• Apolipoprotein A-IMilano
• atherosclerosis
• non-alcoholic steatohepatitis

This project has been inserted involves the development and validation of a new delivery system for a biological drug for the treatment of atherosclerosis and other inflammatory disorders, such as non-alcoholic steatohepatitis. In previous studies, genetically modified rice plants were produced so that they express the human protein APOA-IMilano (AIM), a natural variant of Apolipoprotein A-I with a well-known athero-protective properties (induction of cholesterol efflux, among others). The genetically modified seeds of rice were used to produce the so-called "AIM Rice Milk", which was tested firstly in an in vitro macrophages model and then orally delivered to atherosclerotic mice. In the macrophages (exposed to the oxLDL stimulus), the AIM rice milk was shown to be effective in inhibiting lipids accumulation, while in the mice (atherosclerosis models: Apoe-/- mice exposed to a high fat diet), it was effective in reducing the extent of atherosclerotic plaques and lipid accumulation in the aortas and inflammation in the liver.
Atherosclerosis foresees dyslipidemia among the main risk factors and, since the liver is the central organ of cholesterol metabolism, dyslipidemia is also associated to a liver disease called non-alcoholic steatohepatitis (NASH). Therefore, this work started from a model of atherosclerosis, but then focused on a picture of NASH disease, which presents itself as comorbidity of atherosclerosis.
Once the beneficial effects of AIM rice milk treatment were confirmed, I focused on the evaluation of histological and molecular differences in two different models of atherosclerotic mice, which involved a different schedule of administration of the AIM rice milk/wt rice milk (3 weeks once a day and 4 weeks twice a day). First, on mouse liver sections that had received 4 weeks of treatment, I evaluated macrophage activation and neutrophil infiltration and in both cases was obtained a significant reduction in mice treated with Apo AIM milk compared to control mice (atherosclerotic models given wild type milk). For macrophages, the same result was obtained in previous studies on mice that had received 3 weeks of treatment.
After the in situ study, I evaluated the response at a molecular level. Starting from a literature research on the relationship between hepatic metabolism and microbiome, I learned that the microbiome mainly alters the pathways of the immune system and lipid metabolism and, through a correspondence with the genes of a microarray made on the livers of mice that had received 3 weeks of treatment, several genes belonging to these pathways were differentially expressed between AIM-treated mice and control mice (14 genes implicated in lipid metabolism and 11 in the immune system). Some of these genes and others differentially expressed in the microarray were found to be under the control of 3 miRNAs (miR-122, miR-155 and miR-342), known to have a protective role in models of atherosclerotic / NASH disease.
For the validation with RT-PCR of what has been obtained with the microarray, we chose 21 genes from those connected to the microbiome and those target of miR-122, miR-155 and miR-342 and some of them have confirmed the same trend.