Inhibition Of Oxidative Stress By RNAi Targeting Cyp2e1 Alleviates Alcoholic Liver Disease In Mice And Its Combined Effect With Silybin

Background: Alcoholic liver disease(ALD)is primarily a series of liver injuries caused by heavy alcohol consumption,and the disease burden and potentially violent injuries caused by alcohol abuse are now a significant burden on society.However,there are no clinically effective drug treatments for ALD,and there is an urgent need to develop new therapeutic approaches oriented to the pathological mechanisms of ALD.The research of Chinese medicine and its active ingredients against ALD has been extensively studied at the cellular and animal levels,and the "multi-component;multi-target;multi-pathway" characteristics of Chinese medicine provide great potential for the development of therapeutic drugs for ALD.The development of ALD has been inhibited by constructing CYP2E1 knockout(CYP2E1 KO)mice,and alcohol-induced liver injury has been exacerbated in humanized CYP2E1 knock-in(CYP2E1 KI)mice,suggesting that the therapeutic effect on ALD may be achieved by inhibiting CYP2E1.However,CYP2E1 KO cannot be directly knocked down in vivo,which has limitations for clinical application.RNAi is a technique to silence specific target genes and can be used to regulate the expression of target genes in vivo for the purpose of treating related diseases.Therefore,knockdown of hepatic CYP2E1 expression by RNAi was considered to be able to prevent and treat ALD.However,CYP2E1 is not the only reactive enzyme to metabolize alcohol and generate reactive oxygen species(ROS),and when the expression of CYP2E1 is inhibited that it increases the compensatory pathway of ROS production.Silybin has antioxidant effect and can neutralize the ROS produced by the organism,therefore,it is considered that inhibition of CYP2E1 expression by RNAi technology and combined use of silybin can coordinate to enhance the therapeutic effect on ALD and perfect the antioxidant capacity of the liver.Objective: In this project,we propose to use various alcohol treatment methods by combining chronic alcohol feeding and acute alcohol binges to induce different degrees of ALD models in C57BL/6N mice,and to verify the protective effects of silybin and Dendrobium hosannicum polysaccharides on ALD;to achieve in vivo expression regulation through lipid nanoparticle delivery siRNA targeting hepatic Cyp2e1,and to investigate its effect on the prevention and treatment of ALD and related mechanisms,as well as the therapeutic effect of combining with silybin on ALD.Methods:(1)Construction of ALD model: 6-8 weeks old C57BL/6N female mice were selected for acute gavage three times with 5 g/kg alcohol model(3B);5% alcohol liquid diet ad libitum for 10 days plus 5 g/kg alcohol binges for the last three days(E10d+3B);4% alcohol liquid diet ad libitum plus 5 g/kg alcohol binges twice a week were treated for 4 weeks(E4w +n B),8 weeks(E8w+n B),or 12 weeks(E12w+n B),respectively.Serum and liver were collected 9 hours after the last alcohol gavage.Then alcohol-induced liver injury,oxidative stress,liver lipid metabolism and liver pathological histological sections were examined to assess the different degrees of ALD in mice.Real-time quantitative PCR and western blot were performed to detect hepatic Cyp2e1 expression.Each index assay was operated according to the assay kit instructions.(2)Protective effect of active ingredients of Chinese medicine on ALD: 6-8 weeks old C57BL/6N female mice were selected to construct a model of ALD by chronic alcohol feeding for 10 days plus 3 acute alcohol gavage,and gavage of silybin glucosamine(100 mg/kg),and the mice were executed 9 hours after the last gavage,and the serum and liver were collected to test the indexes related to ALD to verify the therapeutic effects of silybin and Dendrobium Huo Shan polysaccharide on ALD.(3)Production of si-Cyp2e1 LNPs and investigation of anti-ALD mechanism of action: Design of siRNA sequences targeting Cyp2e1,construction and culture of stable cell lines overexpressing Cyp2e1 for in vitro screening of a sequence with the best "silencing" effect.The siRNA was encapsulated in lipid nanoparticles(si-Cyp2e1 LNPs)by microfluidics technology.The mice were selected from 6-8 weeks old C57BL/6N female mice,and the model of ALD was constructed by chronic alcohol feeding for 10 days plus 3 acute alcohol gavage,and si-Cyp2e1 LNPs were injected into tail vein once a week,and the mice were executed 9 hours after the last gavage,and serum and liver were collected to detect indicators related to ALD;liver tissue sections were subjected to F4/80 fluorescence immunohistochemistry;real-time quantitative PCR and western blot were used to detect the expression of Cyp2e1 in the liver.Real-time quantitative PCR was performed to detect the expression levels of related metabolic genes:NOX4,P47 phox,P67phox,GP91 phox genes related to oxidative system;Gsh-Px,Gsh-Rd,Sod1 genes related to antioxidant system;Srebp-1c,Acc,Fasn genes related to lipid synthesis;Pgc-1α,Cpt1 genes related to fatty acid oxidative metabolism;Tgf-β,Tnf-α,IL-6 genes related to inflammation.