The Mechanism By Which Sinomenine Relieves Cerebral Ischemia Reperfusion Injury

Objective:The ischemic cerebrovascular disease is a central nervous system disease that is caused by transient or permanent reduction or depletion of blood supply to some area because of an embolism or thrombus in the cerebral artery.The main clinical treatment is the thrombolytic therapy.However,due to recovered blood supply after thrombolysis,reperfusion injury often occurs and aggravates the inj ure of brain.An important pathological process of the cerebral ischemia/reperfusion injury is that microglia(MG)are inflammatorily activated,the microglia which activated after cerebral ischemia can secrete inflammatory factors,so neurons are exposed to inflammatory factors and injured.Studies have shown that the inflammatory activation of MG and transcription factor NF-?B play a dominant role in the inflammatory response.As a result,it is important to search for effective NF-?B inhibitors and elucidate their anti-inflammation action,for improving clinical treatments and developing new therapies of cerebral ischemia/reperfusion injury.Previous studies have shown that sinomenine,an anti-inflammatory compound extracted from sinomenium acutum,could directly protect neurons against cerebral ischemia injury.No reports whether it could resist ischemia/reperfusion induced inflammatory activation of MG have been published.In the present study,we develop an in-intro ischemia/reperfusion model,a model of oxygen-glucose deprivation/reperfusion(OGD/R),with mouse BV-2 microglia cells and confirm the inhibitory effect of sinomenine pretreatment on inflammatory activeation measured by inflammatory indicators.On the basis of the results,we investigate the mechanism for sinomenine to suppress OGD/R induced inflammatory activation in MG and analyze the regulating pathway at various levels,then verification of the regulatory mechanism is carried out at various levels.So we can clarify the anti-inflammatory mechanism of sinomenine,and provide the laboratory basis for the clinical application of sinomenine.Methods:1.The anti inflammatory effects of sinomenine on OGD/R-induced BV-2 cells BV-2Cells were cultured in vitro,with different concentrations of sinomenine,24 hours after culture,the cell viability was determined with CCK-8 method.When the experimental concentrations of sinomenine were identified,Cells were divided into five groups:normal group,DMSO+OGD/R group,sinomenine(25?M)+OGD/R group,sinomenine(50?M)+OGD/R group,sinomenine(100?M)+OGD/R group.After being cultured overnight,cells were pretreated with 25,50,or 100?M sinomenine for two hours and subjected to OGD/R treatment.24 hours after reperfusion,supernatant was collected for TNFa,IL-1?and IL-6 assays.And cells were collected and total RNA and protein were isolated and used to measure mRNA of TNF?,IL-1? and IL-6 by real time PCR and phosphorylated p50 and p65,I?B? by western blotting.2.Research on the mechanism of sinomenine inhibits OGD/R-induced NF-?B activation in BV-2 cells BV-2 cells were divided into five groups:normal group,DMSO+OGD/R group,sinomenine (25?M)+OGD/R group,sinomenine(50?M)+OGD/R group,sinomenine(100?M)+OGD/R group,and incubated for 24 hours under normal conditions,then they were collected,total RNA and protein were extracted for examination of I?B? mRNA and protein by real-time PCR and western blotting,respectively."TargetScan" was used to predict the theoretic target(seed region)of mmu-miRNA-183-5p in the mRNA sequence of I?B-?.293 cells were transfected with the miRNA-183-5p-mimics,inhibitor,or NC as well as pGL-wt-I?B? and pGL-mt-I?B? using Lipofectamine 2000 transfection system.48 hours after transfection,the cells were harvested and luciferase assays were performed.Subsequently we analyzed the binding sites of miR-183-5p promoter and transcription factor SP1,we constructed the shRNA recombinant expression vector and cDNA expression vector of mouse SP1 gene,wild type and mutant miRNA-183-5p plasmid expression vector,then transfect them into 293 cells,so we could target silencing and overexpress SP1 gene,and confirm the transcriptional activity of SP1 by luciferase assay.In order to verify the SP1/miR-183-5p/I?B? regulatory pathway,SP1 or miRNA183-5p were expressed using a lentiviral approach in BV-2 cells.Twenty four hours after transfection,BV-2 cells were seeded to 6-well plates,and then incubated in medium containing 100?M of sinomenine overnight under normal conditions,and then subjected to OGR/R treatment.Cells were collected 24 hours after reperfusion for measuring relevant genes and proteins.Cells were divided into six groups,normal group,model(OGD/R)group,sinomenine+model group,pcDNA vector+sinome-nine+model group,pcDNA-SP1+sinomenine+model group,and miR-183-5P-mimics+sinomeni-ne+model group.Cells were