| With the rapid development of economy in China,and the population aging,as well as changes in lifestyles,the incidence of cardio-and cerebro-vascular diseases is increasing.Stroke is a major cerebrovascular disease,with the characteristics of high incidence,mobility,mortality and high recurrence rate,which brings a heavy burden to patients’ families and the society.Epidemiological studies have shown that ischemic stroke is the most common of types of stroke accounting for about 75%.Furthermore there is a gender differences in the incidence,severity,and post-stroke disability that postmenopausal women is more likely than young women and same age men to suffer from ischemic stroke.Therefore,it is very important to explore effective strategies to protect ischemic brain.Existing research shows that remote limb ischemia postconditioning(RLIP),as a non-invasive treatment,can reduce the damage of the blood-brain barrier and reduce the effects of inflammation.Strong implementation and other characteristics,but its mechanism needs further research.SDF-1 / CXCR4 plays an important role in the central nervous system.Studies have shown that SDF-1 / CXCR4 is upregulated in the ischemic region after stroke,but whether SDF-1 / CXCR4 plays a regulatory role in post-ischemic limb processing is unclear.In this study,we used a model of whole cerebral ischemia in postmenopausal rats to investigate whether RLIP can protect cerebral ischemia through SDF-1 / CXCR4 and its possible molecular mechanism.Methods:Three-month-old female SD rats(SDF grade)were subjected to bilateral ovariectomy to establish surgical menopausal model.One week later,global cerebral ischemia(GCI)was induced by four-vessel occlusion.The animals were randomly divided into sham group(Sham),ischemia reperfusion group(IR),remote limb ischemic postconditioning group(RLIP),and AMD3100-treatment group,a specific antagonist of SDF-1/CXCR4(RLIP + AMD3100).Double Immunofluorescence staining and western blot analysis were used to detect the protein expression of SDF-1 and glial fibrillary acidic protein(GFAP,a marker of astrocyte),CXCR4 and Iba1(a microglia marker),as well as phosphorylation levels of ERK1/2 and P38 in the hippocampus CA1 region of the rats.The content of SDF-1 in serum and hippocampus CA1 region was detected by ELISA analysis.Results:1.The results of immunofluorescence double staining for SDF-1 and GFAP showed that compared with the sham group,the fluorescence intensity of GFAP and SDF-1 was significantly enhanced(P<0.01)at 3d after ischemia(IR)in the hippocampal CA1 region,while RILT markedly decreased the enhancement compared with IR group(P<0.01).AMD3100,a specific antagonist of SDF-1/CXCR4 axis could remarkedly decrease fluorescence intensity of the both proteins compared to RLIP animals(P<0.01).2.Consistent with the observation by immunofluorescence staining,western blot analysis results of SDF-1 and GFAP also showed a same trend in the both protein expression at reperfusion 3d of the hippocampal CA1 region.Compared with the sham group,IR significantly enhanced protein expression of SDF-1 and GFAP(P<0.01),while RLIP perfectly reversed the effect induced by IR treatment(P<0.01).AMD3100-administration significantly reduced protein expression of SDF-1 and GFAP compared to RLIP animals(P<0.01).3.ELISA protein quantitation showed that in the hippocampal CA1 region of reperfusion 3d rats,SDF-1 concentration significantly increased compared with the sham group(P<0.01),while RLIP effectively revised the change caused by IR(P<0.01).Antagonist AMD3100 expectantly reduced SDF-1 concentration compared to RLIP group(P<0.01).However,the SDF-1 concentration in serum derived from reperfusion 3d animals was significantly reduced compared to sham group(P<0.01),and compared with the IR group,whereas RLIP significantly increased SDF-1 concentration(P<0.01).Administration of AMD3100 inhibited the increase of SDF-1 induced by RLIP(P<0.01).4.The results of immunofluorescence double staining for CXCR4 and Iba1 showed that the fluorescence intensity of CXCR4 and Iba1 in the hippocampal CA1 region of 3d reperfusion rats were significantly increased compared to sham group,and CXCR4 co-localized with Iba1-positive microglia(P<0.01).Whereas the fluorescence intensity of Iba1 and CXCR4 in the RLIP group was markedly reduced compared to IR group(P<0.01).AMD3100-treatment further reduced the CXCR4 and Iba1 fluorescence intensity compared to RLIP group(P<0.01).5.The western blot results of CXCR4 and Iba1 showed that compared with the sham group,the gray values of CXCR4 and Iba1 in the IR group were significantly enhanced(P<0.01).Compared with the IR group,the gray values of CXCR4 and Iba1 in the RLIP group Compared with the RLIP group,the gray values of CXCR4 and Iba1 in the AMD3100 group were significantly reduced(P<0.01).6.The results of p-ERK1 / 2 and p-p38 immunofluorescence double staining showed that p-ERK1 / 2 co-localized with p-p38.The fluorescence result of p-ERK was that compared with sham group,p-ERK1 / 2 of IR group the fluorescence intensity was significantly reduced(P<0.01)Compared with the IR group,the fluorescence intensity of p-ERK1 / 2 in the RLIP group was enhanced(P<0.01).Compared with the RLIP group,the fluorescence intensity of p-ERK1 / 2 in the AMD3100 group was significant.Decrease(P<0.01).The fluorescence results of p-p38 were: Compared with the sham group,the fluorescence intensity of p-p38 in the IR group was significantly enhanced(P <0.01)and the fluorescence intensity of p-p38 in the RLIP group was reduced(P<0.01)compared with the IR group.Compared with the RLIP group,the fluorescence intensity of p-p38 in the AMD3100 group was not statistically significant(P>0.05).7.Western blot results of p-ERK1 / 2,ERK1 / 2,and p-p38 showed that in the hippocampus:p-ERK1 / 2 expression was reduced in the IR group compared with the sham group(P<0.01).Compared with the IR group,the IR The expression in the RLIP group was increased(P<0.01).Compared with the RLIP group,the content of p-ERK1 / 2 in the AMD3100 group was reduced(P<0.05).The western blot results between the ERK1 / 2 groups showed that the content of ERK1 / 2 no statistical significance(P>0.05);compared with the sham group,p-p38 expression in the IR group was increased(P<0.01),compared with the IR group,RLIP group was reduced,(P<0.01),compared with the RLIP group,AMD3100 The content of p-p38 in group was not statistically significant(P>0.05).Conclusion:1.RLIP may reduce neuroinflammation damage by inhibiting the SDF-1 / CXCR4 axis of hippocampal CA1 region after IR,and exert neuroprotective effect on female ovariectomized rats.2.The activation of ERK1 / 2 pathway and the inhibition of P38 signal may be the molecular mechanism by which RLIP regulates the SCF-1 / CXCR4 axis to reduce inflammation damage after ischemia. |
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