Pretreatment with electroacupuncture (EA) attenuates cerebral ischemic injury through the endocannabinoid system, although the molecular mechanisms mediate this neuroprotection are unknown. It is well-known that signal transducer and activator of transcription 3 (STAT3) plays an essential role in cell survival and proliferation. Therefore, we investigated whether STAT3 is involved in EA pretreatment-induced neuroprotection via cannabinoid CB1 receptors (CB1R) after transient focal cerebral ischemia in rats. Two hours after EA pretreatment, focal cerebral ischemia was induced by middle cerebral artery occlusion (MACO) for 120 min. The expression of pSTAT3(Ser727), which is necessary for STAT3 activation, was examined in the ipsilateral ischemic penumbra. Infarct volumes and neurological scores were evaluated at 72 h after MACO in the presence or absence of the STAT3 inhibitor peptide (PpYLKTK). Neuronal apoptosis and the Bax/Bcl-2 ratio were also evaluated 24h after reperfusion. Our results showed that EA pretreatment significantly enhanced neuronal expression of pSTAT3(Ser727) in the ischemic penumbra 6h after reperfusion. Moreover, EA pretreatment reduced infarct volume, improved neurological outcome, inhibited neuronal apoptosis and decreased the Bax/Bcl-2 ratio following reperfusion. The beneficial effects of EA were attenuated by PpYLKTK administered 30 min before MACO, and PpYLKTK effectively reversed the increase in pSTAT3(Ser727) expression. Furthermore, CB1R antagonist or CB1R knockdown with siRNA blocked the elevation of pSTAT3(Ser727) expression by EA pretreatment, whereas the two CB1R agonists increased STAT3 activation. In conclusion, EA pretreatment enhances STAT3 activation via CB1R to protect against cerebral ischemia, suggesting that STAT3 activation may be a novel target for stroke intervention.

OBJECTIVES: Acupuncture has been known to be effective in ischemia, and glutamate excitotoxicity is an important factor for the neuronal cell death. We examined the effect of acupuncture on glutamate level in the ischemic stroke model. METHODS: A global ischemia was induced using the method of 11-vessel occlusion in rat. Rats were randomly divided into two groups: the control group (n=5, with 11-vessel occlusion) and the acupuncture group (n=5, with 11-vessel occlusion + acupuncture). The extracellular glutamate level was assessed using an intracerebral biosensor system measuring 256 samples per second. Simultaneously, the cerebral blood flow was measured and the electroencephalogram data were recorded. The time schedule of the experiment was as follows: 10 minutes of baseline measurement, 10 minutes of occlusion and 30 minutes of follow-up monitoring. Acupuncture stimulation was applied to the acupuncture points GB34 (Yangneungcheon) and GB39 (Hyeonjong) during the occlusion period. RESULTS: In the control group, the extracellular glutamate level was changed as 135.19 +/- 23.76 microM (mean +/- standard deviation) from the baseline level. However, this increase was suppressed in the acupuncture group (72.20 +/- 27.15 microM, p<0.01 versus the control group). The changes of cerebral blood flow and electroencephalogram were not significantly different between the groups. DISCUSSION: This result suggests that the effect of acupuncture might be closely associated with modulation of the brain glutamate release in the ischemic condition.

OBJECTIVES: Acupuncture has been known to be effective in ischemia, and glutamate excitotoxicity is an important factor for the neuronal cell death. We examined the effect of acupuncture on glutamate level in the ischemic stroke model. METHODS: A global ischemia was induced using the method of 11-vessel occlusion in rat. Rats were randomly divided into two groups: the control group (n=5, with 11-vessel occlusion) and the acupuncture group (n=5, with 11-vessel occlusion + acupuncture). The extracellular glutamate level was assessed using an intracerebral biosensor system measuring 256 samples per second. Simultaneously, the cerebral blood flow was measured and the electroencephalogram data were recorded. The time schedule of the experiment was as follows: 10 minutes of baseline measurement, 10 minutes of occlusion and 30 minutes of follow-up monitoring. Acupuncture stimulation was applied to the acupuncture points GB34 (Yangneungcheon) and GB39 (Hyeonjong) during the occlusion period. RESULTS: In the control group, the extracellular glutamate level was changed as 135.19 +/- 23.76 microM (mean +/- standard deviation) from the baseline level. However, this increase was suppressed in the acupuncture group (72.20 +/- 27.15 microM, p<0.01 versus the control group). The changes of cerebral blood flow and electroencephalogram were not significantly different between the groups. DISCUSSION: This result suggests that the effect of acupuncture might be closely associated with modulation of the brain glutamate release in the ischemic condition.

