The Role Of HMGB1/ERS Signaling Pathway In Rifampin Induced Liver Injury In Mice And Its Possible Mechanism

Objective:Rifampicin,as an antituberculous drug,can cause different degrees of liver damage,but the specific mechanism is not clear.Therefore,the main purpose of this study is:(1)to establish rifampicin induced liver damage model in mice,to study the relationship between HMGB1 and ers;(2)to establish animal experiments and cell models,to observe the role of hmgb1-rage and hmgb1-tlr4 signaling pathway in ers induced by rifampicin,The possible mechanism of HMGB1 / ERS in liver damage model induced by rifampin was discussedMethods:(1)Thirty two C57 BL / 6 mice were reared adaptively in SPF environment,and then randomly divided into four groups: normal group,model group,HMGB1 antibody intervention group and Ig G antibody intervention group.The mice in model group,HMGB1 antibody intervention group and Ig G antibody intervention group were given rifampicin 200 mg /(kg · d)by gavage every day,and those in normal group were given the same amount of solvent by gavage.On the 1st,3rd and 5th day after the establishment of the model,the mice in the HMGB1 antibody intervention group and Ig G antibody intervention group were intraperitoneally injected with5 mg / kg of HMGB1 polyclonal antibody and 5mg / kg of Rabbit anti mouse Ig G respectively,and the mice in the normal group and model group were intraperitioneally injected with the same amount of solvent.The mice were anesthetized with propofol 7 days after modeling,the mice were sacrificed by cervical dislocationafter blood was taken from eyeball,and then liver was taken after laparotomy.The serum levels of TBA,ALT,TBIL,DBIL and ALP were detected.The liver pathological changes were observed.The expression of HMGB1,GRP78,CHOP,IRE1 ? and NF-?B p65 protein in liver tissue was detected by WB.(2)Sixteen SPF TLR4 knockout C57 BL / 6 mice were randomly divided into two groups,tlr4 ko group and tlr4 ko + RFP group,8 in each group.Then 16 C57 BL / 6mice of the same batch were randomly divided into two groups,namely normal group and RFP group,8 in each group.The mice in tlr4 ko + RFP group and RFP group were given rifampin 200 mg / kg · d by gavage every day,and the mice in the normal group and tlr4 ko group were given the same amount of solvent by gavage.The mice were anesthetized seven days after modeling.the mice were sacrificed by cervical dislocationafter blood was taken from eyeball,and then liver was taken after laparotomy.Serum TBA,alt,TBIL,DBIL and ALP were detected,and liver pathological changes were observed.The expression of HMGB1,TLR4,GRP78,chop,IRE1 ? and NF-?B p65 were detected by Western blot.(3)HepG2 cells were cultured in l-dmem(10% FBS)at 37 ? and 5% CO2.When the cells grow to about 80% of the bottom of the culture bottle,they are subcultured and transferred to the 6-well plate,and divided into normal group,model group,TLR4 receptor blocking group and RAGE receptor blocking group.The model group,TLR4 receptor blocking group and RAGE receptor blocking group were treated with rifampin 200 ? m(100mg / ml)for 48 h,and the normal group was treated with the same amount of solvent.In the TLR4 receptor blocking group,TLR4 receptor blocking agent 1 ? m tak242 was given before rifampin treatment for 12 h,and in the RAGE receptor blocking group,cells were cleaved after 3 times of cold PBS rinsing.Western blot was used to analyze the expression of HMGB1,TLR4,rage,GRP78,chop,IRE1? and NF-?B p65.Results:(1)Compared with the normal group,TBA,alt,TBIL,DBIL and ALP in the model group and Ig G antibody intervention group were significantly higher(P< 0.05);under light microscope,steatosis and vacuolar degeneration of hepatocytes were obvious,with a large area of