| Objective: Ischemic stroke has become a common disease that seriously threatens human health.In order to alleviate or avoid brain damage caused by ischemia,in principle,blood supply should be restored as soon as possible,and after a period of cerebral ischemia,local blood flow recovery will cause severe secondary injury,which is ischemia and reperfusion injury(I/R).The inflammatory response plays a key role in I/R injury,so it is urgent to find a solution to inhibite the post-ischemic inflammatory response.It will be great significance for the treatment of ischemic brain injury.Experimental research found that delta-opioid recepter(DOR)plays an important protective role in brain I/R and participates in electroacupuncture against acute brain I/R injury,but the reation between DOR and inflammation,there is still no relevant literature reports.In this work,Oxygen-Glucose Deprivation-Reperfusion(OGD/R)model of rat pheochromocytoma cell(PC-12)and Middle Cerebral Artery Occlusion(MCAO)model of SD rat were used to explore the neuroprotective effect of DOR in brain I/R and its anti-inflammatory mechanism via in vivo and in vitro.Method:(1)In vitro experiments: OGD/R model of PC12 cells was used to simulate I/R injury.PC12 cells were induced by different OGD/R time combined with hypoxia chamber,and the morphology and survival rate of PC12 cells were detected by inverted microscope and CCK-8 reagent to select the appropriate time.In the subsequent experiments,we utilize oxygen deprivation for 6 h and reperfusion for 24 h.The PC12 cells in logarithmic growth phase were randomly divided into 5 groups: control group,OGD/R group(ie model group),OGD/R+Tan67 group(ie OGD/R + Tan67 DOR agonist group),OGD/R+NTI group(ie OGD/R + NTI DOR antagonist group),OGD/R+NTI+Tan67 group(ie OGD/R+NTI+Tan67 group).The morphological changes and survival rate of cells in each group were observed and detected by inverted microscope and CCK-8 reagent.The leakage rate of LDH,produced by cells in each group were detected by LDH kits.The apoptosis of cells in each group was detected by In situ cell Death Detection kit.The expression of DOR protein in PC12 cells after pretreatmen was detected by Western blot.The expression of BDNF protein in the supernatant and cells of each group was detected by ELISA.The m RNA expressions of DOR,BDNF,Trk B,IL-1?,IL-6,TNF-?,IL-4 and IL-10 were detected by q RT-PCR.(2)In vivo experiments: MCAO model of SD rats was used to simulate I/R injury.According to random number double-blind method,the healthy male SD rats were randomly divided into 5 groups: Sham group(Control group,n=8),MCAO group(Model group,n=8),Sham EA group(EA+S,n=8),EA group(EA,n=8),EA+Inhibitor group(EA+NTI,n=8).The changes of weight before operation and after operation 24 h were detected.The neurological impairment was measured by neurological function score.The volume of cerebral ischemia was measured by CV staining.The expressions of IL-1? and IL-10 in ischemic infarction area were detected by fluorescence immunostaining.(3)For further explore the protection of DOR in ischemic stroke,small interfering RNA(si RNA)technology was used to knockdown DOR at the gene level and inhibit the expression of DOR protein.Firstly,si RNA-NC-Cy3 transfection experiment was used to detect the transfection efficiency and conditions of si RNA transfection into PC12 cells.Then three DOR-si RNA(Oprd1-rat-356,Oprd1-rat-509,Oprd1-rat-604)were designed to transfect PC12 cells.The expression of m RNA of DOR was detected by q RT-PCR and the expression of DOR protein was detected by Western blot after transfection of PC12 cells for 24 h and 48 h.Determining the transfection efficiency and inhibition of DOR,which is used for further explore the neuroprotective effect of DOR in cerebral I/R injury.Results:(1)In vitro experiments results showed that the cell viability of PC12 cells was reduced after sustained OGD/R injury.The morphological changes showed that the synapses became shorter and its number also reduced,cell body opacity decreased,and the cell morphological damage increased with the prolongation of OGD/R time.The survival rate of CCK-8 results was consistent with the morphological change.The survival rate of PC12 cells was about 60%(P<0.01)at 6 h/24 h of OGD,and this time was selected for subsequent experiments.CCK-8,LDH and TUNEL apoptotic staining test indicated that DOR agonist Tan67 could significantly improve the survival