Effects Of C5a On The Expression Of Tissue Factor And The Potential Mechanism

Sepsis is a major challenge in medicine.Despite enhanced antibiotics and the best available supportive care,sepsis remains one of the most common life-threatening complications in intensive care units.According to studies from North America,the incidence is a bout 3 cases per 1,000 population,an annual burden of 750,000 cases in the United States resulting in 215,000 deaths.It is estimated that severe sepsis is responsible for 2% to 11% of all admissions to intensive care units. The hospital mortality rate associated with severe sepsis and septic shock (30% and more than 60% ,respectively) has not changed much during the last decades.It maybe related to its complex pathophysiological process and the cognition of the diseases.Sepsis is characterised by the activiation of a wide range of cells in succession, then release multiple inflammatory factors and cause tissue damage,multiple organs dysfunction syndromes,even multiple organs failure.During sepsis many biological systems has been activated,such as complement system,coagulation system, immunologic system,apoptosis related system et cetera.Disseminated intravascular coagulation is a severe complication due to coagulation disturbance.Once DIC emerge,a wide range of micro-thrombosis will cause ischemia and hypoxia in tissues and accelerate MODS progression.A study revealed that the proportion of DIC in sepsis especially septic shock is approximately 30%-50%. Coagulation disorder has already become an outstanding feature in sepsis pathophysiologic process.Many kinds of inflammatory mediators and cytokines can lead to coagulation disorders,for instance:LPS,TNF-a,ILs,et cal.Among these,Complement 5a has been drew more and more attentions.C5a as a common cleave product of three complement activation pathways owns wide biological activities.In the new therapeutic strategies of sepsis,不得C5a-antagonist therapy has been mentioned and many studies demonstrate its effectiveness.Our team has screened and synthetized a new type of C5a-antagonist:C5a antisense peptide.Related experiment results show that it can antagonize C5a' functions and ameliorate inflammatory response in sepsis.While the role of C5a in coagulation disorders cannot be ignored.Previously experiment date showed that C5a can up-regulation tissue facotor(TF) on the membrance of vascular endothelial cells,neutrophils and monocytes/macrophages,cause hyper-coagulation activation;down-regulation antithrombin III and thrombomodulin,cause anticoagulation system impaired;effect the expression of tissue plasminogen activator and plasminogen activator inhibitor,cause fibrinolytic system unbalanced. since tissue factor is the initiator of extrinsic coagulation pathway and holds the central status in the modern activation of blood coagulation modle.More attention were paid to the relationship of C5a and TF.Because coagulation disoder was mainly occur in microvascular and lung was the first targeted organ,the effects and mechanisms of C5a on TF in pulmonary microvascular endothelial cells worthy to be focused on.NF-κB is a hot spots of all the transcription factor associated with inflammations.It exists in various kinds of cells and plays a vital role in regulating many functional protein expression.Activiated NF-κB travel to the cell nucleus in the form of dipolymer,then combine to theκB binding site in the gene promoter region and regulate gene and protain expression such as pro-inflammation cytokines(TNF-α,ILs),adhesion molecules,acute phase proteins and enzymes.TF gene promoter region also has anκB binding site so the role of NF-κB in the regulation of TF gene expression deserve to be researched.Tissue factor pathway inhibitor is an endogenous inhibitor of TF-induced coagulation.Normal coagulation depends on the balance of TF and TFPI.The abnormal concentrations of TF and TFPI had been detected in many diseases:coronary heart disease,cerebral infarction,leukocythemia and so on.We consider them a reason of coagulation abnormality.As coagulation disoders also an outstanding clinical manifestation,weather there is a change in the concentration of TF and TFPI in sepsis animal model? Can the newly synthetized reagent antagonize C5a' functions in the aspect of coagulation disorder in vivo?This study was funded by the Project of Science & Technology of Guangzhou City (07Z3-E0121).Experimental content is divided into three parts as follows:First,we use C5a with different concentration (100ng/ml,200ng/ml,300ng/ml)and time (4h,8h,12h)to stimulate human pulmonary microvascular endothelial cells (HPMEN).Real-time fluorescent quantitative PCR and western blotting was used to detect the gene and protein changes in each groups.Second,chose the group C5a 200ng/ml,stimulation time 8h to analyse the potential mechanism. Use C5a antisense peptide to blockade the link between C5a and C5aR and observe the TF changes.Simultaneously,we use immunohitochemistry