| With the gradual aging of the population,cerebrovascular disease has become a common disease and morbidity that endanger human life and health.Stroke is one of the leading causes of death and disability in the world.Ischemic stroke accounts for more than80%of all stroke.It has brought serious economic and mental burden to the community and the patients.Ischemic has high incidence,high mortality,high recurrence rate,and high disability characteristics,and tend to younger in recent years.To date,recombinant tissue plasminogen activator?rt-PA?is the only drug that has been proven for acute stroke treatment.However,only a limit number of patients benefit from it due to its time-limited narrow therapeutic window for treatment as well as its potential side effects.The occurrence of stroke is due to the lack of blood supply to the brain tissue,causing ischemia combined therapy.Stroke occurs due to a loss of blood supply to part of the brain,initiating he ischemic cascade.As oxygen or glucose becomes depleted in ischemic brain tissue,the production of high energy phosphate compounds such as adenosine triphosphate?ATP?fails leading to failure of energy dependent processes?such as ion pumping?necessary for tissue cell survival.This sets off a series of interrelated events that result in cellular injury and death by necrosis.Dead cells by necrosis produce the release of all cytoplasm content into the extracellular space activating the corresponding inflammatory response.With the rapid development of modern immunology and biomolecules,the research on the pathophysiological mechanism of cerebral ischemia and reperfusion has made great progress.Pathological mechanisms that related to stroke are oxidative stress,inflammatory response,apoptosis,intracellular Ca2+overload,free radical damage and so on.In these pathologic damage mechanisms,inflammatory response and oxidative stress become a hotspot in neuroscience research,antioxidant stress,reduce inflammatory injury,anti-apoptosis as one of the main ways to treat stroke.Transcription factors can regulate a variety of cell function genes,as a therapeutic target of molecular repair.Transcriptional activation is considered as a double-edged sword because individual transcription factors can induce protective genes or neurotoxic genes.The transcription factor NF-E2-related factor 2?Nrf2?is a major regulator of endogenous antioxidant defense.In response to oxidative stress,Nrf2 promotes the expression of a wide variety of antioxidant genes,including both enzymatic and non-enzymatic antioxidants-by translocating into the nucleus,binding to antioxidant response elements?AREs?in the nucleus,and regulating the transcription of downstream targetgenes.Nrf2seems to play an important role in the protection of brain cells against cerebral ischemic injury.Loss of Nrf2 function increases the size of cerebral infarct and neurological deficits after an ischemic event.In the last years,heme oxygenase-1?HO-1?has been recognized by many groups as a potential target in ischemic damage.HO-1 is the enzyme responsible for the conversion of the heme group to bilirubin,carbon monoxide and iron;a highly regulated cytoprotective enzyme able to respond to numerous chemical or physical stressors,many of which decrease oxygen availability and generate oxidative stress.CDDO-EA is an acetamide derivative of the triterpenoid compound 2-cyano-3,12-dioxo cyclopentene-1,9?11?-dien-28-acid.Studies have shown that CDDO-EA is a potent inducer of heme oxygenase-1 and Nrf2/ARE signaling,playing a neuroprotective role by activating the Keap1-Nrf2/ARE pathway.In this study,male C57/BL mice were used as the research object,and the model of cerebral ischemia-reperfusion was established by modified Longa suture method.The expression of Ho-1 and M1/M2 microglia markers,the evaluation of neurological deficit,the infarct volume and the inhibition of inflammatory reaction after ischemia were observed in this model.The study was divided into two parts as below.Part one Protective effect of HO-1 on cerebral ischemia-reperfusion in mice Objective:This study is to observe the changes of Ho-1 after cerebral ischemia-reperfusion injury,to investigate the inflammatory response induced by Ho-1 on cerebral ischemia-reperfusion injury,to explore the mechanism of Ho-1 inhibition of neuroinflammation.Methods:In this study,male C57/BL mice were used as the research object,and the model of cerebral ischemia-reperfusion was established by modified Longa suture method.C57/BL mice were randomly divided into 4 groups:sham group,vehicle group,CDDO-EA group,Sn-PPIX group,and neurological score was performed before divided.Measuring Neurological function score,measuring cerebral infarction volume by TTC method,measuring the expression of M1 and M2 markers in microglia by immunofluorescence,and monitoring the expression of Ho-1by western blot before groups divided.Results:1 Compared with sham group,the expression of Ho-1 in vehicle group was increased at 48h and 72h,indicating that ischemia-reperfusion injury resulted in elevated Ho-1expression.Compared with vehicle group,the expression of Ho-1 in CDDO-EA group?25ug,50ug and 100ug groups?increased significantly at 48h,the difference was statistically significant.The increase of Ho-1 in each concentration group was not obvious after 72h,which due to the decrease of plasma concentration2 Mice in sham group had no palsy and a neurological score of zero.There was a serious neurological deficit in the vehicle group both at 24h and 48h.Compared with the vehicle group,CDDO-EA group has slight neurological deficits in the 24h group,but there is no significant difference.There was significant neurological deficit in the 48h group,and the difference was statistically significant;3 There was a significant improvement in infarct volume in the CDDO-EA group at48 h compared