| ObjectiveAcetaminophen(APAP)is a commonly used antipyretic and analgesic drug.Taking too much APAP can cause severe acute liver injury(ALI)and eventually induce acute liver failure(ALF).At present,APAP overdose is the most common cause of ALF cases and has become a public health issue of general concern.In the study of APAP liver toxicity mechanism,mitochondria are considered to be the key targets of APAP.Mitochondrial damage not only affects the production of ATP,but also causes the dynamic imbalance of mitochondrial division and fusion and activates the inflammatory response mediated by inflammasomes,further aggravating liver damage.In the early stage,the research group found that the dynamic indicators of mitochondrial division-fusion have changed through APAP-infected animals.The level of mitochondrial fission-promoting protein was significantly increased,and the expression of mitochondrial fusion protein was reduced.At the same time,the mitochondrial dynamic protein was degraded and the lysis level increased.The previous results suggest that the dynamic changes of mitochondria may play a role in APAP liver toxicity.Therefore,in this study,we used C57/BL6 male mice exposed to APAP as an animal model to explore whether the mitochondrial division intervention agent Mdivi-1 and the proteasome intervention agent MG-132 have a preventive effect on APAP-induced acute liver injury in mice.Methods1.Establishment of animal model(1)Mitochondrial division inhibitor 1(Mdivi-1)intervention model:40 healthy male C57BL/6 mice were randomly divided into 4 groups:control group,Mdivi-1 control group,APAP group,APAP+ Mdivi-1 group,10 per group.After fasting for 12 hours,the control group was intraperitoneally injected with normal saline,the Mdivi-1 control group was intraperitoneally injected with 50 mg/kg.bw Mdivi-1,the APAP group was intraperitoneally injected with 300 mg/kg.bw APAP,and the Mdivi-1 intervention group was given 50 mg/kg.bw 30 minutes in advance.After intraperitoneal injection of Mdivi-1 at kg.bw dose,300 mg/kg.bw APAP was given.After 24 hours,the eyeballs were removed to collect blood,and the mice were sacrificed and liver tissues were removed.(2)Proteasome inhibitor MG-132 intervention model:40 healthy male C57BL/6 mice were randomly divided into 4 groups:control group,MG-132 control group,APAP group,APAP+MG-132 group,each with 10 only.After fasting for 12 hours,the control group was injected with normal saline,the MG-132 control group was injected with 3 mg/kg.bw MG-132,the APAP group was injected with 300 mg/kg.bw APAP intraperitoneally,and the MG-132 intervention group was injected with 3 mg/kg 30 minutes in advance.After intraperitoneal injection of MG-132 at a dose of bw,300 mg/kg.bw APAP was administered.After 24 hours,the eyeballs were removed for blood,and the mice were sacrificed and liver tissues were removed.2.Detection of biochemical indicators,liver pathology and proteins expression(1)Take serum,use automatic biochemical analyzer to detect changes in ALT and AST indicators.The liver is picked and weighed,and the liver coefficient is calculated.The liver at the same position was taken to prepare paraffin sections and stained with HE to observe the pathological changes of APAP-induced acute liver injury in mice.(2)Mdivi-1 intervention model mice,homogenize liver tissue,extract total protein,mitochondrial protein and mitochondrial-associated endoplasmic reticulum membrane(MAMs)protein,evaluate mitochondrial dynamic-related proteins,mitochondrial autophagy-related proteins,and internal Changes in the expression of plasma reticulum stress proteins and inflammation-related proteins.(3)MG-132 intervention model mice,homogenize liver tissue,extract total protein and mitochondrial protein,and evaluate the expression changes of mitochondrial dynamic-related proteins and inflammation-related proteins by Western Blot.3.Observation of mitochondrial dynamic disorder by transmission electron microscopyTake 1 mm3 of liver tissue from the same part and quickly put it into pre-cooled 3%glutaraldehyde for 2h.After soaking,it was fixed in 1%OsO4 for 1 hour,and dehydrated with gradient ethanol and embedded in Epon812 resin.Ultrathin sections were double stained with uranyl acetate and lead citrate,and JEOL-1200EX transmission electron microscope was used to evaluate the mitochondrial damage and mitochondrial fission-fusion process of APAP-infected animals.4.Immunofluorescence staining of liver tissueRoutinely prepare paraffin sections of mouse liver tissues,deparaffinize them to water,immerse them in citrate buffer,and heat them in a microwave oven for antigen retrieval.The sections were sequentially permeabilized with 0.3%TritonX-100 at room temperature for 10 minutes,blocked with 5%BSA at room temperature for 30 minutes,and then incubated with the primary antibody overnight.The next day,the fluorescent secondary antibody and the slice were incubated for 1h at room temperature and protected from light.Hoechst 33343 stains the nucleus and mounts it with SlowFade Antifade Mountant.Fluorescence microscope observation was performed to further evaluate the expression of key proteins in APAP treatment of liver tissue and the shift of mitochondrial division protein Drpl from the cytoplasm to the mitochondrial level.5.Co-immunoprecipitationTo prepare tissue lysate,add 1-5 μg of primary antibody