| Objective:CD8~+T cell plays a major role in anti-infection,autoimmune and anti-tumor immunity.In the thymus,the immature T cells were selectively differentiated into mature CD8~+T cells,and under the stimulation of exogenetic antigens presented by MHC I molecules,CD8~+T cells were transformed from resting state to antigen-specific effective cells,migrating to infection sites to clean infection.In this process,the functional state of CD8~+T cells is closely related to the effective function,which in turn affects the disease process in the body.mTOR(mechanistic target of Rapamycin target protein)is a key signaling pathway that regulates cell growth and metabolism by microenvironment factors such as glucose,amino acid content,stress condition,energy level and so on.It plays a major role in the growth,development,activation,differentiation and metabolism of T cells.DAPK1 is a member of the serine/threonine protein kinase family,which also is a kind of calcium ion and calcium-modulated protein.Studies have shown that DAPK1can combine with TSC2 through its death domain,which is an upstream inhibitor of mTORC1,to reduce formation of TSC1-TSC2 complex,thus promoting mTORC1activity.However,how DAPK1 is activated,as well as its function in immune cells is still unknown.The purpose of this study was to explore how DAPK1 was activated in CD8~+T cells,and whether it could affect the function of CD8~+T cells by regulating the mTORC1pathway,thus affecting the anti-infection and anti-tumor ability in bodies.Methods1.The mechanism of Calcineurin activates DAPK11.1 Effect of Calcineurin inhibitors on the DAPK1 activity of CD8~+T cells induced by TCR:Isolated the spleen from wild-type mice,cracked red blood cells to obtain single cell suspension,cultured with 0.5?g/ml anti-CD3 antibody for 48h,and then cultured with 20ng/ml IL-2 for 6 days,differentiated lymphocytes into cytotoxic CD8~+T(CTL)cells,stravated with serum-free 1640 medium for 12h,and then stimulated with2?g/ml anti-CD3/CD2 antibodies and Calcineurin inhibitors(2?M FK506)or DAPK1inhibitor(2?M DAPK1i)together for 1h,collected cell samples,extracted proteins and detected NFAT signaling pathways and DAPK1 activity by immunoblotting.1.2 Detection of co-localization of Calcineurin and DAPK1:Cultured HEK 293T cells,transferred with DAPK-Flag and Calcineurin-HA retrovirus,then continuous cultured cells for 36h,collected cell samples and extracted protein.The co-expression of DAPK1and Calcineurin in 293 T cells was detected by Flag and HA immunoprecipitation antibody respectively.1.3 Detection of co-localization of Calcineurin and DAPK1 by immunofluorescence:Separatd the spleen from wild-type mice,cracked red blood cells to obtain single-cell suspension,cultured lymphocytes with 0.5?g/ml anti-CD3 antibody for 48h,and then amplificated cells with 20ng/ml IL-2 for 6 days,differentiated into CTL cells.Stravated CTLs with serum-free 1640 medium for 12h,and stimulated with 2?g/ml anti-CD3/CD28antibody or without for 1h,collected cell samples,fixed and disrupted the cell membrane to stain fluorescent antibody.Detected co-location of Calcineurin and DAPK1 in CD8~+T cells with confocal fluorescence microscopy.1.4 Detection of the combination of Calcineurin and DAPK1 different domains:Cultured HEK 293T cells,transferred with different domains of DAPK1 which labeled with Flag and Calcineurin-HA retrovirus,then continuous cultured cells for 36h,collected cell samples and extracted protein.The co-expression of DAPK1 and Calcineurin in 293 T cells was detected by Flag and HA immunoprecipitation antibody respectively.2.The mechanism of DAPK1 regulates TSC2Detection of immunofluorescence co-localization between TSC2 and DAPK1:Separatd the spleen from wild-type mice,cracked red blood cells to obtain single-cell suspension,cultured lymphocytes with 0.5?g/ml anti-CD3 antibody for 48h,and then amplificated cells with 20ng/ml IL-2 for 6 days,differentiated into CTL cells.Stravated CTLs with serum-free 1640 medium for 12h,and stimulated with 2?g/ml anti-CD3/CD28antibody or without for 1h,collected cell samples,fixed and disrupted the cell membrane to stain fluorescent