A ação anti-inflamatória do lítio é influenciada por fatores genéticos, nutricionais e fármacos antidepressivos: estudos in vitro

Abstract

Population aging has increased the prevalence of chronic non-communicable diseases, including psychiatric disorders such as depression and bipolar spectrum disorders (BSDs). Evidence suggests that such disorders are associated with chronic processes of inflammation and oxidative stress. Lithium is one of the main drugs of the psychiatric clinic, and its main mechanism of action is anti-inflammatory by inhibition of the enzyme GSK-3β. However, about 40% of patients do not respond satisfactorily to Lithium therapy. And, this absense of response may involve genetic polymorphisms, or even drug-food interaction, or drug-drug, since lithium is often associated with antidepressants or antipsychotics. Therefore, in this work we emphasize the action of the polymorphism Val16Ala-SOD2 which causes imbalance between the levels of Superoxide and Hydrogen Peroxide and is involved with oxidative and inflammatory metabolism. Bioactive molecules such as xanthines and catechins present in foods, which have an anti-inflammatory and antioxidant effect, and therefore, they could also influence the pharmacological response to lithium and the interaction lithium-antidepressants, often used in TEBs. To evaluate in vitro the influence of genetic and nutritional factors and pharmacological interaction with antidepressants on the anti-inflammatory response of lithium. Three experimental designs were conducted. The first work evaluated the influence of the anti-inflammatory response to the Lithium of peripheral blood mononuclear cells (PBMCs) through two complementary studies. Initially, a study was conducted to confirm that such genetic variation could cause a differentiated inflammatory response evaluating the immunosenescence profile of these cells. Therefore, PBMCs were obtained from volunteers with different Val16Ala-SOD2 genotypes and were cultured for 15 cell passages (60 days) under standardized conditions. At each passage the PBMCs were activated with phytohemagglutinin (PHA), an antigen that triggers inflammatory response. At each passage the PBMCs were activated with phytohemagglutinin (PHA), an antigen that triggers inflammatory response. Each passage was started at a concentration of 1 x 105 cells. The rate of cell proliferation at each passage was determined via MTT spectrophotometric assay and, eventually, by cell cycle analysis by flow cytometry. Pro-inflammatory markers and oxidative markers were also analyzed. We performed a second study, which evaluated the influence of the Val16Ala-SOD2 polymorphism on the modulation of the GSK-3β enzyme by Lithium through the analysis of the gene expression via qRT-PCR and the levels of protein via immunoassay ELISA. We also performed analyzes with the RAW 264.7 macrophages commercial lineage in order to confirm the results obtained in PBMCs. Supplementation of cultures with paraquat to generate VV-like cells (with high levels of superoxide) was used to simulate the S-HP imbalance And porphyrin generating AA-like cells (with elevated PH levels). The second protocol of this work evaluated the potential isolated effect and a mixture of the xanthines and catechins in the anti-inflammatory response of lithium in macrophages RAW 264.7, through the analysis of oxidative and inflammatory markers. Finally, the third protocol evaluated the effect of the interaction between Lithium, Imipramine, Nortriptyline, Fluoxetine and Escitalopram also using RAW 264.7 macrophages as an experimental model. In this latter protocol, an inflammatory index (II) was created in order to aggregate the results obtained. In order to validate the II, a similar analysis was performed to the cells in vitro, in a database of 154 volunteers. This additional analysis allowed to evaluate the accuracy of the II and its potential similarity with inflammatory situations that occur in vivo. In in vitro studies the results were compared by analysis of variance (one-way or two-way, as appropriate) followed by post hoc Dunnet, Tukey or Bonferroni, according to the case. In the in vivo study, a Roc curve was performed to assess whether IF was representative of an actual inflammatory condition. A pattern of immunosenescence was observed in all cultures up to the 15th passage, when there was an interruption in the cell cycle in the G0 / G1 phase. However, prior to that, from the 10th cell passage, there was a state of inflammatory hyperactivation with high rates of cell proliferation and increased levels of proinflammatory cytokines and oxidative markers. Despite this general pattern, AA PBMCs showed greater intensity in the initial inflammatory response, whereas VV PBMCs tended to maintain the inflammatory response longer. Therefore, the set of these results corroborated the suggestion that the S-PH imbalance has a direct influence on the modulation of inflammation. The second study of the first protocol observed that the S-PH imbalance, both genetically determined by the Val16Ala-SOD2 polymorphism and pharmacologically induced influenced the anti-inflammatory response of the Lithium-modulating GSK-3β enzyme. Imbalance induced a more intense anti-inflammatory effect of lithium on AA or AA-like cells. In the second protocol, which evaluated the effect of nutritional factors on the anti-inflammatory action of lithium, especially the mixture of xanthines-catechins intensified the anti-inflammatory response of Lithium via decrease in the levels of pro-inflammatory cytokines, increase in IL-10 levels and decrease in oxidative markers. Finally, the last protocol investigated the influence of the interaction between lithium and antidepressants. The II was validated via analysis of the ROC curve showed that the II is an accurate index in relation to the inflammation with an area on the curve (AUC) of 0.803 (95% confidence interval of 0.715-0.890). Values of II > 2.0 relative to Reactive Protein C levels> 0.6 μg / mL had a sensitivity of 0.915 and specificity of 0.486. Interaction with antidepressants has shown that when Lithium is associated with Imipramine, or Nortriptyline or Fluoxetine, there is an intensification of the anti-inflammatory effect, with a decrease in the II, However, the association with Escitalopram induces pro-inflammatory effects, with an increase in IF indicating the proinflammatory effect of this interaction. In summary, despite the methodological limitations related to the in vitro studies, the set of results confirmed that Lithium has an important anti-inflammatory effect, but that this effect is not universal since it can be influenced by genetic, nutritional factors and even by interaction with antidepressant drugs. These results may be relevant in the psychiatric clinic.