Effects Of FK506 On Microglia Activation And Retinal Inflammation

ObjectiveNeuroinflammation mediated by microglia has a very important role in the pathogenesis of many central nervous system diseases.In this study,we investigated the effect of FK506 on LPS-induced microglial activation and LPS-induced rat retinal inflammation.And explore the potential role of FK506 in diseases related to retinal inflammation.MethodsBV2 microglial cells were cultured in vitro,and the appropriate concentration range of FK506 on BV2 microglial cells was detected using the CCK-8 method.Lipopolysaccharide(LPS)was used to induce the activation of BV2 microglial cells.Study the effect of FK506 on activated microglia by morphological observation.And the positions and expression levels of NF-κB signaling pathway-related proteins between different groups were detected by immunofluorescence detection and western blot method respectively.Construction of a rat retinal inflammation model,using random number table method to randomize 36 male SD rats divided into CON group,LPS group and LPS+FK506 group,the same volume of PBS,LPS and LPS+FK506 were injected into the vitreous cavity respectively,and HE staining and immunofluorescence were used to observe the changes of retinal structure and iNOS expression between different groups.Statistical analysis of the experimental results,P<0.05 was considered statistically significant.Results1.The results of CCK-8 showed that when the FK506 drug concentration reached 15 μg/mL,the cell viability began to decrease(P<0.05).When the FK506 drug concentration was in the range of 0-10 μg/mL,it has no obvious cytotoxic effect on BV2 microglia(P>0.05).2.In different concentrations of FK506 group,LPS-induced microglia activation ratios were:CON group(42.6±5.4)%,LPS group(85.2±7.5)%,1μg/mL FK506 group(74.3±3.8)%,5μg/mLFK506 group(67.0±3.9)%,10μg/mLFK506 group(51.4±5.6)%,comparison between different groups,P<0.05.It showed that FK506 dose-dependently inhibited LPS-induced microglial activation and morphological changes.3.Immunofluorescence results showed that FK506 inhibited P65 nuclear transfer in microglia NF-κB signaling pathway.Western blot results showed that FK506 reduced the degradation of IκB-a in microglia.4.HE staining results of rat retinal slices between different groups showed that there was no obvious abnormality in the retina structure of the CON group.And the ganglion cell layer,inner plexiform layer,inner core layer,outer plexiform layer,and outer nucleus layer in the retina of the LPS group all have different degrees of structural disturbance.And the overall structure of the retina layer in the LPS+FK506 group was relatively neat.5.The Immunofluorescence results in the rat retina between different groups showed that FK506 can inhibit the expression of iNOS in the retina during retinal inflammation.Conclusions1.FK506 reduces LPS-induced microglial activation,and its mechanism may be to inhibit microglia NF-κB signaling pathway.2.FK506 can improve retinal inflammation-induced structural disorder of the retina.3.FK506 inhibited the expression of iNOS and in retinal inflammation,suggesting that FK506 may reduce retinal damage caused by inflammatory mediator-related toxic effects.4.FK506 may reduce retinal inflammation damage by inhibiting the activation of microglia and play a role in protecting the retina.