Anti-inflammatory and antioxidative actions of tacrolimus (FK506) on human microglial HMC3 cell line.

Microglial cells are indispensable for the normal development and functioning of neurons in the central nervous system, where they play a crucial role in maintaining brain homeostasis by surveilling the microenvironment for signs of injury or stress and responding accordingly. However, in neurodegenerative diseases, the density and phenotypes of microglial cells undergo changes, leading to chronic activation and inflammation. Shifting the focus from neurons to microglia in drug discovery for neurodegenerative diseases has become an important therapeutic target. This study was aimed to investigate the potential of Tacrolimus (FK506) an FDA-approved calcineurin inhibitor, to modulate the pathology of neurodegenerative diseases through anti-inflammatory and antioxidative effects on microglial activation. The human microglia clone 3 (HMC3) cells were exposed to 1 µg/mL LPS in the presence and absence of doses of FK506. Survival rates of cells were determined using the 3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide (MTT) method. Morphological evaluation of cells showed that FK506 restored the normal morphology of activated microglia. Furthermore, FK506 treatment increases the total antioxidant capacity and reduces the total oxidative capacity, indicating its potential antioxidant effects. Data from ELISA and RT-PCR analyses showed that LPS abolished its promoting effects on the release of proinflammatory IL-1ß and IL-6 cytokines in HMC3 cells, reflecting the anti-inflammatory effect of FK506. These findings support the idea that FK506 could be a promising therapeutic agent for neurodegenerative diseases by modulating microglial activation and reducing inflammation and oxidative stress.

Microglia cells treated with synthetic vasoactive intestinal peptide or transduced with LentiVIP protect neuronal cells against degeneration.

A common pathological hallmark of neurodegenerative disorders is neuronal cell death, accompanied by neuroinflammation and oxidative stress. The vasoactive intestinal peptide (VIP) is a pleiotropic peptide that combines neuroprotective and immunomodulatory actions. The gene therapy field shows long-term promise for treating a wide range of neurodegenerative diseases (ND). In this study, we aimed to investigate the in vitro efficacy of transduction of microglia using lentiviral gene therapy vectors encoding VIP (LentiVIP). Additionally, we tested the protective effects of the secretome derived from LentiVIP-infected "immortalized human" microglia HMC3 cells, and cells treated with Synthetic VIP (SynVIP), against toxin-induced neurodegeneration. First, LentiVIP, which stably expresses VIP, was generated and purified. VIP secretion in microglial conditioned media (MG CM) for LentiVIP-infected HMC3 microglia cells was confirmed. Microglia cells were activated with lipopolysaccharide, and groups were formed as follows: 1) Control, 2) SynVIP-treated, or 3) LentiVIP-transduced. These MG CM were applied on an in vitro neurodegenerative model formed by differentiated (d)-SH-SY5Y cells. Then, cell survival analysis and apoptotic nuclear staining, besides measurement of oxidative/inflammatory parameters in CM of cells were performed. Activated MG CM reduced survival rates of both control and toxin-applied (d)-SH-SY5Y cells, whereas LentiVIP-infected MG CM and SynVIP-treated ones exhibited better survival rates. These findings were supported by apoptotic nuclear evaluations of (d)-SH-SY5Y cells, alongside oxidative/inflammatory parameters in their CM. LentiVIP seems worthy of further studies for the treatment of ND because of the potential of gene therapy to treat diseases effectively with a single injection.