In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress.

In the current study, we aimed to investigate whether NADPH oxidase, a major ROS-producing enzyme, was involved in the antioxidant effect of acupuncture on cognitive impairment after cerebral ischaemia. The cognitive function, infract size, neuron cell loss, level of superoxide anion and expression of NADPH oxidase subunit in hippocampus of two-vessel occlusion (2VO) rats were determined after 2-week acupuncture. Furthermore, the cognitive function and production of O2(-) were determined in the presence and absence of NADPH oxidase agonist (TBCA) and antagonist (Apocynin). The effect of acupuncture on cognitive function after cerebral ischaemia in gp91phox-KO mice was evaluated by Morris water maze. Acupuncture reduced infarct size, attenuated overproduction of O2(-), and reversed consequential cognitive impairment and neuron cell loss in 2VO rats. The elevations of gp91phox and p47phox after 2VO were significantly decreased after acupuncture treatment. However, no differences of gp91phox mRNA were found among any experimental groups. Furthermore, these beneficial effects were reversed by TBCA, whereas apocynin mimicked the effect of acupuncture by improving cognitive function and decreasing O2(-) generation. Acupuncture failed to improve the memory impairment in gp91phox KO mice. Full function of the NADPH oxidase enzyme plays an important role in neuroprotective effects against cognitive impairment via inhibition of NAPDH oxidase-mediated oxidative stress.

Turmeric has been in use since ancient times as a condiment and due to its medicinal properties. Curcumin, the yellow colouring principle in turmeric, is polyphenolic and major active constituent. Besides anti-inflammatory, thrombolytic and anticarcinogenic activities, curcumin also possesses strong antioxidant property. In view of the novel combination of properties, neuroprotective efficacy of curcumin was studied in rat middle cerebral artery occlusion (MCAO) model. Rats were subjected to 2 h of focal ischemia followed by 72 h of reperfusion. They were pre-treated with curcumin (100 mg/kg, po) for 5 days prior to MCAO and for another 3 days after MCAO. The parameters studied were behavioural, biochemical and histological. Treatment with curcumin could significantly improve neurobehavioral performance compared to untreated ischemic rats as judged by its effect on rota-rod performance and grid walking. A significant inhibition in lipid peroxidation and an increase in superoxide dismutase (SOD) activity in corpus striatum and cerebral cortex was observed following treatment with curcumin in MCAO rats as compared to MCAO group. Intracellular calcium levels were decreased following treatment with curcumin in MCAO rats. Histologically, a reduction in the infarct area from 33% to 24% was observed in MCAO rats treated with curcumin. The study demonstrates the protective efficacy of curcumin in rat MCAO model.

OBJECTIVE:To observe the effect of acupuncture (Acupunct) on cerebral infarction volume and expression of poly ADP ribose polymerase 1 (PARP1), apoptosis-inducing factor (AIF) and endonuclease G (Endo-G) in the cerebral cortex tissue at different time-points after cerebral ischemia (CI) in acute cerebral infarction rats, so as to explore its underlying mechanisms in prolonging time window of thrombolysis.

METHODS:Forty-eight SD rats were randomly divided into sham operation, model, intravenous thrombolysis (IVT)-4.5 h, IVT-6 h, IVT-9 h, Acupunct＋IVT-4.5 h, Acupunct ＋IVT-6 h, Acupunct＋IVT-9 h groups (＝6 in each group). The CI model was established by using modified autologous thromboembolism via the right common carotid artery. Two hours after modeling, rats of the Acupunct groups received Acupunct stimulation of"Shuigou"(GV26) and bilateral"Neiguan"(PC6) for 30 min. Thrombolysis was conducted by injection of recombinant human tissue-type plasminogen activator (rt-PA, 10 mg/kg) via caudal vein. The neurological deficit was assessed with reference to Bederson's methods. 2，3，5-triphenyltetrazolium chloride (TTC) staining was used to assess the cerebral infarction volume, and the expression of cerebral PARP1, AIF and Endo-G proteins detected by Western blot.

