ideas for the prevention and treatment of acute lung injury and new drug development.The main research contents are summarized as follows: 1.Expression of NLRC5 in lung tissues and primary alveolar macrophages of acute lung injury.In this study,the mouse ALI model was induced by LPS though intratracheal instillation,and the primary alveolar macrophage(AM)was obtained and purified by bronchoalveolar lavage fluid.Pathological HE staining was performed on the lung tissue sections of mice ALI model.The specific phenotype marker CD68 immunofluorescence staining was used to identify AM.The localization of NLRC5 was observed by immunofluorescence double standard staining.The results showed that the mouse ALI model was established and the primary alveolar macrophages were separated and cultured successfully.Compared with the control group,NLRC5 m RNA and protein expression were significantly increased in the lung tissues and alveolar macrophages of mice,and mainly was expressed in alveolar macrophages.2.The regulatory effect of NLRC5 on the secretion of inflammatory cytokines in MH-S cells.LPS was used to stimulate mouse alveolar macrophages(MH-S cells)with different concentrations(0,100,200,500,1000,2000 ng/ml)and at different times(0,2,4,6,8,10,12 h).The optimal stimulation concentration and time of LPS were selected to establish the MH-S cell model of acute lung injury in vitro.The results showed that NLRC5 m RNA was highest at 6h,and NLRC5 protein expression was highest at 8h in the LPS induced group with the final concentration of 200 ng/ml.p EGFP-C2-NLRC5/NLRC5 si RNA were transfected into MH-S cells by Lipofectamine TM 2000 respectively.According to q RT-PCR and Western blot results,it was suggested that we successfully transfected p EGFP-C2-NLRC5/NLRC5 si RNA into MH-S cells and NLRC5 were overexpressed and silenced at m RNA and protein levels.In order to study the regulation role of NLRC5 on the secretion of inflammatory cytokines in MH-S cells of acute lung injury,this experiment observed the effects of TNF-α、IL-1β and IL-6 expressions in LPS-induced MH-S cells through specific overexpression and silence NLRC5 method.When NLRC5 was overexpressed on LPS induced MH-S cells,q RT-PCR and ELISA results suggest that p EGFP-C2-NLRC5 can inhibit the expression of TNF-α、IL-1β and IL-6 by the secretion of MH-S cells.It is revealed that NLRC5 can negatively regulate the expression of TNF-α、IL-1β and IL-6 by the secretion of MH-S cells.When NLRC5 was silenced on LPS induced MH-S cells,q RT-PCR and ELISA results suggest that NLRC5 si RNA can promote the expression of TNF-α、IL-1β and IL-6 by the secretion of MH-S cells.It is revealed that NLRC5 can negatively regulate the expression of TNF-α、IL-1β and IL-6 by the secretion of MH-S cells once again.3.The regulation effect of NLRC5 on the signal transduction pathways of NF-κB and p38 MAPK.In order to confirm the role of NLRC5 and NF-κB signal transduction pathway in acute lung injury,the experiment observed the effects of the key protein expressions of NF-κB signaling pathway in LPS-induced MH-S cells through specific overexpression and silence NLRC5 method.The results showed that overexpression NLRC5 can inhibit the expression of pp65 and pp IκBα,while NLRC5 silencing can promote the expression of pp65 and pp IκBα.It is revealed that NLRC5 can negatively regulate NF-κB signaling pathway by affecting pp65 and pp IκBα.Secondly,in order to explore the role of NLRC5 and p38 MAPK signal transduction pathway furtherly in acute lung injury,the experiment observed the effects of the key protein expressions of p38 MAPK signaling pathway in LPS-induced MH-S cells through specific overexpression and silence NLRC5 method.Western blot results showed that overexpression NLRC5 can inhibit the expression of pp38,while NLRC5 silencing can promote the expression of pp38.It is revealed that NLRC5 can negatively regulate p38 MAPK signaling pathway.In conclusion,NLRC5 can negatively regulate the expression of TNF-α、IL-1β and IL-6 by the secretion of MH-S cells,and its mechanism may be related with the negative regulation of NF-κB and p38 MAPK signaling pathways.4.Effects of inhibitors on the expressions of NLRC5.In view of the negative regulatory effect of NLRC5 on NF-κB,we used NF-κB specific inhibitor PDTC to stimulate LPS-induced MH-S cells.The results of q RT-PCR and Western blot showed that PDTC can significantly reduce the expression of NLRC5.Furthermore,these results pointed out that the expression of NLRC5 was related with NF-κB signaling pathway,and NLRC5 itself was also controlled by NF-κB pathway.In order to furtherly observe the relationship between p38 MAPK signaling pathway and NLRC5,we then also used p38 MAPK specific inhibitor SB203580 to stimulate LPS-induced MH-S cells.The results of q RT-PCR and Western blot showed that SB203580 also can significantly reduce the expression of NLRC5.These results furtherly pointed out that the expression of NLRC5 was related with p38 MAPK signaling pathway.