antibody.Detected co-location of TSC2 and DAPK1 in CD8~+T cells with confocal fluorescence microscopy.3.Effect of Calcineurin and DAPK1 on the activity of mTORC1 pathway3.1 Effects of Calcineurin and DAPK1 inhibitors on the mTORC1 activity of CD8~+T cells:Isolated the spleen from wild-type mice,cracked red blood cells to obtain single cell suspension,cultured with 0.5?g/ml anti-CD3 antibody for 48h,and then cultured with 20ng/ml IL-2 for 6 days,differentiated lymphocytes into cytotoxic CD8~+T(CTL)cells,stravated with serum-free 1640 medium for 12h,and then stimulated with 2?g/ml anti-CD3/CD28 antibodies and different concentrations of Calcineurin inhibitors(2?M FK506,500nM Cyclosporin A)or DAPK1 inhibitor(2?M DAPK1i)together for 1h,collected cell samples,extracted proteins and detected the expression of phosphorylated and total protein of the mTORC1 downstream effect substrates ribosomal protein S6 and P70S6K by immunoblotting.3.2 Detection of the effects of Calcineurin and DAPK1 shRNA on the mTORC1activity of CD8~+T cells:Isolated the spleen from wild-type mice,cracked red blood cells to obtain single cell suspension,purified CD8~+T cells by positive selection,then cultured with 5?g/ml anti-CD3/CD28 antibody and T cell culture medium for 24h.Calcineurin shRNA retrovirus,DAPK1 shRNA retrovirus and blank vectors were transfected to CD8~+T cells respectively,after 24h,re-stimulated with 20ng/ml IL-2 for60min.Collected cell samples,extracted proteins and detected the expression of phosphorylated and total protein of the mTORC1 downstream effect substrates ribosomal protein S6 by immunoblotting.3.3 Detection of mTORC1 activity in CD8~+T cells in DAPK1-KD knockout mice(CD4-Cre;DAPK1-KDfl/fl):Isolated the spleen from wild-type and DAPK1-KD knockout mice,cracked red blood cells to obtain single cell suspension.Single cells cultured with 2?g/ml anti-CD3/CD28 antibodies and T cell culture medium for 0,15,30,60min,the expression of phosphorylated and total protein of the mTORC1 downstream effect substrates ribosomal protein S6 was detected by flow cytometry.3.4 Detection of mTORC1 activity in CD8~+T cells in DAPK1-DD knockout mice(CD4-Cre;DAPK1-DDfl/fl):Isolated the spleen from wild-type and DAPK1-DD knockout mice,cracked red blood cells to obtain single cell suspension.Single cells cultured with 2?g/ml anti-CD3/CD28 antibodies and T cell culture medium for 0,15,30,60min,the expression of phosphorylation of the mTORC1 downstream effect substrates ribosomal protein S6 was detected by flow cytometry.4.Effects of DAPK1 on signaling pathways PLC-?,Erk,MAPK,NF-?B and STAT5induced by TCR or IL-24.1 Detection of signaling pathways activities such as PLC-?,Erk,MAPK,NF-?B and STAT5 induced by TCR or IL-2 in CD8~+T cells in DAPK1-KD knockout mice:Isolated the spleen from wild-type and DAPK1-KD knockout mice,cracked red blood cells to obtain single cell suspension,cultured with 0.5?g/ml anti-CD3 antibody for 48h,and then cultured with 20ng/ml IL-2 for 6 days,differentiated lymphocytes into cytotoxic CD8~+T(CTL)cells,stravated with serum-free 1640 medium for 12h,and then stimulated with 2?g/ml anti-CD3/CD28 antibodies or 20ng/ml IL-2 for 60min.Collected cell samples,extracted proteins and detected the expression of phosphorylated and total protein of PLC-?,Erk,P38,P65 and STAT5.4.2 Detection of signaling pathways activities PLC-?,Erk,MAPK,NF-?B and STAT5 induced by TCR or IL-2 in CD8~+T cells in DAPK1-KD knockout mice:Isolated the spleen from wild-type and DAPK1-DD knockout mice,cracked red blood cells to obtain single cell suspension,cultured with 0.5?g/ml anti-CD3 antibody for 48h,and then cultured with 20ng/ml IL-2 for 6 days,differentiated lymphocytes into cytotoxic CD8~+T(CTL)cells,stravated with serum-free 1640 medium for 12h,and then stimulated with 2?g/ml anti-CD3/CD28 antibodies or 20ng/ml IL-2 for 60min.Collected cell samples,extracted proteins and detected the expression of phosphorylated and total protein of PLC-?,Erk,P38,P65 and STAT5.5.Effect of DAPK1 on CD8~+T cell function in mice with acute Lymphocytic choriomeningitis virus infection(LCMV)5.1 Establishment of acute LCMV viral infection mouse model:Injected