collected 24 hours after reperfusion for measuring SP1,IKBa,phosphorylated p50 and p65.Results:1.CCK-8 assay showed that co-cultured for 24 hours with BV-2 cells,200?M sinomenine sig-nificantly inhibited the cell proliferation(P<0.01),therefore,the concentration of sinomenine was determined to be 25,50,100?M.ELISA assay showed that TNF-?,IL-1? and IL-6 in supernatant from the OGD/R model group was increased compared with the control group(P<0.01),indicating inflammatory activation of BV-2 cells.Pretreated with 100 ?M sinomenine significantly decreased TNF-?,IL-1? in supernatant,compared with the model group(P<0.01).The trend for sinomenine to reduce these inflammatory factors was concentration dependent.Compared with the control group,OGD/R treatment can significantly increase the mRNA level of TNF-?,IL-1? and IL-6(P<0.01),pretreated with sinomenine,the mRNA level of TNF-?,IL-1? and IL-6 were decreased,among these groups,the mRNA level of TNF-?,IL-1? decreased significantly(P<0.01).The data showed that OGD/R treatment could stimulate the phosphorylation of p50 and p65,and reduce I?B? protein(P<0.01).Sinomenine pretreatment could reverse this effect in a concentration-dependent manner,reduce phosphorylated p50 and p65,enhance I?B? protein(P<0.01).2.Cells were exposed to sinomenine at different concentrations for 24 hours,IKBa mRNA and protein were examined.The results showed that there was no apparent difference in I?B? mRNA level between the groups treated with sinomenine and without sinomenine(P>0.05),but IKBa protein was observed to be increased in the group treated with sinomenine(P<0.01).Luciferase reporter gene data showed that miRNA-183-5p-mimic significantly inhibited the activity of luciferase in the cells transfected with the reporter vectors carrying the wild type 3'UTR(p<0.05),and miR-183-5p-inhibitor increase luciferase activity(p<0.05).The co-transfection of miR-183-5p-NC had no obvious effects on the luciferase expressed by both vectors(P>0.05).Bioinformatics analysis showed that SP1 did have a theoretic binding site in miR-183-5p promoter.We then verified that site using luciferase reporter assay.Luciferase activity assay showed that SP1 increased the activity of luciferase in cells transfected with the wild-type reporter gene expression vector(P<0.01)but had no apparent effect on the reporter vector carrying a mutant promotor(P>0.05).we expressed SP1 or miR-183-5p-mimics using a lentiviral approach in BV-2 cells,and examined the resulted changes of SP1,I?B-? and phosphorylation of p50 and p65 to investigate the role of the pathway in sinomenine protection against ischemia reperfusion.The results showed that OGD/R could increase the expression of SP1 protein,reduce the expression of IKBa,and promote the phosphorylated p50 and p65(P<0.01)compared with the control group.Pretreatment of 100?M of sinomenine could inhibit the increase in SP1 protein,phosphorylation of p50 and p65,and the decrease in I?B?(P<0.01).SP1 overexpression increased SP1 protein content(P<0.01),and miR-183-5p-mimics overexpression had no observed effects on SP1(P>0.05).Both the expression of SP1 and miR-183-5p-mimics decreased I?B? expression(P<0.01),and increased phosphorylation of p50 and p65(P<0.01).Conclusions:In the present study,we develop an in-intro ischemia/reperfusion model,a model of oxygen-glucose deprivation/reperfusion(OGD/R),with mouse BV-2 microglia cells,and confirm the inhibitory effects of sinomenine pretreatment on inflammatory activation measured by inflammatory indicators.Next,we investigated the mechanism for sinomenine to suppress OGD/R-induced inflammatory activation in BV-2 microglia cells and confirme the regulating pathway at various levels.Then we come to the following conclusions,1.Sinomenine can effectively inhibit the inflammation of BV-2 cells induced by OGD/R,reduce the expression level of TNF-?,IL-1? and IL-6 mRNA,inhibit the phosphorylation of p50 and p65,promote the expression of I?B? protein in BV-2 cells.2.Sinomenine plays an anti-inflammatory role by promoting I?B? protein expression and inhibiting NF-?B activity.Sinomenine regulates the expression of I?B? protein at post-transcriptional level.3.The luciferase reporter assay show that mmu-miR-183-5p can combine with I?B? mRNA 3'-UTR,then inhibit the expression of I?B? at the post-transcriptional level.4.The transcription factors SP1 has a binding site on the miR-183-5p promoter and positively regulate its transcription.5.Sinomenine reduce inflammatory activation of BV-2 cells induced by OGD/R via the SP1/miRNA183-5p/I?B? pathway,that is,it can inhibit the expression of SP1 protein,then inhibit the transcription of miR-183-5p,and thus directly increase the expression of I?B?,which in turn limits the phosphorylation of p50 and p65,and ultimately inhibit the activation of NF-?B.