| Objective:Stroke is a common clinical central nervous system injury disease that endangers human health.Stroke can be ischemic or hemorrhagic,of which ischemic stroke accounts for80%with high mortality and disability rate.Together with coronary atherosclerotic heart disease and cancer,they are three major diseases threatening human health.At present,the clinical treatment of ischemic stroke mainly focuses on thrombolytic therapy in the acute stage and anti-inflammatory,promoting angiogenesis and nerve repair in the recovery stage.In fact,the vast majority of patients have exceeded the time window of thrombolytic therapy,which limits the clinical application of thrombolytic therapy.Therefore,anti-inflammatory response,promoting angiogenesis,improving blood flow in the injured area and repairing nerve become the research focus.Acupuncture is an important part of traditional Chinese medicine.For a long time,electroacupuncture(EA)has achieved satisfactory effect on stroke.Previous studies of our group have shown that delta opioid receptors(DOR)play an important role in the anti-ischemic stroke of electroacupuncture,but whether it is related to anti-inflammatory response,promoting angiogenesis and nerve repair has not been reported so far.Microglia is an innate immune cell in the central nervous system.Microglia activation plays an important role in the pathophysiology of neuroinflammation induced by ischemic stroke,and is a key factor in inflammatory response,angiogenesis and nerve repair.Therefore,the aim of this study is to establish the inflammatory injury model induced by lipopolysaccharide(LPS)in mouse microglia(BV2),the oxygen glucose deprivation/reperfusion(OGD/R)model in BV2 and the middle cerebral artery occlusion/reperfusion model in rats(MCAO/R),to explore the role of DOR in anti-inflammatory response of microglia and promoting angiogenesis in electroacupuncture treatment of cerebral I/R,and to explore the potential mechanism,so as to provide more powerful basis for the mechanism of electroacupuncture treatment of ischemic brain injury.Methods:1.To detect the expression of DOR in BV2,normal mice and human microglia.The changes of DOR expression in BV2 cells stimulated by lipopolysaccharide(LPS) and in activated microglia after LPS injection into mice brain were also observed.Immunofluorescence staining and Western blot were used.2.In vitro experiment2.1 Establish the inflammatory injury model of BV2 cells induced by LPS stimulation and observe the effects of different concentrations of LPS(50,100,200,500 ng/ml) on the viability and morphology of BV2 cells.In order to ensure the success of inflammatory injury model and reduce the adverse effects of LPS on cells,BV2 cells were stimulated with different concentrations of LPS to observe the cell viability.Combined with the experimental results,the appropriate concentration of LPS was selected to stimulate BV2 cells for subsequent experiments.2.2 The effects of TAN-67 and Naltrindole on LPS induced activation of BV2 cells were studied.The specific groups were as follows:normal control group,LPS(500ng/ml)group,LPS(500 ng/ml)+TAN-67(1,3,10μm)group,LPS(500 ng/ml)+Naltrindole(1,3,10μm)group,LPS(500 ng/ml)+Naltrindole(10μm)+TAN-67(1,3,10μm)group.CCK-8 kit was used to detect the cell viability,RT-q PCR and ELISA were used to detect the m RNA and protein changes of IL-1β,IL-6 and IL-10, respectively.Immunofluorescence staining and Western blot were used to detect the expression of i NOS.Western blot was used to detect the expression of MAPK signal related pathway protein p-p38/p38,p ERK/ERK,p JNK/JNK and apoptosis related protein cleaved caspase-3.Apoptosis was also detected by TUNEL staining.2.3 OGD/R(oxygen glucose deprivation 6 h/reperfusion 24 h)of BV2 cells simulated the activation of microglia during cerebral ischemia-reperfusion injury in vitro.The specific groups were as follows:normal control group,OGD/R group,OGD/R+TAN-67(10μm)group,OGD/R+Naltrindole(10μm)group,OGD/R+Naltrindole(10μm)+TAN-67(10μm)group.CCK-8 kit was used to detect cell viability.RT-q PCR and ELISA were used to detect the m RNA and protein changes of IL-1β,IL-6 and IL-10,respectively.3.In vivo experiment:SD rats were randomly divided into five groups:sham operation group(sham,n=8),MCAO/R group(model group,n=8),MCAO/R+EA+S group(sham EA group,n=8),MCAO/R+EA group(EA group,n=8),MCAO/R+Naltrindole+EA group(Naltrindole group,n=8).For Naltrindole group,10μL Naltrindole(100 nm/L)was injected into the lateral ventricle of SD rats half an hour in advance.The changes of body weight before and after operation were measured,and the neurological function of rats in each group was evaluated.CV staining was used to detect the size of cerebral infarction;Iba1 staining was used to detect the activation of microglia around infarct area of cerebral cortex and striatum;Western blot and immunohistochemistry were used to detect the expression of DOR around cerebral infarction;RT-q PCR and immunohistochemistry were used to detect the expression of M1/M2 phenotype related protein;The changes of mitogen activated protein kinases(MAPK)and caspase-3 signaling pathway proteins were detected by Western blot;the injury of neurons and axons was detected by TUNEL and immunohistochemistry;the activation of astrocytes was detected by GFAP staining; the changes of vascular density and vascular endothelial growth factor(VEGF)were detected by Western blot and immunohistochemistry.Results:1.Immunofluorescence staining and Western