Treatment with β-sitosterol ameliorates the effects of cerebral ischemia/reperfusion injury by suppressing cholesterol overload, endoplasmic reticulum stress, and apoptosis

β-Sitosterol is a type of phytosterol that occurs naturally in plants.Previous studies have shown that it has anti-oxidant,anti-hyperlipidemic,anti-inflammatory,immunomodulatory,and anti-tumor effects,but it is unknown whetherβ-sitosterol treatment reduces the effects of ischemic stroke.Here we found that,in a mouse model of ischemic stroke induced by middle cerebral artery occlusion,β-sitosterol reduced the volume of cerebral infarction and brain edema,reduced neuronal apoptosis in brain tissue,and alleviated neurological dysfunction;moreover,β-sitosterol increased the activity of oxygen-and glucose-deprived cerebral cortex neurons and reduced apoptosis.Further investigation showed that the neuroprotective effects ofβ-sitosterol may be related to inhibition of endoplasmic reticulum stress caused by intracellular cholesterol accumulation after ischemic stroke.In addition,β-sitosterol showed high affinity for NPC1L1,a key transporter of cholesterol,and antagonized its activity.In conclusion,β-sitosterol may help treat ischemic stroke by inhibiting neuronal intracellular cholesterol overload/endoplasmic reticulum stress/apoptosis signaling pathways.

The action mechanism by which C1q/tumor necrosis factor-related protein-6 alleviates cerebral ischemia/reperfusion injury in diabetic mice

Studies have shown that C1q/tumor necrosis factor-related protein-6 (CTRP6) can alleviate renal ischemia/reperfusion injury in mice. However, its role in the brain remains poorly understood. To investigate the role of CTRP6 in cerebral ischemia/reperfusion injury associated with diabetes mellitus, a diabetes mellitus mouse model of cerebral ischemia/reperfusion injury was established by occlusion of the middle cerebral artery. To overexpress CTRP6 in the brain, an adeno-associated virus carrying CTRP6 was injected into the lateral ventricle. The result was that oxygen injury and inflammation in brain tissue were clearly attenuated, and the number of neurons was greatly reduced. In vitro experiments showed that CTRP6 knockout exacerbated oxidative damage, inflammatory reaction, and apoptosis in cerebral cortical neurons in high glucose hypoxia-simulated diabetic cerebral ischemia/reperfusion injury. CTRP6 overexpression enhanced the sirtuin-1 signaling pathway in diabetic brains after ischemia/reperfusion injury. To investigate the mechanism underlying these effects, we examined mice with depletion of brain tissue-specific sirtuin-1. CTRP6-like protection was achieved by activating the sirtuin-1 signaling pathway. Taken together, these results indicate that CTRP6 likely attenuates cerebral ischemia/reperfusion injury through activation of the sirtuin-1 signaling pathway.

A matrix metalloproteinase-responsive hydrogel system controls angiogenic peptide release for repair of cerebral ischemia/reperfusion injury

Vascular endothelial growth factor and its mimic peptide KLTWQELYQLKYKGI(QK)are widely used as the most potent angiogenic factors for the treatment of multiple ischemic diseases.However,conventional topical drug delivery often results in a burst release of the drug,leading to transient retention(inefficacy)and undesirable diffusion(toxicity)in vivo.Therefore,a drug delivery system that responds to changes in the microenvironment of tissue regeneration and controls vascular endothelial growth factor release is crucial to improve the treatment of ischemic stroke.Matrix metalloproteinase-2(MMP-2)is gradually upregulated after cerebral ischemia.Herein,vascular endothelial growth factor mimic peptide QK was self-assembled with MMP-2-cleaved peptide PLGLAG(TIMP)and customizable peptide amphiphilic(PA)molecules to construct nanofiber hydrogel PA-TIMP-QK.PA-TIMP-QK was found to control the delivery of QK by MMP-2 upregulation after cerebral ischemia/reperfusion and had a similar biological activity with vascular endothelial growth factor in vitro.The results indicated that PA-TIMP-QK promoted neuronal survival,restored local blood circulation,reduced blood-brain barrier permeability,and restored motor function.These findings suggest that the self-assembling nanofiber hydrogel PA-TIMP-QK may provide an intelligent drug delivery system that responds to the microenvironment and promotes regeneration and repair after cerebral ischemia/reperfusion injury.

