The Regulatory Mechanisms Of HIF-1α Signaling Pathway In Sepsis Induced Acute Lung Injury

Sepsis is a common and complex disease that is caused by host’s unbalanced response to infection and is associated with acute organ dysfunction and high risk of death.It is common in patients with severe trauma,burns,and surgery.The continued development of sepsis can lead to septic shock and the failure of multiple organs such as the heart,lungs,liver,and kidneys until death.The incidence of sepsis is very high.The high mortality rate caused by sepsis is more serious than breast cancer,prostate cancer,AIDS and hepatitis.The mortality rate of sepsis is 20%,and the septic’s shock mortality rate is an astonishing45%.Sepsis and its related diseases are the leading cause of death worldwide.With 19million deaths each year,the pressure of sepsis is a load burden for public health and socioeconomics.Infection is the main cause of sepsis and can occur anywhere in the body,especially in the urinary system,digestive system,blood system or respiratory system.Among them,lung infection is the most likely to cause sepsis.As the body’s gas exchange center,the lung is the organ that most susceptible to sepsis and the first organ to be affected.Therefore,the biological changes of lung in the occurrence and development of sepsis have important significance of research.Lung injury caused by sepsis usually manifests as acute lung injury（ALI）.It is often caused by sepsis induced systemic inflammatory response.Continuous and uncontrolled pulmonary inflammatory response will cause alveolar damage,manifested as acute respiratory failure,progressive hypoxemia and diffuse inflammation of the lungs.Sepsis-induced ALI is often accompanied by Acute Respiratory Distress Syndrome（ARDS）,which increases the incidence and mortality of sepsis.In the early stages of sepsis,microbial invasion activates the host’s immune system,recruits monocytes and initiates inflammatory response.These monocyte macrophages prevent further invasion and reproduction of microorganisms by phagocytosing to kill pathogens.Excessive activation of the inflammatory response is a prominent feature of sepsis,and the activation and damage of monocyte macrophages is the kernel of this feature,which can be caused by bacteria and/or its main product lipopolysaccharide（LPS）.LPS is the main component of Gram-negative bacterial membrane,and it is the representative of the pathogen-associated molecular patterns（PAMPs）,which is currently the most studied.It is known that LPS activates Toll-like receptor-4（TLR4）on the surface of monocytes,then the recruitment and binding of ligand of TLR4 initiate the TLR4 signaling cascade,which ultimately leads to the activation of NF-κB signaling pathway in monocytes.Activation of NF-κB leads to nuclear transport and binding to its target genes,which directly lead to the up-regulation of a series of pro-inflammatory cytokines such as IL-1β,IL-6,IL-8,TNF-α,and other molecules such as COX-2,E-Selectin as well as MCP-1 and i NOS.These inflammatory factors can further activate the NF-κB signaling pathway,causing positive feedback cascade amplification effects,leading to various damages such as oxidative stress,autophagy,and apoptosis,and eventually leading to multiple organ dysfunction.It can be seen that monocytes play an important role in the immune response of sepsis.Inhibiting the activation of monocyte macrophages and their associated NF-κB signaling pathways as well as the transformation of monocyte macrophage phenotypes are the key to reducing the uncontrolled inflammatory response,and it provides an important therapeutic target for the further treatment of sepsis and its induced acute lung injury.Hypoxia-inducible factor 1α（HIF-1α） is a highly conserved transcription factor.It is currently known to exist in almost all cell types.HIF-1αis strictly regulated by O₂concentration,which is the main response molecules to low oxygen levels and regulate the expression of hundreds of genes simultaneously.In the hypoxic state,HIF-1αandβsubunits transfer to the nucleus,and then combine with cofactor P300 to initiate the transcription of downstream genes,which could regulate cell metabolism and immune response.Under normoxic conditions,the half-life of HIF-1αis only 5 minutes,because O₂ activates the proline hydroxylase PHD.HIF-1αis hydroxylated to bind to the E3ubiquitin ligase complex and is degraded by ubiquitination.It can be seen that the stability of HIF-1αis very important for its function.Previous studies have found that the HIF complex promotes the expression of genes such as erythropoietin（EPO）by combining with hypoxia response enhancer elements（HREs）.At the same time,some literatures