ETD

• ApoA-I Milano variante
• NASH-HCC
• oral drug delivery
• plant based
• STAM model

Non-alcoholic fatty liver disease (NAFLD) encompasses a highly heterogeneous spectrum of liver disorders, all characterized by hepatic fat accumulation in the absence of significant alcohol consumption. It has rapidly become a subject of great scientific and biological interest, specifically its chronic and progressive form, non-alcoholic steatohepatitis (NASH).
NASH is primarily characterized by steatosis, resulting in hepatic inflammation, hepatocyte ballooning, and varying degrees of fibrosis.
Given the multifactorial nature of NASH, a definitive pharmacological cure is currently lacking, leaving lifestyle modification as the primary therapeutic strategy. Furthermore, this condition can progress to Hepatocellular Carcinoma (HCC) through a process involving DNA damage, oxidative stress, metabolic dysregulation, and significant genetic vulnerability.
The role of High-Density Lipoproteins (HDLs) is decisive in maintaining lipid homeostasis and influencing disease progression. Thanks to their anti-inflammatory effects, HDLs can contribute to containing pathological mechanisms. Moreover, HDLs play a key role in reverse cholesterol transport from peripheral tissues to the liver. Apolipoprotein A-I (ApoA-I), the most important protein component of HDL, is the primary mediator of its anti-atherogenic activity, acting as the preferential cellular acceptor of cholesterol.
The allelic variant investigated in this project is ApoA-I Milano (AIM), originally discovered in a small community in Northern Italy near Lake Garda. Its effects result in reduced plasma HDL concentrations without an increased risk of cardiovascular disease, suggesting a unique therapeutic potential for this variant in protecting against atherosclerosis.
To this end, a "plant-based" system was studied, in which genetically engineered rice grains express the ApoA-I Milano variant. This allows us to produce a liquid extract that can be administered orally without the need for further protein purification. This approach was previously tested by the research group in a murine model of atherosclerosis, showing significant improvement in the primary hallmarks of the disease.
To evaluate and quantify the therapeutic efficacy of this apolipoprotein variant, the STAM murine model was employed. This model faithfully reproduces the progression from NAFLD to NASH and, ultimately, HCC, making it the most reliable preclinical model available today. The mice were fed into a High-Fat Diet (HFD) to induce steatosis and were subsequently treated with the AIM protein variant via oral administration.
The primary objective of this thesis project is to verify the efficacy of the AIM protein, administered as a lyophilized rice product, in preventing the progression from NAFLD to NASH and subsequently to HCC. To achieve this, multi-level investigations were conducted.
First, monitoring data from the AIM-rice treatment on STAM mice was analyzed and compared to the control group (treated with Wild-Type rice), evaluating growth curves, survival rates, and phenotypic changes throughout the experiment. Subsequently, ex vivo macroscopic analyses were performed to assess tumor mass volume.
Finally, ex vivo microscopic analyses were conducted to compare the two experimental groups regarding the number and area of lesions, their primary morphological characteristics, inflammatory infiltration, and hepatic steatosis.
Taken together, these data serve to provide a more accurate evaluation of the therapeutic potential of ApoA-IM-expressing rice, with the future prospect of oral administration for patients suffering from this specific pathology.