Cav3.2 channel regulates cerebral ischemia/reperfusion injury:a promising target for intervention

Calcium influx into neurons triggers neuronal death during cerebral ischemia/reperfusion injury.Various calcium channels are involved in cerebral ischemia/reperfusion injury.Cav3.2 channel is a main subtype of T-type calcium channels.T-type calcium channel blockers,such as pimozide and mibefradil,have been shown to prevent cerebral ischemia/reperfusion injury-induced brain injury.However,the role of Cav3.2 channels in cerebral ischemia/reperfusion injury remains unclear.Here,in vitro and in vivo models of cerebral ischemia/reperfusion injury were established using middle cerebral artery occlusion in mice and high glucose hypoxia/reoxygenation exposure in primary hippocampal neurons.The results showed that Cav3.2 expression was significantly upregulated in injured hippocampal tissue and primary hippocampal neurons.We further established a Cav3.2 gene-knockout mouse model of cerebral ischemia/reperfusion injury.Cav3.2 knockout markedly reduced infarct volume and brain water content,and alleviated neurological dysfunction after cerebral ischemia/reperfusion injury.Additionally,Cav3.2 knockout attenuated cerebral ischemia/reperfusion injury-induced oxidative stress,inflammatory response,and neuronal apoptosis.In the hippocampus of Cav3.2-knockout mice,calcineurin overexpression offset the beneficial effect of Cav3.2 knockout after cerebral ischemia/reperfusion injury.These findings suggest that the neuroprotective function of Cav3.2 knockout is mediated by calcineurin/nuclear factor of activated T cells 3 signaling.Findings from this study suggest that Cav3.2 could be a promising target for treatment of cerebral ischemia/reperfusion injury.

Lactiplantibacillus plantarum AR113 alleviates microbiota dysbiosis of tongue coating and cerebral ischemia/reperfusion injury in rat

Stroke is one of the leading causes of death and disability worldwide.However,information on stroke-related tongue coating microbiome(TCM)is limited,and whether TCM modulation could benefit for stroke prevention and rehabilitation is unknown.Here,TCM from stroke patients(SP)was characterized using molecular techniques.The occurrence of stroke resulted in TCM dysbiosis with significantly reduced species richness and diversity.The abundance of Prevotella,Leptotrichia,Actinomyces,Alloprevotella,Haemophilus,and TM7_[G-1]were greatly reduced,but common infection Streptococcus and Pseudomonas were remarkably increased.Furthermore,an antioxidative probiotic Lactiplantibacillus plantarum AR113 was used for TCM intervention in stroke rats with cerebral ischemia/reperfusion(I/R).AR113 partly restored I/R induced change of TCM and gut microbiota with significantly improved neurological deficit,relieved histopathologic change,increased activities of antioxidant enzymes,and decreased contents of oxidative stress biomarkers.Moreover,the gene expression of antioxidant-related proteins and apoptosis-related factors heme oxygenase-1(HO-1),superoxide dismutase(SOD),glutathione peroxidase(GSH-Px),nuclear factor erythroid 2-related factor 2(Nrf2),NAD(P)H:quinone oxidoreductase-1(NQO-1),and Bcl-2 was significantly increased,but cytochrome C,cleaved caspase-3,and Bax were markedly decreased in the brain by AR113 treatment.The results suggested that AR113 could ameliorate cerebral I/R injury through antioxidation and anti-apoptosis pathways,and AR113 intervention of TCM may have the application potential for stroke prevention and control.

Endoplasmic reticulum stress and autophagy in cerebral ischemia/reperfusion injury:PERK as a potential target for intervention

Several studies have shown that activation of unfolded protein response and endoplasmic reticulum(ER)stress plays a crucial role in severe cerebral ischemia/reperfusion injury.Autophagy occurs within hours after cerebral ischemia,but the relationship between ER stress and autophagy remains unclear.In this study,we established experimental models using oxygen-glucose deprivation/reoxygenation in PC12 cells and primary neurons to simulate cerebral ischemia/reperfusion injury.We found that prolongation of oxygen-glucose deprivation activated the ER stress pathway protein kinase-like endoplasmic reticulum kinase(PERK)/eukaryotic translation initiation factor 2 subunit alpha(e IF2α)-activating transcription factor 4(ATF4)-C/EBP homologous protein(CHOP),increased neuronal apoptosis,and induced autophagy.Furthermore,inhibition of ER stress using inhibitors or by si RNA knockdown of the PERK gene significantly attenuated excessive autophagy and neuronal apoptosis,indicating an interaction between autophagy and ER stress and suggesting PERK as an essential target for regulating autophagy.Blocking autophagy with chloroquine exacerbated ER stress-induced apoptosis,indicating that normal levels of autophagy play a protective role in neuronal injury following cerebral ischemia/reperfusion injury.Findings from this study indicate that cerebral ischemia/reperfusion injury can trigger neuronal ER stress and promote autophagy,and suggest that PERK is a possible target for inhibiting excessive autophagy in cerebral ischemia/reperfusion injury.