RESULTS:Compared with the sham operation group, the neurological score and percentage of cerebral infarction volume, expression levels of PARP1, AIF and Endo-G proteins were significantly increased in the model group (<0.01，<0.05). After the intervention, modeling-induced increase of the aforementioned indexes was reversed in the IVT-4.5 h, Acupunct＋IVT-4.5 h and Acupunct＋IVT-6 h groups (<0.05), except PARP1 expression of the Acupunct＋IVT 6 h group (>0.05). The levels of neurological score, percentage of cerebral infarction volume, and AIF expression were significantly lower in both the Acupunct＋IVT 4.5 h and Acupunct＋IVT-6 h groups than in the simple IVT-4.5 h and simple IVT-6 h groups, respectively (<0.05), and the expression levels of PARP1 and Endo-G proteins were obviously lower in the Acupunct＋IVT-4.5 h group than in the IVT-4.5 h group (<0.05). Endo-G proteins were obviously lower in the Acupunct＋IVT-9 h group than in the IVT-9 h group (<0.05).

CONCLUSION:Acupuncture may improve neurological function, reduce cerebral infarction volume and prolong the time window of thrombolysis in CI rats, which may be associated with its effect in suppressing AIF/PARP1/ Endo-G signaling.

OBJECTIVE: To observe the effect of electroacupuncture (EA) and acupuncture (A) on the proliferation of stem cells in the subependymal zone (SPZ) of the lateral ventricle and the frontal lobe cortex in hyperlipemia(HL) combined with cerebral ischemia (CI) rats. METHODS: A total of 72 male SD rats were randomized into control, HL, HL+EA, CI, CI+A, HL+CI, HL+CI+EA I and HL+CI+EA II groups (n=9 /group). HL model was established by feeding the animals with high fat forage for 6 weeks and CI model was established by FeCl3-induced occlusion of the unilateral middle cerebral artery. EA was applied to "Sanyinjiao" (SP 6) and "Fenglong" (ST 40) once daily for 17 days for HL+ EA group; and acupuncture to "Baihui" (GV 20) and "Shuigou" (GV 26) once daily for 7 days for CI + A group. For HL+CI+EA I group, EA was applied to SP 6 + ST 40 first before CI, once daily for 10 days, followed by EA of SP 6+ST 40 and acupuncture of GV20+GV26 for 7 days after CI. For HL+CI+EA II group, no treatment was given before CI, then, acupuncture of GV 20 + GV 26 and EA of SP 6 + ST 40 were given once daily for 7 days after CI. The immunoactivity of Nestin and proliferation cell nuclear antigen (PONA) of SPZ was detected by immunohistochemistry. RESULTS: In comparison with normal control group, the numbers of both Nestin and PCNA immunoreaction (IR) positive cells in the dorsolateral extension and the wall of the lateral ventricle of the brain increased significantly in CI and HL+CI groups (P<0.01). Compared with CI group, the numbers of Nestin and PCNA IR positive cells in the dorsolateral extension and the wall of the lateral ventricle in CI + A group increased considerably (P<0.01). In comparison with HL+CI group, both Nestin and PCNA IR positive cell numbers in the dorsolateral extension and the wall of the lateral ventricle of the brain in HL+CI+EA I and HL+CI+EA II groups increased significantly (P<0.01), and the effect of HL+CI+EA I group was markedly superior to that of HL+CI+EA II group in upregulating the numbers of Nestin and PCNA IR positive cells in the aforementioned regions of the lateral ventricle (P<0.01). No significant differences were found between HL and control groups, and between HL+EA and HL groups in the numbers of Nestin and PCNA IR positive cells in the dorsolateral extension and the wall of the lateral ventricle of the brain (P>0.05). CONCLUSION: EA can upregulate Nestin and PCNA expression of the dorsolateral extension and the wall of the lateral ventricle of the brain on the ischemic side in rats with CI, and with HL+CI, which may contribute to its effects in promoting the proliferation and migration of neural stem cells in the brain.

