Reducing Oxidative Stress-Mediated Alcoholic Liver Injury by Multiplexed RNAi of Cyp2e1, Cyp4a10, and Cyp4a14

Abstract

The prevalence of excessive drinking-related alcoholic liver disease (ALD) is rising, yet therapeutic options remain limited. High alcohol consumption and consequent oxidative metabolism by cytochrome P450 (CYP) can lead to extremely high levels of reactive oxygen species, which overwhelm cellular defenses and harm hepatocytes. Our previous investigations showed that inhibiting Cyp2e1 using RNA interference reduced the incidence of ALD. However, compensatory mechanisms other than CYP2E1 contribute to oxidative stress in the liver. Therefore, we coupled triple siRNA lipid nanoparticles (LNPs) targeting Cyp2e1 with two isoenzymes Cyp4a10 and Cyp4a14 to treat ALD mouse models fed with Lieber-Decarli ethanol liquid diet for 12 weeks at the early (1st week), middle (5th week), and late (9th week) stages. The administration of triple siRNA LNPs significantly ameliorated chronic alcoholic liver injury in mice, and early treatment achieved the most profound effects. These effects can be attributed to a reduction in oxidative stress and increased expression of antioxidant genes, including Gsh-Px, Gsh-Rd, and Sod1. Moreover, we observed the alleviation of inflammation, evidenced by the downregulation of Il-1β, Il-6, Tnf-α, and Tgf-β, and the prevention of excessive lipid synthesis, evidenced by the restoration of the expression of Srebp1c, Acc, and Fas. Finally, triple siRNA treatment maintained normal metabolism in lipid oxidation. In brief, our research examined the possible targets for clinical intervention in ALD by examining the therapeutic effects of triple siRNA LNPs targeting Cyp2e1, Cyp4a10, and Cyp4a14. The in vivo knockdown of the three genes in this study is suggested as a promising siRNA therapeutic approach for ALD.

Keywords: alcoholic liver disease; cytochrome P450s; ferroptosis; in vivo RNAi; inflammation.

Figure 1

Diagram showing the administration of drugs for chronic alcoholic liver injury. LDC: Lieber–DeCarli; LNP: Lipid nanoparticle.

Figure 2

Knockdown effects of triple siRNA LNPs in vivo. (a) Representative transmission electron microscopy images of LNPs encapsulated with triple siRNA. (b,c) Effect of knocking down Cyp2e1 gene in an alcohol mouse model on CYP4A. (n = 4). ** p < 0.01 versus pair-fed. (d) Changes over time in Cyp2e1, Cyp4a10, and Cyp4a14 mRNA levels in mice injected with a single dose of triple siRNA LNPs. ** p < 0.01 versus pair-fed. (e,f) Changes in CYP2E1 and CYP4A protein levels over time in mice injected with a single dose of triple siRNA LNPs. Data are expressed as mean ± standard deviation (SD) (n = 8). * p < 0.05 and ** p < 0.01 compared with Dulbecco’s phosphate-buffered saline (DPBS) as control; n.s. means not significant. LNPs: lipid nanoparticles.

Figure 3

The role of triple gene knockdown in the treatment of alcoholic liver injury. (a) Gross morphology of the liver of mice in each group. (b) Effect of triple siRNA LNP administration on the liver index. (c,d) Effect of triple siRNA LNPs on serum AST and ALT levels in mice with alcoholic liver injury. (e,f) The protein expression levels of CYP2E1 and CYP4A. (g) Histopathological sections of the mouse liver were stained with hematoxylin and eosin (200×). (h) Sirius scarlet staining of the histopathological sections of mouse liver (200×). Data are expressed as the mean ± SD (n = 8). ** p < 0.01 versus pair-fed; # p < 0.05 and ## p < 0.01 versus alcohol-fed. PF/AF/ET/MT/LT: Pair-fed/Alcohol-fed/Early-treatment/Mid-treatment/Late-treatment.

Figure 4

Antioxidant levels in liver tissue and the effect of triple siRNA LNPs on blood lipid levels in mice. (a–f) Effect of si-Cyp2e1 LNP on hepatic glutathione peroxidase (GSH-PX), glutathione (GSH), reactive oxygen species (ROS), catalase (CAT), superoxide dismutase (SOD), and malondialdehyde (MDA) levels in alcoholic liver-injured mice. (g,h) Effect of triple siRNA LNPs on hepatic triglyceride (TG) and total cholesterol (TC) levels in mice with alcoholic liver injury. (i) Quantification of Oil Red O staining in different groups. (j) Histopathological sections of the mouse liver stained with Oil Red O (200×). Orange-red represents obvious fat accumulation in the liver. Data are expressed as the mean ± SD (n = 8). ** p < 0.01 versus pair-fed; # p < 0.05 and ## p < 0.01 versus alcohol-fed. PF/AF/ET/MT/LT: Pair-fed/Alcohol-fed/Early-treatment/Mid-treatment/Late-treatment.

Figure 5

Fluorescence immunohistochemistry of F4/80 (red) in the mouse liver; the nuclei are stained with DAPI (blue) (200×). ET/MT/LT: Early-treatment/Mid-treatment/Late-treatment.

Figure 6

Role of triple siRNA LNPs in oxidative stress and levels of lipid- and inflammation-related genes and proteins. (a) mRNA expression levels of Il-1β, Il-6, Tnf-α, and Tgf-β. (b) mRNA expression levels of Gsh-px, Gsh-rd, and Sod1. (c) The mRNA expression levels of Cpt1 and Pgc-1α. (d) The mRNA expression levels of Fas, Srebp1c, and Acc. (e,f) Relative protein expression levels of CPT1, SREBP1c, FAS, and PGC-1α. Data are expressed as mean ± SD (n = 8). * p < 0.05 and ** p < 0.01 versus pair-fed; # p < 0.05 and ## p < 0.01 versus alcohol-fed. The mRNA levels are relative to the levels of the reference gene Gapdh, and the protein levels are relative to the levels of the reference protein GAPDH. ET: Early-treatment.

Figure 7

Effects of triple siRNA LNPs on alcohol-induced ferroptosis in hepatocytes. (a) The mRNA expression levels of Nrf2, HO-1, and GPX4. (b,c) The relative protein expression levels of GPX4. Data are expressed as mean ± SD (n = 8). ** p < 0.01 versus pair-fed; # p < 0.05 and ## p < 0.01 versus alcohol-fed. The mRNA levels are relative to the levels of the reference gene Gapdh, and the protein levels are relative to the levels of the reference protein GAPDH. ET: Early-treatment.