| Background and SignificanceChronic inflammatory process in the brain, triggered by microglia,may play a significant role in the pathologies of several neurological disorders,including brain tumors,cerebral ischemia,brain trauma,brain infections,neurodegenerative disorders.Microglia,the primary resident immune surveillance cell in the brain,lie in the core of these pathogensis.In addition to their phagocytic role--clearing died cell debris,activated miroglia can synthesize and secrete potential neurotoxins that may cause neuronal damage or aggravate underlying pathology.These neurotoxins include nitric oxide (NO),tumor necrosis factor-α(TNF-α),interleukin-lβ(IL-lβ),inter- leukin-6 (IL-6) and free radicals etc.Therefore,in recent years,it is a emerging conception that inhibition of microglial activation may be a novel therapeutic target to slow down the progress of these diseases.Translocator protein (TSPO,18 kDa),an 18 kDa protein,was initially identified as a peripheral binding site for diazepam and previously known as the peripheral-type benzodiazepine receptor (PBR). TSPO is primarily localized at outer mitochondrial membranes.In the normal brain,overall TSPO expression is low,and TSPO is mainly found in glia and at very low levels in neurons.In addition,different forms of neural injury and different neuropathological conditions as inflammation result in the induction of the exptession of TSPO in the nervous system,mainly in microglia.The induction of TSPO expression in reactive microglia suggests that TSPO may be involved in the regulation of reactive microglia.Furthermore,TSPO ligands-PK 11195 and Ro5-4864 (PK 11195,an antagonists of PBR;Ro5-4864,an agonists of PBR) have been intensively investigated in regard to its anti-inflammatory action in peripheral system.However,the precise role of TSPO in reactive microglia is unknown.Moreover,in the last few years,there has been an increased interest in understanding which associations between TSPO expression and microglia activation are,whether TSPO ligands can inhibit microglia activation and reduce secretion of inflammatory cytokines or not,and what the exact mechanisms by which TSPO ligands confer protection are.Therefore,lastly,it is critically important to understand the function and how TSPO expression is regulated in the microglia function in order to indentify of TSPO as a potential target for development of new therapeutic strategies to decrease inflammation associated to some central nervous system degenerative diseases.Objectives1.To determine the associations between TSPO expression and microglia activation.2.To explore the inhibitory effect of TSPO ligands on microglia activation.3. To study the role of TSPO gene in inflammatory process induced by microglia.4.To explore the exact mechanisms by which TSPO ligands confer inhibition effect on microglia activation.Research Contents1.Levels of proinflammatory cytokines in microglial cells after LPS treatment.2.TSPO expression in the LPS-induced microglia activation.3.Effects of TSPO ligands on levels of proinflammatory cytokines in microglial cells after LPS treatment.4.Effects of TSPO gene silenced by TSPO siRNA on levels of proinflammatory cytokines in microglial cells after LPS treatment.5.The inhibitory effect of TSPO ligands on levels of proinflammatory cytokines in microglial cells transfected with TSPO siRNA after LPS treatment.6.Signaling pathways in effect on TSPO ligands on levels of proinflammatory cytokines in microglial cells after LPS treatment.7.Effects of TSPO ligands on mRNA stability of proinflammatory cytokines in microglial cells after LPS treatment.Methods1.We treated BV-2 microglial cells cultures with LPS (l00 ng/ml) to induce microglia activation and tested the levels of proinflammatory cytokines (TNF-α,IL-1βand IL - 6) and the morphology of BV-2 cells in different time (30 min,1 h,2 h,4 h,6 h,12 h,24 h) of LPS treatment with ELISA and inverted phase contrast microscope.2.We treated BV-2 microglial cells cultures with LPS (l00 ng/ml) to induce microglia activation and tested TSPO