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In a previous study by our group, we demonstrated that electroacupuncture (EA) activates the class I phosphoinositide 3-kinase (PI3K)/Akt signaling pathway. There is considerable evidence that the downstream mammalian target of rapamycin complex 1 (mTORC1) plays an important role in autophagy following ischemic stroke. The aim of the present study was to determine whether EA exerts a neuroprotective effect through mTORC1-mediated autophagy following ischemia/reperfusion injury. Our results revealed that EA at the LI11 and ST36 acupoints attenuated motor dysfunction, improved neurological deficit outcomes and decreased the infarct volumes. The number of autophagosomes, autolysosomes and lysosomes was decreased following treatment with EA. Simultaneously, the levels of the autophagosome membrane maker, microtubule-associated protein 1 light chain 3 beta (LC3B)II/I, Unc-51-like kinase 1 (ULK1), autophagy related gene 13 Atg13) and Beclin1 (ser14) were decreased, whereas mTORC1 expression was increased in the peri-infarct cortex. These results suggest that EA protects against ischemic stroke through the inhibition of autophagosome formation and autophagy, which is mediated through the mTORC1-ULK complex-Beclin1 pathway.

Electroacupuncture (EA) has been widely used to treat cognitive impairment following cerebral ischemia. However, the functional mechanisms of EA have not been fully elucidated. The aim of the present study was to investigate whether EA at the GV 20 and DU 24 acupoints can improve the learning and memory ability via alteration of the neurochemical metabolism in the hippocampus (HPC) and prefrontal cortex (PFC) of rats with ischemia and reperfusion (I/R) injury. Sprague-Dawley male rats were randomly divided into three groups, namely the sham group (n=12), the middle cerebral artery occlusion (MCAO) group (n=12) and the EA treatment (MCAO + EA) group (n=12). MCAO was performed to establish the left focal cerebral I/R injury model, and the GV 20 and DU 24 acupoints were then stimulated with EA for 30 min per time, once daily, for 7 consecutive days. The Morris water maze (MWM) test was used to assess learning and memory ability. T2-weighted imaging was used to assess the cerebral infarct volume. Magnetic resonance spectroscopy was used to assess neurochemical metabolism of HPC and PFC. The neurological scores of the MCAO + EA group were significantly reduced compared with those of the MCAO group 7 days after EA treatment (P0.05). The ratios of NAA/Cr, Cho/Cr and Glu/Cr of left-to-right PFC were elevated (P

Electro-acupuncture (EA) is a novel therapy based on combining traditional acupuncture with modern electrotherapy, and it is currently being investigated as a treatment for ischemic stroke. In the present study, we aimed to investigate the mechanisms through which EA regulates the proliferation of neural progenitor cells (NPCs) in the cortical peri‑infarct area after stroke. The neuroprotective effects of EA on ischemic rats were evaluated by determining the neurological deficit scores and cerebral infarct volumes. The proliferation of the NPCs and the activation of the Wnt/β‑catenin signaling pathway in the cortical peri‑infarct area were examined. Our results revealed that EA significantly alleviated neurological deficits, reduced the infarct volume and enhanced NPC proliferation [nestin/glial fibrillary acidic protein (GFAP)‑double positive] in the cortex of rats subjected to middle cerebral artery occlusion (MCAO). Moreover, the Wnt1 and β‑catenin mRNA and protein levels were increased, while glycogen synthase kinase‑3 (GSK3) transcription was suppressed by EA. These results suggest that the upregulatory effects of EA on the Wnt/β‑catenin signaling pathway may promote NPC proliferation in the cortical peri-infarct area after stroke, consequently providing a therapeutic effect against cerebral ischemia.

The aim of this study was to investigate the effect of electroacupuncture (EA) on cell proliferation and its molecular mechanisms. Sixty rats were randomly divided into 5 groups: sham operation control (SC), ischemia control (IC), EA, EA and DMSO injection (ED), EA and U0126 injection (EU). All the groups, with the exception of SC, underwent middle cerebral artery occlusion (MCAO), and DMSO or U0126 was injected into the rat in the ED or EU group 30 min prior to MCAO. Cell proliferation was evaluated by proliferating cell nuclear antigen (PCNA) immunostaining. The changes of cell cycle proteins (cyclin D1, CDK4, cyclin E, CDK2, p21 and p27) and the ERK1/2 pathway activation were examined by RT-PCR and western blot analysis. The results showed that the positive cell numbers of PCNA immunostaining in the EA and ED groups were more than those in the IC group (P

