| Objective:To investe the mechanism of improving coagulation disorder by Xuebijing injection,which aims to provide experimental basis for the treatment of coagulation dysfunction in sepsis.Methods:1.Endothelial cell was divided randomly into control group(0 ng/mL LPS)and 62.5ng/mL LPS group,125 ng/mL LPS group,250 ng/mL LPS group,500 ng/mL LPS group,and 1000 ng/mL LPS group,CCK 8 method is used to detect cell proliferation after culturing for 24 h,48h,and 72 h,in the meantime,the release amount of lactic dehydrogenase(LDH)was detected.,we screened for the most valid dose of LPS.Then,we detected the cell proliferation,the amount of LDH,tissue factor(TF)and platelet active factor(PAF)in supermant by various concentrations Xuebijing injection.2.All groups were divided into control group,LPS group,the Xuebijing group(1,5,and 25 u l/ml)+ LPS group,inhibitor group(50 nmol/l,5 nmol/l,0.5 nmol/l)+ LPS group,24,48 and 72 h after cultivating,Ca2 + and blood coagulation factor(coagulation factor,F)Ⅶ and F Ⅹ,activation FⅩa level was detected with chromophore substrate method;at 72 h,the expression level of IRE1α,uXBP-1,sXBP1,and PDI was dectected by Western blot technique in control group,LPS group and Xuebijing group(1,5,and 25ul/ml)+LPS group.3.Model of sepsis in rats was built by cecal ligation and puncture(CLP),All rats were divided into five groups: control group,SHAM group,CLP group,Xuebijing group,and inhibitor groups,each group owns 6 rats,Xuebijing group was injected Xuebijing after CLP,and inhibitor groups was injected IRE1αinhibitor,we cut the tail to test blood time and took blood through abdominal aortic to measure blood clotting function at the 24 h,48h,72 h respectively,then we collected the serum to test the expression of FⅩa.And the protein expression of IRE1α,uXBP1,sXBP1,and PDI was dectected in abdominal aorta by western.In addition,CLP model was constructed to detect the fatality rate of 7 days in each group.Results:1.Different concentrations of LPS stimulated endothelial cells to imitate the microenvironment of sepsis:compared with control group,the proliferation and LDH of 62.5 ng/mL LPS group was no obvious difference(P>0.05);but in 125 ng/mL LPS group,the 250 ng/mL LPS group,500 ng/mL LPS group,the 1000 ng/mL LPS group,proliferation ability was lower and LDH,TF,PAF of spernatant were higher than control group(P<0.05 or P<0.01),especially the 500 ng/mL was ramarkable,1000ng/ml LPS group cell floating increased,we considered the cell was death.Compared with the other groups,the proliferative activity was lower and the secretion TF and PAF were higher in 500 ng/mL group.2.The research of FⅩa and IRE1α expression level on endothelial cell with different concentrations of Xuebingjing injection:at 24 h,there was no significant difference among all groups;48 h,compared with control group,expression of FⅩa in LPS group increased significantly(P<0.01);compared with LPS group,expression of FⅩa in LPS+5 nmol/L inhibitor group was lower(P < 0.05);at 72 h,compared with control group,expression of FⅩa in LPS group increased significantly(P <0.001),Compared with LPS group,LPS+5 nmol/L FⅩa inhibitor group was obviously lower(P < 0.01).Compared with control group,the expression of IRE1α,uXBP1,sXBP1,and PDI were raised in 500 ng/ml LPS group,there were markedly difference(P< 0.05);Compared with the LPS group,the LPS+Xuebijing group showed obviously higher the cell proliferation and lower release of LDH(P<0.05 or P<0.01),expressions of IRE1α,uXBP1,sXBP1,and PDI were significantly reduced(P<0.01);meanwhile,FⅩa generation decreased with IRE1α,XBP1 and PDI expression levels(P<0.05),and 5μl/ml Xuebijing was the optimal dose in down-regulation of FⅩa.3.The research on the mechanism of Xuebijing to improve coagulation function and survival rate in sepsis rats: the coagulation function mainly refer to the plasma thrombin time(PT),activated partial prothrombin time(APTT),serum FXa,rat tail bleeding time.Compared with control group,sham group,model group,Xuebiing group and inhibitor group were no significantly increased at 24 h for PT and APTT,and compared with control group,sham group were no remarkable difference at all points;compared with control group,model group blood coagulation function was apparently unusual,compared with model group,PT,APTT,tail bleeding time of the Xuebijing group and inhibitor group were shorten,expression of FⅩa was lower.TheSAHM group,CLP group,Xuebijing group,inhibitor group rates were 98.18%,61.77%,90.32% and 85.71% respectively,the Log-rank(Mantel-Cox)Test statistics method,compared with the CLP group,the blood will net group survival rate increased significantly(P = 0.007,n = 20),inhibitors improve group survival(P= 0.012,n = 20)Conclusion: Xuebijing injection can reduce the expression of PDI by blocking IRE1α-XBP1 signaling pathway to enhance the procoagulation of TF in endothelial cells and improve the coagulation function in sepsis rat. |