In the present study, we evaluated the curative effect of dipeptidyl peptidase-IV (DPP-IV) inhibitor alogliptin combined with motor imagery under hyperbaric oxygen in diabetic nephropathy (DN) with silent cerebral infarction (SCI). Two-hundred newly diagnosed DN patients with and without SCI were included. The SCI patients were divided into two treatment groups: Alogliptin (A group, n=50) and alogliptin combined with motor imagery under hyperbaric oxygen (B group, n=50). The degrees of neurocognitive dysfunction were evaluated at baseline and after 6 months of treatment. Thromboelastograms (TEGs) mapping were conducted. Serum glycoprotein VI (GPVI) mRNA expression and urine 11-DH-TXB2 levels were determined. Compared to group A patients, the severity of neurofunctional defects, GPVI mRNA expression and 11-DH-TXB2 levels were significantly lower in group B (P<0.05), while comprehensive, MoCA scores were higher in group B. The MoCA subscores of visuospatial/executive function, attention and concentration were significantly higher compared to group A (P<0.05). The sub-scores of computation, abstract thinking, language competence, memory and orientation were also higher in group B but the differences were not significant (P>0.05). TEG indexes were improved in both groups after treatment as manifested by increased R and K values, but there was significant improvement in group B. Intra-group comparisons revealed a time-dependent effect of treatment. In conclusion, the treatment of alogliptin combined with motor imagery under hyperbaric oxygen can better promote thrombolysis absorption, restore brain damage and improve neurocognitive function in DN with silent cerebral infarction.

Accumulating evidence demonstrates that acupuncture and electroacupuncture (EA) can exert a neuroprotective role for cerebral ischemia, but their precise mechanism remains largely unknown. Therefore, in this study, the effects of EA stimulation on cerebral ischemia reperfusion and its neuroprotective mechanisms were investigated. A rat model of middle cerebral artery occlusion (MCAO) was developed, and EA stimulation (2Hz, 1mA) at Baihui and Siguan acupoints was applied 30min after MCAO and then once daily for 7 consecutive days. The results indicated that EA stimulation significantly reduced the cerebral infarct area and neurological deficit scores, decreased the number of apoptotic cells, up-regulated Bcl-2 protein expression, and down-regulated Bax protein expression. EA stimulation resulted in a significant increase of proliferative cells in the cerebral tissues. Additionally, EA stimulation significantly down-regulated the expression levels of matrix metalloproteinase -9 (MMP-9) mRNA and protein, and simultaneously up-regulated the expression levels of tissue inhibitor of metalloproteinases-1 (TIMP-1) mRNA and protein, which resulted in an imbalance of MMP-9/TIMP-1expression, although it did not significantly change MMP-2 and TIMP-2 expression. These findings indicate that EA stimulation at Baihui and Siguan acupoints exerts a neuroprotective role against cerebral ischemia-reperfusion injury, which is probably associated with the inhibition of apoptosis and altering the balance of MMP-9/TIMP-1 expression.

Background. The therapeutic mechanisms of cerebral ischemia treatment by acupuncture are yet not well addressed. Objective. We investigated the effects of electroacupuncture (EA) at GV26 observing the expression of autophagy-related proteins Beclin-1 and LC3B and proportion of apoptotic cells and Bcl-2 positive cells in MCAO/R model rats. Methods. Sprague-Dawley (SD) male rats were randomly assigned to 7 groups: model groups (M6h, M24h, and M72h), EA treatment groups (T6h, T24h, and T72h), and sham operation group (S). Neurological deficit and cerebral infarction volume were measured to assess the improvement effect, while the expression of Beclin-1 and LC3B and proportion of Tunel-positive and Bcl-2 positive cells were examined to explore EA effect on autophagy and apoptosis. Results. EA significantly decreased neurological deficit scores and the volume of cerebral infarction. Beclin-1 was significantly decreased in T24h, while LC3B-II/LC3B-I ratio markedly reduced in 6th hour. EA groups markedly reduced the number of Tunel positive cells, especially in T24h. Meanwhile, the number of Bcl-2 positive cells obviously increased after EA treatment, especially in T6h and T24h. Conclusions. The alleviation of inadequate autophagy and apoptosis may be a key mechanism involved in the reflex regulation of EA at GV26 to treat cerebral ischemia.