blot were used to detect the expression of DOR in normal BV2 cells.After LPS stimulation,the expression of DOR was significantly up-regulated.The expression of DOR in microglia was also found in normal human brain tissue.The expression of DOR in activated microglia was significantly up-regulated under LPS induced central nervous system inflammation in mice.2.The results of in vitro experiments were as follows(1)In the experiment of LPS induced inflammatory reaction of mouse microglia BV2 cells,by detecting the effect of different concentrations of LPS on the survival rate of BV2 cells,500 ng/ml LPS was selected as the concentration of BV2 cell inflammation model induction.At this concentration,the survival rate of BV2 cells was about 60%,which was significantly different from that of the control group(P<0.001).CCK-8 showed that TAN-67(10μm),a DOR agonist,significantly increased the survival rate of BV2 cells in LPS induced inflammatory response(P<0.01),and pre-incubation with Naltrindole,a DOR antagonist,blocked the protective effect of TAN-67.RT-q PCR test showed TAN-67 significantly inhibited the expression of IL-1βand IL-6 m RNA in a dose-dependent manner,increased the expression of IL-10 m RNA(all P<0.05).Naltrindole could block this effect.ELISA results showed that TAN-67 also inhibited the expression of IL-1β,IL-6 and promoted the expression of IL-10 protein in a dose-dependent manner,and there were significant differences between TAN-67 and LPS control group at the concentrations of 3 and 10μm(all P<0.01).Naltrindole also blocked this effect.These results suggest that TAN-67 may promote the transformation of activated microglia into M2 phenotype.By detecting the expression of M1 marker CD16/32and M2 marker CD206,it was also found that TAN-67 inhibited the expression of CD16/32 and promoted the expression of CD206 in LPS induced BV2 cells activation(P<0.01),suggesting that TAN-67 can regulate the phenotypic transformation of microglia under inflammatory stimulation.Immunofluorescence staining and Western blot analysis showed that TAN-67 pretreatment significantly inhibited LPS induced i NOS expression in BV2 cells(P<0.001,P<0.05),which was blocked by Naltrindole.It further indicates that TAN-67 can inhibit the inflammatory response of microglia.Western blot showed that TAN-67 could inhibit the expression of p-p38,p-JNK and p-ERK proteins in LPS induced BV2 cells(P<0.05,P<0.01,P<0.01,respectively),and the inhibition of these phosphorylated proteins could be blocked by Naltrindole.The results suggest that the anti-inflammatory effect of DOR in LPS induced BV2 cells may be related to the regulation of MAPK pathway.In addition,TAN-67 significantly down regulated the expression of cleaved Caspase-3(P<0.05),the key protein of apoptosis pathway,and Naltrindole pretreatment could reverse the inhibition of TAN-67 on apoptosis pathway.TUNEL staining also showed that TAN-67 could significantly reduce the apoptosis of BV2 cells(P<0.001),and Naltrindole could inhibit this effect.These results indicate that DOR activation can promote the transformation of microglia to M2 phenotype,and reduce LPS induced inflammatory response and apoptosis of BV2 microglia.DOR mediated neuroprotection is closely related to M2 phenotype transformation through down regulating MAPK and caspase-3 signaling pathway.(2)In order to better simulate the inflammatory response induced by hypoxic-ischemic brain injury,the experiment of BV2 cells inflammatory response induced by OGD/R was further verified.The results of cell viability test indicated that TAN-67(10μm)pretreatment could significantly increase the cell viability of BV2 cells(P<0.01).This effect can be blocked by Naltrindole.RT-q PCR and ELISA results showed that TAN-67(10μm)could significantly inhibit the expression of IL-1βand IL-6 in BV2 cells induced by OGD/R at m RNA and protein levels,and upregulate the expression of IL-10(all P<0.05),while Naltrindole pretreatment could reverse these effects.The results suggest that TAN-67,an agonist of DOR,can also inhibit the inflammatory response of BV2 cells and promote M2 polarization in the model of OGD/R-induced inflammatory response of BV2 cells.3.The results of in vivo experiments were as follows(1)Compared with MCAO/R group,the body weight of EA group increased significantly from day 1 to day 4(P<0.05).The results showed that compared with the MCAO/R group,the neurological function score and the infarct volume of the ischemic side in the EA treatment group decreased significantly(P<0.05)after 4 days of treatment(P<0.05).There was no significant difference in neurological function score and infarct volume between Naltrindole intervention group and MCAO/R group(P>0.05).The results suggest that EA may protect MCAO/R rats from brain injury through DOR activation pathway.