Microglial activation plays an important role in nero- inflammation following ischemic stroke. Activated microglial cells can then migrate to the site of injury to proliferate and release substances which induce secondary brain damage. It has been shown that microglial migration is associated with the activation of the mitogen-activated protein kinase (MAPK) signaling pathways. The Chinese formula, GuaLou GuiZhi decoction (GLGZD), has long been administered in clinical practice for the treatment of post-stroke disabilities, such as muscular spasticity. In a previous study, we demonstrated that the anti-inflammtory effects of GLGZD were mediated by the TLR4/NF-κB pathway in lipopolysaccharide (LPS)-stimulated microglial cells. Therefore, in this study, we evaluated the role of GLGZD in microglial migration by performing scratch wound assays and migration assays. We wished to elucidate the cellular and molecular mechanisms elicited by this TCM formula in microglial-induced inflammation by evaluating the release and expression of chemotactic cytokines (monocyte chemo attractant protein-1 (MCP-1), macrophage inflamma - tory protein-1α (MIP-1α) and interleukin (IL)-8) by ELISA and quantitative PCR. Our results revealed that the migra- tion of microglial cells was enhanced in the presence of LPS (100 ng/ml); however, GLGZD (100 µg/ml) significantly inhib - ited cell motility and the production of chemokines through the inhibition of the activation of the p38 and c-Jun N-terminal protein kinase (JNK) signaling pathway. We demonstrate the potential of GLGZD in the modulation of microglial motility by investigating the effects of GLGZD on microglial migration induced by LPS. Taken together, our data suggest that GLGZD per se cannot trigger microglial motility, whereas GLGZD impedes LPS-induced microglial migration through the acti- vation of the MAPK signaling pathway. These results provide further evidence of the anti-inflammatory effects of GLGZD and its potential for use in the treatment of ischemic stroke.

The PI3K/Akt pathway, a critical mediator of cell survival, is suppressed in cerebral ischemia/reperfusion (I/R) injury; therefore, it is a major focus in treatment of ischemic stroke. Acupuncture has long been used in China to clinically treat stroke. However, the precise mechanism of its neuroprotective activities remains largely unknown. Using a focal cerebral I/R injured rat model, in the present study we evaluated the in vivo therapeutic efficacy of electroacupuncture and investigated the underlying molecular mechanisms. We found that electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints on the contralateral paralyzed limb significantly improved neurological deficits and cerebral infarction. In addition, electroacupuncture profoundly activated PI3K/Akt signaling in ischemic cerebral tissues. Consequently, the upregulatory effect of electroacupuncture on PI3K/Akt activation resulted in the inhibition of cerebral cell apoptosis. Moreover, electroacupuncture increased the serum secretion levels of the PI3K activators BDNF and GDNF, as well as upregulated the anti-apoptotic Bcl-2/Bax ratio in ischemic cerebrum. Our data suggest that electroacupuncture at Quchi and Zusanli acupoints exerts neuroprotective function in ischemic stroke via activation of the PI3K/Akt pathway.

The liver X receptor α (LXRα)/ATP-binding cassette transporter A1 (ABCA1) pathway and LXR-modulated cytokines play an important role in macrophages which mediate lipid engulfment and the inflammatory response, and participate in the process of atherosclerosis. Therefore, lipid-lowering and anti-inflammatory therapy through the activation of the LXRα/ABCA1 pathway and LXRα-modulated cytokines may prove to be one of the main treatment strategies for atherosclerosis. Alisma Decoction (AD) has long been used in China to clinically treat cardiovascular and cerebral diseases; however, the precise mechanisms involved remain to be elucidated. In the present study, we evaluated the regulation of lipids and the anti-inflammatory effects exerted by AD and investigated the underlying molecular mechanisms using oxidized low-density lipoprotein (ox-LDL)-stimulated foam cells derived from rat peritoneal macrophages. We first found that AD markedly relieved lipid deposition in foam cells as it increased LXRα and ABCA1 expression and decreased the ox-LDL-induced expression of inflammatory cytokines, such as matrix metalloproteinase-9 and interleukin-1β. Collectively, our findings suggest that blocking lipid deposition and inhibiting inflammatory response through the activation of the LXRα pathway may be one of the main mechanisms through which AD exerts its anti-atherosclerotic effects.

Gua Lou Gui Zhi decoction (GLGZD), a traditional Chinese medicine consisting of different herbal medicines, has been used for centuries in the treatment of muscular spasticity following stroke, epilepsy or spinal cord injury. However, the precise mechanisms involved remain poorly understood. In the present study, we investigated the neuroprotective effects of GLGZD on glutamate-induced apoptosis in cultured BV-2 cells, as well as the underlying mechanisms. A 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay was applied to assess the viability of the cells. An Annexin V/propidium iodide (PI) assay was utilized to analyze cellular apoptosis. Mitochondrial membrane potential (MMP) was evaluated by flow cytometry and laser scanning confocal microscopy. The gene and protein expression of the apoptosis-related genes, Bcl-2 and Bax, was analyzed by RT-PCR and western blot analysis, respectively. Furthermore, the expression of cleaved caspase-3 protein was detected by immunofluorescence. Glutamate treatment induced the loss of BV-2 cell viability, which was associated with an increase in the apoptotic rate, as well as an increase in the Bax/Bcl-2 ratio and the extracellular levels of cleaved caspase-3. Treatment with GLGZD significantly reversed these phenotypes, with its maximum protective effects observed at the concentration of 1,000 µg/ml. These results indicate that GLGZD protects BV-2 cells from glutamate-induced cytotoxicity. These protective effects may be ascribed to its anti-apoptotic activities, in part, associated with the decrease in the Bax/Bcl-2 ratio and caspase-3 expression, as well as with the stability of high mitochondrial membrane potential.