expression in different time (4 h,8 h,24 h,48 h) of LPS treatment with Real-time PCR.3.We explored effects of TSPO ligands (PK 11195 and Ro5-4864) at different dose (10μM,50μM,100μM) on productions and mRNA expressions of proinflamma- tory cytokines in BV-2 cells after LPS treatment (l00 ng/ml) with ELISA and Real-time PCR.4.We transfected TSPO siRNA into BV-2 cells and analysed the expression of TSPO in BV-2 cells after transfected with TSPO siRNA in order to determined effect of silenced TSPO gene in BV-2 cells after transfected with TSPO siRNA with Real-time PCR and Western blot.After that,we studied effects of silenced TSPO gene on productions and mRNA expressions of proinflammatory cytokines in BV-2 cells after LPS treatment (l00 ng/ml) with ELISA and Real-time PCR.5.We studied effects of TSPO ligands (PK 11195 and Ro5-4864) at different dose (10μM,50μM,100μM) on productions and mRNA expressions of proinflamma- tory cytokines in BV-2 cells transfected with TSPO siRNA after LPS treatment (l00 ng/ml) with ELISA and Real-time PCR.6.We explored effects of TSPO ligands (PK 11195 and Ro5-4864) at different dose (10μM,50μM,100μM) on MAPK pathway (Erk,JNK and p38) and IκB degra- dation in BV-2 cells after LPS treatment (l00 ng/ml) with Western blot.7.We studied effects of TSPO ligands (PK 11195 and Ro5-4864) at the dose of 100μM for 24 h on a single VB-2 intracellular calcium concentration ([Ca2 +] i) by flow cytometry.8.We treated BV-2 microglial cells cultures with LPS (l00 ng/ml) for 2 h to induce microglia activation.Following,LPS-induced reactive microglia were exposed for 1 h and 2 h to ACTD+PK 11195,ACTD+Ro5-48643 and ACTD+DMSO,respectively,for measurement of mRNA expressions of IL-1βand IL–6 with Real-time PCR,and analysis of mRNA degradation of IL-1βand IL–6.Results1.LPS induced BV-2 microglial cells activation,as follows:dynamic morphology changes in cell growth to Amoeba-like appearance,levels of proinflammatory cytokines increased,such as,TNF-α,IL-lβand IL -6. Levels of proinflamma- tory cytokines increased in time and dose-dependent way of LPS.2.The TSPO expression was detected in resting BV-2 cells at low level,but increased in LPS-induced BV-2 cells in time-dependent way,increasing signifycantly in 24 h and 48 h after LPS treatment(P <0.01).3.TSPO ligands (PK 11195 and Ro5-4864) showed anti-inflammatory properies. They inhibited reactive BV-2 cells after LPS treatment and reduced levels of IL-l βand IL -6, except for TNF-α.On IL-1β, Ro5-4864 showed the same inhibitory effects as PK 11195, and Ro5-4864 showed more strong inhibitor than PK 11195 on IL-6.So,Ro5-4864 showed more strong anti-inflammatory properies than PK 11195 on IL-6.4. The expression and protein of TSPO were less in BV-2 cells after transfected with TSPO siRNA than in BV-2 cells after transfected with control siRNA.The difference between the two groups was significant (P <0.01).The results showed that TSPO siRNA can downregulate effectively the expression and protein of TSPO.However,the products of TNF-α,IL-lβand IL-6 did not decrease in BV-2 cells transfected with TSPO siRNA relative to those in BV-2 cells transfected with control siRNA after LPS treatment.In other words,down- regulation of TSPO had no effect on levels of proinflammatory cytokines,such as,TNF-α,IL-lβand IL-6 in BV-2 cells treated by LPS.The results showed that TSPO gene does not directly mediate inflammation,or play a second role in inflammatory process induced by reactive BV-2 cells.5. TSPO ligands (PK 11195 and Ro5-4864) had reduced levels of IL-lβand IL-6 in BV-2 cells transfected with TSPO siRNA and control groups (BV-2 cells transfected with control siRNA) after LPS treatment. The difference between the two groups was not significant (P >0.01).TSPO gene did not mediate the inhibitory effect of TSPO ligands (PK 11195 and Ro5-4864) on levels of proinflammatory cytokines, such as, IL-lβand IL-6 in BV-2 cells treated by LPS. The result showed that TSPO gene does not directly regulate or play a role for transmitting information to the other subunits in the anti-inflammatory