Periodic Reporting for period 1 - Food-PPP-NAFLD (Discovery and characterization of food bioactive compounds modulating the Pentose Phosphate Pathway against non-alcoholic fatty liver disease.)
Reporting period: 2019-10-01 to 2021-09-30
Non-alcoholic fatty liver disease (NAFLD) epidemic is a public health concern with a severe healthcare burden. It affects about 25% of adults and currently there is no approved pharmacological treatment. In the general adult population, NAFLD prevalence is higher in men than in women. It has been demonstrated that female and male livers are metabolically distinct organs. Despite the current knowledge on the topic, there is no evidence to allow sex-specific personalized therapies. Thus, it is key to identify sex-dependent metabolic pathways in order to address a personalized therapy.
Oxidative stress and inflammation have been recognised as important factors in the pathological progression of NAFLD. The accumulation of hepatic lipids derives into oxidative damage, manifested in the liver as an increased lipid peroxidation, which subsequently can result in hepatocyte injury and cell death. This scenario can trigger an inflammatory response and progression to non-alcoholic steatohepatitis (NASH). Therefore, therapies to protect against liver redox imbalance in the progression of this disease are urgently needed. In this regard, the overall objective of this new Marie-SkŁodowska-Curie Action was to dissect the role of the pentose phosphate pathway (PPP) as a therapeutic strategy to prevent oxidative stress and inflammation as the primary target against NASH progression. To achieve this aim, the gender dimension has been considered using both genetic and pharmacological approaches in a mouse diet intervention inducing NASH. In addition, to find new bioactive compounds with the ability to modulate the PPP as pharmacological drugs to treat NASH, a screening was performed.
Overall, we have observed that using both the genetic and the pharmacological strategies, females were more protected than males against the oxidative and inflammatory damage induced by the mouse diet intervention inducing NASH. The pharmacological approach reduced more efficiently the NASH liver parameters, including an amelioration of the histological evaluation, the oxidative stress markers and reduction of the liver triglycerides.
Oxidative stress and inflammation have been recognised as important factors in the pathological progression of NAFLD. The accumulation of hepatic lipids derives into oxidative damage, manifested in the liver as an increased lipid peroxidation, which subsequently can result in hepatocyte injury and cell death. This scenario can trigger an inflammatory response and progression to non-alcoholic steatohepatitis (NASH). Therefore, therapies to protect against liver redox imbalance in the progression of this disease are urgently needed. In this regard, the overall objective of this new Marie-SkŁodowska-Curie Action was to dissect the role of the pentose phosphate pathway (PPP) as a therapeutic strategy to prevent oxidative stress and inflammation as the primary target against NASH progression. To achieve this aim, the gender dimension has been considered using both genetic and pharmacological approaches in a mouse diet intervention inducing NASH. In addition, to find new bioactive compounds with the ability to modulate the PPP as pharmacological drugs to treat NASH, a screening was performed.
Overall, we have observed that using both the genetic and the pharmacological strategies, females were more protected than males against the oxidative and inflammatory damage induced by the mouse diet intervention inducing NASH. The pharmacological approach reduced more efficiently the NASH liver parameters, including an amelioration of the histological evaluation, the oxidative stress markers and reduction of the liver triglycerides.
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.
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.
NAFLD is considered the hepatic component of the metabolic syndrome and it is associated to obesity, insulin resistance and hyperlipidaemia. It is now recognized as the most prevalent chronic liver disease worldwide and it is estimated that the NASH prevalence could increase by up to 56% in the next decade. Thus, any effort to reduce its prevalence will have a great socio-economic impact.
From a scientific point of view, the outcomes obtained from this Project will contribute towards a better understanding of the role of the pentose phosphate pathway in the context of the redox status and inflammation physiopathology of NAFLD progression. This research may lead to the use of the redox status as a surrogate marker of NAFLD progression, which could have great implications in the future diagnosis and therapy of patients.
There is evidence about the sex difference in NAFLD; but more consideration is urgently needed to fill the gaps in the current knowledge of this disease and implement precision medicine for patients suffering NAFLD. In our study we have identified how the sexual dimorphism affected this hepatic disease. We found that our pharmacological approach ameliorated the progression of NASH in females, but it had a minor effect in male mice. This may increase the awareness towards a more personalized medicine approach to treat individuals more effectively.
Finally, we have developed a screening of bioactive compounds that increase the hepatic NADPH levels. These new molecules (bioactive compounds), will be evaluated as therapeutic strategies for the treatment of NAFLD and other pathologies related to redox status with a high prevalence in the current society. Those compounds that display the best efficacy improving NAFLD progression will be susceptible of patent application or to be disclosed with the pharmaceutical Industry for its exploitation.
In all the project contributes towards a preventive approach in the progression of the NAFLD disease, which will promote the sustainability and efficiency of National Health Systems worldwide.
From a scientific point of view, the outcomes obtained from this Project will contribute towards a better understanding of the role of the pentose phosphate pathway in the context of the redox status and inflammation physiopathology of NAFLD progression. This research may lead to the use of the redox status as a surrogate marker of NAFLD progression, which could have great implications in the future diagnosis and therapy of patients.
There is evidence about the sex difference in NAFLD; but more consideration is urgently needed to fill the gaps in the current knowledge of this disease and implement precision medicine for patients suffering NAFLD. In our study we have identified how the sexual dimorphism affected this hepatic disease. We found that our pharmacological approach ameliorated the progression of NASH in females, but it had a minor effect in male mice. This may increase the awareness towards a more personalized medicine approach to treat individuals more effectively.
Finally, we have developed a screening of bioactive compounds that increase the hepatic NADPH levels. These new molecules (bioactive compounds), will be evaluated as therapeutic strategies for the treatment of NAFLD and other pathologies related to redox status with a high prevalence in the current society. Those compounds that display the best efficacy improving NAFLD progression will be susceptible of patent application or to be disclosed with the pharmaceutical Industry for its exploitation.
In all the project contributes towards a preventive approach in the progression of the NAFLD disease, which will promote the sustainability and efficiency of National Health Systems worldwide.