Autofagia como mecanismo subjacente ao efeito imunomodulador do canabidiol em micróglia humana: relevância para efeito tipo-antidepressivo

Abstract

Cannabidiol (CBD) is a non-psychostimulant compound from Cannabis sativa with immunoregulatory effects and therapeutic potential in several brain disorders. Since autophagy has been elected as an important mechanism for the control of microglial functions, including inflammatory responses, the aim of this study was to investigate the role of (macro)-autophagy in the CBD effects in pro-inflammatory activated human microglia. To do this, human microglia cells (HMC3 lineage) were treated with CBD (1, 10, 50 and 100 µM) and 24h later they were stimulated with lipopolysaccharide (LPS) for more 24h (prevention protocol or pretreatment). Also, other batch of cells were firstly exposed to LPS for 24 h, and 24h later treated with CBD (reversal protocol or post-treatment). Hydroxychloroquine (HCQ-10 µM) and starvation (STV) (2h) were used as autophagy inhibitor and inducer, respectively. Cell viability was evaluated through MTT and LDH assays. The formation of acidic vesicular organelles (AVOS) was determined through acridine-orange flow cytometry. Autophagy-related proteins (ATGs) gene expression (ATG5, ATG12, BECLIN-1 and p62/SQSTM1) was determined through RT-PCR. The protein expression of the isoforms LC3B-I and LC3B-II, an index of maturation of autophagossomes, and p62/SQSTM1, an index of degradation of the autophagic cargo, were determined through western blott. Supernatants were collected for determination of nitrite, TNFα, IL-1β, IFNγ, IL-4 and TGF-β, brain-derived neurotrophic levels (BDNF) and arginase activity. Also, the expression of the enzymes inducible nitric oxide synthase (iNOS) and Arginase1 (Arg1) was determined through immunofluorescence and their colocalization was evaluated. Phagocytosis was evaluated through fluorescein-labeled beads intracellular detection; migration-mobility was evaluated through scratch assay. In the in vivo protocol, adult Swiss mice were treated with HCQ (60 mg/kg, IP.), daily, for three days. On the second day, the animals were injected with LPS (0.5 mg/kg, IP) or saline (SAL), and on the third day, they were treated with CBD (10 mg/kg, IP), S-ketamine (10 mg/kg, IP) or SAL. Several behavioral assays were performed to evaluate the CBD’s antidepressant-like effect, and the mouse brain areas: prefrontal cortex (PFC) and hippocampus (HC) were collected. CBD increased the % of AVOS+ cells just in the reversal protocol compared to LPS, which was blocked by HCQ. STV increased AVOS+ cells in both protocols compared to controls. CBD increased some ATGs mRNA levels: ATG5 and p62/SQSTM1 in the prevention, and ATG12 and p62/SQSTM1 in the reversal protocol, compared to LPS. Additionally, CBD in the reversal protocol did not significantly increase the LC3B-I or II protein expression, but it significantly decreased the p62/SQSTM1 levels compared to control and LPS groups. CBD (10 µM) attenuated LPS-induced increase in nitrite levels, TNF-α and IL-1β in both protocols. CBD increased IL-4 and BDNF levels in both protocols, while arginase activity and TGF-β were increased only in the reversal one. STV in both protocols similarly increased the levels of anti-inflammatory mediators: IL-4, arginase activity and TGF-β compared to the LPS group. HCQ co-treatment attenuated CBD effects in the reversal protocol on nitrite and TNF-α levels, and significantly reduced the rise in IL-4, BDNF and arginase activity. CBD increased phagocytic activity in the reversal protocol, and increased cell migration in both protocols compared to LPS group. These effects were equally blocked by HCQ in the reversal protocol. In the in vivo protocol, CBD partially reversed LPS-induced depressive-like behavior, which was attenuated by co-treatment with HCQ. CBD increased the expression of LC3B-II in the PFC, while S-ketamine increased this protein in the HC compared to the LPS group. Only one S-ketamine was able to reduce TNFα concentrations in the HC of LPS-challenged animals. Therefore, this study contributes to a better understanding of the effects of CBD in microglia by providing evidence of the participation of autophagy-related mechanism to its immunoregulatory/anti-inflammatory actions. Finally, autophagy-inducer agents, possibly CBD, can be promising tools for microglia-related neuroinflammatory disorders, such as depression.