| With great change in populational structure for growing problem of aging, theincidence of stroke in China is the first one in the world. Stroke, which ischemia strokeaccounts for80%, has an annual growth rate of8.7%. Stroke has become the first cause ofdeath and the primary causes of disability in adults, its enormous harm seriously declinetheir quality of life. The family and society have been suffered great spiritual andeconomic burden. Classic thrombolytic therapy, namely recombinant tissue-type tissueplasminogen activator (rtPA), because of its short therapeutic time window (after a stroke3-4.5h) and increasing the risk of intracranial hemorrhage during treatment, was limited inclinical application. Only about5%of stroke patients can get timely and effectivethrombolytic therapy. Additionally, antiplatelet therapy of the stroke patient withcardiovascular disease and recurrent stroke patient should be administrated at strictly accurate dosage to prevent more serious complications. Thus, it requires more scientificstrategy to find more effective measures for the prevention and treatment of these strokepatients.As researching on the pathogenesis of stroke further, we recognized thatexcitotoxicity, depolarization surrounding infarct, neuroinflammatory and programmedcell death mechanism lead ischemia injury. All these ischemia-induced damagemechanism overlap closely. Such cascade reflection cause irreversible neuronal damagewithin minutes after cerebral ischemia. The response of neuroinflammatory in centralnervous system involved in various pathological stage after the blood supply disruptionscaused by the acute cerebrovascular events, not only lead to brain damage but thesubsequent process of neuroregeneration. Thus, stroke induced nerve inflammation is animportant factor in ischemia-reperfusion injury and neurological recovery, the study ofcerebral ischemic injury induced inflammatory mechanism of neuroprotection will be theemphasis in future research. Previous studies in our laboratory demonstrated thatelectacupuncture pretreatment increased α7nAChR expression in rat neurons afterreperfusion and significantly reduced plasma HMGB1levels, alleviated cerebralreperfusion injury, which may act on proinflammatory cytokines and inhibit inflammationin the nervous system. However, the anti-inflammatory mechanism of acupunctureresearch in the nervous system is far from being revealed. In addition, we confirmed thatelectacupuncture precondition take a role of upregulation of CB1R receptor surroundingcerebral ischemia tissue and activate endogenous protective mechanism. The latest studyin vitro confirmed that cannabinoids involved neuroinflammation, which have significantanti-inflammatory effect. Mechanism of the endogenous cannabinoid system ininflammatory response is not entirely clear, the crosstalk between inflammatory mediatorsand inflammatory factors are complex and multi-directional.Transforming growth factor beta (TGF-β) proteins are multifunctional cytokineswhose neural functions are increasingly recognized. TGF-β plays an pleiotropic action inthe central nervous system and the development of certain peripheral organs and formation,have neuroprotective activities too. TGF-β may affect functional status of ischemia-activated microglia, which playing a protective role in the brain. A variety ofbrain injury and disease induce the expression of TGF-β, while exhibiting itsneuroprotective effect. The interaction between endocannabinoid system and the TGF-βacting on microglia produce the neuroprotective effect. Thus, this study focuses on thatTGF-β may be involved in EA-indued neuroprotection. On the other hand, invesgatingrelationship between the endogenous cannabinoid system and the TGF-β could providemore adequate theory for the clinical treatment of stroke.Experiment IEffects of EA pretreatment on TGF-β1expression after cerebral ischemia inratsObjective To investigate the effects of electroacupuncture precondition onexpression of TGF-β1in cerebral penumbra after ischemia-reperfusion in rats.Method1. Neuroprotection of Electrical stimulation preconditioningSame as previous researches.2. After cerebral ischemia-reperfusion injury in the penumbra of the expression