The Role And Its Mechanism Of Calcium-dependent Proteolytic Enzyme Calpain In Acetaminophen-induced Acute Liver Injury

ObjectiveAcetaminophen(APAP)is an over-the-counter antipyretic and analgesic drug commonly used in clinical practice.Overdose of APAP can induce severe acute liver injury and is the most common cause of acute liver failure.Mitochondrial damage is considered to be a key event in APAP liver toxicity.It not only affects ATP production,but also causes calcium homeostasis imbalance in liver cells and activates inflammatory responses to further increase liver damage.Calpain is a class of calcium-dependent proteolytic enzymes present in the cytoplasm,which is widely involved in a variety of pathophysiological processes.However,so far,whether Calpain is involved in APAP-induced liver injury has not been reported at home and abroad.In this study,the APAP dose-response model and the Calpain inhibitor ALLN intervention model were established to detect changes in Calpain degradation system,mitochondrial dynamics and NF-?B-NLRP3 signaling pathway and mitochondrial autophagy-related proteins in the liver,to explore the role and molecular mechanism of Calpain in APAP-induced hepototoxicity.Methods1 The establishment of Animal modelDose-response model of liver injury in APAP-treatment mice:40 male C57BL/6 mice were adaptively fed for 3 days and randomly divided into 4 groups,control group,APAP low-dose group(180mg/kg.bw),and medium-dose group(300 mg/kg.bw)and high dose group(500 mg/kg.bw).After fasting for 12 hours,the mice in the exposure group were injected with APAP solution intraperitoneally,and the mice in the control group were injected with an equal volume of normal saline.After 24 hours,the animals were sacrificed.Calpain inhibitor(ALLN)intervention model:40 male C57BL/6 mice were adaptively fed for 3 days and randomly divided into 4 groups:control group,20mg/kg.bw ALLN group,300mg/kg.bw APAP model group,20mg/kg.bw ALLN+300mg/kg.bw APAP group,10 mice in each group.After fasting for 12 hours,the control group was given normal saline,the ALLN group was injected intraperitoneally with an ALLN solution at a dose of 20 mg/kg.bw,the APAP model group was intraperitoneally injected with a 300 mg/kg.bw APAP solution,and the ALLN intervention group was given a dose of 20 mg/kg.bw 1 h before After intraperitoneal injection of ALLN solution,300 mg/kg.bw APAP was given.After 24 hours,the animals were sacrificed.2 Detection of biochemical indicators,liver pathology,and protein expression levels(1)All mice were tested for serum ALT and AST biochemical indicators.The liver was stripped and weighed to calculate the liver coefficient.The same location of the liver was used for pathological sectioning and HE staining was used to detect pathological damage.(2)APAP dose-response model mice,the total protein was extracted from the homogenized liver tissue,and the expression of Calpain-related protein was detected by Western Blot.(3)In ALLN intervention model mice,after homogenizing the liver tissues of mice,GENMED tissue calpain activity fluorescence quantitative detection kit was used to detect changes in Calpain activity,and total protein,mitochondrial protein,cytoplasmic protein,and nuclear protein were separately extracted.Western Blot was used to detect the expression changes of Calpain-related protein,inflammation-related protein,mitochondrial dynamic-related protein and mitochondrial autophagy-related protein.3 Observation of mitochondrial damage by transmission electron microscopeTake 1mm3 of tissue from the same part of the mouse liver and quickly immerse it in a pre-chilled 3%glutaraldehyde fixing solution for 2 h.After immersion,it was fixed in 1%OsO4 for 1 h,and then dehydrated with ethanol gradient and embedded in Epon812 resin.The ultra-thin sections were double-stained with uranyl acetate and lead citrate,and then observed with a JEOL-1200EX transmission electron microscope and a field of view was selected for photographing.4 Immunofluorescence staining of liver tissueParaffin sections of mouse liver were prepared routinely,and paraffin sections of mouse liver(5?m)were dewaxed and hydrated.The sections