| Part I Short-term use of MyD88 inhibitor TJ-M2010-5 prevents infection-related acute liver injury in mice[Objective] To investigate the effectiveness of MyD88 inhibitor for the treatment of D-gal/LPS-induced acute liver injury.[Methods] 1.Mice received intraperitoneal(i.p.)injection of D-gal and LPS at a dose of 800 mg/kg and 80 ?g/kg body weight,respectively,to induce acute liver injury.(1)The MyD88 level from hepatic tissue was determined via western blotting and real-time PCR after D-gal/LPS stimulation.(2)The mice were divided into four groups: saline group: BALB/c mice were injected i.p.with saline as a control with an equal volume of D-gal/LPS;D-gal/LPS group: BALB/c mice were treated with 0.5% CMC before D-gal/LPS injection;TJ-M2010-5 group: BALB/c mice were pre-treated with TJ-M2010-5 and then injected with D-gal/LPS;MyD88 knock-out(KO)group: BALB/c MyD88-/-mice were injected D-gal/ LPS without any other intervention.The survival of each group mice was observed within 48 hours after D-gal/LPS injection.Serum samples were collected from mouse at 12 hours after D-gal/LPS injection and used to measure the levels of ALT,AST,IL-1?,TNF-?,IL-6,i NOS and COX2.Liver tissues were obtained at 12 hours and 24 hours after D-gal/LPS injection and stained with TUNEL and H&E to evaluate the numbers of apoptotic cells in liver and the degree of acute liver injury,respectively.The levels of IL-1?,TNF-?,IL-6,i NOS and COX2 in liver tissue were detected by real-time PCR.Liver tissues were also used to perform immunofluorescence to evaluate the expression of TNF-? at 6 hours and 12 hours after D-gal/LPS injection.Western blots were used to detect the NF-?B nuclear translocation and the expression of TLR4,I?B-?,TNFR1,p-P38,p-ERK,p-JNK in liver tissues.The mononuclear cells were isolated from liver tissues at at 6 hours after D-gal/LPS injection and stained with the antibodies of F4/80,CD80,CD86 to detect the percentage of actived macrophages in liver via flow cytometry.(3)Clophosome was used to deplete macrophages in mice.Then mice were injected with D-gal/LPS.H&E was performed to evaluate the degree of acute liver injury and real-time PCR was used to detect the levels of IL-1?,TNF-?,IL-6,i NOS and COX2 in liver.2.Bone marrow-derived macrophages(BMDMs)were isolated and cultured.At 6 days,Before stimulation with LPS(500ng/ml),BMDMs were incubated with TJ-M2010-5 at different concentrations(0,5,10,30 ?mol/L)for 2 hours.12 hours later,the levels of IL-1?,TNF-?,IL-6,i NOS and COX2 were measured by ELISA and real-time PCR.And the F4/80+CD80+ or F4/80+CD86+ BMDMs was detected by flow cytometry.[Results] 1.(1)The expression of MyD88 was significantly higher in D-gal/LPS mice liver.(2)Mice were all died within 36 hours after D-gal/LPS injection,but pretreatment with TJ-M2010-5 and knock-out of MyD88 in mice dramatically improved their survival rate,which reached to 73.3% and 80.0% at 48 hours,respectively.ALT and AST levels were dramatically elevated in D-gal/LPS group rather than the saline group(P < 0.001).Strikingly,pretreatment with TJ-M2010-5 or knock-out of MyD88 efficiently prevented an increase in ALT and AST levels at 12 h(P < 0.001,P < 0.001).TUNEL staining revealed a small amount of hepatocyte apoptosis at 12 hours and numerous apoptotic hepatocytes at 24 hours after D-gal/LPS stimulation.TJ-M2010-5 pretreatment or knock-out MyD88 could significantly reduce the number of apoptotic hepatocytes at 12 hours and 24 hours upon D-gal/LPS stimulation.H&E staining showed slight liver congestion,hepatocyte necrosis and inflammatory cells infiltration at 24 hours in D-gal/LPS group.However,these pathological alterations were significantly ameliorated,except for mild edema of hepatocytes,by TJ-M2010-5 pretreatment or knocking out MyD88.The levels of IL-1?,TNF-?,IL-6,i NOS and COX2 were significantly increased in both liver tissues and serum after D-gal/LPS injection,but which could be reduced by TJ-M2010-5 pre-treatment or knocking out MyD88.The results of immunofluorescence also showed that the levels of TNF-? were significantly increased in D-gal/LPS group,and application of TJ-M2010-5 or MyD88 knock-out minimized this increase at both 6 hours and 12 hours after D-gal/LPS injection.The results of flow cytometry showed the massive amounts of liver macrophages were activated in the D-gal/LPS group and pretreatment with TJ-M2010-5 or knock-out of MyD88 significantly suppressed such activation.After D-gal/LPS injection,the content of NF-?B nuclear transcription and the expression of TLR4,TNFR1,p-P38,p-ERK,p-JNK was increased,but the expression of I?B-? was decreased in liver tissue.However,pretreatment with TJ-M2010-5 or MyD88 knock-out could suppress NF-?B nuclear transcription,the increasing of TLR4,TNFR1,p-P38,p-ERK,p-JNK,and the decreasing of I?B-?.