ofTGF-β1and electricity for the expression of TGF-β1According to ischemia-reperfusion injury time points, SD rats were randomly dividedinto eight groups (n=6): sham operation group (Sham), after reperfusion2h,4h,6h,12h,24h,48h and72h, at each time point the animals were sacrificed, and the penumbra weredetected the expression of TGF-β1at different points in time by Western Blot.Grouping male SD rats same to1, the animals were sacrificed24h after reperfusion(n=6), Western Blot detected expression of TGF-β1in penumbra each group.3. Electrical stimulation impact on neuronal expression of TGF-β1in penumbraMale SD rats were randomly divided into3groups (n=4), the animals were killed24hafter ischemia-reperfusion, and perfused with4%paraformaldehyde (PFA). We performedImmunofluorescence staining of frozen tissue sections. While the marker of NeuN TGF-β1and neurons were labeled by immunofluorescence double staining.ResultAfter reperfusion72h, EA group infarct volume compared with I/R group wassignificantly smaller (P<0.05), and neurobehavioral score was higher than I/R group (P<0.05). Western blot showed that occurs after reperfusion6h, ischemia-reperfusion groupexpression of TGF-β1significantly increased compared with Sham group (P<0.05), theexpression of which24h is higher than others.24h after ischemia-reperfusion, penumbratissue of EA group shown that TGF-β1levels were significantly higher compared with I/Rgroup (P<0.05). Using of TGF-β1and neuronal marker NeuN double labeling, the resultsshow that TGF-β1expression in neuronal cells increased significantly at24h after cerebralischemia-reperfusion injury by EA pretreatment.ConclusionTGF-β1expression increased in brain tissue surrounding ischemia field, EApretreatment further upregulated it and play a protective role in the brain, neurons TGF-β1expression may be upregulated by EA pretreatment24hours after ischemia.Experiment IIInvolvement TGF-β1in EA pretreatment-induced neuroprotection in stroke ratsObjective To investigate downregulation of the expression of TGF-β1effect on theprotective effect of electroacupuncture and exogenous TGF-β1effects on cerebralischemia-reperfusion injuryMethodSD rats were randomly divided into six groups (n=8): Sham group, I/R group, EAgroup and EA+siRNA interference group, EA+MAB240group, I/R+recombinant TGF-β1group. Before ischemia30min, drugs were performed intracerebroventricular injection(ICV), the animals experienced after ischemia-reperfusion72h2h sacrificed neurologicalbehavior scores and infarct volume was measured. Particularly, ICV of TGF-β1-siRNAwas administrated48h before ischemia. ResultEA+siRNA and EA+MAB240significant increase in infarct size compared withthe EA group (P<0.05), while the I/R+rhTGF-β1significantly reduced infarct volumecompared with the I/R group (P<0.05), produced a EA pretreatment-like neuroprotectiveeffects. Compared with the EA group, behavior score of EA+siRNA group andEA+MB240were significantly declined(P<0.05), and administration of recombinantexogenous TGF-β1can significantly improve neurobehavioral score at72h after cerebralischemia and reperfusion in rats compared the I/R group (P<0.05).ConclusionProtective effect of EA pretreatment can be reversed by downregulation of TGF-β1,and exogenous TGF-β1can simulate EA induced cerebral ischemic toleranceExperiment IIITGF-β1was involved in anti-apoptotic effects by EA pretreatment in stroke ratsObjective To observe the TGF-β1role on EA pretreatment induced anti-apoptosisrules after cerebral ischemia and apoptosis-related protein expression after reperfusion.Method1. TGF-β1impacting on the number of nerve cell apoptosis after ischemia andreperfusion.SD rats, according to a random number table, were randomly divided into5groups(n=4): Sham group, I/R group, EA group, EA+siRNA group and I/R+rhTGF-β1group..Before ischemia48h, conducted TGF-β1-siRNA (25nmol) through intracerebroventricularinjection; before ischemia0.5h, conducted rhTGF-β1(0.8ug/Kg) throughintracerebroventricular injection, the volume is10ul.The animals were sacrificed24h afterischemia and reperfusion. Perfusion with4%paraformaldehyde, paraffin sections weremade for TUNEL staining to observed the number of apoptotic neurons each group.2Effect of TGF-β1protein