Study On The Role Of Lipid Nanoparticles Transporting Multiple SiRNAs In Alcoholic Liver Disease

Alcoholic liver disease（ALD）is a liver disease caused by damage to hepatocytes resulting from heavy alcohol consumption,and depending on the degree of clinical progression,alcoholic liver damage may lead to the development of alcoholic liver fibrosis.RNA interference（RNAi）is a technique for silencing specific genes to regulate the expression of genes targeted by complex mechanisms of disease.RNA interference（RNAi）is a technique for silencing specific genes to regulate the expression of genes targeted by complex mechanisms of disease in vivo,leading to therapeutic effects.The molecular mechanisms underlying the development of alcoholic liver injury are still unclear,and there is a need to identify key targets for the treatment of ALD.Cyp2e1 knockout(Cyp2e1^-/-)mice can reduce alcoholic liver injury,demonstrating the critical role of CYP2E1 in the development of ALD research and treatment.Given that Cyp2e1^-/-mice do not completely block alcohol-induced oxidative stress and that the remaining oxidative stress pathways are still present in mice after alcohol administration as a surrogate,it was found that CYP4A10 and CYP4A14,two key factors in CYP4A,showed increased surrogation when CYP2E1 was lowly expressed,and therefore the simultaneous inhibition of the triple genes in vivo by RNAi technology was considered to Therefore,the simultaneous inhibition of the expression of these three genes in vivo by RNAi technology could synergistically increase the therapeutic effect on ALD.Objectives:To investigate the combined use of lipid nanoparticles（triple siRNAs LNPs）targeting the hepatic Cyp2e1,Cyp4a10 and Cyp4a14 triplet genes in the treatment of two models of subacute and chronic alcoholic liver disease.We also investigated the regulation of long-term alcohol-induced hepatic fibrosis and iron death in mice,the expression of regulatory genes and the flexible control of target gene silencing,and the mechanisms involved in the prevention and treatment of different severity of ALD.Methods:（1）Preparation and characterization of triple siRNAs LNPs.The triple siRNA sequences were encapsulated in lipid nanoparticles by microfluidic technology.The particle size of the triple siRNAs LNPs was measured and their appearance was observed by transmission electron microscopy to determine the encapsulation rate and drug loading.In vivo experiments were carried out to screen for optimal dosing conditions,both in terms of dose and time.（2）The effect of triple siRNAs LNPs on the treatment of subacute alcoholic liver disease and chronic alcoholic liver disease.Sub-acute ALD and chronic ALD mouse models were constructed respectively,and the chronic model was administered in 3 stages.The serum and liver of the mice were collected,and the liver indexes such as biochemical indexes,oxidative indexes and fat metabolism were measured by index test kits,and liver tissue sections were analysed for H&E staining,Sirius scarlet staining,oil red O staining and F4/80immunofluorescence staining to determine the damage caused by acute alcoholism to the liver of the mice.The effect of triple siRNAs LNPs administered at different stages of alcohol consumption on chronic drinking mice was analysed.（3）Triple siRNAs LNPs were administered prophylactically to study the mechanism of liver damage in chronic mice.RT-PCR and Western Blot were used to detect the expression levels of oxidative stress,lipid synthesis and inflammation related factors.The severity of liver fibrosis in mice was studied and the expression of m RNA and protein of liver fibrosis-related factors was analysed after administration.Tissue iron content was analysed and the expression levels of factors associated with iron death were measured after administration.Results:（1）The triple siRNAs LNPs were characterized with particle size around 80 nm,spherical or oval shape,high drug loading and encapsulation rate,high gene silencing could be maintained at0.5 mg/kg dose in the in vivo study,and gene silencing could be maintained for at least 7 days after dosing.（2）Both subacute and chronic model groups of mice showed abnormal liver appearance,with significantly higher liver index,serum aspartate aminotransferase（AST）and alanine aminotransferase（ALT）levels,malondialdehyde（MDA）,triacylglycerol（TG）and total cholesterol（TC）values measured after liver homogenization,and significantly higher liver homogenization glutathione peroxidase（GSH-PX）and Superoxide dismutase（SOD）values were significantly lower;the H&E results were analysed with more fat vacuoles.The oil red O results showed a significant increase in fat content,along with the accumulation of lipid droplets.A small amount of collagen fibrils could also be observed in the Sirius Scarlet stain and a positive fluorescent expression in the F4/80 immunofluorescence results with an inflammatory response.Triple siRNAs LNPs protect the liver from acute alcohol damage.In chronic alcohol model studies,triple siRNAs LNPs were initially most effective in prevention and triple siRNAs LNPs were effective in treating ALD at different stages.（3）The initial administration of triple siRNAs LNPs formed a preventive and therapeutic effect.Compared with the model group,the expression of antioxidant-related factors（Gsh-px,Gsh-rd and Sod1）was significantly increased,the expression of related lipid metabolism and synthesis factors（Srebp-1c,Acc and Fasn）was significantly decreased,and the expression of inflammatory factors（Tgf-β,Tnf-α,Il-6 and IL-1β）were significantly reduced.The balance between oxidative and antioxidant systems was restored,liver fat accumulation was significantly reduced and inflammatory factor metabolism was normalized in the mice.In the model group,liver fibrosis was formed in the liver and m RNA and protein expression of liver fibrosis markers（α-SMA,Collenge-Ⅰand Collenge-Ⅲ）were increased,while triple siRNAs LNPs administration reduced alcoholic liver fibrosis.In the model group,hepatocytes showed significant iron death and increased iron content in liver tissues,which affected the normal metabolism of hepatocytes,while the expression of iron death indicators（Nrf2,HO-1 and GPX4）in hepatocytes decreased due to alcohol.Conclusions:（1）The triple siRNAs LNPs achieved the target gene targeting in vivo,successfully constructed gene silencing drugs,regulated the expression of triple genes in vivo,inhibited ROS production,lipid synthesis and regulated lipid metabolism,and were able to inhibit liver inflammation even,significantly reducing acute and chronic alcohol-induced liver injury.（2）The therapeutic efficacy of triple siRNAs LNPs administration was demonstrated to be superior to that of the positive drug metadoxine.（3）Administration of triple siRNAs LNPs at all three stages of ALD resulted in a better initial intervention than mid-stage administration of ALD than late administration.（4）Triple siRNAs LNPs can attenuate alcoholic liver fibres,reduce fibrotic response,modulate the occurrence of iron death and treat alcohol-induced cellular iron death.