wild type and DAPK1-KD knockout mice by i.p.with 2×10~5 PFU LCMV-Armstrong virus fluid.5.2 Detection of the effects of DAPK1 on mTORC1 activity in CD8~+T cells in LCMV model mice:Isolated the spleen from wild-type and DAPK1-DD knockout LCMV model mice at 3,5,and 8 dpi,cracked red blood cells to obtain single cell suspension.The expression of phosphorylation of the mTORC1 downstream effect substrates ribosomal protein S6 in activated CD8~+T(CD44~+CD8)cells was detected by flow cytometry.5.3 Detection of the effects of DAPK1 on proliferation and apoptosis of CD8~+T cells in LCMV model mice:(1)At 8 and 30 dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.Numbers and frequencies of total CD8~+T cells and activated CD8~+T cells were detected.The expression of Ki-67,Annexin V and PI were measured in activated CD8~+T cells by flow cytometry.(2)At 8 dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.Activated CD8~+T cells were purified by magnetic beads,RNA was extracted,and the expression of mRNA of anti-apoptosis molecular Bcl-2 in activated CD8~+T cells was detected by real-time quantitative PCR.5.4.Detection of the effects of DAPK1 on effective function of CD8~+T cells in LCMV model mice:(1)At 8 dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.The expression of IL-2,TNF-?,IFN-?,Granzyme B and T-bet in activated CD8~+T cells was detected by flow cytometry.(2)At 8 dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.Activated CD8~+T cells were purified by magnetic beads,RNA was extracted,and the expression of mRNA of Granzyme B,Granzyme K,Ifng,Tbx21 and Klrg1 were detected by quantitative PCR5.5 Detection of the effects of DAPK1 on CD8~+T cell differentiation in LCMV model mice:(1)At 8 and 30 dpi,isolated the spleen from LCMV model mice,cracked red blood cells to obtain single cell suspension.Stained with KLRG1 and CD127 in activation CD8~+T cells,the proportion and number of terminal effect(TE)CD8~+T cells and Memory precursors(MP)CD8~+T cells were detected by flow cytometry.(2)At 8 and 30 dpi,isolated the spleen from LCMV model mice,cracked red blood cells to obtain single cell suspension.Expression of memory-related factors CD127,CXCR3and transcription factor TCF-1,Eomes in activated CD8~+T cells was detected by flow staining.5.6 Detection of the effects of DAPK1 on the glucose metabolism of CD8~+T cells in LCMV model mice:(1)At 8 and 30 dpi,isolated the spleen from LCMV model mice,cracked red blood cells to obtain single cell suspension.The expression of 2-NBDG in activated CD8~+T cells was detected by flow cytometry.(2)At 8 dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.Activated CD8~+T cells were purified by magnetic beads,RNA was extracted,and the expression of mRNA of glycolysis related molecules Eno1,Hif1?,Ldha,Pdk1,Scl2a,Tpi1,Pfkm,Pgk1 and oxidative phosphorylation related molecules Atp5i,Cox5a,Ndufa2,Nrf1 in activated CD8~+T cells were detected by quantitative PCR.5.7 Detection of the effects of DAPK1 on anti-infection function of CD8~+T cells in LCMV model mice:At 5 dpi,mice were executed,serum,spleen,liver and lung tissue were separated and tissue viral RNA was extracted.Viral titer was detected by quantitatively PCR.6.Effect of DAPK1 on antigen-specific CD8~+T cells in LCMV mouse model6.1 Establishment of adoptive immunity acute LCMV model:P14 CD8~+T cells were isolated and purified from WT P14(CD45.2)and CD4-Cre;P14;DAPK1-KDfl/fl(CD45.2)mice.5×10~5 WT(CD45.2)or DAPK1-KDfl/fl(CD45.2)P14 CD8~+T cells were intravenous injection into the receptor(CD45.1 WT)mice,after 16h,2×10~5 PFU LCMV-Armstrong virus fluid was injected by i.p.to the receptor mice.6.2 Detection of the effects of DAPK1 on proliferation and apoptosis of antigen-specific CD8~+T cells in LCMV model mice:o At 8 and 30 dpi,isolated the spleen from LCMV model mice,cracked red blood cells to obtain single cell suspension.Numbers and frequencies of P14 CD8~+T cells were detected.The expression of Ki-67,Annexin V and PI were measured in P14 CD8~+T cells by flow cytometry.6.3 Detection of the effects of DAPK1 on effective function of antigen-specific CD8~+T cells in LCMV model mice:At 8 dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.The expression of Granzyme B,IFN-?,T-bet,KLRG1 and CD127 in P14 CD8~+T cells was detected by flow cytometry.6.4 Detection of the effects of DAPK1 on antigen-specific CD8~+T cell differentiation in LCMV model mice:At 8 dpi,isolated the spleen from LCMV model mice,cracked red blood cells to obtain single cell suspension.Expression of memory-related factors CD127,CXCR3 and transcription factor TCF-1 in P14 CD8~+T cells was detected by flow staining.7.Effect of DAPK1 knockout environment on the function of antigen-specific CD8~+T cells in LCMV model mice7.1 Establishment of endogenous adoptive immunity acute LCMV model:P14 CD8~+T cells were isolated from the spleen of WT P14(CD45.1~+CD45.2~+)mice.5×10~5 P14CD8(CD45.1~+CD45.2~+)T cells were intravenous injection into the receptor(CD45.1 WT and CD45.2;CD4-Cre;P14;DAPK1-KDfl/fl)mice,after 16h,2×10~5 PFU LCMV Armstrong virus fluid was injected by i.p.to the receptor mice.7.2 Detection of the effects of DAPK1 knockout environment on the proliferation and apoptosis of antigen-specific CD8~+T cells in LCMV model mice:At 8dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.Numbers and frequencies of P14 CD8~+T cells were detected.The expression of Ki-67,Annexin V and PI were measured in activated CD8~+T cells by flow cytometry.7.3 Detection of the effect of DAPK1 knockout environment on antigen-specific CD8~+T cell effect function in LCMV model mice:At 8dpi,mice were executed and single cell suspension was made from the spleen cracking red blood cells of mice.Expression of Granzyme B in P14 CD8~+T cells from different receptor mice was detected by flow Cytometry.Results1.The mechanism of Calcineurin activation DAPK1:Calcineurin can be combined with DAPK1 calcium-modulated protein region(CaM),stimulate with TCR,the combination of Calcineurin and DAPK1 increases,which leads to the enhanced activity of DAPK1.2.DAPK1 mechanism for regulating TSC2:The combination of DAPK1 and TSC2,stimulate with TCR,the combination of DAPK1 and TSC2 increased.3.Effect of Calcineurin and DAPK1 on mTORC1 pathway activity:In CD8~+T cells,Calcineurin and DAPK1 can regulate the activity of mTORC1.The activity of mTORC1decreased when knocked out the DAPK1 kinase region(KD)and the death domain(DD)specifically in CD8~+T cells.4.Effect of DAPK1 on signaling pathways PLC-?,Erk,MAPK,NF-?B and STAT5induced by TCR or IL-2:Immunoblot results show that there was no effect on the activity of TCR or IL-2 induced PLC-?,ERK,MAPK,NF-?B and STAT5 signaling pathways inDAPK1-deficient CD8~+T cells.5.Effect of DAPK1 on CD8~+T cells in LCMV mouse model:Deficiency of DAPK1will lead to less mTORC1 activity,decreased cell proliferation and increased apoptosis,weaker effective function and the ability to differentiate into memory progenitor cells decreased in CD8~+T cells.Besides,deficient DAPK1 also leads to reduced glucose metabolism and impaired anti-infection ability in CD8~+T cells.6.Effect of DAPK1 on antigen-specific CD8~+T cells in LCMV mouse model:Deficiency of DAPK1 leads to reduced proliferation,increased apoptosis,decreased effective function and the diminished ability to differentiate into memory progenitor cells of antigen-specific CD8~+T cells.7.Effects of DAPK1-deficient environment on antigen-specific CD8~+T cell function in LCMV model mice:DAPK1-deficient environment had no effect on the proliferation,apoptosis and effect function of antigen-specific CD8~+T cells.8.Effect of DAPK1 on the function of CD8~+T cells in bone marrow transplantation mouse model:In the bone marrow transplantation mouse model,deficiency of DAPK1reduced the proliferation and activation and weakened the effect function of CD8~+T cells.9.Effect of DAPK1 on antitumor function of CD8~+T cells:Deficiency of DAPK1leads to faster growth of tumor and weaker effect function of CD8~+T cells.Conclusion1.Calcineurin activated DAPK1 by combining with calcium-modulated protein region(CaM)of DAPK1.2.DAPK1 inhibited the formation of TSC1/TSC2 complexes and activated mTORC1signaling pathway by combining death domain(DD)with TSC2.3.Deficiency of DAPK1 resulted in reduced CD8~+T cell