of TSPO ligands.6. TSPO ligands (PK 11195 and Ro5-4864) had no affect on Iκ- B degradation and phosphorylation of Erk,JNK and p38 in BV-2 cells after LPS treatment. However,PK 11195 and Ro5-4864 had affect on [Ca2+]i in BV-2 cells. PK 11195 increased significantly [Ca2+]i from 1.95±0.16 to 3.37±0.14(MFI). The difference between PK 1195 treatment and control groups was significant (P <0.01);Ro5-4864 decreased significantly [Ca2+]i in BV-2 cells from 1.95±0.16 to 1.56±0.09(MFI).The difference between Ro5-4864 treatment and control groups was significant (P <0.01).7. TSPO ligands (PK 11195 and Ro5-4864) accelerated degradation of IL-1βand IL- 6 mRNA.The reductions of IL-1βand IL -6 mRNA were 60.2 %,64.3 % and 69.4 %,74.1 % at 1 h and 2 h treatment of PK 11195,and they were 58.2 %,60.2 % and 66.9 %,70.0 % with Ro5-4864 treatment in LPS-induced BV-2cells. The half-life of mRNA of IL-1βand IL-6 in LPS-induced BV-2cells after PK 11195 or Ro5-4864 treatment were significantly shorter that that in control groups,from 2 h to 1 h.The findings showed PK 11195 and Ro5-4864 decreased mRNA stability of IL-1βand IL -6.Conclusions1.LPS can induce microglia activation to grow with Amoeba-like appearance,and synthesize and secrete proinflammatory cytokines,such as,TNF-α,IL-lβand IL -6 through MAPKs signal transduction pathways and NF-κB transcription factor.2.In resting microglia,TSPO expression is low.But in activated microglia after LPS treatment , TSPO expression increases in time-dependent way , increasing significantly in 24 h and 48 h. Combined with the first study finding, the results show that increased TSPO expression is closely associated with micrglia activation,and may be related to the secretion of inflammatory cytokines.So,The induction of TSPO expression in reactive microglia suggests that TSPO may be involved in the regulation of reactive microglia.3.TSPO ligands are of anti-inflammatory properies.They can inhibite reactive microglia after LPS treatment and reduce levels of proinflammatory cytokines. Nevertheless,TSPO sub-unit does not mediate the inhibitory effect of TSPO ligands on levels of proinflammatory cytokines,and only play a role for transmitting information to the other sub-units (VDAC and ANT).So,the other two sub-units (VDAC and ANT) in PBR complex may involve in anti- inflammatory properies of TSPO ligands. These findings suggest that the three sub-units (TSPO,VDAC and ANT) are interlinked and interacted to constitute a complex of PBR. Isoquinoline carboxamides (such as PK 11195) can be indivi- dually bind the isoquinoline binding site (TSPO).However,interactions among TSPO,VDAC and ANT are considered to play a role in the process that benzodiazepine-type drugs (such as Ro5-4864 ) bind specifically TSPO.So,Ro5- 4864 showed more strong inhibitor than PK 11195 on IL-6,and on IL-1β, Ro5-4864 showed the same inhibitory effects as PK 11195.In general,Ro5-4864 has showed more strong anti-inflammatory properies than PK 11195.These findings support the concept on renaming the PBR to TSPO,which reflect more accurately the cellular and physiological functions in which TSPO has important role.4.TSPO ligands can inhibit reactive microglia after LPS treatment and reduce levels of proinflammatory cytokines (IL-1βand IL-6) through decreasing mRNA stability of proinflammatory cytokines and Ca2+-mediated signaling pathways,except through MAPKs signal transduction pathways and NF-κB transcription factor.5. PK 11195 and Ro5-4864 play different roles in intercellular concentration of calcium([Ca2 +]i) in microglial cells.PK 11195 increased significantly [Ca2+]i ,and Ro5-4864 decreased significantly [Ca2+]i in microglial cells.Combined with their results in inflammation induced by microglia,it is inaccurate that PK 11195 is generally known as PBR antagonists and Ro5-4864 as PBR agonists.Further,it is relatively exact that PK 11195 is known as voltage-dependent anion channel (VDAC) antagonists and Ro5-4864 as VDAC agonists according to their properties.We will further study the work in the future.
|
PDF Full Text Request