Experimental Study Of Simvastatin On Regulating The Polarization Of Microglia

PART Ⅰ The Identification of Classical or Alternative Activation on BV2MicrogliaObjective:To observe the changes in metabolic molecule of BV2microglia after it is classical or alternative activated, and judge the methods to identify the Ml or M2-type polarized microglia cells. Methods:BV2microglia cells were classical activated by LPS in the concentration of100ng/ml and alternative activated by IL-4in the concentration of20ng/mL for24hours, then ELISA was used to measure the levels of IL-12, IL-10, TNF-a, and Real-time PCR was used to detect the mRNA expression of iNOS,Arg-1,IL-10and IL-12. Flow cytometry(FCM) was used to measure the levels of MMr membrane protein. Results:Microglia presented M1-type polarization state after it were classical activated, the secretion of IL-12and TNF-a, the expression of iNOS mRNA and IL-12mRNA were significantly increased,and the difference were significant compare with that of in normal control group(P<0.05). Microglia presented M2-type polarization state after it were alternative activated, and the secretion of IL-10, the expression of Arg-1mRNA and IL-10mRNA were significantly increased, and the difference were significant compared with that of in normal control group(P<0.05). The levels of MMr were also increased significantly measured by Flow cytometry(FCM). Conclusion:BV2microglia could present Ml-type polarization state after it were classical activated, M2-type polarization state after alternative activated. The two type polarization could be identified detected from three aspects including inflammatory factors, arginine metabolic molecular and specific cell membrane proteins. PART Ⅱ The Regulation of Notch Signaling Pathway on Classical and Alternative Activation of BV2MicrogliaObjective:To study the influence of Notch signal system on classical and alternative activation of microglia cells. Methods1. Microglia cells were classical activated by LPS and alternative activated by IL-4respectively, then Real-time PCR was used to detect the mRNA expression of Notch1/Jagged-1/Hesl/Hes5.2. DAPT was treated to block the Notch signaling pathway before the BV2cells were stimulated by LPS and IL-4respectively. ELISA was used to measure the levels of IL-12, IL-10, TNF-α, and Real-time PCR was used to detect the mRNA expression of iNOS,Arg-1,IL-10and IL-12. Flow cytometry(FCM) was used to measure the levels of membrane protein CD16/32and CD206. Results1. Notch signal molecules of microglia were expressed in normal control group, LPS inducing group and IL-4stimulating group, and the expression of Notchl/Jagged-1/Hesl mRNA in LPS inducing group was enhanced.2. After the Notch signaling pathway was blocked by DAPT, the secretion of IL-12/TNF-a and the expression of iNOS/IL-12mRNA were significantly decreased compared with LPS inducing group (p <0.05), while the secretion of IL-10and the expression of Arg-1mRNA were significantly increased compared with IL-4stimulating group (p<0.05). Flow cytometry detection also presented lower expression of membrane protein CD16/32and higher expression of membrane protein CD206than LPS inducing group. Conclusion:Notch signaling molecules were all expressed whether the BV2microglia cells were in quiet state, classical activated and alternative activated. When microglia is in classical activated state, Notch signal channel was also activated. If the Notch signaling pathway was blocked, classical activation of BV2microglia cells would be accordingly be inhibited, and the M2-type polarization molecules would express more, showing certain trend of grade transition from M1to M2polarization. PART Ⅲ Effects of Simvastatin on Notch Signaling Pathway in BV2MicrogliaObjective:To investigate the effects of simvastatin on Notch signaling pathway in BV2microglia. Methods:Some BV2cells were pretreated with simvastatin in20ug/mL concentration, while some other cells were pretreated with simvastatin in20ug/mL concentration plus Jagged1/Fc in0.5μg/ml concentration. After all the pretreatment exist for24hours, LPS in100ng/ml concentration were treated to the BV2cells for stimulation. Western blot was used to analyze the levels of Notch1/Hes1protein, and Real-time PCR was applied to detection the expression of Notch1/Jagged-1/Hesl/Hes5mRNA. Results: The expression of Notch1/Jagged-1/Hes1/Hes5mRNA in LPS solely inducing group were all significantly increased compared with the normal control group (p<0.05). In the simvastatin retreating group, the levels of Hesl/Notch1protein, the expression of Notch1/Hes1/Hes5mRNA were decreased significantly compared with the LPS solely inducing group (p<0.05). But the expression of Jagged-1mRNA didn’t present the same decrease like Notch1mRNA. And in the simvastatin plus Jagged-1/Fc pretreating group, the expression of Notch1/Hesl mRNA is higher than simvastatin pretreating group. Conclusion:LPS could activate the Notch signaling pathway in BV2microglia. Simvastatin could inhibit the activity of the Notch signaling pathway. According to the reversion of Jagged-1/Fc protein in simvastatin’s inhibition on the Notch signaling pathway, we can infer that the regulation of a simvastatin on Notch signaling pathway may happened extracellular. PART Ⅳ Regulation of Simvastatin on BV2Microglia’s Polarization stateObjective:To investigate the regulation of simvastatin on BV2microglia polarization state. Methods:BV2cells were pretreated with simvastatin in high dose of20ug/ml or lowe dose of5ug/ml. After the pretreatment exist for24hours, BV2cells were classical activated by LPS or stimulated by Jagged-1/Fc protein respectively. ELISA was used to measure the levels of IL-12, IL-10, TNF-α, and Real-time PCR was used to detect the mRNA expression of iNOS,Arg-1,IL-10and IL-12. Flow cytometry(FCM) was used to mesure the levels of membrane protein CD16/32and CD206. Results:1. Compared with LPS classical activated group, the secretion of IL-12/TNF-a and the expression of iNOS/IL-12mRNA in Simvastatin pretreating group were significantly decreased; and the expression of IL-10mRNA was increased; while the flow cytometry detection presented lower level of membrane protein CD16/32and higher level of membrane protein CD206. The difference between this two groups was significant (p<0.05).2. Compared with Jagged1/Fc protein stimulating group, the secretion of IL-12/TNF-a and the expression of iNOS/IL-12mRNA in Simvastatin pretreating group were significantly decreased; and the expression of IL-10/Arg-1mRNA was increased. The difference between this two groups was significant (p<0.05). Conclusion:Simvastatin could inhibit the classical activation of BV2microglia induced by LPS. In the meanwhile simvastatin can restrain the classical activation of cells induced by Jagged1/Fc protein, the Notch signal activator. From all above, we can conclude that simvastatin can regulate the polarization state of microglial cells through Notch signaling pathway.