Inflammation after stroke is the main cause of cerebral ischemia/reperfusion injury. Cascading events after injury can lead to cell death. Heat shock protein 70 and other endogenous injury-signaling molecules are released by damaged cells, which can lead to systemic stress reactions. Protecting the brain through repair begins with the stress-injury-repair signaling chain. This study aimed to verify whether acupuncture acts through this chain to facilitate effective treatment of ischemic stroke. Rat models of cerebral ischemia/reperfusion injury were established by Zea Longa's method, and injury sites were identified by assessing neurological function, 2,3,5-triphenyltetrazolium chloride staining, and hematoxylin-eosin staining. Electroacupuncture at acupoints Baihui (DU20) and Zusanli (ST36) was performed in the model rats with dilatational waves, delivered for 20 minutes a day at 2-100 Hz and an amplitude of 2 mA. We analyzed the blood serum from the rats and found that inflammatory cytokines affected the levels of adrenotrophin and heat shock protein 70, each of which followed a similar bimodal curve. Specifically, electroacupuncture lowered the peak levels of adrenocorticotrophic hormone and heat shock protein 70. Thus, electroacupuncture was able to inhibit excessive stress, reduce inflammation, and promote the repair of neurons, which facilitated healing of ischemic stroke.

BACKGROUND: Cerebral ischemia increases neurogenesis in the subventricular zone (SVZ) and in the subgranular zone (SGZ) of the dentate gyrus, and this might be modulated by an enriched environment including voluntary physical activity. We examined whether enforced physical training (EPT) influences neurogenesis in the SVZ and SGZ after cerebral ischemia.

METHODS: Adult male Sprague-Dawley rats were subjected to focal cerebral ischemia for 2 h, and divided into an EPT and a non-EPT group. All rats in the EPT group were trained using a rota-rod for 14 days. 5-bromo-2'-deoxyuridine (BrdU) was injected to determine levels of cell proliferation. Functional recovery was assessed using a set of behavioral test batteries. Extents of endogenous neurogenesis in the SVZ and SGZ were quantified by immunofluorescence staining. Although final infarction volumes were not significantly different in the groups, functional recovery was better in the EPT group at 10 and 17 days after ischemia. In the SVZ, BrdU labeling and double labeling of BrdU/Dcx and of BrdU/NeuN were not significantly different in the two groups. However, in the SGZ, EPT significantly increased the number of BrdU-positive cell numbers (EPT vs. non-EPT: 159.1+/-19.9 vs. 101.8+/-7.8, p=0.04), and the number of BrdU/Dcx double-labeled cells (130.6+/-16.9 vs. 73.6+/-7.2, p=0.01).

CONCLUSIONS: The results obtained indicate that EPT promotes neurogenesis in the SGZ of the dentate gyrus after ischemia, but not in the SVZ. The biochemical mechanism that determines the differential effects of EPT remains to be clarified.

Previous studies have demonstrated that autophagy induced by caloric restriction (CR) is neuroprotective against cerebral ischemia. However, it has not been determined whether intermittent fasting (IF), a variation of CR, can exert autophagy-related neuroprotection against cerebral ischemia. Therefore, the neuroprotective effect of IF was evaluated over the course of two weeks in a rat model of focal cerebral ischemia, which was induced by middle cerebral artery occlusion and reperfusion (MCAO/R). Specifically, the role of autophagy modulation as a potential underlying mechanism for this phenomenon was investigated. It was demonstrated that IF reduced infarct volume and brain edema, improved neurobehavioral deficits, and rescued neuronal loss after MCAO/R. Furthermore, neuronal apoptosis was decreased by IF in the rat cortex. An increase in the number of autophagosomes (APs) was demonstrated in the cortices of IF-treated rats, using immunofluorescence staining and transmission electron microscopy. Using immunoblots, an IF-induced increase was detected in microtubule-associated protein 1 light chain 3 (LC3)-II, Rab7, and cathepsin D protein levels, which corroborated previous morphological studies. Notably, IF reduced the accumulation of APs and p62, demonstrating that IF attenuated the MCAO/R-induced disturbance of autophagic flux in neurons. The findings of the present study suggest that IF-induced neuroprotection in focal cerebral ischemia is due, at least in part, to the minimization of autophagic flux disturbance and inhibition of apoptosis.

