| Objective: To investigate the effect of liraglutide on the regulation of the inflammatory response of BV2 cells and thus the production of A1-type reactive astrocytes,and the process on the inflammatory response of microglial cells and A1-type astrocytes in SD rats after TBI Impact and mechanisms.method:(1)Culture BV2 cells,observe their morphology under a light microscope,and identify the expression of surface-specific marker Iba-1 and GLP-1R by immunofluorescence.(2)Adjust the BV2 cell condition to inoculate the laser confocal dish when it is better,and divide the cells into 3 groups,which are normal group,LPS activation group,liraglutide pretreatment + LPS activation group.After 24 hours of LPS treatment,the cell morphology of each group was observed by light microscopy.Immunofluorescence was used to detect the expression of cell surface antigens i NOS and Iba-1.Cell protein was extracted from each group to detect PKA and CREB protein expression.q RT-PCR was used to detect the expression of TNF-α,IL-1α and C1 q.(3)Culture primary astrocytes,observe their morphology under light microscope,and use immunofluorescence to identify the expression of surface specific antigens GFAP and GLP-1R.Adjust the cell state to a better level when inoculated in a laser confocal dish,divide the cells into 3 groups,add the culture medium of BV2 cells to the astrocyte medium for 24 hours,and then perform immunofluorescence detection Expression of cell surface GFAP and C3.(4)Sixty male adult healthy SD rats were randomly divided into three groups: sham group,TBI group and lilalutide group(TBI + lilalutide).Sham group opened bone window;TBI group used e CCI for severe TBI modeling(speed 5 m / s,depth 1.8mm,hammer dwell time 130 ms),liraglutide pretreatment group can be injected subcutaneously after TBI.Lypodermic injection 200 mg / kg for 14 consecutive days.(5)The m NSS scores were performed at 7,14 and 28 days after TBI,and the Morris water maze test was performed at 14 days after modeling to compare the neurological deficits in each group.(6)After the rats were sacrificed,the brain was perfused with 4% paraformaldehyde,and the sections were stained with HE to detect the inflammation of the brain tissue.Immunofluorescence was used to detect the expression of GLP-1R in the brain of the TBI group.Activation and A1 astrocyte expression.(7)After the brain was perfused with saline,WB was used to detect the expression of PKA and CREB protein,and q RT-PCR was used to detect the expression of proinflammatory factors TNF-α,IL-1α and C1 q.Result:(1)Observe BV2 cells under a microscope,which is basically spherical.The results of immunofluorescence showed that the cell surface expressed Iba-1 and GLP-1R,which proved that the cell line was of high purity and expressed the presence of liraglutide receptor GLP-1R.(2)24 hours after LPS treatment,cells in the LPS group significantly expressed INOs compared to cells in the Sham group,while the expression of i NOS in the liraglutide group was significantly lower than that in the LPS group(P <0.05),and the difference was statistically significant.WB results showed that the ratios of p-PKA / PKA,p-CREB / CREB in the Sham group were significantly higher than those in the LPS group and the liraglutide group,while those in the liraglutide pretreatment group were higher than those in the LPS group(P <0.05).q RT-PCR results showed that the expression of TNF-α,IL-1α and C1 q was significantly increased after LPS treatment,and liraglutide could inhibit the increase.(3)The extracted primary astrocytes protruded from the axons under a light microscope.GFAP staining proved that their purity was 99% and could be used for subsequent experiments,and astrocytes did not express GLP-1R protein.(4)The culture medium of 4 kinds of microglia was used to culture astrocytes for 24 hours.Observe the axon elongation of the LPS group,and the cell body enlarged to express a large amount of A1 astrocyte marker C3.Cells produced by the liraglutide pretreatment group produced less C3.(5)The results of HE showed that local inflammatory cells infiltrated into the brain tissue of TBI rats,and brain tissue edema was heavier.Pretreatment with liraglutide improved the local inflammatory infiltration in rat brain tissue.Immunofluorescence results showed that liraglutide reduced the expression of i NOS,IBA-1,C3 and GFAP after TBI in rat brain tissue.(6)WB results of rat brain tissues showed that the ratios of p-PKA / PKA,p-CREB / CREB in the Sham group were significantly higher than those in the TBI group and the liraglutide group,while those in the liraglutide group were higher than those in the TBI group(P <0.05).q RT-PCR results showed that the expression of TNF-α,IL-1α and C1 q was significantly increased after LPS treatment,and liraglutide could inhibit the increase.(7)The results showed that the m NSS score of TBI group was higher than that of sham on the 7th and 14 th day after modeling.The scores of rats in the lilalutide group were significantly lower than those in the TBI group(P < 0.05).The results of water maze showed that the times of platform crossing and the time of staying in target quadrant of Morris water maze test in TBI group were lower than those in sham group and lilalutide group,and the escape latency of lilalutide group was longer than that in TBI group,and the times of platform crossing and the time of staying in target quadrant were longer.conclusion: 1.Liraglutide can inhibit LPS-induced microglial activation and can also reduce the inflammatory response around the brain injury area after TBI.2.Liraglutide can regulate microglial inflammation through PKA / CREB signaling pathway,and then exert liraglutide’s anti-inflammatory effect.3.After the inflammation of microglia is inhibited,the release of a large number of inflammatory cytokines can be reduced,thereby inhibiting the generation of A1-type reactive astrocytes and improving the recovery of neural function after TBI... 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