The Effect And Mechanism Of The TLR4 Inhibitor TAK-242 On Acute Liver Failure

Background:Acute liver failure is a serious clinical disease with poor prognosis and high mortality.However,the mechanism of fulminant hepatitis is not fully understood.Studies have shown that adverse inflammatory response plays an important role in the acuete liver failure.A large number of immune cells infiltrate the liver to produce a large number of pro-inflammatory cytokines,thereby inducing apoptosis and necrosis of liver cells.Currently,many studies have suggested that liver function can be restored;however,liver transplantation is still the more effective method.Owing to liver origin,transplant rejection,surgical complications,and other reasons,mostpatients cannot be effectively treated.Therefore,new and effective treatment methods are needed.TAK-242 is a newly developed highly selective TLR4 signaling pathway inhibitor that can treat lung inflammation and kidney damage.However,it is not clear whether TAK-242 has a therapeutic effect on acuete liver failure.Myeloid-derived suppressor cells(MDSC)are a group of heterogeneous immune cells derived from bone marrow that play an important immunosuppressive and protective role in human hepatitis,liver cancer,liver fibrosis and other diseases through different mechanisms.Therefore,based on the above research,we proposed the following hypothesis:whether TAK-242 can exert a protective effect on explosive hepatitis through the regulation of MDSC.Ojective:To investigate the effect of TLR4 inhibitor TAK-242 on LPS/D-GalN model induced Acuete liver failure in mice,as well as its mechanism of action onMDSC in Acuete liver failure,so as to provide theoretical basis for basic research and clinical intervention of Acuete liver failure.Method:1.C57BL/6 mice were intraperitoneally injected with LPS(10 μg/kg)/D-GalN(250 mg/kg)to establish a acuete liver failure model,and 3 hours later,they were intraperitoneally injected with TAK-242(5 mg/kg)and observed Whether TAK-242 can treat acuete liver failure in mice;mice are pretreated with TAK-242(5 mg/kg)for 3 hours,intraperitoneally injected with LPS(10 μg/kg)/D-GalN(250 mg/kg),and then Observe the effect of TAK-242 on acuete liver failure,and explore the appropriate administration time of TAK-242.2.Male C57BL/6 mice were pretreated with TLR4 inhibitor TAK-242(5 mg/kg)for 3 h,then injected intraperitoneally with LPS(10 μg/kg)/D-GalN(250 mg/kg)at 3 h,6 h and 12 h to establish the model of Acuete liver failure.(1)The serum of mice was collected for the detection of ALT and AST.(2)The expression levels of cytokines IL-6,TNF-α and IL-12 in serum of mice were determined by ELISA.(3)10%liver homogenate was collected from mice liver to detect the expressions of SOD,MDA GSH and MPO in liver.(4)The liver of mice was collected and HE staining was performed to observe the damage of liver tissue.(5)The mRNA levels of IL-6,TNF-α and IL-12 in mice liver were detected by qRT-PCR.(6)The mononuclear cells from liver were extracted,and the proportion of myeloid inhibitory cells(CD11b+Gr-1+)was detected by flow cytometry.(7)The proportion of CD11b+Gr-1+ in spleen cells was determined by flow cytometry.3.Male C57BL/6 mice were pretreated with TLR4 inhibitor TAK-242(5 mg/kg)for 3 h and injected intraperitoneally with LPS(15 μg/kg)/D-GalN(400 mg/kg)to observe the survival of the mice.4.C57BL/6 mice were injected with Gr-1 depletion antibody through tail vein and the MDSC adoptive transfer for mice to observe the liver injury of mice.5.MDSC cultured in vitro:(1)Different concentrations of TAK-242 were used to treat MDSC in vitro,and the proportion of MDSC was observed after 4 days of culture.(2)MDSC was cultured for 4 days and treated with different concentrations of TAK-242 for 6 hours,then the mRNA levels of IL-10,iNOS,Arg-1 and NOx 1 in MDSC were detected byqRT-PCR.(3)MDSC was cultured for 4 days and treated with different concentrations of TAK-242 for 1 hto extract proteins.The relative expression levels of phosphorylated p38,ERK1/2,JNK and p65 in MDSC were detected by Western blot.Results:1.TAK-242 has no therapeutic effect on acuete liver failure in mice,but TAK-242 pretreatment has a protective effect on acuete liver failure.2.TAK-242 pretreatment groups:(1)the serum ALT,AST levels significantly reduced;(2)the serum proinflammatory cytokines reduced;(3)the higher expression of SOD and GSH in liver tissue,MPO and MDA decreased;(4)H&E staining showed that hepatic injury reduced;(5)proinflammatory cytokines expression in liver tissue reduced;(6)The proportion of MDSC(CD11b+Gr-1+)in spleen and liver of mice was increased.3.The survival rate of mice was improved by TAK-242 pretreatment.4.Adoptive transfer of MDSC can ameliorate Acuete liver failure in mice.In vitro,TAK-242 inhibited the phosphorylation of MAPK and NF-κB signaling pathways,and increased the recruitment of MDSC and the expression of related immunosuppressive cytokines.Conclusion:TAK-242 alleviates LPS/D-GalN-induced Acuete liver failure by promoting the accumulation of MDSC.