Toll-like receptor 4 (TLR4)/nuclear factor-κB (NF-κB) signaling-mediated neuroinflammation contributes to secondary brain damage in ischemic stroke; therefore, anti-inflammatory therapy via suppression of the TLR4/NF-κB pathway could be a promising strategy for the treatment of stroke and post-stroke disabilities. Gua Lou Gui Zhi decoction (GLGZD) has long been used in China to clinically treat dysfunction after stroke such as muscular spasticity, but the precise mechanisms are largely unknown. In the present study, we evaluated the anti-inflammatory effect of GLGZD and investigated the underlying molecular mechanisms using lipopolysaccharide (LPS)-stimulated BV-2 microglial cells as an in vitro inflammatory model of neural cells. We found that GLGZD inhibited the inflammatory response in microglial cells as it significantly reduced LPS-induced expression of pro-inflammatory nitric oxide, tumour necrosis factor-α, interleukin (IL)-6 and IL-1β in BV-2 cells, in a dose-dependent manner. In addition, GLGZD treatment significantly decreased the protein expression of TLR4 and myeloid differentiation factor 88, inhibited the phosphorylation of IκB and blocked the nuclear translocation of NF-κB in BV-2 cells, demonstrating its inhibitory effect on the activation of TLR4/NF-κB signaling. Collectively, our findings suggest that inhibition of the inflammatory response via suppression of the TLR4/NF-κB pathway may be one of the mechanisms through which GLGZD ameliorates the damage in ischemic cerebral tissues.

Inflammatory response has been shown to play a critical role in brain damage after cerebral ischemia-reperfusion (I/R) injury, which is tightly regulated by the Toll-like receptor (TLR)4/nuclear factor (NF)-κB pathway; therefore, suppression of TLR4/NF-κB signaling has become a promising target for the anti-inflammatory treatment in ischemic stroke. Acupuncture has been used as a complementary and alternative therapy practice that supplements conventional medicine. Numerous studies have demonstrated the clinical efficacy of acupuncture in stroke rehabilitation. However, the precise mechanism of its neuroprotective effect remains poorly understood. Using a focal cerebral I/R injured rat model, in the present study we evaluated the neuroprotective and anti-inflammatory activities of electroacupuncture at Quchi and Zusanli, and investigated the underlying molecular mechanisms. We found that electroacupuncture at Quchi (LI11) and Zusanli (ST36) acupoints significantly improved the ischemia-associated scores of neurological deficits, reduced cerebral infarction and alleviated inflammatory responses. Moreover, the crucial signaling molecules in the TLR4/NF-κB signaling pathway were regulated by acupuncture, which coincided with suppressed secretion levels of inflammatory cytokines such as TNF-α, IL-1β and IL-6. Our data suggest that electroacupuncture exerts a neuroprotective function in ischemic stroke through inhibition of TLR4/NF-κB-mediated inflammation.

Neurogenesis is regulated by a number of signaling pathways, including the retinoic acid (RA) pathway, a key regulator of neurogenesis in the subventricular zone (SVZ) and hippocampus. Acupuncture has been used to treat neurological conditions and is known to potentially enhance cell proliferation in the neurogenic area (hippocampal dentate gyrus and the SVZ of the lateral ventricle walls) in pathological conditions, which is associated with improved brain function. However, whether or not the neuroprotective effects of electroacupuncture (EA) are mediated by the regulation of the RA signaling pathway remains to be determined. Using a transient middle cerebral artery occlusion model, in the present study we evaluated the effect of EA on the neurological functional recovery, infarction volume and investigated the underlying molecular mechanisms. Two hundred and sixteen SD rats were randomly divided into 3 groups: sham, model group (ischemic rats without EA stimulation) and EA group (ischemic rats with EA stimulation on ST36 and LI11). Behavioral deficits were detected with high-resolution digital analysis of 24-h home-cage video recordings. Infarct volume was determined by triphenyltetrazolium hydrochloride staining and the expression of RA mRNA and protein was measured using RT-PCR and western blotting, respectively. We found that EA decreased the infarct volume, promoted neurological functional recovery and increased the RA mRNA and protein expression, compared with the model group. Findings of this study suggest that promoting neurological functional recovery by modulating RA expression in the post-ischemic brain is one of the mechanisms by which EA can be effective in the treatment of ischemic stroke.