were then immersed in citrate buffer and microwave-heated to 92 ? for 10 minutes for antigen retrieval.For immunofluorescence staining,first permeate the membrane with 0.3%TritonX-100 at room temperature for 10 min,block the sections with 5%BSA for 30 min at room temperature,and then incubate with the primary antibody overnight.The next day,the sections were incubated with fluorescent secondary antibody at room temperature in the dark for 1-1.5 h.Nuclei were stained with Hoechst 33343 and mounted on a SlowFade Antifade Mountant.Finally,observe the stained sections and select a field of view with a Nikon fluorescence microscope to take pictures.Results1 APAP-induced liver injury and Calpain activation in miceExcessive APAP results in severe liver damage in mice,manifested by increased serum ALT and AST activity and extensive hepatic lobular necrosis.A dose-dependent increase in ALT and AST was observed in the APAP-treated group(p<0.05).Histopathological examination showed that the liver sections of the APAP group had varying degrees of lobular hepatocyte necrosis,accompanied by granular degeneration of hepatocytes and inflammatory cell infiltration.More importantly,APAP treatment activated the calpain degradation pathway.Compared with the control group,the levels of ?-Calpain,m-Calpain and Calpastatin cleavage products increased significantly,and there was a clear dose-response relationship.2 ALLN intervention can reduce APAP-induced liver injury and inhibit Calpain activationPretreatment with ALLN can significantly reduce ALT and AST levels in APAP-exposed mice and reduce the area of liver necrosis.The activity of serum ALT and AST in mice treated with 20 mg/kg ALLN before APAP exposure was significantly reduced to levels close to normal mice.Moreover,histopathological analysis further confirmed the biochemical evaluation of the ALLN intervention experiment.In addition,ALLN pretreatment could significantly inhibit the activation of calpain degradation pathway in APAP-exposed mice.3 The effects of ALLN intervention on mitochondrial dynamic related proteins and mitochondrial damageThe results of Western blot showed that the level of Drpl in the APAP group was significantly increased,and the expression of Drpl was significantly decreased after ALLN intervention.This result was consistent with the immunofluorescence results.Compared with the control group,the expression levels of mitochondrial fusion proteins MFn2 and OPA1 in the model group were reduced,and ALLN The increased expression of MFn2 and OPA1 in the pretreatment group showed that APAP treatment promoted mitochondrial division and weakened mitochondrial fusion,and increased mitochondrial damage was observed by transmission electron microscopy,and ALLN intervention could reverse this situation.4 ALLN intervention can inhibit the occurrence of mitochondrial autophagyWestern Blot results suggest that the expression of autophagy and mitochondrial autophagy-related proteins P62,P-P62,LC3,Pink1,and Parkin in the APAP model group are significantly increased.The expression levels of these proteins were significantly reduced.5 ALLN intervention can inhibit APAP-induced activation of NF-?B-NLRP3 inflammatory pathwayAPAP exposure resulted in a significant increase in IKK levels and a significant nuclear translocation of NF-kB(p65).In contrast,ALLN pretreatment significantly inhibited the level of IKK,the upstream signal of NF-?B,and the transport of NF-?B from the hepatocyte cytoplasm to the nucleus.More importantly,compared with the control group,APAP exposure significantly increased the levels of NLRP3,caspase-1,and IL-1? in the liver of mice.Moreover,the active form of caspase-1 and mature IL-1? also increased significantly after APAP exposure.Compared with the model group,the intervention of ALLN significantly reduced the increase of NLRP3,caspase-1 and IL-1? caused by APAP infection.Conclusions1.Excessive APAP causes acute liver injury in mice and activates the Calpain degradation system.2.Calpain inhibitor ALLN can inhibit APAP-induced mitochondrial fragmentation and liver inflammation.3.ALLN can effectively protect APAP-induced acute liver injury.