expression of apoptosis-relatedMale SD rats were randomly divided into5groups (n=6): Sham group, I/R group,EA group, EA+siRNA group and I/R+rhTGF-β1group.24h after reperfusion, penumbra were detected by western blot for Bcl-2and Bax protein quantitative analysis.ResultEA group, the number of TUNEL(+) cells compared with I/R group significantlydecreased (P<0.01). TUNEL(+) cells significantly increased (P<0.01) in EA+siRNAgroup compared with the EA group. I/R group compared with Sham group Bax/Bcl-2ratioincreased (P<0.05). EA group Bax/Bcl-2ratio was significantly less than I/R group(P<0.05). EA pretreatment group given TGF-β1-siRNA intracerebroventricular injectionscan significantly decrease the expression of Bcl-2and increase the expression of Bax toincrease the ratio between them (P<0.05). The I/R group received intracerebroventricularinjection of recombinant TGF-β1can significantly reduce the ratio (P<0.05).ConclusionReduced TGF-β1can increase neuronal apoptosis after ischemia-reperfusion injury inrat brain, which antagonize the protective effect of EA pretreatment; and thereorganization of TGF-β1reducing ischemic injury in the nervous system result in theproposed EA protective effects of cerebral ischemia, indicating that TGF-β1plays animportant role in EA neuroprotection.Experiment IVEA pretreatment regulated expression of TGF-β1in cerebral ischemiathrough CB1R in rats.Objective To investigate the effects of cannabinoids on the expression of TGF-β1inEA pretreatment cerebral ischemia-reperfusion injuryMethod1. CB1receptor antagonist AM251, SR141716A influence the expression of TGF-β1after EA pretreatmentSD rats, according to a random number table randomly divided into four groups(n=6): I/R group, EA+I/R group, EA+AM251(SR141716A) group and EA+DMSO group.EA pretreatment2h before cerebral ischemia, and0.5h before making models conductedintraperitoneal injection of AM251(SR141716A) with a dose of1mg/kg (1.5mg/kg).24hafter reperfusion injury in the cortex, penumbra were detected by Western Blot for determination of TGF-β1expression.2. The impact of interference RNA of CB1receptors for TGF-β1expression after EApretreatmentSD rats, with the former group (n=6): I/R group, EA+I/R group, EA+CB1R-siRNAgroup and EA+Vehicle group. EA pretreatment producting2h before cerebral ischemiamodel,48h prior to making models intracerebroventricular injection of CB1R-siRNA(10μg/10μl) were performed, rats were sacrificed at24h after reperfusion injury forcortical penumbra. Western Blot determinated TGF-β1levels in penumbra.3. CB1receptor agonist ACEA expression of TGF-β1after EA pretreatmentSD rats were randomly divided into3groups (n=6): I/R group, I/R+ACEA group andI/R+Vehicle group.0.5h before Making models injected ACEA intraperitoneally, with adose of2.5mg/kg,24h after reperfusion injury rats were sacrificed for cortical penumbra.Western Blot determinated TGF-β1levels in penumbra.Result24h after reperfusion expression of TGF-β1in penumbra of EA+AM251,EA+SR141716A and EA+CB1-siRNA groups significantly lower than EA group andEA+DMSO(or Vehicle) groups (P<0.05); I/R+ACEA group’s TGF-β1relative contentwas significantly increased than I/R group and I/R+vehicle group (P<0.05).ConclusionEA pretreatment while giving CB1receptor antagonist AM251, SR141716A andinterfering RNA of CB1receptor, which inhibiting the biological activity of CB1receptors, found that two types of drugs can reverse the EA pretreatment inducedupregulation of TGF-β1expression; the contrast phenomenon was that given experimentCB1receptor agonist ACEA, it is able to simulate the increase of TGF-β1expressionsimilarly with EA pretreatment, suggesting that EA pretreatment may regulate TGF-β1viaCB1receptor, thus play a protective role in the brain.Summary1. EA pretreatment can improve neurological behavior scores and infarct volume in cerebral ischemia-reperfusion injury, increased expression of TGF-β1in ischemicpenumbra.2. Inhibiting TGF-β1expression can reverse neuroprotection EA pretreatment.3. Downregulation of TGF-β1can increase apoptotic of nerve cells in penumbra aftercerebral ischemia-reperfusion.4. EA pretreatment regulate TGF-β1by CB1receptor upstream. |
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