inflammatory infiltration and inflammatory focus,local nucleolysis and nuclear pyknosis;HMGB1,GRP78 The expressionof chop,IRE1? and NF-?B p65 increased(P < 0.05).Compared with the model group,the serum TBA,alt,TBIL,DBIL and ALP of the HMGB1 antibody intervention group were decreased(P < 0.05);the pathological changes of the liver were significantly improved under the light microscope;the HMGB1,GRP78 and The expression of chop,IRE1? and NF-?B p65 protein decreased(P < 0.05);compared with the Ig G antibody intervention group,the HMGB1 antibody intervention group had similar results with the model group,and all improved significantly(P < 0.05).Compared with the Ig G antibody intervention group,there was no significant difference in the model group(P> 0.05).(2)Compared with the normal group and tlr4 ko group,TBA,alt,TBIL,DBIL and ALP were significantly increased in RFP group and tlr4 ko + RFP group(P < 0.05);vacuolar degeneration and fatty degeneration were obvious in some hepatocytes,local inflammatory infiltration,nuclear lysis and nuclear pyknosis were found;HMGB1,GRP78,chop,IRE1 ? and NF-?B p65 protein expression were increased(P < 0.05).Compared with RFP group,TBA,alt,TBIL,DBIL and ALP in tlr4 ko + RFP group were increased(P < 0.05),liver cell pathological changes were aggravated;HMGB1,GRP78,chop,IRE1 ? and NF-?B p65 protein expression were increased(P < 0.05).Compared with the normal group,the serum indexes of tlr4 ko group had no signifcant change(P > 0.05);the liver cell pathology had no abnormali-ty;the expression of HMGB1,GRP78,chop,IRE1 ? and NF-?B p65 protein had no significant difference.(3)Compared with the normal group,the intracellular calcium concentration in the model group,TLR4 receptor blocking group and RAGE receptor blocking group increased significantly(P < 0.05);the expression of HMGB1,GRP78,chop,IRE1 ?and NF-?B p65 protein increased significantly(P < 0.05).Compared with the model group,the intracellular calcium concentration of TLR4 receptor block-ing group has not differencei(P> 0.05);the expression of HMGB1,GRP78,chop,IRE1 ? and NF-?B p65 protein increased(P < 0.05).Compared with the model group,the expression of HMGB1,GRP78,chop,IRE1 ? and NF-?B p65 decreased signifi-cantly(P <0.05).Compared with TLR4 receptor blocking group,the intracellular calcium concentration of RAGE receptor blocking group decreased(P < 0.05);the expression of HMGB1,GRP78,chop,IRE1 ? and NF-?B p65 protein decreased(P < 0.05).Conclusion:(1)Rifampicin induced liver injury in mice showed the increase of HMGB1 expression intensity and endoplasmic reticulum stress.Blocking HMGB1 can significantly reduce the intensity of ER stress,improve liver function and reduce liver injury,suggesting that there is an internal relationship between HMGB1 and ERS in rifampicin induced liver injury.(2)TLR4 gene knockout aggravated significantly rifampin induced liver injury in mice and up regulate the expression of HMGB1,ERS-related protein and NF-? B p65 in liver.Blocking TLR4 can increase the expression of HMGB1,ERS-related protein and NF-? B p65 protein in HepG2 cells.It is suggested that TLR4 may play a protective role in liver injury induced by rifampicin.The mechanism may be that TLR4 gene knockout and blocking of TLR4 leads to the up regulation of ER stress,NF KB and HMGB1 expression.(3)Rifampicin can induce HepG2 cell injury and increase the expression of Ca2 +,HMGB1,GRP78,CHOP,IRE1 ? and NF-?B p65 in HepG2 cells.Blocking TLR4 receptor and RAGE receptor can increase or decrease Ca2 + concentration,HMGB1,GRP78,CHOP,IRE1 ? and NF-?B p65 protein expression in HepG2 cells,respextively.It is suggested that RAGE / ERS / NF-KB signaling pathway is involv-ed in rifampicin-induced HepG2 cell injury,in which Ca2 + plays an important role.