(3)H&E staining showed depleting macrophages in mice could protect liver against D-gal/LPS-induced lesion.And the levels of Inflammatory factors in liver were decreased in D-gal/LPS mice after depleting macrophages.2.LPS stimulation increased BMDMs expression in CD80 and CD86.TJ-M2010-5 suppressed BMDMs expression in a dose-dependent manner,especially 30 ?mol/L TJ-M2010-5 significantly inhibited BMDMs activation.In addition,the levels of IL-1?,TNF-?,IL-6,i NOS and COX2 at 12 hours after LPS stimulation were increased and this increase were inhibited by TJ-M2010-5 in a dose-dependent manner.[Conclusion] MyD88 is highly expressed and has a key role in D-gal/LPS-induced liver injury.Inhibiting or knocking out MyD88 protects liver against D-gal/LPS-induced injury,which is caused from inhibiting macrophage activation via inactivating NF-?B and MAPK signaling pathway and decreasing pro-inflammatory factors in liver.Part II Short-term use of MyD88 inhibitor TJ-M2010-5 prevents autoimmune liver injury in mice[Objective] To investigate the effectiveness of MyD88 inhibitor for the treatment of Con A-induced acute liver injury,and to investigate the influences of MyD88 inhibitor on T lymphocyte and macrophage.[Methods] Mice received intravenous injection of ConA to induce acute liver injury.The mice were divided into four groups: normal control group: BALB/c mice were injected with saline as a control with an equal volume of Con A;Model group: BALB/c mice were treated with 0.5% CMC before Con A injection;TJ-M2010-5 group: BALB/c mice were pre-treated with TJ-M2010-5 and then injected with Con A;MyD88 knock-out(KO)group: BALB/c MyD88-/-mice were injected Con A without any other intervention.The survival of each group mice was observed within 24 hours after lethal dose of Con A(25mg/kg)injection.Mice received non-lethal dose of Con A(15mg/kg)and were sacrificed at 12 hours.Serum samples were collected to measure the concentrations of ALT,AST and ALP.Liver tissues were obtained and stained with H&E and TUNEL to evaluate the degree of acute liver injury and the numbers of apoptotic cells in liver,respectively.The levels of IL-4,IFN-?,IL-1? and TNF-? in liver tissue were detected by real-time PCR.The levels of MDA and SOD in liver were measured using kits according to the manufacturer's instructions.The mononuclear cells were isolated from liver tissues to detect the percentage of activated macrophages and T lymphocytes in liver tissues via flow cytometry.Lymphocyte and BMDM were isolated from mouse lymph nodes and bone marrow,respectively,and cultured in vitro.After resuscitation,the hepatocyte line of mouse,AML-12,was also cultured in vitro.After stimulation with Con A and incubation with TJ-M2010-5,respectively,the activity of these three cells was measured by CCK-8 kit.Lymphocytes were stimulated with Con A,meanwhile pre-treatment with TJ-M2010-5.The activation of lymphocytes was measured by flow cytometry and the levels of IL-4,IFN-?,IL-1? and TNF-? were detected by real-time PCR.BMDMs were stimulated with Con A.The polarization of BMDMs was measured by flow cytometry and the levels of TNF-? and IL-1? were detected by real-time PCR.BMDMs were cultured with the supernatant of activated lymphocytes,meanwhile pre-treatment with TJ-M2010-5.The polarization of BMDMs and the expression of TNF-? were measured by flow cytometry;the levels of TNF-? and IL-1? were detected by real-time PCR;the expression of MyD88 and the content of NF-?B nuclear transcription were measured by WB.AML-12 was incubated with the supernatant of activated lymphocytes.The apoptosis and the activity of AML-12 were measured by flow cytometry and CCK-8 kit,respectively;the level of HMGB1 was measured from cytosolic protein and nuclear protein;the level of HSP60 was measured from total protein;after pre-treatment with TJ-M2010-5,the level of HMGB1 was measured from cytosolic protein.After,co-culture of lymphocytes,BMDMs and Con A,the supernatant was used to stimulate with AML-12,meanwhile pre-treatment with TJ-M2010-5,the apoptosis of AML-12 was measured by flow cytometry.