An Experiment About The Effect Of Hypoxia And P38 MAPK Inhibitor SB203580 On Lipopolysaccharide Induced TNF-α Expression In Murine Macrophages

Background and objectives:Sepsis is common in infection,severe trauma,operation and other stress state. The incidence of sepsis is on the rise in recent years and the possible reasons include occurrence of tumor,diabetes;population aging;microbial resistance due to antibiotics abuse;increased use of invasive procedures and transplantation;the intra-hospital infection etc.Pediatric sepsis is sill prevelant with high mortality.An international panel of 20 experts in sepsis and clinical research was convened to describe the specific definitions of pediatric SIRS,infection,sepsis,severe sepsis,and septic shock and pediatric organ failure and the validity of pediatric organ failure scores.The definition was published in 2005 after three years of practice.Severe sepsis has become a major threat to human health and burden to ecmonic development due to its mortality and medical cost. Gram-negative bacteria initiate the transduction of inflammatory signal after the binding of the bacterial membrane component lipopolysaccharide(LPS)to pattern recognition receptors,Toll-like receptor(TLR)2 and TLR4.Then via the intracellular co-receptor MD88 to trigger signal transduction and induce cytokines and inflammatory mediators.TNF-α,a pro-inflammatory cytokine,is included and has been proved to be the vital inflammatory medium in sepsis and septic shock. Macrophages are responsible for most of TNF-αrelease in sepsis.Large amount of TNF-αis irregularly produced and results in the over-expression and release of various cytokines and inflammatory mediators.The systemic inflammatory reaction brings in high permeability of blood vessels,leaking body fluid and lymphocytes migrating to inflammatory area.The ensuing multi-organ dysfuntion affects the cardiovascular,respiratory,and haematopoietic systems etc.Of these,the commonest organ affected is the lung and patients with sepsis frequently develop acute lung injury(ALI)or its extreme manifestation,the acute respiratory distress syndrome (ARDS).The defects of lung gas exchange result in tissue and cellular hypoxia evitablely.Micro-environmental conditions found in areas of inflammation are characterized by hypoxia,hypo glucose and high levels of inflammatory cytokines and metabolites of reactive oxygen.Hypoxia inducible factor-1(HIF-1)is a principal transcription factor of homeostasis in cells and tissue exposed to hypoxia.HIF-1α,the oxygen reactive subunit can generally only be detected under low oxygen concentrations and activates the expression of hypoxia responsive genes(HRG). Although hypoxia and septic shock are related,the mechanism through which hypoxia exacerbates septic shock and TNF-αhas not been fully investigated.p38 MAPK(p38),regarded as "stress-induced MAPK",is a critical pathway contributing to the LPS-induced TNF-αexpression.Previous studies illustrated p38 MAPK controlled the expression of TNF-αboth in transcriptional(mRNA)and post-transcriptional(mRNA stability and translation).Inhibition of this pathway has been proposed for therapeutic strategy in the treatment of inflammatory diseases.The pyridinylimidazole compounds,exemplified by SB203580,were found to be selective inhibitors of p38 MAP kinase.SB203580 has been used broadly in cellular level study in p38 MAPK pathway analysis.However,the in vivo effects of the SB203580 in serum TNF-αexpression in septic shock animal model are still controversial.Few reports relate to hypoxia and p38 activity.In this study,the pathophysiological relationship between hypoxia and TNF-αbiosynthesis has been explored using SB203580.We observed TNF-αsecretion pattern in RAW264.7 cells and mouse peritoneal macrophage influenced by p38 via hypoxia.The treatment effect of SB203580 on the serum level of TNF-αin a mouse model of LPS induced endotoxemia was invstigated.We try to elucidate the role of hypoxia in TNF-αbiosynthesis and the cross-talk between p38 and HIF-1αin sepsis. These observations are relevant to the identification of novel molecular targets and strategies for the clinical management of sepsis and septic shock.Methods:1.The influence of oxygen concentration on the effects of SB203580 on TNF-αproduction(1)The dosage experiment of SB203580 under normoxiaThe mouse macrophage cells RAW264.7 were pretreated with 25μM and 10μM SB203580 for 2 h before 10-1000 ng/ml LPS was added.For other groups,cells was treated with 1-20μM SB203580 or 0.2-2μM SB203580 for 2 h before lng/ml LPS or 0.1 ng/ml was added.The cultures were then maintained in conditions of normoxia for 18h and the supernatant was collected.