| ObjectiveTaking GSK(Glycogen Synthase Kinase)-3β signaling pathway as the research target,the therapeutic effect of Xuebijing(XBJ)on patients with blood stasis syndrome in septic acute liver injury(SALI)was studied,and the protective effect of XBJ and ferulic acid(FA),one of the monomer components,on SALI was also studied,so as to explore the mechanism of XBJ regulating GSK-3β signaling pathway in the treatment of SALI.Methods1.Clinical ResearchUsing the random number method,the 60 patients with SALI were randomly divided into XBJ intervention group and non XBJ intervention group.Both groups were given corresponding anti infection regimen and related sepsis cluster therapy.XBJ intervention group:on the basis of non XBJ intervention group,XBJ injection was added.Specific usage:50ml each time,twice a day,intravenous injection for 7 days.The main indicators for observation are as follows:①Syndrome integrals for blood stasis in traditional chinese medicine(TCM).②modern medical scoring indicators:APACHE-Ⅱ score and SOFA score.③liver function related indicators:alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(AKP),y-Glutamyl Transferase(y-GT),total bilirubin(TBIL),albumin(ALB).④Infection related indicators:white blood cell count(WBC),percentage of neutrophils(N),C reactive protein(CRP),interleukin(IL)-6,procalcitonin(PCT).⑤coagulation related indicators:platelet count(PLT),prothrombin time(PT),activates partial thromboplastin time(APTT),fibrinogen(FIB),D-dimer(D-D).2.Network PharmacologyThe molecular mechanism of XBJ in the treatment of SALI was studied by network pharmacology.Firstly,XBJ small molecule and related target protein data set,SALI related target protein data set,XBJ biological network and the interaction network of XBJ target protein were constructed,then the function and biological pathway enrichment of XBJ target protein were analyzed.3.Experimental Section3.1 XBJ sectionAnimal experiments:C57BL/6 mice were randomly divided into 5 groups with 18 mice in each group.Control group(Con group):sham operation and intraperitoneal injection of normal saline.Model group(CLP Group):cecum ligation and puncture operation(CLP)plus intraperitoneal injection of normal saline.Low dose XBJ group(L-XBJ):CLP operation+low dose XBJ group(intraperitoneal injection of 4ml XBJ per kilogram of body weight per day).High dose XBJ group(H-XBJ):CLP operation+high dose XBJ group(intraperitoneal injection of 8ml XBJ per kilogram of body weight per day).Dexamethasone group(DXM):CLP operation+DXM(intraperitoneal injection of 5mg DXM per kilogram of body weight per day).XBJ intervention experiment lasted for 5 days,during which mice could eat and drink freely.All groups of mice received CLP surgery on the 6th day after the end of all interventions,except the control group of mice that underwent sham surgery.After CLP operation,mice in each group were intraperitoneally injected with corresponding concentrations of XBJ or DXM in three time periods,respectively,after operation,2 hours after operation and 12 hours after operation.And mice were sacrificed on day 7 to obtain liver tissue and serum.The following indexes of each group were tested:①Draw the survival curve of mice in each group.②The liver of mice in each group was analyzed histologically and the pathological changes of liver tissue were observed.③GSK-3β(Ser9)in liver tissue of mice in each group was detected by immunofluorescence.④Biochemical indexes in liver tissue,such as Myeloperoxidase(MPO),ALT,AST,were tested according to the corresponding kit instructions.⑤Indicators in serum,such as IL-10,tumor necrosis factor-α(TNFα),IL-6,IL-12,IL-1β,were detected by ELISA kit.⑥Proteins in liver tissue of mice in each group,such as GSK-3β(Ser9),GSK-3β,Nuclear Factor kappa-B(NF-κB),p-NF-κB,cAMP-Response Element-Binding protein(CREB),p-CREB,were detected by Western blot.Cell experiments:RAW264.7 cells were used in cell experiment and divided into five groups.Control group(Con group):no drug treatment.Model group(LPS group):1μg/ml lipopolysaccharide(LPS).Low concentration XBJ group(100-XBJ group):treated with 1μg/ml LPS and XBJ diluted 100 times.Medium concentration XBJ group(50-XBJ group):treated with 1μg/ml LPS and XBJ diluted 50 times.High concentration XBJ group(25-XBJ group):treated with 1μg/ml LPS and XBJ diluted 25 times.Different concentrations of drugs were given for 2 hours before modeling,and then the control group was given normal culture medium,LPS group and XBJ groups were given lμg/ml LPS culture medium.The incubation was continued for 24 hours,and then the follow-up experiments were carried out:①The viability of cells in each group was measured.