Hypoxic-ischaemic encephalopathy remains a global health burden. Despite medical advances and treatment with therapeutic hypothermia, over 50% of cooled infants are not protected and still develop lifelong neurodisabilities, including cerebral palsy. Furthermore, hypothermia is not used in preterm cases or low resource settings. Alternatives or adjunct therapies are urgently needed. Exendin-4 is a drug used to treat type 2 diabetes mellitus that has also demonstrated neuroprotective properties, and is currently being tested in clinical trials for Alzheimer's and Parkinson's diseases. Therefore, we hypothesized a neuroprotective effect for exendin-4 in neonatal neurodisorders, particularly in the treatment of neonatal hypoxic-ischaemic encephalopathy. Initially, we confirmed that the glucagon like peptide 1 receptor (GLP1R) was expressed in the human neonatal brain and in murine neurons at postnatal Day 7 (human equivalent late preterm) and postnatal Day 10 (term). Using a well characterized mouse model of neonatal hypoxic-ischaemic brain injury, we investigated the potential neuroprotective effect of exendin-4 in both postnatal Day 7 and 10 mice. An optimal exendin-4 treatment dosing regimen was identified, where four high doses (0.5µg/g) starting at 0 h, then at 12 h, 24 h and 36 h after postnatal Day 7 hypoxic-ischaemic insult resulted in significant brain neuroprotection. Furthermore, neuroprotection was sustained even when treatment using exendin-4 was delayed by 2 h post hypoxic-ischaemic brain injury. This protective effect was observed in various histopathological markers: tissue infarction, cell death, astrogliosis, microglial and endothelial activation. Blood glucose levels were not altered by high dose exendin-4 administration when compared to controls. Exendin-4 administration did not result in adverse organ histopathology (haematoxylin and eosin) or inflammation (CD68). Despite initial reduced weight gain, animals restored weight gain following end of treatment. Overall high dose exendin-4 administration was well tolerated. To mimic the clinical scenario, postnatal Day 10 mice underwent exendin-4 and therapeutic hypothermia treatment, either alone or in combination, and brain tissue loss was assessed after 1 week. Exendin-4 treatment resulted in significant neuroprotection alone, and enhanced the cerebroprotective effect of therapeutic hypothermia. In summary, the safety and tolerance of high doseexendin-4 administrations, combined with its neuroprotective effect alone or in conjunction with clinically relevant hypothermia make the repurposing of exendin-4 for the treatment of neonatal hypoxic-ischaemic encephalopathy particularly promising.

Electroacupuncture (EA) is an important treatment method in Chinese traditional medicine. The main purpose of the investigation was to explore whether EA could improve cerebral ischemia/reperfusion (CI/R) via neurons' autophagy. 4-vessel occlusion (4-VO) was applied to establish cerebral ischemia model and reperfusion 12h, 24h and 48h respectively were conduct 3h later. TTC staining was applied to assess cerebral infarction area and the concentrations of IL-6, TNF-α, IL-1β, SOD and MDA were detected by commercial kits. The expressions of autophagy-related protein LC3, mammalian target of rapamycin (mTOR) and Beclin1 were measured by Western blot. EA treatment was given at"BaiHui","MingMen"and"ZuSanLiXue". The obtained results from TTC showed that the severity of cerebral ischemia-reperfusion was improved with EA treatment. Oxidative and inflammatory damages were also alleviated with EA intervention. Meanwhile, western blot analysis revealed the decreased levels of LC3 and Beclin1 in EA rats, as well as the elevated level of mTOR. Besides, our previous study found that EA can enhance mitochondrial respiratory chain enzyme activity, reduce mitochondrial damage and inhibit neuronal apoptosis further. Thus, it is assumed that 4-VO-induced cerebral ischemia/reperfusion might be alleviated by EA through inhibiting neurons' excessive autophagy in reperfusion period.

