| Background/ Objective Acetaminophen(APAP)is a common antipyretic analgesic in children.Acute liver injury caused by excessive or improper use is one of the common causes of drug-induced liver injury in children.However,there are few reports on the difference in APAP acute liver injury between children and adults.The core of its pathogenesis is APAP initiated by cytochrome P450 metabolic enzymes CYP2E1 metabolized into toxic products N-acetyl-p-benzoquinone imine(NAPQI)which results in depletion of reduced glutathione(GSH)in hepatocytes,further causing oxidative stress,mitochondrial dysfunction,finally leading to hepatocytes death.Recent studies have suggested that APAP-induced liver injury has “two hits”.TLR4 signaling may mediate the programmed cell death of hepatocytes during the “two hits” of APAP acute liver injury..Exploring the difference of the susceptibility between different ages and genders mice to APAP-induced acute liver injury,in order to provide a theoretical basis for clinical treatment of APAP-induced liver injury in different populations.Comparing the difference of APAP-induced acute liver injury between wild-type C3 H / He N and TLR4 mutant C3 H / He J mice,In order to explore the role of TLR4 at different stages of APAP induced acute liver injury.Methods Research one has two experiments.Experiment one: we used immature(3 weeks)CD-1 mice(10 males and 10 females),and adult(8 weeks)CD-1 mice(10 males and 10 females)in our experiments.All mice were divided into four groups: immature male mice,immature female mice,adult male mice and adult female mice.After a 12-h fast,all mice were intraperitoneally(i.p.)injected with a single dose of APAP(300 mg/kg).Observing survival rate of four groups mice to the 7th day after APAP injection.Experiment two: we used immature(3 weeks)CD-1 mice(40 males and 40 females),and adult(8 weeks)CD-1 mice(40 males and 40 females)in our experiments.All mice were divided into two groups: APAP treatment group and control group.APAP treatment group mice were i.p.injected with APAP(300mg/kg)after fasted for 12 hours,while control group mice were i.p.saline.APAP treatment group mice were sacrificed at different time points(0,1,4 or 24 h or 7d)after APAP injection.Comparing liver index,serum ALT,detecting liver GSH,GSSG,GSSG / GSH ratio.Observing pathological liver injury by HE staining.Detecting hepatocytes cell death by TUNEL staining.Detecting liver nitration of tyrosine 3-NT expression by immunohistochemical.Detection liver metabolic enzymes Cyp1a1,Cyp2e1,Cyp3a11,Ugt1a1,Gpx,Gr,Gst by RT-PCR.Testing GPX,GR and GST activity by Kits.Testing liver CYP2E1,p-JNK,JNK,RIP1 and RIP3 protein level by western blot.Research two has two experiments.Experiment one: we used wild-type group(C3H / He N)mice and mutant group(C3H / He J)mice in our experiments.Each group had ten mice.After a 12-h fast,all mice were intraperitoneally(i.p.)injected with a single dose of APAP(300 mg/kg).Observing survival rate of two groups mice to 72 h after APAP injection.Experiment two: We used wild-type C3 H / He N mice and mutant C3 H / He J mice in our experiments.All mice were divided into two groups: APAP treatment group and control group.APAP treatment group mice were i.p.injected with APAP(300mg/kg)after a 12-h fast,while control group mice were i.p.injected with equal volume saline.Some mice were sacrificed at 0,4,12 h after APAP injection.Comparing liver index,serum ALT.Observing pathological liver injury by HE staining.Detecting hepatocyte cell death by TUNEL staining.Testing liver p-JNK,JNK,RIP1 and RIP3 protein level by western blot.Results Immature mice were more susceptible than adult mice to APAP-induced acute liver injury.The results of survival curves suggested that survival rate of immature mice(male 50%,female 60%)were significantly lower than adult mice(80% male,90% female).Compared with the same gender adult mice,liver index of immature mice were significantly high at 4h after APAP injection,serum ALT of immature mice were higher both at 4h and 24 h after APAP injection.We observed hepatocyte congestion and centrilobular necrosis significantly by HE staining.Liver necrotic areas of immature mice were larger than the same gender adult mice after APAP injection.TUNEL assay prompted liver TUNEL positive cells in immature mice were significantly higher than the same gender adult mice.Futhermore,p-JNK and RIP3 protein level of immature mice were significantly higher than the same