(2)The inhibitory effect of SB203580 under condition of different oxygen concentrationRAW264.7 cells were pretreated by 10μM SB203580 for 2 h before 1 ng/ml LPS was added.Culture was maintained under normoxia,hypoxia(0.5%oxygen)or treated with CoCl₂(100μM)to mimic hypoxia for 18h and the supernatant was collected. (3)The effect of SB203580 on TNF-αmRNA transcription and stability under different oxygen conditionRAW264.7 cells were treated like above.Then cells were collected in Trizol at 2 h after the addition of LPS.2μg/ml Actinomycin D,a transcription inhibitor,was added 2 h after LPS treatment and this time point was taken as time 0 in another group of cells.Cells were collected in Trizol at 10,30 and 60 min.(4)Kinetics of TNF-αbiosynthesisRAW264.7 cells were exposed to LPS 10μM SB203580 for 2 h before 1 ng/ml LPS was added.The supernatant was collected at 1,2,4,6 and 8h after LPS treatment under normoxia or hypoxia(CoCl₂)conditions.(5)The effect of hypoxia on p38 MAPK activity and the down-stream target gene MAPKAPK2RAW264.7 cells were treated like above.The protein was harvested at 10,30 and 60min after LPS activation under normoxia or hypoxia(CoCl₂)conditions.2.The effect of SB203580 in mouse peritoneal macrophageMouse peritoneal macrophages in 6-well culture plates were treated with 20μM and10μM SB203580 for 2h.Then cells were stimulated with 1 ng/ml or 10 ng/ml LPS.Culture was maintained under normoxia or hypoxia(CoCl₂)conditions and the supernatant was collected after 18 h.Untreatment control groups and vehicle control groups were set in all cellular experiments.3.Animal model experiments(1)The induction of LPS on TNF-αin mouse model under normoxiaBALB/c mice accepted i.p.injection of 0.5-3.5 mg/kg LPS.Then mice were sacrificed to obtain blood samples at 90 min after LPS injection.(2)The effect of SB203580 on mouse serum TNF-αlevel under normoxia and hypoxia SB203580 15 mg/kg,25 mg/kg was administrated by i.v.injection.LPS 0.5 mg/kg was injected i.p.2 h later.Mice lived in normoxia or hypoxia(10%O₂).They were sacrificed 90 min after LPS injection.(3)The effect of SB203580 on the expression of HIF-1αin mouse lungMice accepted SB203580 15 mg/kg by i.v.injection 2 h before i.v.injection of LPS 0.5 mg/kg.Mice were sacrificed at 90 min and 240 min after LPS injection to collect blood and lung samples.The vehicle control group and saline control group were set for the animal experiments and each group had three mice.4.Sample examination(1)The TNF-αlevel in cell culture supematant and mice serum were inspected by ELISA.(2)The effect of SB203580 on TNF-αmRNA transcription and stability was compared by Real-time PCR.(3)Western blot was adopted to analyze the effect of SB203580 on the activity of p38 and MAPKAPK2.(4)The expression of HIF-1αmRNA in mouse lung was examined by PCR and Real-time PCR.(5)IHC and Western blot method were used to observe the HIF-1αprotein morphologically and quantitatively.Results:1.Under normoxia,biosynthesis of TNF-αwas markedly increased from baseline(1237.33±132.61pg/ml,n=3)to＞10 fold(18777±1481.68pg/ml,n=3)after exposure to LPS.The administration of SB203580 to normoxic LPS treated macrophage cultures completely abrogated TNF-αproduction,about 90%.The inhibition of TNF-α.production by SB203580 was dose-dependently.SB203580 dosage higher than 10μM and 1μM could effectively inhibit TNF-αinduced by LPS 0.1 mg/kg and 1 mg/kg.Otherwise,no obvious inhibition was observed. 2.Hypoxia and CoCl₂ treatment in the absence of LPS increased the level of TNF-αto nearly two fold(p＜0.05,n=3)above the baseline.In contrast,the TNF-αproduction was only slightly increased(statistically not significant)with LPS stimulation under hypoxia.Exposure to SB203580 also reduced hypoxia induced TNF-αto baseline level.However,the inhibitory effects of SB203580 on TNF-αexpression by LPS stimulation were not observed under hypoxia or CoCl₂ treatment3.SB203580 treatment effectively inhibited TNF-αtranscription under normoxia (p＜0.05,n=3).Hypoxia and CoCl₂ treatment augment LPS-induced TNF-αmRNA expression which was resistant to the inhibitory effects of SB203580.4.Hypoxia accelerated TNF-αsecretion by 2 h and the peak level was observed