with the vehicle group.4 Compared with the sham group,the expression of Ho-1 in vehicle group increased significantly,indicating that ischemia-reperfusion injury resulted in elevated Ho-1expression.Ho-1 was in the high expression level both in the cytoplasm and nuclear.The expression of Ho-1 was increased after dealing with 50ugCDDO-EA,the difference was statistically significant.Ho-1 expression was significantly reduced after dealing with Sn-PIXX inhibitor,the difference was statistically significant.5 Tunel immunofluorescence staining showed that there was a large number of staining positive cells in the damage area.Apoptosis was significantly improved in CDDO-EA group,the number of apoptotic cells significantly reduced,and the difference was statistically significant.6 The immunofluorescence staining showed that there were little amounts of microglia markers?Iba1?in sham group;M2 type microglia expression rate was significantly increased in CDDO-EA group,the difference was statistically significant.The positive rate of M1-type microglia increased in vehicle group,the difference was statistically significant,and the positive rate of M1 microglia in CDDO-EA group was significantly lower,the difference was statistically significant.Conclusion:Ho-1 has neuroprotective effect on ischemia-reperfusion mice,improving the neurological deficit after cerebral ischemia and reperfusion.And it has effect on reducing the infarct size,inhibiting the expression of M1 glial,and promoting the expression of M2type microglia cell.Thereby Ho-1 can reduce the post-ischemic inflammatory response,to achieve neuroprotective effects after ischemia.Part two Ho-1 participates in neuroprotective effects by regulating the polarization state of microgliaObjective:The study was to observe the effect of CDDO-EA on the up-regulation of Ho-1 in LPS-induced central nervous system inflammation model.To investigate the effect of Ho-1on M1/M2 type microglia polarization state,to explore the mechanism of Ho-1 inhibition of nerve inflammation.Method:Inducing BV2 activation in vitro by Lipopolysaccharide?LPS?,co-culturing with CDDO-EA.MTT assay was used to detect the activity of BV2 cells,excluding the cytotoxicity effect of LPS.Griess reagent method was used to detect the content of NO,the expression of inflammatory factor was detected by ELISA,and the expression of Ho-1 was detected by western blot.Real-time Quantitative PCR and QPCR)were used to detect the expression of microglia in M1 and M2 microglia in vitro.The effect of CDDO-EA on the phagocytosis of BV2 in vitro was analyzed by Nile red microspheres.Result:1 BV2 cells in control group were in good condition.When LPS concentration was lower than 100ng/ml,the cell viability was higher than 90%.With the increase of LPS concentration,the cell viability decreased.When the concentration was 1ug/ml,LPS had toxic effects on BV2 cell,the cell activity was less than 55%.2 NO detection and expression of inflammatory factors:the expression of inflammatory factors increased after stimulating with LPS.Compared with control group,the content of NO,TNF-?and IL-6 in LPS+PBS group were significantly increased,the content of LPS+CDDO-EA group was higher than that of control group,which was lower than that of LPS+CDDO-EA group.Compared with control group,IL-10 content in LPS+PBS group and LPS+CDDO-EA group increased.Compared with LPS+PBS group,IL-10 content in LPS+CDDO-EA group increased significantly,and there was significant difference.3 The expression of Ho-1 was detected by western blot.Compared with LPS group,the expression of Ho-1 in CDDO-EA group was increased.Compared with LPS group,the expression of Ho-1 was increased after treatment with 50ug,100ug and 200ugCDDO-EA,the difference was statistically significant.Compared with control group,the expression of Ho-1 in LPS group increased,the difference was statistically significant.Compared with LPS group,the expression of Ho-1 in CDDO-EA group was significantly increased.Compared with CDDO-EA group,the expression of Ho-1 was significantly decreased in the Sn-PPIX group,the difference was statistically significant.4 Real-time quantitative PCR?Q-PCR?was used to detect the expression of microglia in M1 and M2 microglia in vitro.The expression of CD16 mRNA which is M1 marker,and CD206 which is M2 marker of BV2 in vitro was detected by RT-PCR.The results showed that the CD16 mRNA expression in LPS group was higher than that of control group.The expression level of CD16 mRNA in CDDO-EA group was higher than that in control group,which was lower than that in LPS group.The expression of CD16 mRNA in CDDO-EA group was increased compared with that in the control group,which was higher than that in CDDO-EA group.The expression of CD206 mRNA which is the M2 phase marker in LPS group was higher than that of control group.The expression of CD206 mRNA in CDDO-EA group was higher than that in control group,which was higher than that in LPS group.The expression of CD206 mRNA in the CDDO-EA group was higher than that in the control group.The expression of CD16 mRNA in CDDO-EA group was lower than that in CDDO-EA group.5 The effect of CDDO-EA on the phagocytosis of BV2 in vitro was analyzed by Nile red microspheres.Compared with control group,the phagocytosis of BV2 in LPS group was enhanced,and the phagocytosis of BV2 in CDDO-EA group was higher than that in LPS group.The number of phagocytic microspheres in the Sn-PPIX group was significantly lower than that in the CDDO-EA group,the difference was statistically significant.The results suggest that Ho-1 can enhance BV2 phagocytosis,reduce the inflammatory response.Conclusions:Ho-1 regulates the polarized state of activated microglia,inhibits M1-type microglia,promoting M2-type microglia,thereby reducing the inflammatory response. |
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