Effect And Mechanism Of Salt-Induced Kinase 2 On Microglia Cell Polarization In Cerebral Ischemia-Reperfusion Injury

Objectives:To investigate whether salt-induced kinase 2(SIK2)is involved in polarization of microglia in cerebral ischemia-reperfusion injury and to explore the effects of overexpression or suppression of SIK2 in rat brains on microglia and its possible molecular mechanisms.Methods: Rats were randomly divided into four groups: sham,ischemia-reperfusion(I/R),Bosutinib(low SIK2 expression),and Ad-h SIK2(high SIK2 expression)groups.Bosutinib was administered via tail vein injection to suppress SIK2 expression 1 day before the experiment.Adenovirus was injected into the right lateral ventricles of rats to construct an SIK2 overexpression model in the brain tissue 1 week before the experiment.The rats were subjected to middle cerebral artery occlusion to establish a rat model of cerebral ischemiareperfusion and simulate the clinical treatment of cerebral ischemia-reperfusion injury.The Zea Longa score was used to evaluate neurological deficits in rats.The expression levels of SIK2,CD86,CD206,nuclear and cytoplasmic CREB-regulated transcriptional coactivator 3(CRTC3),phosphorylated CREB(p-CREB),and total CREB were detected using western blot analysis.Immunofluorescence double staining was used to detect CD86+/Iba-1+ and CD206+/Iba-1+ positive cells.The TTC staining method was used to evaluate the volume of brain infarction in rats.The ELISA method was used to measure the expression levels of interleukin-6,tumor necrosis factor-α,and interleukin-10 in brain tissue.Results:(1)Expression of SIK2 in rat brain tissue and microglia: The expression of SIK2 after cerebral ischemia-reperfusion injury first decreased and then increased.The expression of M1 and M2 microglia(CD86+/Iba-1+ and CD206+/Iba-1+)was significantly increased in the I/R(2/6h),I/R(2/12h),I/R(2/24h),and I/R(2/72h)groups compared with the sham group(P<0.05).The ratio of M1 to M2 cells began to decrease in the I/R(2/24h)group.(2)Correlation between SIK2 expression and microglia expression: Compared with the sham group,the percentage of brain infarction volume(13.39±1.09)and neurological deficit score were significantly increased(P<0.05),the expression of CD86 protein and the number of CD86+/Iba-1+ cells were significantly increased(P<0.05),and the quality fraction of proinflammatory factors IL-6 and TNF-α were significantly increased(P<0.05)in the I/R(2/24h)group.Compared with the I/R(2/24h)group,the percentage of brain infarction volume(6.52±0.73),neurological deficit score,expression of CD86 protein,and the number of CD86+/Iba-1+ cells were significantly decreased(P<0.05),but the expression of CD206 protein and the number of CD206+/Iba-1+ cells were significantly increased(P<0.05),the quality fraction of pro-inflammatory factors IL-6 and TNF-α were significantly decreased(P<0.05),and the quality fraction of anti-inflammatory factor IL-10 was significantly increased(P<0.05)in the Bosutinib(suppression of SIK2)group.Compared with the I/R(2/24h)group,the percentage of brain infarction volume(13.39±1.09),neurological deficit score,expression of CD86 protein,and the number of CD86+/Iba-1+ cells were significantly increased(P<0.05),but the expression of CD206 protein and the number of CD206+/Iba-1+cells were significantly reduced(P<0.05),the quality fraction of pro-inflammatory factors IL-6 and TNF-α were significantly increased(P<0.05),and the quality fraction of antiinflammatory factor IL-10 was significantly decreased(P<0.05)in the Ad-h SIK2(high SIK2expression)group.(3)Signaling pathway by which SIK2 affects microglia polarization:Compared with the sham group,the ratio of nuclear to cytoplasmic CRTC3 and p/T-CREB was significantly increased in the I/R(2/24h)group(P<0.05).Compared with the I/R(2/24h)group,the N/C-CRTC3 and p/T-CREB ratio were significantly increased in the Bosutinib group(P<0.05),while the expression of N/C-CRTC3 and p/T-CREB were significantly decreased in the Ad-h SIK2 group(P<0.05).Conclusions:(1)SIK2 is involved in microglia polarization in cerebral ischemia-reperfusion injury.(2)Suppression of SIK2 results in improved neurological deficits,decreased brain infarction volume,downregulation of M1 microglia expression,decreased interleukin-6 and tumor necrosis factor-α,increased interleukin-10,and improved cerebral ischemiareperfusion injury in rats.(3)SIK2 regulates microglia polarization through the CRTC3-CREB pathway.