Characterization of the biological effects of natural compounds against inflammation, metabolic syndrome and cancer
- Autores: Sara Tomás Hernández
- Directores de la Tesis: Miquel Mulero Abellán (dir. tes.), Santiago Garcia Vallvé (codir. tes.)
- Lectura: En la Universitat Rovira i Virgili ( España ) en 2017
- Idioma: español
- Tribunal Calificador de la Tesis: Mayte Blay Olivé (presid.), Francesc Puiggròs Llavinés (secret.), Olivier R.Baris (voc.)
- Programa de doctorado: Programa de Doctorado en Nutrición y Metabolismo por la Universidad Rovira i Virgili
- Materias:
- Enlaces
- Tesis en acceso abierto en: hdl.handle.net TDX
- Resumen
Natural products (NPs) have been used since ancient times for the prevention and treatment of many diseases and illnesses. Humans have relied on nature for the cure and prevention of a wide spectrum of diseases using traditional medicines, remedies, potions and oils with bioactive natural compounds. Many of these natural molecules have gone on to become current drugs.
The main goal of this doctoral thesis is to characterize selected bioactive natural compounds that could be used for the prevention or treatment of some major significant diseases, such as metabolic syndrome, neurodegenerative diseases and cancer. These medical conditions constitute an important health problem worldwide since they are included in the world leading causes of death.
In recent years, NPs and their active metabolites have become increasingly important on pharmaceutical research since they are a very important source when looking for novel molecules that could be used as lead compounds during the development of new drugs. Unfortunately, bioactivity screening in extracts exclusively by in vitro or in vivo approaches is a complex and expensive process which is difficult to afford. In that sense, computer-aided drug design (CADD) methodologies like Virtual Screening (VS) have been successfully used to screen large NPs databases in order to identify new bioactive molecules for specific targets.
The first part of this thesis was focused on the identification and characterization of natural compounds with anti-inflammatory properties. Since numerous studies have shown that the chronic inflammation process is directly involved in the onset of metabolic syndrome and neurodegenerative diseases we aimed to find a natural compound that could revert this harmful and persistent inflammation state.
In these initial studies, firstly, we applied previously validated VS workflow developed by our group to a natural products database in order to identify potential IKK-2 inhibitors. One of this predicted IKK-2 inhibitors was o-orsellinaldehyde, a molecule contained in the Grifola frondosa mushroom specie.
In that sense, in order to validate the in silico predictions we performed a kinase assay that confirmed that o-orsellinaldehyde directly targets IKK-2 and reduces its IKK-2 kinase activity in a dose-response manner. Additionally, we validate that o-orsellinaldehyde significantly inhibited IKβα phosphorylation in LPS-stimulated RAW 264.7 macrophage cells. Moreover, the anti-inflammatory properties of the studied molecule have also been demonstrated by the obtained results referred to the reduction of nitrites, IL-6 and iNOS expression in the in vitro model used.
To study if this compound was also effective in vivo we induced an LPS endotoxic shock model in Balb/c mice. In both accomplished studies (intraperitoneal administration and oral administration of o-orsellinaldehyde) the molecule significantly reduced the serum IL-6 concentration. All this findings provide strong evidences that o-orsellinaldehyde possesses anti-inflammatory properties and that it exerts its activity by influencing NF-kB activity, more concretely, acting as IKK-2 inhibitor.
We wanted to study the citotoxic effect of this molecule described by other groups. Our results also show that o-orsellinaldehyde exhibits a cytotoxic effect on HepG2 cells and that this effect was mediated through an apoptotic process. Bearing in mind that NF-kB complex is also involved in propagating the cellular response to apoptosis and carcinogenesis we suggest that some of the cytotoxic and apoptotic effects of o-orsellinaldehyde observed against the cancer cells could be due to NF-kB inhibition.
Finally, Grifola frondosa mushrooms extracts were analyzed by LC-QTOF-MS in order to identify and quantify the amount of this compound and evaluate the feasibility of preparing extracts from this natural source that were enriched on o-orsellinaldehyde. The results of this experiments show that, although we were able to isolate the molecule, the amount of this compound in the extracts prepared was very low, suggesting that further studies would be necessary in order to isolate enough amount of the anti-inflammatory molecule from the mushroom specie.
In summary this first study presented the potential anti-inflammatory activity of o-orsellinaldehyde, suggesting that it may be a potential preventive or therapeutic candidate for the treatment of inflammatory disorders such as metabolic syndrome. (Manuscript 1) Once examined the beneficial properties of the o-orsellinaldehyde in RAW 264.7 cells and in Balb/c mice we wanted to elucidate if this molecule could also exert this potent anti-inflammatory effect in a neuroinflammation context. In that sense, we evaluate the effects of o-orsellinaldehyde in LPS-activated glia. (Manuscript 2).
The results obtained show that o-orsellinaldehyde caused an inhibition on Nitrite oxide (NO) production and iNOS expression after LPS-stimulation in both mixed glia and microglia cells. We also evaluated the effect of o-orsellinaldehyde in the expression of the stress protein heme oxygenase-1 (HO-1), observing that o-orsellinaldehyde cell pretreatment decreases HO-1 protein over-expression in LPS-activated mixed glia and microglia cells.
