| Objective: To observe the effect of liraglutide on the expression of microglia/macrophage surface markers and the secreted inflammatory cytokines,and key proteins of the Nrf2/HO-1 signaling pathway in the ischemic side brain tissue of the permanent cerebral ischemia model of diabetic rats,to further explore the potential mechanism of liraglutide brain protection.Methods: Seventy-five healthy male SD rats aged 8-10 weeks and weighing 280-320 g were first injected intraperitoneally with streptozotocin to simulate the diabetes model.After the diabetes model was successfully established,they were randomly divided into groups: sham operation group(cerebral ischemia group),Diabetic cerebral ischemia(DCI)group,insulin(Ins)group,liraglutide(Lir)group and liraglutide + HO-1 inhibitor tin protoporphyrin(Lir + Sn PP IX)group,15 rats in each group.Then use the suture method to establish a permanent middle cerebral artery occlusion(p MCAO)model.The Lir group and the Ins group were intraperitoneally injected with liraglutide(700 μg/kg)and insulin(<22 mmol/L,1IU/kg;22-28 mmol/L,1.5IU/kg;>28mmol /L,2 IU/kg)at 1 hour after p MCAO.The other three groups were intraperitoneally injected with the same amount of saline at the same time period.In the Lir+Sn PP IX group,Sn PP IX(20μg)was injected into the left lateral ventricle after p MCAO,and liraglutide(700 μg/kg)was injected intraperitoneally 1 hour after the operation.The remaining four groups of rats were also injected with the same amount of normal saline in the left lateral ventricle.After 3 days of p MCAO operation in each group of rats,the neurological deficit was assessed,and the brain was taken for TTC staining to measure the cerebral infarct volume;Western blot was used to detect the protein expression of Nrf2,HO-1,CD16,and CD206 on the ischemic side of each group,and fluorescence quantitative PCR was used to detect the expression levels of IL-10,IL-4,IL-1?,TNF-ɑ m RNA on the ischemic side;Double immunofluorescence method was used to detect the co-labeling of M1 type marker CD16/32 and M2 type marker CD206 and Iba-1 in the ischemic brain tissue.Results: 1.Changes in fasting blood glucose: Before STZ intervention,there was no significant change in blood glucose in each group,but blood glucose levels changed significantly after intraperitoneal injection of STZ(P<0.05),and the blood glucose in the Ins group was lower than before(P < 0.05);Lir group The blood sugar of Lir+Sn PPIX group was lower than before(P<0.05).There was no significant change in blood glucose before and after the CI group.2.Neurological function score: Compared with the DCI group,the neurological function score of the Lir group was significantly decreased(P<0.05),and there was no significant difference in the Ins group(P>0.05);compared with the Lir group,the neurological function score of the Lir+Sn PP IX group was obvious Increase(P<0.05).3.Cerebral infarct volume: white areas were seen in each group after TTC staining.Compared with the CI group,the infarct volume in the DCI group was significantly increased(P<0.05);compared with the DCI group,the infarct volume on the ischemic side of the Lir group was significantly reduced(P<0.05),there was no significant difference in the Ins group(P>0.05).Compared with the Lir group,the infarct volume on the ischemic side of the Lir+Sn PP IX group was significantly increased(P<0.05).4.TNF-ɑ,IL-1?,IL4,IL-10 m RNA expression: Compared with the CI group,the DCI group TNF-ɑ,IL-1? expression was up-regulated,and IL4,IL-10 expression was downregulated(P<0.05).Compared with the DCI group,the expressions of TNF-ɑ and IL-1? in the Lir group were down-regulated,and the expressions of IL4 and IL-10 were up-regulated(P<0.05).There was no significant difference between the Ins group and the DCI group(P>0.05).Compared with the Lir group,the expressions of TNF-ɑ and IL-1? in the Lir+Sn PP IX group were up-regulated,and the expressions of IL4 and IL-10 were down-regulated(P<0.05).5.Expression of Nrf2 and HO-1 protein: Compared with the CI group,the expression of Nrf2 and HO-1 was down-regulated in the DCI group(P<0.05).Compared with the DCI group,the expression of Nrf2 and HO-1 in the Lir group was increased(P<0.05).Compared with the Lir group,the expression of Nrf2 and HO-1 in the Lir+Sn PP IX group decreased(P<0.05).There was no significant change between the DCI group and the Ins group(P>0.05).6.Expression of CD16 and CD206 protein: Compared with the CI group,the expression of CD16 in the DCI group increased,and the expression of CD206 decreased(P<0.05).Compared with the DCI group,the expression of CD16 was lower in Lir group,and the expression of CD206 was higher(P < 0.05).Compared with the Lir group,the expression of CD16 in the Lir+Sn PP IX group increased,and the expression of CD206decreased(P<0.05).The DCI group was compared with the Ins group(P>0.05).7.Polarized expression of microglia/macrophages: Compared with the CI group,the DCI group M1 microglia/macrophage marker CD16/32/Iba-1 positive cells significantly increased,M2 microglia /macrophage marker CD206/Iba-1 positive cells was significantly reduced(P<0.05);compared with the DCI group,the Lir group M1microglia/macrophage marker CD16/32/Iba-1 was significantly reduced,and M2microglia/macrophage marker CD206/Iba-1 positive cells was significantly increased(P<0.05);compared with the Lir group,the Lir+Sn PP IX group M1 microglia/macrophage marker CD16/32/Iba-1 positive cells increased significantly,and M2microglia/macrophage marker CD206/Iba-1 positive cells significantly decreased(P<0.05).Conclusion: 1.Liraglutide has direct brain protection on diabetic rats with cerebral ischemia injury.2.By activating the Nrf2/HO-1 signaling pathway,thereby inhibiting the polarization of M1microglia/macrophages,and regulating the conversion of microglia/macrophages to M2 type,it may be one of the important mechanisms of liraglutide’s neuroprotection against diabetic cerebral ischemia injury. |
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