Protective Effect And Mechanism Of Histone Deacetylase 6 Inhibitor ACY-1215 On Liver Injury Induced By Gefitinib

Background and Objectives:According to the 2019 guidelines of the Chinese Medical Association,drug-induced liver injury(DILI)refers to liver damage induced by various prescription or non-prescription chemical drugs,biological preparations,traditional Chinese medicine,natural remedies,health supplements,dietary supplements,their metabolites,and excipients.DILI,also known as drug-induced liver disease,is one of the most common and serious adverse drug reactions.Gefitinib,a tyrosine kinase inhibitor(TKI)of the epidermal growth factor receptor(EGFR),is currently a first-line treatment for non-small cell lung cancer.However,its most common adverse reaction is liver toxicity,and there is currently a lack of recognized safe and effective treatments to prevent or treat gefitinib-induced liver injury.ACY-1215 is a selective inhibitor of histone deacetylase 6(HDAC6)and has been shown to have potent anti-inflammatory effects.To date,ACY-1215 has not been applied to DILI.Therefore,this study aims to explore the mechanism of gefitinib-induced liver injury and to verify the protective effect and mechanism of ACY-1215 on gefitinib-induced liver injury,in order to provide new therapeutic strategies for gefitinib-induced liver injury.Methods:(1)AML 12 cells and MIHA cells were cultured in media containing different concentrations of gefitinib for 24 hours.The cell viability was detected using the Cell Counting Kit-8(CCK-8)assay kit.The lactate dehydrogenase(LDH)level in the cell supernatant was measured using the assay kit.Using q RT-PCR to measure m RNA expression levels of TLR4,My D88,IL-6 and TNF-α,and TLR4/My D88/MAPK/NFκB signaling pathway protein expression levels were detected using Western blotting to evaluate the effect of gefitinib on liver cells and explore the mechanism.(2)Male C57BL/6J mice aged 7-8 weeks were orally administered different concentrations of gefitinib.The body weight and liver wet weight of the mice were measured,and the liver index was calculated.Hematoxylin-eosin(H&E)staining was used to observe the histological changes of the mouse liver.The levels of serum alanine aminotransferase(ALT),aspartate aminotransferase(AST),LDH,and total bile acid(TBA)were measured using assay kits.m RNA expression levels were measured using q RT-PCR,and protein expression levels were detected using Western blotting to evaluate the effect of gefitinib on mouse liver and explore the mechanism.(3)AML 12 cells and MIHA cells were co-cultured with gefitinib and ACY-1215 for 24 hours.The cell viability was detected using the CCK-8 assay kit.The LDH level in the cell supernatant was measured using the assay kit.Using q RT-PCR to measure m RNA expression levels,and protein expression levels were detected using Western blotting to evaluate the effect of ACY-1215 on gefitinib-induced liver cell damage and explore the mechanism.(4)Male C57BL/6J mice aged 7-8 weeks were orally administered gefitinib and ACY-1215.The body weight and liver wet weight of the mice were measured,and calculating the liver index.H&E staining was used to observe the histological changes of the mouse liver.The levels of serum ALT,AST,LDH,and TBA were measured using assay kits.Using q RT-PCR to measure m RNA expression levels,,and protein expression levels were detected using Western blotting to evaluate the effect of ACY-1215 on gefitinib-induced mouse liver injury and explore the mechanism.Results:(1)When the concentration of gefitinib exceeded 20 μM,it significantly inhibited the growth of liver cells in vitro and increased the LDH level in the cell supernatant.(2)Oral administration of gefitinib to mice resulted in slower body weight gain,increased infiltration of inflammatory cells in the liver,and elevated serum levels of ALT,AST,LDH,and TBA.(3)Gefitinib could activate the TLR4/My D88/MAPK/NFκB signaling pathway and increase the expression of inflammatory cytokines IL-6 and TNF-α in liver cells in vitro and in mouse liver.(4)ACY-1215 improved the growth inhibition of liver cells induced by gefitinib in vitro and reduced the LDH level in the cell supernatant.(5)Compared with the gefitinib-gavaged group,ACY-1215 restored the rate of body weight gain in mice,reduced infiltration of inflammatory cells in the liver,and decreased serum levels of ALT,AST,LDH,and TBA.(6)ACY-1215 could partially reverse gefitinib-induced liver cell damage and mouse liver injury by inhibiting the TLR4/My D88/MAPK/NFκB signaling pathway and reducing the expression of inflammatory cytokines IL-6 and TNF-α in vitro and in mouse liver.Conclusions:(1)Gefitinib could cause liver injury in AML 12 cells,MIHA cells,and mice,and its mechanism of injury involved the activation of the TLR4/My D88/MAPK/NFκB signaling pathway and increased inflammatory response.(2)ACY-1215 could protect against gefitinib-induced liver cell damage and mouse liver injury.(3)ACY-1215 could partially reverse gefitinib-induced liver cell damage and mouse liver injury by inhibiting the TLR4/My D88/MAPK/NFκB signaling pathway and reducing the expression of inflammatory cytokines.