Furthermore, we observed that cell pre-treatment with o-orsellinaldehyde suppressed the phosphorylation of Ikβα in LPS-activated microglia. Thus confirming that o-orsellinaldehyde exerts its anti-inflammatory effects by acting as IKK-2 inhibitor and modulating NFkB activity.
Moreover, MAPKs, including p38 and JNK play an important role in the inflammatory response since they are involved in the microglia activation. In that sense, we investigated the effect of o-orsellinaldehyde in the phosphorylation state of these MAPKs in LPS-stimulated microglia. Our results indicates that o-orsellinaldehyde markedly inhibit the activation of both, p38 and JNK, after LPS stimulation in microglial cells. Thus suggesting that the anti-inflammatory effect of this molecule could also be exerted by the inhibition of the MAPKs signaling pathway.
Regarding the potential of o-orsellinaldehyde on promoting M2 polarization in LPS-activated microglia we studied the expression of several genes associated with either M1 or M2 phenotypes. On the basis of our results we can state that the molecule tested, o-orsellinaldehyde, is able to promote the macrophage switch from inflammatory M1 type to the anti-inflammatory M2 phenotype.
In summary, with this study we demonstrated that o-orsellinaldehyde is a potent modulator of microglia activation and possesses strong neuroprotective effects. Thus suggesting that this molecule could improve some neuroinflammatory states and could be used as a hypothetical candidate for the treatment of several pathologies that deal with neuroinflammation.
The second part of this doctoral thesis was focused on the identification and characterization of novel PPARγ partial agonists from natural origin that present anti-diabetic properties without owning the side effects associated with the current existing drugs. (Manuscript 3 and Manuscript 4) Novel PPARγ ligands or modulators are being developed as new antidiabetic drugs and have revealed a new mechanism of action for the anti-diabetic effect of some PPARγ agonist. This mechanism is completely independent of the classical PPARγ transactivation and consists on the inhibition of the phosphorylation of PPARγ on Ser 273, thereby preventing the deregulation of a several PPARγ beneficial related genes involved in metabolism homeostasis. It is has been suggested that a substantial portion of the therapeutic benefits of PPARγ ligands in metabolic disease is through the inhibition of the phosphorylation of PPARγ at Ser 273, whereas at least some of the undesirable side effects caused by current full agonist drugs may occur due to classical agonist actions.
With this purpose, our group in a previous study, designed and applied a virtual screening workflow to identify novel PPARγ partial agonists among natural products databases. This in silico experiment successfully identified several molecules with high chance of being effective PPARγ partial agonists. One of this predicted molecules was2,4,6-Trimethoxybenzophenone a molecule contained in the C. sumatranum ssp. Neriifolium plant.
On the basis of this in silico results, we wanted to examine the effect of this novel partial agonist on the Cdk5-mediated phosphorylation of PPARγ as well as its influence on adipogenesis. In that sense, we developed and performed a kinase assay and we further study the effect of the test compound in the in the adipogenic differentiation process of 3T3-L1 cells. Our results demostrated that the natural compound 2,4,6-Trimethoxybenzophenone could be a novel PPARγ ligand that would retain the benefits of improving insulin resistance since its able to inhibit Cdk5-mediated PPARγ phosphorylation at ser273, but minimizes the common side effects of TZDs, such as adipogenesis, by alleviating PPARγ agonistic activity. Although further characterization of the ant-diabetic properties of this compound is needed, these preliminary results suggest that the molecule studied could be a promising candidate drug for the treatment of some metabolic diseases, such as T2DM.
The last part of this thesis aimed to clarify the combined effect of the two natural compounds Quercetin and Resveratrol, which have been previously described as potent autophagic inducers, on this important catabolic process in HepG2 cells. (Manuscript 5) Unexpectedly we discovered that the combination of both polyphenols has an opposite effect on autophagy as well as on ER-stress. QCT induced a potent autophagic activation in HepG2 cells that was confirmed through acridine orange staining and conversion of LC3- II . This QCT-mediated activation of autophagy decreased ER-stress induced by RSV. On the other hand, RSV was also able to promote a significant but weaker (compared to QCT) autophagic process. Besides, QCT promoted a “starvation-like” state characterized by a potent signaling through AMPK which could be, at least partially, responsible for this autophagic process. Interestingly, although both compounds were not equally able to inhibit mTOR phosphorylation, the combination of QCT and RSV was the treatment that induced the most potent inhibition on the phosphorylation of mTOR.
Nevertheless, in a cancerous context, such as our HepG2 in vitro model or others, QCT in a combined treatment with an autophagy inhibitor, may be an excellent therapeutic approach to reduce cancer cell proliferation and could be a promising strategy to sensitize cells to QCT treatment. In conclusion, RSV will act differentially on the autophagic process depending on the cellular energetic state. We have further characterized the molecular mechanisms that are related to this effect and we observed that the AMP-activated protein kinase (AMPK) phosphorylation and heme oxygenase 1 (HO-1) downregulation could be important modulators of such RSV related effect, and could globally represent a promising strategy to sensitize cancer cells to QCT treatment.