[Results] The mortality of mice injected with the lethal dose of Con A reached 73.3% within 24 hours.Pretreatment with TJ-M2010-5 and knock-out of MyD88 in mice significantly decreased their mortality,which down to 13.3% and 0,respectively.The expression of MyD88 was significantly higher in Con A mice liver and the levels of ALT,AST and ALP were dramatically elevated.Strikingly,pretreatment with TJ-M2010-5 or knock-out of MyD88 efficiently prevented an increase in ALT,AST and ALP levels.H&E staining showed hepatocyte necrosis and inflammatory cells infiltration at 12 hours after Con A injection.However,these pathological alterations were significantly ameliorated,except for mild edema of hepatocytes,by TJ-M2010-5 pretreatment or knocking out MyD88.TUNEL staining revealed numerous apoptotic hepatocytes at 12 hours after Con A stimulation.TJ-M2010-5 pretreatment or knock-out MyD88 could significantly reduce the number of apoptotic hepatocytes at 12 hours upon Con A stimulation.The levels of IL-4,IFN-?,IL-1? and TNF-? were significantly increased in liver tissues after Con A injection,but which could be reduced by TJ-M2010-5 pre-treatment or knocking out MyD88.After Con A stimulation,the level of MDA was increased and the level of SOD was decreased in liver tissues.Pretreatment with TJ-M2010-5 or knock-out MyD88 significantly restored both MDA and SOD levels.Macrophages and T lymphocytes in liver tissues were both activated after Con A stimulation,and TJ-M2010-5 pretreatment significantly suppressed the activation of macrophages,but could not suppress the activation of T lymphocytes.The results of CCK-8 showed Con A promoted T lymphocytes proliferation but had no influence on BMDMs and AML-12,and TJ-M2010-5 had no any cytotoxicity on T lymphocytes,BMDMs and AML-12 within 30 ?mol/L.The percentage of CD3+CD4+CD69+ and CD3+CD8+CD69+ Cells was increased after Con A stimulation in a dose-dependent manner in vitro.This increase could not be suppressed by TJ-M2010-5.Real-time PCR results revealed the levels of IL-4,IFN-?,TNF-? were up-regulated with or without TJ-M2010-5,but the level of IL-1? had no obviously changed.The percentage of F4/80+CD11c+ BMDMs and the levels of TNF-?,IL-1? m RNA had no changed after Con A stimulation.The percentage of F4/80+CD11c+ BMDMs and the expression of TNF-? in BMDMs were increased after culturing with the supernatant of Con A-induced activated lymphocytes.Pre-treatment with TJ-M2010-5 could suppress such increase in a dose-dependent manner.The up-regulated levels of TNF-? and IL-1? m RNA were also inhibited by TJ-M2010-5 in a dose-dependent manner.WB results showed the expression of MyD88 and the content of NF-?B nuclear transcription were increased after the supernatant of Con A-induced activated lymphocytes stimulation,and TJ-M2010-5 could suppress NF-?B nuclear transcription.The number of apoptotic AML-12 was increased and the activity of AML-12 is decreased while incubation with the supernatant of Con A-induced activated lymphocytes.And the level of HMGB1 in cytoplasm was increased with or without TJ-M2010-5,while the level of HMGB1 in karyon was decreased.The HSP60 was overexpressed after stimulation.But the apoptosis of AML-12 was suppressed by TJ-M2010-5 in a dose-dependent manner after the supernatant of co-culture of lymphocytes,BMDMs and Con A stimulation.[Conclusion] MyD88 is highly expressed and has a key role in Con A-induced liver injury.Inhibiting or knocking out MyD88 protects liver against Con A-induced injury,which is caused from inhibiting macrophage but not T lymphocytes activation.Macrophage activation maybe result from DAMPs released by damaged hepatocytes and activated T lymphocytes. |
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