function in acute viral infections.Objective: Sepsis is a clinically infectious disease with a high mortality rate.It starts with a strong inflammatory response in the initial stage,which causes severe immunosuppression,damaged organ functions and leading to an increase in the course of the disease.The liver plays a major role in regulating the immune response of sepsis patients and is also the main target organ for immune injury.Current studies show that CD8~+ T cells play a critical role in infection,but the immune response of CD8~+ T cells in the liver of sepsis is unclear.Studies have shown that enhancing the effective function of CD8~+ T cells can curb the persistence of infection and gradually remove infection in infective diseases.Therefore,the study of the immune response of CD8~+ T cells in sepsis is of profound significance for the treatment of sepsis.The purpose of this study was to study the immune response of CD8~+ T cells in the liver of the main target organ of sepsis,and to improve the course of disease and treat sepsis patients by enhancing the anti-infective ability of CD8~+ T cells in the organ.Methods:1.Detection of CD8~+ T cell function in peripheral blood of healthy volunteers and sepsis patientsThe expression of Tim-3 and Annexin V in peripheral blood CD8~+ T cells: lymphocytes were isolated from the peripheral bllod of healthy volunteers(n=20)and sepsis patients(n=35)and stained with CD8,Tim-3 and Annexin V.The cells were analyzed by flow cytometry.2.Detection of CD8~+ T cell function in sepsis and sham model mice2.1 Construction of sepsis(CLP)mouse model: Narcotized and fixed normal healthy wild mice in a supine position,cleaned abdomen hair and disinfected.Cutted open the abdominal cavity,then ligated and perforated the cecum,sutured the abdominal cavity and resuscited the mice,induced sepsis response in mice.The control group(Sham)mice also cutted open and sutured the abdominal cavity,but did not do any treatment of the cecum.The remaining processing methods are consistent with the experimental group.At different time points after the operation,mice were executed and follow-up experiments were carried out.2.2 Separation of lymphocytes from the spleen: Killed mice and took the spleen,cutted and grinded the filter,and made a single cell suspension.After centrifugation,the erythrocyte lysate was added to crack the cell mass to remove the red blood cells.The remaining cells were lymphocytes,which are re-suspended with 1×PBS.2.3 Isolation of lymphocytes in the liver: Killed mice and took liver,chopped and grinded filtration,made single-cell suspension,centrifuged with 33.75% Percoll to re-suspend cells,then centrifuged to remove hepatocytes,isolated and purifyied lymphocytes.Joined the erythrocyte lysate to crack the isolated lymphocytes and removed the red blood cells.The remaining cells were purified lymphocytes,which were re-suspended with 1×PBS.2.4 Expression of Tim-3 in the spleen and liver of CD8~+ T cells in mice: The spleen and liver lymphocytes of the septic experimental group and the control group were isolated at different time points,and the cells of each 1×10~6 were used for flow staining,stained CD8 and Tim-3 antibodies respectively,and their expression was detected by flow cytometry.3.Apoptosis of splenic and liver CD8~+ T cells in septic model mice3.1 Detection of the expression of Annexin V in the liver CD8~+ T cells of sepsis model mice: 1×10~6 splenic and liver lymphocytes were used for flow staining,stained CD8 and Annexin V respectively,and detected by flow cytometry.3.2 Construction of Tim-3 mutation(Tim-3mut)mice: Tim-3 mutant mice were selected C57BL/6 background mice that mutated their encoding the end h21883 position of Tim-3gene of the molecular cytoplasmic region to construct the mouse.3.3 Detection of the expression of Annexin V in CD8~+ T cells in the liver of Tim-3mutant mice: 1×10~6 liver lymphocytes were used for flow staining,stained CD8 and Annexin V respectively,and detected by flow cytometry.4.The protective effect of ?-lactose on damaged liver in sepsis mice4.1 Effects of ?-lactose on the expression of Tim-3 in the liver CD8~+ T cells of sepsis mice: Injected 1×PBS or ?