Preclinical Study Of Acanthus Ilicifolius Alkaloid A In The Treatment Of Drug-induced Liver Injury

Objective:This study aims to establish drug-induced liver injury model for preclinical studies,and to evaluate the effect of Acanthus Ilicifolius Alkaloid A(HBOA)on drug-induced liver injury in mice at the preclinical level,thus providing experimental basis for further development and utilization of Acanthus Ilicifolius Alkaloid A.Methods:Sixty male Kunming mice were randomly divided into the normal group,the APAP group(acetaminophen 300 mg/kg),the HBOA high,medium and low dose groups(200,100,50 mg/kg)and the positive group(Bifendate 150 mg/kg).The experimental reagents corresponding to HBOA high,medium and low dose groups and positive groups were given,while the normal group and APAP group were given the same amount of sodium carboxymethyl cellulose solution once a day for a total of ten days.After intragastric administration,the clinical model of drug-induced liver injury was induced by intraperitoneal injection of APAP(300mg/kg)in the high,medium and low dose HBOA group and the positive HBOA group.When the mice were finished the collection of samples,the pathological changes of liver were observed by Hematoxylin-Eosin staining.Terminal Deoxynucleotidyl Transferase-mediated d UTP-biotin Nick End Labeling assay was used to detect the apoptosis of hepatocytes.The contents of AST,ALT and TBIL in serum were determined by automatic biochemical analyzer.The contents of GSH,GSH-PX,CAT,SOD,MDA and ROS in liver tissue were measured with the kit.Western blotting was used to determine the expressions of protein IκBα,p-IκBα,NF-κBp65,p-NF-κBp65,IKKα/β,p-IKKα/β,NF-κBp50,HO-1,Keap1,Nrf2,Bax,Bcl-2 and Cyt C.The m RNA expressions of HO-1,NQO1,GCLC,GCLM and MGST-1 were detected by RT-PCR.Results:1.HBOA had a moderating effect on drug-induced liver injuryFor this experiment,compared with the normal group,the serum levels of AST,ALT and TBIL in the APAP group were significantly increased(P<0.05).Hematoxylin-eosin staining results showed that the liver cells in APAP group had obvious degeneration and necrosis,indicating the model of drug-induced liver injury had been successfully replicated.Compared with the APAP group,the levels of AST,ALT and TBIL in serum of mice in each drug group showed a downward trend(P<0.05).Moreover,the degeneration and necrosis of hepatocytes were alleviated in Hematoxylin-eosin staining results.These results all suggested that HBOA can relieve acute liver injury caused by APAP.2.HBOA could enhance the antioxidant capacity of drug-induced liver injuryThe relevant antioxidant enzymes were detected by the kit,and the contents of MDA and ROS in the liver tissues of mice in the APAP group were obviously increased compared with those in the normal group,the expression levels of GSH,GSH-PX,CAT and SOD were reduced(P<0.05).The levels of MDA and ROS in the liver tissues of the HBOA dose group was lower than the APAP group,while the expression levels of GSH,GSH-PX,CAT and SOD were increased(P<0.05).The above results indicated that HBOA can enhance the expression of antioxidant enzyme in drug-induced liver injury.3.HBOA attenuated hepatocyte apoptosis and mitochondrial damage in drug-induced liver injuryTerminal Deoxynucleotidyl Transferase-mediated d UTP-biotin Nick End Labeling(TUNEL)cell apoptosis kit was used to detect the apoptosis of hepatocytes in mice.Compared with normal mice,the hepatocytes in APAP group showed a large amount of apoptosis.Compared with the APAP group,apoptosis was reduced in each dose of HBOA group.Meanwhile,The expression levels of Bcl-2 and Bax in liver tissues and Cyt C in cytoplasm were determined by western blot.Compared with the APAP group,in each HBOA dose group,the protein expression levels of Bax and Cyt C were decreased and the level of Bcl-2 was increased(P<0.05).According to the above results,HBOA regulated the expression of Bcl-2 family protein and Cyt C protein,and reduced hepatocyte apoptosis and mitochondrial in drug-induced liver injury.4.HBOA inhibited the overexpression of NF-κB signaling pathway on drug-induced liver injuryTo investigated the effect of HBOA on the NF-κB signaling pathway in liver injury induced by excessive APAP,we measured the expression levels of proteins associated with the NF-κB signaling pathway,including IκBα,p-IκBα,NF-κBp65,p-NF-κBp65,IKKα/β,p-IKKα/β and NF-κBp50.These results showed that the expressions of NF-κBp50,p-IκBα,p-NF-κBp65 and p-IKKα/βin the APAP group were obviously higher than the normal group(P<0.05).In comparison with the APAP group,these proteins were decreased in each HBOA dose group(P<0.05).Results suggested that HBOA can inhibit the expression of NF-κB signaling pathway in drug-induced liver injury.5.HBOA could promote the protien expression of Nrf2 signaling pathway in drug-induced liver injuryAccording to protein western blot detection,the expressions of HO-1,Keap1 and Nrf2 in liver tissues of mice in the APAP group were lower than those in the normal group,while the expressions of HO-1,Keap1 and Nrf2 in each dose group of HBOA were higher than those in the APAP group(P<0.05).Compared with the normal group,RT-PCR detection results showed that the expression of downstream genes of Nrf2 signaling pathway in the APAP group were decreased,including m RNA HO-1,NQO1,GCLC,GCLM and MGST-1(P<0.05).While compared with the APAP group,the expression of downstream genes of this signaling pathway were increased in each HBOA dose group(P<0.05).The above results indicated that HBOA could promote the expression of Nrf2 signaling pathway in drug-induced liver injury.Conclusion:Acanthus Ilicifolius Alkaloid A has a relief effect on drug-induced liver injury.It may be associated with lessening abnormally elevated liver enzyme levels in serum of mice,enhancing the effect of antioxidant enzymes,weakening the liver cell apoptosis and mitochondrial damage,promoting Nrf2 signaling pathways and inhibiting the expression of the NF-κB signaling pathways,so as to achieve the therapeutic effect of drug-induced liver injury..