| Background:Drug-induced liver injury is a common adverse reaction to medication use in clinical practice.Overdosing on acetaminophen(APAP)can lead to acute liver injury(ALI).The mechanisms of APAP-induced acute liver injury are complex and studies have confirmed that endoplasmic reticulum stress,oxidative stress,cellular pyroptosis,ferroptosis,and inflammatory response may all be involved in the pathogenesis of APAP-induced ALI.Our previous experimental results have demonstrated that pretreatment with rosiglitazone(RSG),a peroxisome proliferator-activated receptor-γ(PPAR-γ)ligand,can significantly alleviate APAP-induced ALI in mice.This effect may be related to the suppression of liver oxidative stress response caused by APAP.The specific pathway through which RSG inhibits oxidative stress response remains to be elucidated.It has been documented that endoplasmic reticulum stress can induce oxidative stress.Whether RSG can inhibit oxidative stress by suppressing endoplasmic reticulum stress and ultimately achieve its protective effect against APAP-induced acute liver injury warrants further investigation.Objective:This study aims to establish an animal model of APAP-induced acute liver injury,observe the effects of RSG on endoplasmic reticulum stress signaling pathways during APAP-induced acute liver injury,and further explore the protective mechanism of RSG against APAP-induced ALI.Methods:First,48 eight-week-old male CD-1 mice were adaptively fed and randomly divided into six groups: control,RSG,APAP 4h,APAP 24 h,RSG + APAP 4h,and RSG + APAP 24 h.RSG and RSG + APAP groups were given RSG(20 mg/kg)by gavage 48,24,and 1 hour before a single intraperitoneal injection of APAP(300mg/kg).The APAP group received only an intraperitoneal injection of APAP(300mg/kg).All mice were fasted for 12 hours before APAP administration.Mice were sacrificed immediately after APAP injection(RSG and control groups),4 hours(APAP4h and RSG + APAP 4h),or 24 hours(APAP 24 h and RSG + APAP 24h).Liver tissue was collected for hematoxylin-eosin staining,pathological examination,TUNEL staining for apoptosis detection,and immunoblotting to detect the expression of endoplasmic reticulum stress(ERS)-related proteins.Serum alanine aminotransferase(ALT)and aspartate aminotransferase(AST)levels were also measured.An additional20 male CD-1 mice were divided into two groups of 10 each,APAP and RSG + APAP,to observe the survival status within one week after APAP administration and to plot survival curves.Lastly,24 male CD-1 mice were randomly divided into four groups:control,RSG,APAP 1h,and RSG + APAP 1h.Mice were sacrificed one hour after APAP administration,and liver tissue was collected to detect reduced glutathione(GSH)content.Results:1.Protective effects of RSG pretreatment on APAP-induced acute liver injuryAt 4h and 24 h after APAP administration,serum ALT and AST levels were significantly increased.RSG pretreatment resulted in varying degrees of reduction in serum ALT and AST levels compared to the corresponding APAP-only groups,indicating the protective effect of RSG pretreatment on APAP-induced acute liver injury.Pathological observations revealed no significant histological liver damage in the control and RSG groups.Mild hepatocyte degeneration and necrosis(mainly fragmentary necrosis and bridging necrosis)were observed in the APAP 4h group,while conspicuous submassive or massive necrosis was observed in the APAP 24 h group.In RSG pretreated APAP groups,the degree of hepatocyte degeneration and necrosis was significantly alleviated compared to the corresponding APAP-only groups.GSH content measurement in liver tissue revealed that the GSH was depleted in mouse liver tissue 1h after APAP administration,while RSG pretreatment significantly reduced GSH consumption in mouse liver tissue.2.RSG pretreatment improves the survival rate of mice with APAP-induced acute liver injuryThe 7-day survival rate of mice in the APAP-only group was 50%,significantly lower than the RSG + APAP group(80%).3.Effects of RSG on endoplasmic reticulum stress signaling pathways during APAP-induced acute liver injury3.1 Effects of RSG on the expression of endoplasmic reticulum stress marker GRP78 in APAP-induced acute liver injuryWestern blotting was used to detect and analyze the expression of endoplasmic reticulum stress marker GRP78 in mouse liver tissue.The study showed that after 4hours of APAP treatment,GRP78 expression was significantly increased,continuing to rise until 24 hours,while RSG pretreatment significantly reduced the expression of GRP78 protein induced by APAP.3.2 Effects of RSG on the PERK signaling pathway in APAP-induced acute liver injuryWestern blotting was used to detect the expression of p-PERK protein.The results showed that the expression of p-PERK was significantly increased 4 hours after APAP administration and reached its peak at APAP 24 hours.The expression of p-PERK in the liver of RSG pretreated mice was significantly lower than that of the corresponding APAP-only group.Western blotting was used to detect the expression of p-e IF2α.The results showed that the expression of p-e IF2α was significantly increased at 4h and 24 h after APAP administration.RSG pretreatment significantly reduced the expression of p-e IF2αcompared to the corresponding APAP-only group.Western blotting was used to detect the expression of CHOP.The results showed that the expression of CHOP was significantly increased at 4h and 24 h after APAP administration.RSG pretreatment significantly reduced the protein expression level of CHOP.These results suggest that RSG has a significant inhibitory effect on the PERK branch of the endoplasmic reticulum stress signaling pathway during APAP-induced acute liver injury.3.3 Effects of RSG on the IRE signaling pathway in APAP-induced acute liver injuryWestern blotting was used to detect the expression of p-IRE.The results showed that after 4 hours of APAP treatment,the expression of phosphorylated IRE in the liver was significantly increased,and the expression of p-IRE was significantly increased after 24 hours of APAP treatment.The expression of phosphorylated IRE in RSG pretreated APAP mice was significantly reduced compared to the corresponding APAP-only group.Western blotting was used to detect and analyze the expression of p-JNK in hepatocytes.The results showed that the expression of p-JNK was significantly increased 4 hours after APAP treatment and continued to increase for 24 hours,whereas RSG pretreatment significantly reduced the expression of p-JNK.Western blotting was used to detect the expression of nuclear XBP-1.The results showed that the expression of XBP1 protein in mouse liver tissue was significantly increased at 4h and 24 h after APAP administration,and the expression of XBP1 gene in RSG pretreated APAP mice was significantly lower than that in the corresponding APAP-only group.These results suggest that RSG has a significant inhibitory effect on the IRE branch of the endoplasmic reticulum stress signaling pathway during APAP-induced acute liver injury.3.4 Effects of RSG on the ATF6 signaling pathway in APAP-induced acute liver injuryWestern blotting was used to detect the nuclear expression of ATF6 in mouse liver tissue.The results showed that the nuclear expression of ATF6 was significantly increased 4 hours after APAP administration,and the peak was reached 24 hours after APAP administration.The nuclearexpression of ATF6 in the liver tissue of RSG +APAP group mice was significantly lower than that in the corresponding APAP-only group.These findings suggest that RSG has a certain inhibitory effect on the ATF6 branch of the endoplasmic reticulum stress signaling pathway during APAP-induced acute liver injury.Conclusion:Based on the comprehensive experimental results,the following conclusions can be drawn:1.Endoplasmic reticulum stress is present during APAP-induced acute liver injury.2.RSG pretreatment has a certain protective effect on APAP-induced acute liver injury.3.RSG may exert its protective effect on APAP-induced acute liver injury by inhibiting the three branch signaling pathways of the endoplasmic reticulum tress signaling pathway.Figure [17] Table [9] Reference [74]... |
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