Garlic (Allium sativum) is used for culinary and medicinal purposes in diverse cultures worldwide. When fresh garlic is soaked in aqueous ethanol under ambient environment over 4 months or longer, the majority of irritating taste and odor is eliminated and the antioxidant profile in the resulting aged garlic extract (AGE) changes significantly. Recently, AGE and its components have been demonstrated to exert neuroprotective effects in neurodegenerative diseases, includingAlzheimer's disease, Parkinson's disease, Huntington's disease, and cerebral ischemia. Because of its health supporting potential, there is increasing interest in understanding the antioxidant and anti-inflammatory properties and the underlying mechanisms for its protective effects in heath and disease. There is evidence for AGE to exert its action on distinct signaling pathways associated with oxidative stress and neuroinflammation, although the primary molecular mechanisms remain unclear. By utilizing quantitative proteomic approaches, we demonstrated that AGE and two of its major ingredients, S-allyl-L-cysteine and N (α)-(1-deoxy-D-fructos-1-yl)-L-arginine, can attenuate neuroinflammatory responses in microglial cells through modulation of Nrf2-mediated signaling as well as other oxidative stress-related pathways. These experimental data provide information for the molecular targetsof AGE and its components to mitigate neurodegeneration and neuroinflammation and show a promising potential of these compounds as dietary supplements for health maintenance.

BACKGROUND:Zinc finger protein A20 (tumor necrosis factor alpha-induced protein 3) functions as a potent negative feedback inhibitor of the nuclear factor-kB (NF-kB) signaling. It exerts these effects by interrupting the activation of IkB kinase beta (IKKβ), the most critical kinase in upstream of NF-kB, and thereby controlling inflammatory homeostasis. We reported previously that electroacupuncture (EA) could effectively suppress IKKβ activation. However, the mechanism underlying these effects was unclear. Therefore, the current study further explored the effects of EA on A20 expression in rat brain and investigated the possible mechanism of A20 in anti-neuroinflammation mediated by EA using transient middle cerebral artery occlusion (MCAO) rats.

METHODS:Rats were treated with EA at the"Baihui (GV20),""Hegu (L14),"and"Taichong (Liv3)"acupoints once a day starting 2 h after focal cerebral ischemia. The spatiotemporal expression of A20, neurobehavioral scores, infarction volumes, cytokine levels, glial cell activation, and the NF-kB signaling were assessed at the indicated time points. A20 gene interference (overexpression and silencing) was used to investigate the role of A20 in mediating the neuroprotective effects of EA and in regulating the interaction between neuronal and glial cells by suppressing neuronal NF-kB signaling during cerebral ischemia/reperfusion-induced neuroinflammation.

RESULTS:EA treatment increased A20 expression with an earlier peak and longer lasting upregulation. The upregulated A20 protein was predominantly located in neurons in the cortical zone of the ischemia/reperfusion. Furthermore, neuronal A20 cell counts were positively correlated with neurobehavioral scores but negatively correlated with infarct volume, the accumulation of pro-inflammatory cytokines, and glial cell activation. Moreover, the effects of EA on improving the neurological outcome and suppressing neuroinflammation in the brain were reversed by A20 silencing. Finally, A20 silencing also suppressed the ability of EA to inhibit neuronal NF-kB signaling pathway.

CONCLUSIONS:Ischemia/reperfusion cortical neurons in MCAO rats are the main cell types that express A20, and there is a correlation between A20 expression and the suppression of neuroinflammation and the resulting neuroprotective effects. EA upregulated neuronal A20 expression, which played an essential role in the anti-inflammatory effects of EA by suppressing the neuronal NF-kB signaling pathway in the brains of MCAO rats.