rate of PC12 cells injured,reduce the release of LDH and the apoptosis of PC12 cells after OGD/R injury(P<0.01,P<0.01,P<0.001,respectively)and this protective effect could be reversed by NTI,a DOR inhibitor.Western Blot assay presented that DOR agonist Tan67 could partially inhibit the decrease of DOR protein induced by OGD/R(P<0.05)and NTI could reverse this effect.The ELISA results showed that activation of DOR could increase the supernatant and intracellular BDNF content of each group after OGD/R induced PC12 cells injury(P<0.001,P<0.01),which could be reversed by NTI inhibitor.The results of q RT-PCR showed that DOR agonist Tan67 could increase the expression levels of DOR and Trk B after OGD/R injury in PC12 cells(P<0.01,P<0.001),decrease the expression levels of pro-inflammatory factors TNF-?,IL-1? and IL-6(P<0.01,P<0.05,P<0.05),and increase the expression levels of anti-inflammatory factors IL-10 and IL-4(P<0.001,P<0.001).However,this effect was antagonized by first administering the inhibitor NTI.These above results suggest that DOR may achieve neuroprotective effects by activating the BDNF/Trk B pathway to alleviate the post-ischemic inflammatory response and apoptosis.(2)In vivo experiment results showed the changes in body weight between the experimental animals before and 24 h after surgery were no statistical significant.In terms of neurological impairment and cerebral ischemic volume,the neurological impairment and infarct volume of the sham electroacupuncture group showed no statistical significant compared with the model group.Electroacupuncture could improve the neurological function of SD rats after MCAO(P<0.05),which could be blocked by NTI(P<0.05).CV staining indicated that electroacupuncture could reduce the volume of cerebral infarction in SD rats after MCAO and NTI could reversed the protective effect of electroacupuncture.MCAO+NTI group revealed that the volume of infarction was larger.Immunofluorescence assay diaplayed that electroacupuncture could decrease the expression of IL-1?(P<0.05)and increase the expression of anti-inflammatory factor IL-10(P<0.05)in cerebral infarction area of SD rats after MCAO.NTI,a DOR inhibitor,could block this protective effect.The expression of IL-1? in MCAO+NTI was higher,while the expression of anti-inflammatory factor IL-10 was lower.These above results suggest that DOR may participate in electroacupuncture treatment of ischemic brain injury by inhibiting I/R inflammatory response,and provide a theoretical basis for the application of electroacupuncture in the treatment of ischemic stroke.(3)The results of si RNA specific knockdown of DOR gene in PC12 cells showed that the designed si RNA could successfully transfect PC12 cells,and the transfection efficiency was more than 70%.The three DOR-si RNA sequences(Oprd1-rat-356,Oprd1-rat-509,Oprd1-rat-604)were designed.The results of q RT-PCR and Western Blot indicated that the expression of DOR m RNA as well as DOR protein decreased at 24 h and 48 h after transfection,especially 24 h after transfection.The inhibition rate of DOR m RNA and DOR protein could all reach 60% after 24 h of transfection with Oprd1-rat-509.The results suggested that PC12 cells transfected by Oprd1-rat-509 24 h successfully silenced the DOR gene and inhibited the expression of DOR protein,which could be used to further study the specific role and mechanism of DOR in anti-ischemic brain injury.Conclusion:(1)Activation of DOR has a significant protective effect on OGD/R-induced PC12 cells injury.And its neuroprotective effect may be achieved by activating BDNF/Trk B signaling pathway to alleviate inflammation and reduce apoptosis.(2)Electroacupuncture "shuigou" and "Neiguan" treatment of MCAO rats can reduce the volume of cerebral infarction,improve neurological function.increase inflammatory factors IL-1? and reduce anti-inflammatory factors IL-10 in cerebral ischemic area,wich has obvious neuroprotective effect.DOR's inhibitor reverses the protective effects.This suggests DOR may participate in the treatment of ischemic brain injury improving neurological protection by EA through BDNF/Trk B signaling pathway to inhibite post-I/R inflammatory response.(3)After transfecting PC12 cells with Oprd1-rat-509 for 24 h,the DOR gene was successfully knockdown and the expression of DOR protein was inhibited,wich can be used for further reserarch. |
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