Report of the main results achieved during the project.
1- An oxidative stress model based on the detection of free radicals by fluorescence, was established in two hepatic (AML12 and HepG2) cell lines. However, we could not achieve the characterization of the role of G6PD in this in vitro model since the state of alarm for the Covid outbreak was declared.
2- We optimized and set up a screening platform based on the detection of NADPH by bioluminescence in the mouse hepatic AML12 cell line. We identified 83 hits compounds, out of 1303 compounds tested, which were able to increase the NADPH reductive power up to a 20% compared to the control.
3- We performed an in vivo characterization of G6PD function in the progression of NAFLD. For this, we first determined that 3-weeks of a methionine and choline-deficient diet (MCD diet) intervention was the optimal time to induce NASH-like liver damage in our mouse colony. To test the effect of G6PD in the progression of NAFLD, we used a G6PD-transgenic (G6PD-tg) mouse line which increased the total G6pd mRNA expression by about 2 folds relative to wild-type littermates in both genders. We observed that only G6PD-tg female mice ameliorated some of the oxidative stress and inflammatory parameters related to the NASH phenotype induced by the 3-weeks MCD diet intervention.
4- We selected one of the compounds assessed in our screening to assess its properties in the prevention of NAFLD in a mouse model. Since we had a delay in the final validation of the compounds, because of COVID-19 outbreak, we used a positive control of the screening to achieve this aim. Two weeks before the MCD diet intervention, we started the drug treatment through their drinking water, that was continued during the whole MCD diet intervention.
In female mice, the drug treatment significantly reduced different parameters related to oxidative stress, although further analysis to explore the redox status markers are still in progress. In addition, histological evaluation of liver tissue revealed that, only in females, the drug treatment decreased the steatosis and the lobular inflammation induced by the MCD diet. The reduction of the inflammatory markers was accompanied by a modulation of some immune population analysed by flow cytometry.
In order to disseminate the results we currently have a manuscript in preparation to be submitted in a high standard Journal and to a conference. Despite the limited time (due to Covid reason) we have delivered 2 seminars for results dissemination addressed to the scientific community. To engage with the general audience, we have organised 2 webinars in collaboration with IMDEA FOOD and with Madri+d foundation, during the European Research Night 2020, that were also promoted via social media.
Regarding the possibility of results exploitation, since we had a delay in the performance of our screening, we have not had the opportunity to finally assess the applicability of the hits detected in our mouse model. In this line, our results are still in a very initial phase to be disclosed to the Industry or to be patentable. However, we are applying for further grants to develop the use of these compounds in the prevention of NAFLD disease.