techenique to survey NF-κB p65 changes.Finally, establishe sepsis animal model with caecal ligation plus puncture (CLP).Kunming mouse were divided into normal groups,sham-operated groups,model groups and antisense peptide groups.At different time (2h,4h,8h,12h),we use ELISA to detect the concentration of TF, TFPI in the blood and estimate the antagonism of newly synthetized antisense peptides. The main results and conclusions are as follows:l.The effects of C5a on TF expression in HPMECs:Normal HPMECs seemed not express TF.When the concentration of C5a is 200ng/ml,4h,8h,12h gene and protein is (1.00±0.13),(3.04±0.26), (6.58±0.44), p<0.05 and(0.15±0.05),(0.27±0.06),(0.45±0.07), p<0.05.When the stimulation time is 8h,100ng/ml,200ng/ml,300ng/ml gene and protein is (1.00±0.14),(1.80±0.16),(3.09±0.09), p<0.05 and(0.29±0.05),(0.41±20.73), (0.53±0.40),p<0.05.This prove that TF began to express after C5a stimulation.TF gene and protein expression increased dose-dependently and time-dependently in 12h.It suggests that C5a can up-regulate TF exprssion and play a role in hyper-coagulation activation.2.The potential mechanism of C5a up-regulate TF exprssion.With addition of C5a antisense peptide, TF mRNA decreased to(0.66±0.67)of the comparable group(1.00±0.10),p<0.05,protein decreased to(0.27±0.31)of the comparable group(0.33±0.34),p<0.05.Immunohistochemistry images showed that no yellow-brown granular pigment in negative control;there is yellow-brown granular pigment in the cytoplasm of the normal group;in stimulated group,yellow-brown granular increased in cytoplasm and also emerged in cell nucleus.It suggests that the newly synthetized has blocked C5a bind to C5aR. Gene expression decrased to 80% and protain 68%.Immunohistochemistry images showed yellow-brown granular increased in cytoplasm and also emerged in cell nucleus suggests that NF-κB had been activated.Results of these two part show that during 12h, TF increased dose-dependently and time-dependently after C5a stimulation.The protential mechanism may be C5a binds to its receptor then activiates NF-κB through specific signaling pathways and finally regulates TF expression. 3. Blood concentration of TF and TFPI.When the time is 2h,the concentration of TF in each groups(normal group,sham-operative group,model group,C5a antisence peptide intervention group) are(42.44±2.19)pg/ml,(54.12±3.47)pg/ml,(59.81±3.69)pg/ml,(54.23±3.73)pg/ml.Wh en the time is 4h,the concentration of each groups are(42.29±1.98)pg/ml, (53.70±4.14)pg/ml,(82.77±9.11)pg/ml,(70.91±7.19)pg/ml,respectively. When the time is 8h,the concentration of each groups are:(41.45±1.32)pg/ml,(56.33±2.68) pg/ml,(125.34±6.32)pg/ml,(103.36±6.72)pg/ml respectively. When the time is 12h,the concentration of each groups are:(42.75±1.25)pg/ml,(60.73±1.43)pg/ml, (139.81±6.17)pg/ml,(121.41±7.21)pg/ml.The concentration of TF were gradually increased over time;C5a antisence peptide groups are lower than model group at any time points, p<0.05.When the time is 2h,the concentration of TFPI in each groups(normal group,sham-operative group,model group,C5a antisence peptide intervention group) are:(14.60±0.76)ng/ml,(16.67±2.19)ng/ml,(26.2±2.21)ng/ml,(26.18±3.3)ng/ml.Whe-n the time is 4h,the concentration of each groups are:(15.11±0.37)ng/ml, (18.94±0.39)ng/ml,(28.08±2.46)ng/ml,(28.72±1.46)ng/ml,respectively. When the time is 8h,the concentration of each groups are:(15.72±0.92)ng/ml,(18.01±2.92) ng/ml, (27.45±2.52) ng/ml,(29.20±1.61)ng/ml,respectively. When the time is 2h,the concentration of each groups are:(15.00±0.42)ng/ml,(19.1±0.96)ng/ml,(29.2±1.41) ng/ml,(29.4±1.32)ng/ml. The oncentration of TFPI is not increased after 2h.There is no significant difference between model groups and C5a antisence peptide groups, p>0.05.It suggests that coagulation disorders emerged in the early stage of sepsis. Mainly expressed in the imbalance of TF and TFPI. C5a antisence peptide can not change the concentration of TFPI. So the primary mechanism of antisence peptide effects coagulation disorder maybe reducing the production of TF then striking the balance of TF and TFPI.We have discussed the relationship of C5a and TF in cells and in animals.From cell experiments,we found that TF increased dose-dependently and time-dependently after C5a stimulation.The potential mechanism is C5a binds to its receptor then activiates NF-κB through specific signaling pathways and finally regulates TF expression and the newly synthetized reagent has the antagonism in vitro.From animal experiments,we found that there is coagulation disorders in sepsis.Exhibited with persistent hyper-coagulation activation but relative insufficient anti-coagulation.After the treatment of C5a antisense peptide,the coagulation disorders have a certain degree of alleviate.It evidence that the newly synthetized reagent has the antagonism in vivo.If we can improve the experiment conditions and design the experiment more reasonable,potent evidences will support our hypothesis.