The Involvement Of TLR4in Lithium Regulated LPS-induced BV-2Microglia Cells Inflammation

Background: Inflammation of the Central Nervous System (CNS) plays a criticalrole in the occurrence and development of chronic neurodegenerative diseases.Microglial cells which act as scavengers in CNS, can produce cytokines, oxygenradicals and nitric oxide and other inflammatory substances to mediate a variety ofpathological processes under the incentive of trauma, ischemia and infection.Therefore, inhibiting inflammatory cytokines production induced by activatedmicroglia cells may be a new way for prevention of degenerative diseases of thenervous system.As a pathogen-associated molecular patterns recognizing receptor on the surface ofmicroglia, TLR4can identify pathogenic microorganisms as well as endogenousmolecules released after stress or tissue damage, thereby causing the expression ofdownstream signaling proteins and leading to massive release of inflammatorymediators. It follows that TLR4plays a role in the innate immunity in response topathogens and also participate in CNS inflammation and neurodegeneration.Glycogen synthase kinase3β (GSK-3β) is involved in the microglia inflammatoryresponse in the balance between the regulation of inflammatory cytokines andanti-inflammatory cytokines. Lithium chloride is the best-characterizedpharmacological inhibitor of GSK-3β, which can phosphorylate GSK-3β and inhibitits activity, reducing the release of inflammatory factors, thereby to produce aprotective effect on the neuroinflammtion. Our previous study found that lithiumchloride increased phosphorylation of GSK-3β (ser9) in hippocampus, and decreasedthe expression of IL-1β. Meanwhile the Morris water maze test scores had improved significantly, suggesting that lithium chloride pretreatment can reduce the centralactivation of microglia s and inflammation of the CNS.BV-2microglia is commonly used in vitro. Whether Lithium chloride in vitro caninhibit BV-2of microglial inflammatory responses induced by LPS, and theinvolvement of TLR4on lithium chloride in the central inflammation responses havenot been reported.Objective: The experimental is scheduled to compare the effect of lithium chlorideand GSK-3β specific inhibitor SB216763on LPS-induced BV-2microgliainflammatory response in vitro, analysis the relationship with the activity of GSK-3β,and explore the mechanism of lithium chloride and whether TLR4is involved in thereaction with siRNA interference technology.Methods: BV-2microglia cells were seeded in culture plates overnight prior toincubation with drugs. Then the cells were preincubated with a final concentration of1μg/ml LPS and100nM of TLR4siRNA respectively. TLR4mRNA and protein weredetected at the corresponding time points. And then BV-2microglia were randomlydivide into six groups: blank control group, LPS group, LPS+lithium chloride group,LPS+SB216763group, LPS+TLR4siRNA group, LPS+TLR4siRNA+lithiumchloride group. Then the cells were preincubated with the structurally distinctselective GSK-3β inhibitors,10mM lithium chloride and10μM SB216763for30minprior to stimulation with lipopolysaccharide. BV-2cells were treated with LPS,or Liclafter transfecting with TLR4siRNA for48h. Cells and cell culture supernatants werecollected and total RNA and protein were extracted from BV-2cells. IL-1β andTNF-α concentrations in cell culture supernatants were measured by ELISA. Thechanges of TLR4mRNA and protein, total GSK-3β and p-GSK-3β were detected byRT-PCR, qPCR and Western blot. Statistical analysis was performed with GraphPadPrism5.0statistical procedures. Results:(1) The best detection time of TLR4mRNA and protein: TLR4mRNA andprotein level are time-dependent, and peaks respectively at4h and12h followed LPStreatment (P<0.05). BV-2cells were transfected with TLR4-specific siRNA for24hand48h, and levels of TLR4protein were decreased44%and71%,respectively(P<0.05).(2) Compared with the control group, each group of IL-1β and TNF-α proteinexpression were significantly increased (P<0.05); the TLR4mRNA and protein in theLPS group, LPS+lithium chloride group and LPS+SB216763group weresignificantly increased (P<0.05). Compared with the LPS group, IL-1β and TNF-αprotein expression were significantly decreased in LPS+lithium chloride group, LPS+SB216763group, LPS+siRNA group and LPS+TLR4siRNA+lithium chloridegroup(P<0.05); TLR4mRNA and protein levels were significantly decreased in LPS+lithium chloride group and LPS+SB216763group (P <0.05), while p-GSK-3βcontent was significantly increased (P <0.05). Compared with LPS+lithium chloridegroup, IL-1β and TNF-α protein in LPS+TLR4siRNA+lithium chloride group wereincreased (P <0.05).Conclusion: Lithium chloride and SB216763can inhibit GSK-3β activity of BV-2microglial cells, and reduce the amount of expression of LPS-induced inflammatorycytokines IL-1β and TNF-α in inflammatory response of the CNS, and candownregulate the expression of TLR4mRNA and protein. Silence of TLR4cansignificantly attenuate LPS-induced the expression of IL-1β and TNF-α, and inhibitthe regulation of Lithium chloride on inflammatory response.These results suggestedthat TLR4plays a critical role in BV-2microglia activation, and involved in theregulation of Lithium chloride on inflammatory response.The mechanism of Lithiumchloride inhibiting microglia inflammatory response not only related to GSK-3β butalso the TLR4.