Study On Regulation Of Neuroflammation By Clock Gene Period1 And Period2

Aim: To gain insight into the role and the mechanism of clock gene Period1(Per1) and Period2(Per2) in LPS-induced neruoinflammation.Methods: Endogenous Per1 or Per2 was knocked down in BV2 microglial cell. Then the cells were treated with lipopolysaccharides(LPS) 100 ng/ml for 12 hours. Concentration of NO, IL-6 and TNF-α in the culture media were measured by NO reagent kit or ELISA. The protein levels of i NOS and COX2 in BV2 cells were detected by immunoblot. LPS(2 μg) was injected in lateral ventricles of WT, Per1 KO or Per2 KO mice. The m RNA levels of inflammatory cytokines genes IL-6, TNF-α, i NOS and COX2 were estimated by quantitative real-time PCR(q RT–PCR) in the brain of mice. 7 days after LPS(2 μg) was injected in lateral ventricles,the astrocytes of mice brain were labled with anti-GFAP and were observed by immunofluorescent staining. To further identify the regulation of Per1 and Per2 on neuroinflammation, the primary astrocytes cells of WT, Per1 KO and Per2 KO mice were treated with LPS for 24 hours. The culture media were analyzed for IL-6 and TNF-α protein levels by ELISA. Total cell lysates of primary astrocytes were analyzed by inmmunoblot with antibodies to i NOS and COX2. To investigate the regulatory mechanism, WT, Per1 KO and Per2 KO mouse primary astrocytes were treated with LPS and then analyzed by immunoblot with antibodies of p65 and IκBα. The translocation of p65(NF-κB subunit) was observed by immunocytochemistry. The cytoplasmic and nuclear fractions were separated and analyzed by immunoblot with p65 antibodies.Results: The results showed relatively low levels of IL-6, TNF-α, NO, i NOS and COX2 production after LPS treatment for 12 hours in Per 1 knockdown BV2 cells, compared with the control cells. LPS treatment in Per2 knockdown BV2 cells led to a significant increase of the IL-6, TNF-α, NO, i NOS and COX2 levels. After LPS was injected in lateral ventricles of mice, the marked decreased m RNA levels of IL-6, TNF-α, i NOS and COX2 were observed in Per1 KO mice, relative to WT. However, the m RNA levels of these inflammatory cytokines were increased significantly in Per2 KO mice, relative to WT. The levels of IL-6 and TNF-α were dramatically lower in the culture media of primary astrocytes and the expression of i NOS and COX2 was also lower in Per1 KO mice than in WT mice after LPS treatment for 24 hours. By contrast, the levels of IL-6, TNF-α, i NOS and COX2 were up-regulated appreciably in Per2 KO mice. GFAP-immunofluorescent staining showed Per1 KO may reduce LPS induced the activation of astrocytes in vivo, especially at local cortex and corpus striatum. However Per2 KO may strengthen the the LPS induced activation of astrocytes. The action was been seen obviously at local cortex and corpus striatum. Next, we tried to identify the mechanism of Period in neuroinflammation. With prolongation of LPS treatment, the expression of p65 was reduced in the primary astrocytes of Per1 KO mice compared with WT mice. The protein of p65 in the primary astrocytes did not show any significant difference between Per2 KO and WT mice after LPS treatment, but the expression of IκBα was marked decreased after LPS treatment. The immunocytochemistry analysis showed the level of p65 in nuclear was higher in the primary astrocytes of Per2 KO mice than WT mice at 30 min after LPS treatment. In addition, the nuclear content of p65 was increased, and cytoplasmic content was decreased in BV2 cells transfected with Per2 si RNA, compared with control cells.Conclusion: Absence of clock gene Per1 constitutively inhibited LPS-induced neuroinflammation which may result from the reduction of p65. Lack of clock gene Per2 promoted LPS-induced neuroinflammation. Absence of Per2 may increase degradation of IκBα to accelerate NF-κB nucleus translocation and strengthen inflammation during LPS treatment.