②GSK-3β(Ser9)in each group of cells was detected by immunofluorescence.③Inflammatory factors in cell supernatant of each group,such as IL-10,IL-1β,IL-6,IL-12,TNF-α,were detected by the corresponding kits.④Proteins in cells,such as GSK-3β(Ser9),GSK-3β,p-NF-κB,NF-κB,CREB(Ser133),CREB,were measured by Western blot.⑤Cells were first transfected using lentiviral vectors(LeGSK-3β)and negative control vectors(LeCtrl)overexpressing GSK-3β(Total),then p-NF-κB or CREB(ser133)was detected by Western blot.At the same time,the binding rate trends of p-NF-κB and CREB(ser133)to binding protein of CREB(CBP)were detected by Co-immunoprecipitation(Co-IP).3.2 FA sectionAnimal experiments:C57BL/6 mice were randomly divided into 5 groups with 18 mice in each group.Control group(Con group):sham operation and intraperitoneal injection of normal saline.Model group(CLP Group):CLP operation plus intraperitoneal injection of normal saline.Low dose FA group(6-FA):CLP operation+low dose FA group(intraperitoneal injection of 6ml FA per kilogram of body weight per day).High dose FA group(12-FA):CLP operation+high dose FA group(intraperitoneal injection of 12ml FA per kilogram of body weight per day).DXM group:CLP operation+DXM(intraperitoneal injection of 5mg DXM per kilogram of body weight per day).FA intervention experiment lasted for 5 days,during which mice could eat and drink freely.All groups of mice received CLP surgery on the 6th day after the end of all interventions,except the control group of mice that underwent sham surgery.After CLP operation,mice in each group were intraperitoneally injected with corresponding concentrations of FA or DXM or normal saline in three time periods,respectively,after operation,2 hours after operation and 12 hours after operation.And mice were sacrificed on day 7 to obtain liver tissue and serum.The following indexes of each group were tested:①Draw the survival curve of mice in each group.②The liver of mice in each group was analyzed histologically and the pathological changes of liver tissue were observed.③GSK-3β(Ser9)in liver tissue of mice in each group was detected by immunofluorescence.④Biochemical indexes in liver tissue,such as MPO,ALT,AST,were tested according to the corresponding kit instructions.⑤Indicators in serum and supernatant,such as IL-10,TNF-α,IL-6,IL-12,IL-1β,were detected by ELISA kit.⑥Proteins in liver tissue of mice in each group,such as GSK-3β(Ser9),GSK-3β,p-NF-κB,NF-κB,p-CREB,CREB,were detected by Western blot.Cell experiments:RAW264.7 cells were used in cell experiment and divided into five groups.Control group(Con group):no drug treatment.Model group(LPS group):1μg/ml LPS.Low concentration FA group(25-FA group):treated with 1μg/ml LPS and 25μM FA.Medium concentration FA group(50-FA group):treated with 1μg/ml LPS and 50μM FA.High concentration FA group(100-FA group):treated with 1μg/ml LPS and 100μM FA.Different concentrations of drugs were given for 2 hours before modeling,and then the control group was given normal culture medium,LPS group and FA groups were given 1μg/ml LPS culture medium.The incubation was continued for 24 hours,and then the follow-up experiments were carried out:①The viability of cells in each group was measured.②GSK-3β(Ser9)in each group of cells was detected by immunofluorescence.③Inflammatory factors in cell supernatant of each group,such as IL-10,IL-1β,IL-6,IL-12,TNF-α,were detected by the corresponding kits.④Proteins in cells,such as GSK-3β(Ser9),GSK-3β,p-NF-κB,NF-κB,CREB(Ser133),CREB,were measured by Western blot.