indicate that HIF-1αmay mediate reprogramming of macrophages in sepsis,which shifts them from a pro-inflammatory phenotype to an immunosuppressive phenotype.However,research on the role of HIF-1αin sepsis and its induced lung injury is very limited.In this study,we take HIF-1αhomeostasis as the starting point to further explore the role and regulatory mechanism of HIF-1αin sepsis and its induced lung injury,and this study provides a new theory for revealing the pathophysiology of sepsis.Roxadustat（FG-4592）is an oral small molecule HIF-1α-PHD inhibitor,which can inhibit the degradation of HIF-1αby PHD in the normoxic state and improve the body’s ability to withstand hypoxia,which can improve the prognosis of various diseases.Compared with traditional HIF-1α-PHD inhibitors,FG-4592,which has completed phase III clinical trials and is used to treat chronic renal anemia,is more safe and targeting.Current research proves that FG-4592 plays a regulatory role by stabilizing HIF-1αand up-regulating the production of downstream transcription factors,for example:（1）treatment of renal anemia by promoting the synthesis of erythropoietin（EPO）in the kidney;（2）Up-regulation of the transcription factor vascular endothelial growth factor（VEGF）accelerates the vascularization process of subcutaneous tissue engineering models;（3）accelerates epidermal stem cell（Ep SC）nuclear proliferation and motility to promote wound healing.However,the current research on the application of FG-4592 in inflammatory diseases,especially sepsis,is still very limited.Can it participate in the occurrence and development of sepsis-induced lung injury by inhibiting PHD and stabilizing HIF-1αexpression?The exploration on this issue will help to further reveal the pathophysiology of sepsis and broaden the clinical application of FG-4592.miRNAs play an indispensable role in gene regulation at the post-transcriptional level,including stem cell differentiation,embryogenesis,hematopoietic,metabolism,immune response,or infection.Endogenous miRNA is an evolutionarily conserved small molecule non-coding RNAs,which plays a regulatory role in the pathogenesis of various diseases.miRNA can induce degradation of the m RNA of the target gene by forming complementary binding with the 3’UTR of the target gene,or can inhibit the protein translation process of the target gene by incompletely binding to the 3’UTR of the target gene.This study also tried to screen out miRNAs that can regulate HIF-1αexpression after transcription and further explore the role of miRNAs in the pathophysiology of sepsis.Together,this study takes HIF-1αhomeostasis as the entry point,and explores the effect and mechanism of HIF-1αin sepsis and sepsis induced acute lung injury by inhibiting HIF-1αprotein degradation and post-transcriptional regulation of HIF-1αexpression.Purpose（1）To investigate the effect of HIF-1αon LPS-induced sepsis and acute lung injury in vivo and in vitro models.（2）To explore the potential role of FG-4592 in regulating sepsis and acute lung injury by regulating the expression of HIF-1α.（3）Analyze and select endogenous miRNA that regulate HIF-1αexpression and explore the selected miRNA’s role in LPS-induced sepsis and acute lung injury.Methods and Results（1）In the cell experiment section,we selected two types of mouse alveolar macrophage cell line MH-S and epithelial cell line MLE-12.These two cells were stimulated with LPS（1μg/ml）.The change of inflammatory factors（IL-6,IL-1βand TNF-α）at different time point was determined by q RT-PCR,and the peak value of inflammatory response was 12 h.The expression of HIF-1αwas detected by q RT-PCR and western blot assay,and the expression of HIF-1αwas found to be increased after LPS stimulation.We used lentivirus to overexpress or inhibit HIF-1αin two kinds of cells and then LPS was added to stimulate the expression of inflammatory factors.It was found that overexpression of HIF-1αcan significantly reduce the expression of inflammatory factors and inhibition of HIF-1αcan increase the expression of inflammatory factors.（2）In the cell experiment section,different concentration of FG-4592（0μM-100μM）was added to the two cells,and the CCK8 test results suggested that 25μM was a safe dose.After successfully establishing cell modeling,LPS and FG-4592 interacted with the two cells,and the same trend was found at different time points:FG-4592 can clearly reduce the increase of inflammatory factors caused by LPS.The expression of HIF-1αwas detected and it was found that the expression of HIF-1αsignificantly increased after FG-4592 stimulation compared to LPS group.At the same time,the phosphorylation level of NF-κB-related proteins in the classical pathway of inflammatory response was examined.The results showed that the phosphorylation levels of P65 and IKBαprotein were significantly reduced with the participation of FG-4592,which also indicated the decline of inflammatory response.