| Background and objectives In the process of neuropathy of diseases such as Parkinson’s disease,Alzheimer’s disease and traumatic brain injury,the severity of neuroinflammation and oxidative stress are related to the development of the diseases.As the immune cells of the central nervous system,microglia synthesize reactive oxygen species（ROS）,inducible nitric oxide synthase（i NOS）and interleukin-6（IL-6）,which are important markers of oxidative stress and neuroinflammation,and are also the main target of screening therapeutic drugs.Microglia can help form neural circuits through synaptic pruning during development.Howerver,They can play a dual role in some pathological conditions,such as neurodegenerative diseases or central nervous system injuries.On the one hand,the purpose of limiting damage is achieved by phagocytosis and elimination of pathogens,dead cells and protein aggregates in the early stage of the disease.With the progress of the disease,microglia can secrete some anti-inflammatory and neurotrophic factors to promote tissue repair.On the other hand,when this balance is broken,that is,microglia is chronically activated,continuously producing inflammatory mediators,reactive oxygen species and cytotoxic molecules,which will cause different degrees of damage to normal tissues and cells,consequently resulting or even aggravating neuropathological events.Therefore,inhibiting neuroinflammation and oxidative stress mediated by microglia and making them play a proper role are potential therapeutic strategies for neurodegenerative diseases.Triiodothyronine（T3）is an amino acid derivative,which plays an important role in the growth,development and metabolism of the body.In recent years,there are more and more reports about T3’s involvement in oxidative stress,inflammatory reaction and apoptosis events.In combination with our previous research,we found that the PET/CT images of the brain of hypothyroid rats showed that microglia cells were in an activated state,which suggested that T3 might be related to the activation of microglia.In this research,BV2 microglial cell line was used to establish an in vitro inflammatory model,to observe the effect of T3 on the activation of microglial cells under inflammatory conditions and explore its mechanism,in order to provide new ideas and basis for the treatment of neurodegenerative diseases and central nervous system injuries.Methods BV2 microglias were treated with different concentrations of T3(3×10-10mol/L,3×10-9mol/L,3×10-8mol/L,3×10-7mol/L,1×10-6mol/L,1×10-5mol/L)for24h,and then given 1μg/m L lipopolysaccharide（LPS）stimulation for 24h,the level of inflammatory factor TNF-αwas detected by western blot to determine the appropriate T3 action concentration;The number of cells in control group,LPS group,T3 group and T3+LPS group was detected by MTT method;The morphological changes of the four treatment groups were observed by inverted microscope;Use ROS detection kit to observe the changes of ROS in different treatment groups under inverted fluorescence microscope;Use the lipid peroxidation MDA assay kit to detect the optical density value with a microplate reader;Use western blot analysis to detect the expression of inflammatory related proteins i NOS,IL-6,pro-IL-1β,IL-1β,TNF-α,oxidative stress-related proteins SOD1,SOD2 and key signal molecules ERK,p-ERK,NF-κB p65,NLRP3,proccaspase-1,caspase-1.Results 1.Compared with LPS group,T3 of 3×10-10mol/L,3×10-9mol/L,3×10-8mol/L and 3×10-7mol/L did not affect LPS-induced TNF-αlevels.The expression level of TNF-αdecreased while the concentration of T3 was up to 1×10-6mol/L,and T3 could inhibit the content of TNF-αto the most at 1×10-5mol/L.2.MTT experiment results showed that LPS（1μg/m L）significantly reduced the survival rate of BV2 microglias.Although the number of BV2 cells pretreated with T3 of 1×10-5mol/L increased,there was no statistically significant difference.3.The observation results under general light microscope showed that the volume of BV2 cells stimulated by LPS increased significantly,the number of cell processes increased,and the cells transformed into multipolar cells with vesicles inside,while the number of processes of BV2 cells pretreated by T3 decreased obviously,and the proportion of multipolar cells and spindle-like cells decreased.4.Western blot assays showed that the level of inflammatory related proteins i NOS,IL-6,pro-IL-1β,IL-1βand TNF-αin BV2 cells was increased after LPS stimulation,while the contents of these proteins in T3+LPS group decreased significantly.5.ROS detection results showed that T3 significantly inhibited the increase of reactive oxygen species in BV2 cells induced by LPS.6.The detection results of lipid peroxidation（MDA）showed that T3 reduced the degree of lipid peroxidation induced by LPS.7.Western blot showed that T3 could increase the expression of SOD1 and SOD2 compared with LPS group.8.Compared with LPS group,the phosphorylation level of ERK protein in T3+LPS treatment group decreased,the total protein content of NF-κB p65 and the expression of NLRP3,proccaspase-1 and caspase-1 also declined in this group.Conclusions 1.T3 significantly reduced the expression of LPS-induced inflammation related proteins in BV2 microglia.2.T3 has a significant down-regulation effect on LPS-induced oxidative stress in microglia.3.T3 may regulates the activation of microglia through ERK/NF-κB pathway and NLRP3 inflammatory body. |
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