gender adult mice after APAP induced.The expression of nitration of tyrosine 3-NT in immature mice liver were higher than the same gender adlut mice at 4h and 24 h after APAP injection.In order to explore the mechanism of immature mice were susceptible to APAP-induced liver injury,we compared APAP metabolic enzymes levels and GSH metabolism in liver between different ages and genders.Liver GSH consumption of immature mice were more obvious and GSSG/GSH ratio increased significantly after APAP-induced than the same gender adult mice.Hepatic Cyp2e1 and Cyp3a11 m RNAs in immature mice were higher than that in same gender adult mice.Moreover,the levels of hepatic CYP2E1 protein were higher in immature mice than that in same gender adult mice.Gender differences only existed in adult mice.Adult male mice were more susceptible to hepatotoxicity.Serum ALT of adult male mice were higher than adult female mice both at 4h and 24 h after APAP injection.Liver necrotic areas of adult male mice were larger than adult female mice.TUNEL assay prompted liver TUNEL positive cells in adult male mice were significantly higher than adult female mice.Futhermore,p-JNK protein level of adult male mice were significantly higher than adult female mice at 4h after APAP induced.3-NT expression of liver in adult male mice were significantly higher than adult female mice both at 4h and 24 h after APAP injection.Depletion of GSH in adult male mice were larger than adult female mice after APAP induced.GSSG / GSH ratio recovery rate was significantly slowly than adult female mice.GPX and GST activity of liver in adult male mice were lower than adult female mice.The liver injury of C3 H / HeN mice were more serious than C3 H / He J mice at early stage after APAP injection.The results of 72 h survival curves suggested that wild-type C3 H / He N mice occurred death at 4h after APAP injection and 72 h survival rate were 40%.C3 H / He J mice occurred death at 8h after APAP injection and 72 h survival rate were 70%.Therefore,survival rate of wild-type C3 H / He N mice were lower than mutant C3 H / He J mice.Liver index of C3 H / He N mice were significantly higher than C3 H / He J mice at 4h after APAP injection.Serum ALT of C3 H / He N mice were significantly higher than C3 H / He J mice both at 4h and 12 h after APAP injection.Liver necrotic areas of C3 H / He N mice were larger than C3 H / He J mice both at 4h and 12 h after APAP injection.TUNEL assay prompted liver TUNEL positive cells in C3 H / He N mice were significantly higher than C3 H / He J mice both at 4h and 12 h after APAP injection.The expression of p-JNK in C3 H / He N mice were higher than C3 H / He J mice both at 4h and 12 h after APAP injection.The expressing of RIP1 in C3 H / He N mice were higher than C3 H / He J mice at 4h after APAP injection,but had no difference with C3 H / He J mice at 12 h after APAP injection.The expressing of RIP3 in C3 H / He N mice were higher than C3 H / He J mice at 12 h after APAP injection.Conclusions APAP-induced acute liver injury displayed different susceptibility between different ages mice.Immature mice were more susceptible than adult mice to APAP-induced acute liver injury.The mechanism may be related to the hepatic(Cyp2e1,Cyp3a11)m RNAs and CYP2E1 protein levels,which in immature mice were higher than adult mice.Immature mice were more susceptible than adult mice to APAP-evoked hepatic GSH depletion.APAP-evoked hepatic JNK phosphorylation and RIP3 activation were stronger in immature mice than in adult mice.Moreover,Gender differences in APAP-induced acute liver injury were observed only in adult mice.Adult male mice were more susceptible to hepatotoxicity.The mechanism may be related to the GSH metabolic(GPX and GST)enzyme activity,which in adult male mice were lower than adult female mice.Depletion of liver GSH in adult male mice were larger than adult female mice after APAP.These reduced capacity of antioxidant and excretion of toxic metabolites,which exacerbate persistent activation of JNK in liver and lead to cause serious liver damage.C3H / He N mice had more serious liver injury in the early stage of APAP-induced liver injury than C3 H / He J mice.It is suggested that TLR4 gene mutation play a protective role in the early stage of APAP-induced liver injury.We speculated that hepatic TLR4 signaling pathway is involved in activation of hepatic JNK,RIP1 and RIP3,then promoting liver programmed necrosis. |
PDF Full Text Request