-lactose into peritoneal cavity of the mice with sepsis model,killed the mice for experiments in different time points.Separated and purified liver lymphocytes for staining,stained with CD8 and Tim-3 and detected by flow cytometry.4.2 Effects of ?-lactose on the liver pathological tissue in sepsis mice: Injected 1×PBS or ?-lactose into peritoneal cavity of the mice with sepsis model and killed the mice for experiments in different time points.Fixed and sliced liver tissue for immunohistochemical staining.The pathological images of liver tissue were taken under fluorescence microscope.4.3 Effects of ?-lactose on liver function in sepsis mice: Intraperitoneal injection of 1×PBS or ?-lactose to the septic model mice and killed the mice for experiments in different time points.Blood were collected from the eyeball and centrifuged to obtain serum.Expression levels of ALT and AST in serum were detected by ALT and AST kits respectively.4.4 Effects of ?-lactose on the expression of inflammatory factors in the liver of sepsis mice: Intraperitoneal injection of 1×PBS or ?-lactose to the septic model mice,the mice were executed after 24 h,the liver of mice was separated and the liver tissue was homogenized by homogenization instrument to extract tissue RNA by Tizol.The m RNA expression of Il-6,Il-23,Il-27 and Tnf? in liver tissue was detected by quantitative PCR.Result:1.Expression of Tim-3 and Annexin V in peripheral blood of sepsis patients:Compared with normal volunteers,CD8~+ T cells in peripheral blood of sepsis patients showed high expression of Tim-3 and Annexin V.In addition,Annexin V+CD8~+ T cells expressed higher Tim-3 in the peripheral blood of sepsis patients.2.Tim-3 expression of CD8~+ T cells in spleen of sepsis model(CLP)mice: There was no difference in Tim-3 expression of CD8~+ T cells in spleen of sepsis model mice compared with control group(Sham)mice.3.Tim-3 expression of CD8~+ T cells in liver of sepsis model(CLP)mice: Tim-3expression of CD8~+ T cells in liver of sepsis model mice showed a biphasic increase over time compared with that of control(Sham)mice.4.Annexin V expression of CD8~+ T cells in spleen of sepsis model(CLP)mice: There was no difference in Annexin V expression of CD8~+ T cells in spleen of sepsis model mice compared with control group(Sham)mice.5.Annexin V expression in liver CD8~+ T cells of mice with sepsis model(CLP):Compared with control group(Sham)mice,Annexin V expression in liver CD8~+ T cells of mice with septic model increased at the begining and then decreased with time.6.Annexin V expression in liver CD8~+ T cells of tim-3 mutant mice in sepsis model(CLP): Compared with normal wild-type sepsis model mice in the control group,Annexin V expression in liver CD8~+ T cells of Tim-3 mutant sepsis model mice was significantly decreased.7.Effect of ?-lactose on Tim-3 expression of liver CD8~+ T cells in mice with sepsis model(CLP): Compared with sepsis model mice which injected with 1×PBS intraperitoneally in control group,the expression of Tim-3 in CD8~+ T cells of sepsis mice injected with ?-lactose was significantly decreased.8.Effects of ?-lactose on liver function in CLP mice: Compared with sepsis mice injected with 1×PBS by i.p.,liver Injury and inflammation of hepatic lobules were significantly reduced,and liver cell necrosis was even less in sepsis mice injected with?-lactose.9.Effect of ?-lactose on the expression of inflammatory factors in liver of mice with sepsis model(CLP): Compared with sepsis mice injected with 1×PBS intraperitoneally,the expression of IL-23,IL-27 and TNF-? in the liver of sepsis mice injected with?-lactose was significantly decreased,but the expression of IL-6 was not changed.Conclusion:1.In the peripheral blood of sepsis patients,the increase of Tim-3 expression was related to the apoptosis of CD8~+ T cells.2.In the liver CD8~+ T cells of sepsis model mice,the expression of Tim-3 increased bipolar with time.3.In the liver of sepsis model mice,Tim-3 mediated apoptosis of CD8~+ T cells.4.?-lactose can reduce liver damage in sepsis model mice. |
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