（3）In the cell model,we transfected two types of cells with lentivirus to overexpress or inhibit HIF-1αand examined the changes of related inflammatory factors with the participation of FG-4592.We found that overexpression of HIF-1αcan enhance the effect of FG-4592 on reducing inflammatory response while inhibition of HIF-1αimpaired the protective effect against LPS-induced cell injury of FG-4592.（4）In the cell model,miR-155 was found to bind to the 3’UTR region of the HIF-1αpromoter using the luciferase reporter gene assay,suggesting that HIF-1αis a potential target of miR-155 and can be directly regulated by miR-155.q RT-PCR was used to detect the changes of miR-155 at the transcription level after LPS stimulation of MH-S and MLE-12 cells.The results showed that after LPS stimulation,miR-155 was significantly increased.After transfection with lentivirus to overexpress or inhibit miR-155 in both cells,LPS was added to detect the expression of inflammatory factors and HIF-1α.It was found that overexpression of miR-155 can significantly increase inflammation factor expression while decreased HIF-1αexpression and inhibition of miR-155 expression can reduce the expression of inflammatory factors while increase HIF-1αexpression.（5）In the animal experiment part,we constructed a mouse sepsis model by intraperitoneal injection of LPS with overexpressing or inhibiting HIF-1αby tail vein injection of lentivirus.As a result,it was found that overexpression of HIF-1αcan significantly improve the survival rate of mice and decreased the expression of inflammatory factors in sera,alveolar lavage fluid and lung tissues.On the contrary,inhibition of HIF-1αcan significantly decreased the survival rate of mice and increased the expression of inflammatory factors in sera,alveolar lavage fluid and lung tissues.（6）In the animal experiment section,a mouse sepsis model was constructed by intraperitoneal injection of LPS.We observed a significant increase in the survival rate of the septic mouse after intraperitoneal injection of FG-4592.Then ELISA was used to detect the changes of inflammatory factors in mouse serum and alveolar lavage fluid.It was found that the inflammatory factors increased significantly under LPS stimulation,and this trend was reversed after adding FG-4592.The results of pathological sectioning of lung tissue in mice suggest that the alveolar wall is thickened and congested,the lung interstitial and alveolar edema are obvious,and large numbers of inflammatory cells infiltrate with LPS stimulaiton and this trend was relieved after adding FG-4592.（7）In animal experiments,a mouse sepsis model was constructed by intraperitoneal injection of LPS.The expression of HIF-1αwas detected by q RT-PCR and western blot assay,and the expression of HIF-1αwas found to be increased after LPS stimulation while the expression of HIF-1αwas furthur increased after FG-4592 stimulation compared to LPS group.This conclusion was further validated by immunohistochemistry.At the same time,the changes of phosphorylation level of NF-κB related protein in lung tissue were detected,and the same trend was obtained in in vitro cell experiments:FG-4592 can inhibit the amplification of LPS-mediated inflammation.At the same time,we also tested the effect of overexpression or inhibition of HIF-1αon the effect of FG-4592 on reducing inflammatory response of septic mice and found that overexpression of HIF-1αcan enhance the anti-inflammatory effect of FG-4592,while inhibiting HIF-1αdamages the anti-inflammatory effect of FG-4592.（8）In animal experiments,we constructed a mouse sepsis model by intraperitoneal injection of LPS with overexpressing or inhibiting miR-155 by tail vein injection of lentivirus.The results showed that overexpression of miR-155 can reduce mouse survival rate and improve the expression of inflammatory factors in mouse serum,alveolar lavage fluid and lung tissues,while inhibition of miR-155 increased mouse survival rate,and increased expression of inflammatory factors in mouse serum,alveolar lavage fluid and lung tissues.Conclusion（1）HIF-1αhas a protective effect on acute lung injury in LPS induced septic mice.（2）FG-4592 plays a protective role in LPS-induced sepsis and acute lung injury by stabilizing the expression of HIF-1α.（3）miR-155 can directly bind to the 3’UTR region of the HIF-1αpromoter and negatively regulate HIF-1αexpression at the post-transcription level,thereby regulating the role of HIF-1αin LPS-induced sepsis and acute lung injury.