(2)The results of Iba1 immunohistochemistry showed that the number of microglia in MCAO/R group was significantly increased and activated around the infarcted cortex and striatum.The cell body was large and round,and the processes disappeared.There were almost no microglia in the infarct center.Quantitative analysis of Iba1~+cells by integral optical density(IOD)showed that the number of microglia in cortex or striatum around infarcted area in EA group was significantly lower than that in MCAO/R group(all P<0.05).At the same time,a small number of microglia survived in the infarct center.Compared with MCAO/R group,the number of CD16/32~+cells in EA group decreased significantly(P<0.01),while the number of CD206~+cells increased significantly(P<0.01).There was no significant difference in the number of M1/M2 microglia between Naltrindole group and MCAO/R group.RT-q PCR was used to detect the m RNA expression of M1/M2 phenotype related markers in striatal infarction tissues.The results showed that compared with MCAO/R group,the relative m RNA expression of M1 microglia markers IL-6 and TNF-αin EA treatment group was significantly decreased(all P<0.05),and the relative m RNA expression of M2 microglia markers IL-10 and TGF-βwas significantly increased(all P<0.05).However,there was no significant difference in the m RNA of M1/M2 microglia between Naltrindole group and MCAO/R group.In addition,the expression of DOR in M1 and M2 microglia was detected by double immunofluorescence staining.It was found that the activated M1/M2 microglia expressed more DOR in MCAO/R model,but the expression of DOR in M2microglia was more significant than that in M1 microglia.Western blot also showed that the expression of DOR increased significantly in the tissues around the infarct,especially in the EA group,suggesting that the increased expression of DOR may be related to the activation of microglia.This part of the results showed that after 4 days of MCAO/R in rats,a large number of activated microglia gathered around the cerebral infarction focus,mainly M1 type,while EA treatment could promote the phenotypic transformation of microglia and increase M2 type microglia.This effect may be related to the expression of DOR in microglia.EA can promote the expression of DOR in microglia,and high expression of DOR further promotes the polarization of microglia to M2 type.(3)The western blot results showed that compared with MCAO/R group,the expressions of p-p38,p-JNK and p-ERK in EA group were significantly down regulated(P<0.05)in EA group,and the inhibition of these phosphorylated proteins could be blocked by Naltrindole.The results suggested that the regulatory effect of EA on microglia might be related to the regulation of MAPK pathway.At the same time,the expression of cleaved caspase-3 was detected.Compared with MCAO/R group,the expression of cleaved caspase-3 was significantly decreased in EA group(P<0.05).However,there was no significant difference in the expression of MAPK pathway protein and cleaved-caspase-3 protein between Naltrindole group and MCAO/R group.These results suggest that EA may promote the conversion of microglia to M2 phenotype by down regulating MAPK and caspase-3 signaling pathway mediated by DOR.(4)The results of double staining of DOR and microglia markers showed that DOR was also expressed in the microglia near the vascular endothelial cells,and in MCAO/R model,DOR expression in the microglia cells near the vascular endothelial cells was significantly enhanced.The results showed that the vascular density and VEGF protein in electroacupuncture group were significantly increased compared with MCAO/R group(P<0.01),while the vascular density and VEGF protein level of Naltrindole pretreatment group were not significantly changed compared with MCAO/R group.The results suggested that electroacupuncture may promote VEGF secretion by activating DOR expression on microglia near vascular endothelial cells,thus promoting neovascularization in damaged brain tissue.(5)TUNEL and Neu N double staining was used to detect neuronal apoptosis,and NF200(neurofilament 200)+MBP(myelin basic protein)immunofluorescence staining was used to detect axonal injury.The results showed that compared with MCAO/R group,EA treatment group could significantly reduce neuronal apoptosis in cortex and striatum(all P<0.05),and the axonal injury was also significantly improved.GFAP staining was used to detect the activation of astrocytes.It was also found that the response of astrocytes in EA treatment group was significantly less than that in MCAO/R group(P<0.05).However,Naltrindole pretreatment group blocked these neuroprotective effects of EA treatment group.The results suggest that EA therapy may inhibit the inflammatory response of microglia around the infarcted area through DOR activation,and then play a neuroprotective role in inhibiting neuron injury and astrocyte activation.Conclusion:(1)DOR is widely expressed in microglia and up-regulated when microglia are activated.(2)The activation of DOR can inhibit LPS or OGD/R-induced BV2 cell inflammatory effects,inhibit the activation of microglia and the release of inflammatory related factors,inhibit the activation of M1 microglia,promote the polarization of microglia to M2 type,down regulate the phosphorylation of ERK,JNK and p38 induced by LPS,and inhibit caspase-3-mediated apoptosis.It is suggested that the anti-inflammatory and anti-apoptotic effects mediated by DOR activation may be related to the inhibition of MAPK/caspase-3 signaling pathway.(3)EA treatment can significantly reduce the cerebral infarction volume of MCAO/R rat model and improve the neurological deficit score;EA treatment may inhibit the activation of microglia and promote the polarization to M2 microglia through DOR;EA may inhibit the expression of inflammation related proteins;EA may promote the secretion of VEGF,promote vessel angiogenesis in the infarcted area through the regulation of DOR,EA may inhibit MAPK and Caspase-3 through the activation of DOR on microglia so as to inhibit neuronal apoptosis,axon injury and activation of astrocytes. |
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