4-trifluoromethylbromobenzyl Hesperetin Derivative Inhibits Alcohol-induced Liver Injury

Background:Continuous drinking can lead to liver steatosis,which can develop to inflammation,fibrosis and eventually cirrhosis.The pathogenesis of alcoholic liver injury(ALI)is multi-factor.In addition to genetic factors,alcohol can lead to hepatocytes damage,excessive accumulation of oxygen free radicals,overgrowth of bacterial microorganisms to liver steatosis and activation of inflammatory cells(macrophages and neutrophils).Persistent alcohol stimulation and pro-inflammatory cytokine infiltration activate hepatocyte stellate cells,and lead to liver fibrosis.In addition to abstinence from alcohol,liver protection drugs are commonly used to maintain the treatment of patients with ALI.Consequently,drug research and new drug design in alcoholic hepatitis is very necessary.In the theory of Traditional Chinese medicine,the treatment of "alcoholic disease" with tangerine peel as a single medicine has a long history.In addition,with the help of high-frequency drug cluster analysis and analysis technology,it was found that tangerine peel was second only to liquorice in the frequency of 429 antidote prescriptions.Hesperidin is a kind of natural flavonoid derived from citrus fruits of the Rutaceae family.In the previous study in our laboratory,it was found that the hesperidin derivative numbered HD-1L had strong pharmacological activity and little toxic and side effects.Objective:The purpose of this study was to investigate the anti-inflammatory activity and molecular mechanism of HD-1L through mouse alcoholic liver injury model and in vitro cell model.Materials and methods:There five parts in this study.1.To explore hesperidin derivatives HD-1 | efficacy in the mouse model,Binge model was used to construct alcoholic liver injury mice model,all mice were orally treated with drug.The groups were as follows:normal group,model group(alcohol feeding),model+low dose HD-1L group(25 mg/kg),model+medium dose HD-1L group(50 mg/kg),model+high dose HD-1L group(100 mg/kg),normal+high dose HD-1L group(100 mg/kg),and normal+dexamethasone group(1 mg/kg).Pathology(HE staining),serology(determination of alanine aminotransferase and aspartate aminotransferase content),liver/body mass ratio,western blot,real-time fluorescence quantitative PCR and ELISA were used to detect the liver injury and the expression of pro-inflammatory cytokines in mice after alcoholic liver injury model was completed.2.In vitro experiments,RAW 264.7 cells were used to study inflammation in vitro,and the cells were treated with alcohol to obtain an in vitro model of inflammation,and the mechanism was investigated.In the study of the efficacy of HD-1L,the following groups were divided:1.Normal RAW 264.7 cells group;2.Model group(100 mM alcohol);3.Model+HD-1L(5 g/ml);4.Model+HD-1L(10 g/ml);5.Model+HD-1L(20 g/ml);Normal+HD-1L(20 g/mL).The expression levels of proinflammatory factors were investigated by western blotting,quantitative real-time PCR and ELISA3.Discover Studio software was used to conduct molecular simulation pairing experiment,and TR-FRET experiment and ELISA experiment were used to verify the relationship between HD-1L and Brd24.To explore the role of Brd2 in alcoholic hepatitis in the mouse model,the following groups were established:1.Normal group;2.Model(alcohol feeding)group;3.Model+J Q1(Brd2 inhibitor)group.Pathology(HE staining),serology(determination of alanine aminotransferase and aspartate aminotransferase content),liver mass/body mass ratio,western blot,real-time fluorescence quantitative PCR and ELISA were used to detect the liver injury and the expression level of pro-inflammatory factors in mice after alcoholic hepatitis model was completed5.To investigate the role of Brd2 in the pharmacological activity of HD-1L,RAW 264.7 cells were used,and four subgroups were designed:1.Normal group;2.Model group(100 mM alcohol);3.Model+HD-1L;4.Overexpression of model+HD-1L+Brd2.Western blot,real-time fluorescence quantitative PCR and ELISA were used.6.In the study of HD-1L regulating signaling pathway,the pharmacological and biological methods were used to interfere with the Brd2 expression in RAW 264.7 cells.The following groups were established:1.Normal control group;2.Model group(100 mM alcohol stimulation);3.Model+control group;4.Model+SI-BRD2(gene silencing)group.The second part is grouped as follows:1.Normal control group;2.2.Model group(100 mM alcohol stimulation);3.Model+JQ1 drug.Western blotting was used to explore the expression level of key proteins in NF-κB pathway.Results:1.The HD-1L has protective effect on alcoholic liver injury:HE staining,determination of cereal third transaminase,aspertate aminotransferase and mice liver/body mass ratio suggest HD-1L could significantly inhibit liver injury in mice caused by alcohol and edema,and ELISA,protein immunoblot experiment and real-time fluorescent quantitative PCR is pointed out that the HD-1Lcould inhibit the TNF-α caused by alcohol,IL-1β and the high expression of IL-6;2.HD-1L significantly inhibited the inflammatory response of RAW 264.7 cells induced by alcohol:ELISA,western blot(WB)and real-time quantitative PCR(qRT-PCR)showed that HD-1L significantly inhibited the high expression of TNF-α,IL-1β and IL-6 induced by alcohol.3.HD-1L may act on alcoholic hepatitis through Brd2:The simulated molecular pairing experiment,TR-FRET experiment and ELISA experiment all suggested that HD-1L could bind to Brd2 with a high negative correlation.4.The inhibition of Brd2 protein levels can significantly protect liver from damage caused by alcohol:HE staining,cereal third transaminase,aspertate aminotransferase tip used in the determination of HD-1 L can significantly inhibit liver injury in mice caused by alcohol,and ELISA,protein immunoblot experiment and real-time fluorescent quantitative PCR is pointed out that the HD-1L can significantly inhibit alcohol-induced high expression of TNF-α and IL-6,IL-1β;5)HD-1L inhibits Brd2,which inhibits inflammatory responses:qRT-PCR and ELISA results indicated that when Brd2 was overexpressed,and the pharmacological activity of HD-1L disappeared.6.Brd2 affects NF-κB inflammatory signaling pathway:Western blotting indicated that Brd2 inhibitors(JQ1)and SI-BRD2 significantly inhibited p-P65 expression.Conclusion:HD-1L inhibits the activation of NF-κB signaling pathway by inhibiting Brd2,and ultimately inhibits alcohol-induced liver inflammation and plays a protective role in the liver.Conclusion:HD-1L could attenuate inflammatory responses via Brd2-NF-κB axis.