at 4h after LPS stimulation.But in the presence of SB203580 this effect was abrogated and the kinetics of TNF-a synthesis paralleled those observed under conditions of normoxia.As reported above,SB203580 completely blocked TNF-αexpression under normoxia.5.Under normoxia,the active and phosphorylated form of p38 MAPK(p-p38) was detected 10 min after LPS stimulation,peaked at 30 min and had declined by 60 min.A similar pattern of p-p38 expression was observed following CoCl₂ treatment and exposure to SB203580 induced no additional changes in p-p38 expression.For the inactive form of p38 MAPK neither hypoxia nor SB203580 altered its expression level.The phosphorylated MK2 was first detectable at 10 min and reached to the maximum level at 30 min after addition of LPS and declined at 60 min in the end under normoxia condition.Hypoxia increased expression of pMK2 markedly compared to that under normoxia at 30 and 60 min and the pattern of expression was only minimally altered by exposure to SB203580.6.TNF-αmRNA stability was enhanced by hypoxia.The effect of SB203580 was minimized at 60 min both in normoxia and hypoxia compare to that in normoxia. The mRNA level although at 30 min,less than 10%difference can be observed in these three groups.7.Hypoxia stimulation did not affect obviously the baseline of TNF-αin mouse peritoneal macrophage(p＞0.05,n=3).LPS activated TNF-αin a dose-dependent manner under normoxia,while different dosage of LPS worked similarly under hypoxia(p＜0.05,n=3).Although mouse peritoneal macrophage was not sensitive as RAW64.7 to LPS,they reacted to SB203580 and hypoxia similarly.Hypoxia could diminish about 50%inhibitory ability of SB203580.8.Different dosage of LPS could all increase TNF-αlevel in mouse serum significantly(p＜0.05,n=3).The in vivo administration of SB203580 to mice with LPS induced endotoxemia had no effect on serum level of TNF-αcompare to vehicle control(p＞0.05,n=3)either under normoxia or hypoxia.The TNF-αlevel in the serum rose at 90 min after LPS 0.5 mg/kg administration and fell at 240 min,then restored to normal level at 24 h.9.LPS induced the expression of HIF-1αwith no significant difference in SB203580 treated mice or vehicle control(p=0.34).The expression of HIF-1αprotein was detected in mouse lung both at 90 min and reduced at 240 min after LPS injection. No effect of SB203580 can be found on HIF-1αexpression compared with that in vehicle control mice.The immunohistochemical staining has further confirmed the observation by at 90 min after LPS treatment,in lung,the protein was detected in the interstitial tissue.The positive cells reduced at 240 min and similarly to that in western blot,the effect of SB203580 in HIF-1αexpression was not obvious.Conclusions:1.Our data indicate that LPS induced p38 MAPK activation was enhanced by hypoxia and consequently increased TNF-αsecretion.The administration of SB203580 to LPS treated macrophage under normoxia completely abrogated TNF-αproduction,while SB203580 lost the ability under hypoxia. 2.Hypoxia affected the reaction of mouse peritoneal macrophage cells to LPS and SB203580 similarly with RAW264.7.3.Hypoxia could augment the TNF-αtranscription and mRNA stability. SB203580 inhibited TNF-αtranscription under normoxia without affecting TNF-αmRNA stability.4.In the present study we found that hypoxia did not increase the quantity of p38 or p-p38,but enhance the activity of p38 to phosphorlate MK2 to promote TNF-αproduction.5.HIF-1αwas detected in the lung of endoxemia mouse model and its expression was not altered by SB203580.SB203580 failed to reduce the TNF-αlevel in mouse serum.6.The induction of HIF-1αin mice with endotoxemia suggested a synergistic effect on p38 mediated TNF-αexpression.And HIF-1αpathway possibly functions independently of p38 MAPK signaling in vivo.Innovation and significance:1.The present study established that hypoxia played an important role in TNF-αbiosynthesis in sepsis and p38 activity was enhanced by hypoxia.This explained the controversial practice of SB203580.2.Our data indicated that HIF-1αwas involved in TNF-αregulation.Hypoxia may function independently of p38 via HIF-1αand a synergistic signal may exit between the two pathways.3.These findings emphasized that the pathologic hypoxia should be taken into consideration besides TNF-αbiosynthesis mechanism for sepsis and septic treatment strategy.This provided new molecular target in sepsis and septic shock therapy.