(4)The effect and mechanism of si-Cyp2e1 LNPs combined with silymarin on long-term ALD: 6-8 weeks female C57BL/6N mice were selected and treated with 4% alcohol liquid diet plus 5g/kg alcohol gavage twice a week for up to 12 weeks to construct a model of ALD,and given once a week at the early(at the beginning of alcohol treatment),at the middle(after 4 weeks of alcohol treatment)and at the late(after 8 weeks of alcohol treatment).The mice were executed 9 hours after the last gavage,and the serum and liver were collected for the detection of ALD related indexes.The expression levels of metabolic genes related to fat synthesis and metabolism in the liver of each group of mice: Srebp-1c and Fasn genes related to fat synthesis;Pgc-1α and Cpt1 genes related to fatty acid oxidation.Results:(1)Although mice in the 3B model group had significantly higher liver index,serum AST and ALT levels,liver MDA and TC values,and significantly lower liver GSH and SOD values;however,the H&E results showed only a small number of small vacuoles of steatosis;the Oil Red O results showed no significant accumulation of lipid droplets in the liver.The liver injury indexes of E10d+3B model group were higher and more significant than those of 3B model group;the H&E results showed that the liver results were disordered,with a large number of fatty degeneration vacuoles and cell necrosis;the Oil Red O results showed that the liver showed the accumulation of large and small lipid droplets,and the liver fat content was significantly increased.In contrast,mice treated with alcohol for up to 4,8 and 12 weeks showed severe liver injury in each group,and the degree of injury increased more significantly with longer alcohol treatment time,and the fatty degenerative vacuoles and cell necrosis in the liver in the H&E results and the accumulation of hepatic lipid droplets in the Oil Red O results increased positively with increasing alcohol treatment time.The liver fat content exceeded 10%after 4 weeks of alcohol treatment,while extending to 12 weeks of alcohol treatment,the liver fat content exceeded 20%.Sirius red staining showed that after 4 weeks of alcohol treatment failed to induce fibrosis in the liver;while when alcohol treatment was extended to 8 weeks,slight fibrosis appeared in the liver;and after 12 weeks of alcohol treatment,liver fibrosis increased.The results of real-time fluorescence quantitative PCR and western blot showed that alcohol significantly induced the upregulation of Cyp2e1 expression in the liver.(2)ALD in mice using the modeling method of E10d+3B and the administration of silymarin glucosamine intervention resulted in significant improvements in liver injury indicators,oxidative stress indicators,liver lipid metabolism indicators and liver histopathology.(3)The siRNA sequence with 90% inhibition effect was obtained by in vitro screening,and then siRNA targeting to mouse liver Cyp2e1 by lipid nanoparticle delivery achieved 70% protein knockdown effect,and the knockdown effect was effectively sustained until day 10.Alcoholic liver injury was induced by 5% alcohol liquid diet feeding for 10 days plus gavage of 5 g/kg alcohol for the last three days,and administration of si-Cyp2e1 LNPs tail vein significantly inhibited the development of ALD.The results showed that inhibition of Cyp2e1 expression in vivo effectively reduced alcohol-induced liver injury and suppressed oxidation-related genes(Nox4,P47 phox,P67phox and Gp91phox)and promoted the expression of antioxidant-related genes(Gsh-Px,Gsh-Rd and Sod1),inhibited the expression of lipid synthesis-related genes(Srebp-1c,Acc and Fasn)and induced the expression of fat metabolism-related genes,and inhibited the expression of inflammation-related genes(Tgf-β,Tnf-α and Il-6).Improving the body’s antioxidant capacity reduced the hepatic oxidative stress response,thereby inhibiting hepatic fat accumulation and inflammatory response.(4)After 12 weeks of 4% alcohol liquid diet plus twice weekly treatment with 5 g/kg alcohol gavage,alcoholic liver injury,steatosis,fat accumulation,inflammatory response and fibrosis were induced in mice.At the initial administration,the therapeutic effects of si-Cyp2e1 LNPs and silymarin glucosamine administration on ALD were comparable,but the therapeutic effects of the combination were weaker;at the middle administration,the therapeutic effects of ALD were si-Cyp2e1 LNPs>silymarin glucosamine group>combination group;at the later administration,the therapeutic effects of ALD were si-Cyp2e1 LNPs>silymarin glucosamine group and combination group.The effect of ALD treatment was si-Cyp2e1 LNPs>salicylic acid glucosamine group and no significant effect in the combination group.Compared with siCyp2e1 LNPs or silymarin glucosamine alone,the expression of liver fat synthesis-related genes Srebp1 c and Fasn was increased and the expression of fatty acid oxidation-related genes Cpt1 and Pgc-1α was decreased in the coadministered group.Conclusion:(1)Different stages of alcoholic liver injury were successfully constructed by acute ethanol gavage and chronic ethanol feeding.Chronic alcohol feeding combined with acute alcohol gavage could lead to more serious liver injury and fatty liver than alcohol gavage alone;the degree of liver injury induced by increasing the time of alcohol treatment deepened,and up to 12 weeks of alcohol treatment could even lead to slight liver fibrosis.(2)This study was validated that silymarin has a better protective effect on ALD.(3)The siRNA lipid nanoparticles targeting hepatic CYP2E1 were successfully constructed,and the expression of CYP2E1 was knocked down efficiently and stably,and the in vivo regulation of CYP2E1 was realized.By knocking down CYP2E1 expression,we inhibited ROS production and thus inhibited oxidative stress,promoted lipolysis and inhibited lipid neosynthesis to reduce hepatic fat accumulation,suppressed hepatic inflammation and fibrotic response,and thus reduced alcohol-induced liver injury.(4)Using a 12 weeks alcohol treatment model and administering si-Cyp2e1 LNPs and Silymarin alone or in combination at different stages,the therapeutic effect of si-Cyp2e1 LNPs was superior to silymarin superior to the combination,and the early intervention effect of ALD was superior to the mid-term administration superior to the late-term administration.si-Cyp2e1 LNPs in combination with Silymarin instead had an effect This may be related to the fact that the excessive consumption of ROS promotes lipid synthesis and inhibits fatty acid metabolism.