⑤Cells were first transfected using lentiviral vectors(LeGSK-3β)and negative control vectors(LeCtrl)overexpressing GSK-3β,then p-NF-κB or CREB(ser133)was detected by Western blot.At the same time,the binding rate trends of p-NF-κB and CREB(ser133)to CBP(binding protein of CREB)were detected by Co-IP.Results1.Clinical Research①Compared with non XBJ intervention group,the therapeutic effect of TCM syndrome in XBJ intervention group was significantly better than that in non XBJ intervention group,and the difference was statistically significant(P<0.05).②There was no significant difference in the clinical scores of modern medicine(APACHE Ⅱscore,SOFA score)between the two groups before and after treatment,that is,the difference was not statistically significant(P>0.05).③After 7 days of treatment,ALT and AST in XBJ intervention group were significantly improved compared with those in non XBJ group,and the difference was statistically significant(P<0.05).While other liver function-related indicators in the two groups,such as AKP,y-GT,TBIL,ALB,were not statistically significant after treatment(P>0.05).④After 7 days of treatment,PCT in XBJ intervention group was significantly improved compared with that in non XBJ group,and the difference was statistically significant(P<0.05).While other infection-related indicators in the two groups,such as WBC,N,CRP,IL-6,were not statistically significant after treatment(P>0.05).⑤After 7 days of treatment,D-D in XBJ intervention group was significantly improved compared with that in non XBJ group,and the difference was statistically significant(P<0.05).While other coagulation-related indicators in the two groups,such as PLT,PT,APTT,FIB,were not statistically significant after treatment(P>0.05).2.Network PharmacologyThrough the systematic pharmacological study on the molecular mechanism of XBJ injection in the treatment of SALI,it is found that 9 compounds,such as quercetin,luteolin,baicalein,tanshinone IIA,cryptotanshinone,are the main active components.By targeting proteins such as COX-2(PTGS2),iNOS(NOS2),ESR2,VEF-A,JUN,TNF,IFNγ,IL-2、IL-6,and regulating the inflammatory response of TNF,PI3K-Akt,ERK1/2,XBJ can treat SALI.3.Experimental Section3.1 XBJ sectionAnimal experiments showed that:①Liver morphology showed that XBJ alleviated CLP-induced SALI in C57BL/6 mice.②XBJ improved liver function in SALI mice.③XBJ regulated the level of serum inflammatory factors in CLP-induced SALI mice.④XBJ regulated the activity of GSK-3β,NF-κB and CREB in the liver of CLP-induced SALI mice.Cell experiment showed that:①XBJ increased the viability of RAW264.7 cells and the expression of GSK-3β(Ser9).②XBJ regulated the level of cytokines in LPS-induced cells.③XBJ regulated the activity of GSK-3β,NF-κB and CREB in LPS-induced cells.④Overexpression of GSK-3β regulated the binding of CBP to p-NF-κB and p-CREB.By influencing GSK-3β,XBJ regulated the binding of CBP to p-NF-κB or p-CREB.3.2 FA sectionAnimal experiments showed that:①Liver morphology showed that FA alleviated CLP-induced SALI in C57BL/6 mice.②FA improved liver function in SALI mice.③FA regulated the level of serum inflammatory factors in CLP-induced SALI mice.④FA regulated the activity of GSK-3β,NF-κB and CREB in the liver of CLP-induced SALI mice.Cell experiment showed that:①FA increased the viability of RAW264.7 cells and the expression of GSK-3β(Ser9).②FA regulated the level of cytokines in LPS-induced cells.③FA regulated the activity of GSK-3β,NF-κB and CREB(cAMP-response element-binding protein)in LPS-induced cells.④Overexpression of GSK-3β regulated the binding of CBP to p-NF-κB and p-CREB.By influencing GSK-3β,FA regulated the binding of CBP to p-NF-κB or p-CREB.Conclusion① Clinical research shows that XBJ can effectively improve the TCM syndrome,liver function indexes(ALT、AST),infection index(PCT)and coagulation index(D-D)of patients with SALI and blood stasis syndrome.② Network pharmacology research found that XBJ may regulate inflammation by acting on core targets such as TNF-α,IL-6,IL-10 and intervening with the PI3K-AKT-GSK-3β signaling pathway to achieve the effect of treating SALI.③Experimental studies have shown that through the GSK-3β/NF-κB/CREB signaling pathway,XBJ and FA can inhibit the expression of pro-inflammatory cytokines and increase the expression of anti-inflammatory cytokines so as to treatSALI. |
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