to the target protein to the tissue lysate containing 1000 μg of total protein,and mix gently overnight at 4℃.Add Protein A and Protein G breads to obtain immune complex precipitation,wash with Wash Buffer and analyze by Western Blot to detect the specific interaction between mitochondrial dynamic protein and ubiquitinated protein.Results1.The effect of mitochondrial division intervention agent on APAP-induced liver mitochondrial damage,endoplasmic reticulum stress and inflammatory response in mice(1)Mdivi-1 intervention reduces acute liver injury induced by APAP.In APAP-infected mouse animal models,significant liver damage can be observed,manifested by histopathological changes such as hepatocyte necrosis and inflammatory cell infiltration,and the blood biochemical levels of ALT and AST are significantly increased.Mdivi-1 pretreatment can significantly reduce blood ALT and AST levels in APAP-infected mice,improve APAP-induced acute liver injury.(2)The effect of Mdivi-1 intervention on mitochondrial dynamic related proteins and mitochondrial damage.Western Blot detection results showed that APAP-induced liver injury mice had significantly increased levels of Drpl and phosphorylated Drpl(ser616),suggesting increased mitochondrial division.After Mdivi-1 intervention,the expression of Drp1 and phosphorylated Drpl(ser616)decreased,and immunity The fluorescence detection result is consistent with this.At the same time,compared with the control group,the levels of mitochondrial fusion proteins Mfn2 and OPA1 in the liver of APAP-infected mice were reduced,and Mdivi-1 pretreatment could significantly inhibit the decrease of Mfn2 and OPA1 protein expression.In addition,transmission electron microscopy showed that mitochondria were damaged after APAP treatment,which was manifested as excessive division of mitochondria,showing fragmented morphology.The results suggest that mitochondrial division increased and mitochondrial fusion weakened after APAP treatment.The intervention of Mdivi-1 significantly improved mitochondrial dynamic imbalance and mitochondrial damage.(3)The effect of Mdivi-1 intervention on mitochondrial autophagy-related proteins.Western Blot detection results showed that the levels of LC3,p62,p-p62,PINK1,Parkin and other mitochondrial autophagy-related proteins in the liver whole protein and mitochondrial extracts of the APAP model group increased,and transmission electron microscopy observed a significant increase in the number of liver autophagosomes.Compared with the model group in the Mdivi-1 intervention group,the expressions of LC3,p62,p-p62,PINK1,Parkin in the whole liver protein and mitochondrial extract components were significantly reduced.(4)The effect of Mdivi-1 intervention on endoplasmic reticulum stress-related proteins.Western Blot results showed that the expression levels of the endoplasmic reticulum stress-related proteins CHOP and BIP in the liver tissue of the APAP model group were significantly increased.Compared with the model group,the expression of CHOP and BIP decreased significantly after the intervention of Mdivi-1.(5)The influence of Mdivi-1 intervention on inflammation-related proteins.Western Blot results showed that the expression levels of inflammation-related proteins NLRP3,caspase1,and IL-1β in the liver tissue of the APAP model group were significantly increased.Compared with the model group,the expressions of NLRP3,caspasel and IL-1β were significantly reduced after the intervention of Mdivi-1.2.The effect of proteasome inhibitors on APAP-induced liver mitochondrial dynamic-related proteins and inflammatory response in mice(1)MG-132 protects APAP to induce acute liver injury.In mouse animal models,obvious liver damage was observed 24 hours after APAP administration,manifested by histopathological changes such as hepatocyte necrosis and inflammatory cell infiltration,and blood biochemical levels of ALT and AST were significantly increased.MG-132 pretreatment can significantly reduce blood biochemical levels and improve APAP-induced acute liver injury.(2)The effect of MG-132 intervention on mitochondrial dynamic-related proteins Compared with the APAP model group of mice,the level of Mfn2 protein in the MG-132 pretreatment group was significantly increased,and the expression of Drpl was decreased.(3)The effect of MG-132 intervention on inflammation-related proteins.Compared with mice in the APAP model group,the expression levels of inflammation-related proteins NLRP3,caspase 1,and IL-1β were significantly reduced after MG-132 pretreatment.Conclusions1.Excessive APAP causes acute liver injury in mice,leads to mitochondrial division-fusion dynamic imbalance,activates mitochondrial autophagy,endoplasmic reticulum stress and NLRP3 inflammasome-mediated inflammatory response.2.Mitochondrial division inhibitor Mdivi-1 can inhibit APAP-induced excessive mitochondrial division and promote the balance of fusion-fission,thereby inhibiting the activation of mitochondrial autophagy,endoplasmic reticulum stress and NLRP3 inflammasome,and protecting APAP-induced Acute liver injury.3.The proteasome inhibitor MG-132 can inhibit the degradation of mitochondrial dynamic protein,promote the occurrence of mitochondrial fusion,inhibit the activation of NLRP3 inflammasome,and protect APAP-induced acute liver injury. |
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