Selective ischemic-hemisphere targeting Ginkgolide B liposomes with improved solubility and therapeutic efficacy for cerebral ischemia-reperfusion injury

Cerebral ischemia-reperfusion injury(CI/RI)remains the main cause of disability and death in stroke patients due to lack of effective therapeutic strategies.One of the main issues related to CI/RI treatment is the presence of the blood-brain barrier(BBB),which affects the intracerebral delivery of drugs.Ginkgolide B(GB),a major bioactive component in commercially available products of Ginkgo biloba,has been shown significance in CI/RI treatment by regulating inflammatory pathways,oxidative damage,and metabolic disturbance,and seems to be a candidate for stroke recovery.However,limited by its poor hydrophilicity and lipophilicity,the development of GB preparations with good solubility,stability,and the ability to cross the BBB remains a challenge.Herein,we propose a combinatorial strategy by conjugating GB with highly lipophilic docosahexaenoic acid(DHA)to obtain a covalent complex GB-DHA,which can not only enhance the pharmacological effect of GB,but can also be encapsulated in liposomes stably.The amount of finally constructed Lipo@GB-DHA targeting to ischemic hemisphere was validated 2.2 times that of free solution in middle cerebral artery occlusion(MCAO)rats.Compared to the marketed ginkgolide injection,Lipo@GB-DHA significantly reduced infarct volume with better neurobehavioral recovery in MCAO rats after being intravenously administered both at 2 h and 6 h post-reperfusion.Low levels of reactive oxygen species(ROS)and high neuron survival in vitro was maintained via Lipo@GB-DHA treatment,while microglia in the ischemic brain were polarized from the pro-inflammatory M1 phenotype to the tissue-repairing M2 phenotype,which modulate neuroinflammatory and angiogenesis.In addition,Lipo@GB-DHA inhibited neuronal apoptosis via regulating the apoptotic pathway and maintained homeostasis by activating the autophagy pathway.Thus,transforming GB into a lipophilic complex and loading it into liposomes provides a promising nanomedicine strategy with excellent CI/RI therapeutic efficacy and industrialization prospects.

A molecular probe carrying anti-tropomyosin 4 for early diagnosis of cerebral ischemia/reperfusion injury

In vivo imaging of cerebral ischemia/reperfusion injury remains an important challenge.We injected porous Ag/Au@SiO_(2) bimetallic hollow nanoshells carrying anti-tropomyosin 4 as a molecular probe into mice with cerebral ischemia/reperfusion injury and observed microvascular changes in the brain using photoacoustic imaging with ultrasonography.At each measured time point,the total photoacoustic signal was significantly higher on the affected side than on the healthy side.Twelve hours after reperfusion,cerebral perfusion on the affected side increased,cerebrovascular injury worsened,and anti-tropomyosin 4 expression increased.Twenty-four hours after reperfusion and later,perfusion on the affected side declined slowly and stabilized after 1 week;brain injury was also alleviated.Histopathological and immunohistochemical examinations confirmed the brain injury tissue changes.The nanoshell molecular probe carrying anti-tropomyosin 4 has potential for use in early diagnosis of cerebral ischemia/reperfusion injury and evaluating its progression.

Vav1 promotes inflammation and neuronal apoptosis in cerebral ischemia/reperfusion injury by upregulating microglial and NLRP3 inflammasome activation

Microglia,which are the resident macrophages of the central nervous system,are an important part of the inflammatory response that occurs after cerebral ischemia.Vav guanine nucleotide exchange factor 1(Vav1) is a guanine nucleotide exchange factor that is related to microglial activation.However,how Vav1 participates in the inflammato ry response after cerebral ischemia/reperfusion inj ury remains unclea r.In this study,we subjected rats to occlusion and repe rfusion of the middle cerebral artery and subjected the BV-2 mic roglia cell line to oxygen-glucose deprivatio n/reoxygenation to mimic cerebral ischemia/repe rfusion in vivo and in vitro,respectively.We found that Vav1 levels were increased in the brain tissue of rats subjected to occlusion and reperfusion of the middle cerebral arte ry and in BV-2 cells subjected to oxygen-glucose deprivation/reoxygenation.Silencing Vav1 reduced the cerebral infarct volume and brain water content,inhibited neuronal loss and apoptosis in the ischemic penumbra,and im p roved neurological function in rats subjected to occlusion and repe rfusion of the middle cerebral artery.Further analysis showed that Vav1 was almost exclusively localized to microglia and that Vav1 downregulation inhibited microglial activation and the NOD-like receptor pyrin 3(NLRP3) inflammasome in the ischemic penumbra,as well as the expression of inflammato ry facto rs.In addition,Vov1 knoc kdown decreased the inflammatory response exhibited by BV-2 cells after oxygen-glucose deprivation/reoxyge nation.Taken together,these findings show that silencing Vav1 attenuates inflammation and neuronal apoptosis in rats subjected to cerebral ischemia/repe rfusion through inhibiting the activation of mic roglia and NLRP3 inflammasome.

Ischemic accumulation of succinate induces Cdc42 succinylation and inhibits neural stem cell proliferation after cerebral ischemia/reperfusion

Ischemic accumulation of succinate causes cerebral damage by excess production of reactive oxygen species. However, it is unknown whether ischemic accumulation of succinate affects neural stem cell proliferation. In this study, we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery. We found that succinate levels increased in serum and brain tissue(cortex and hippocampus) after ischemia/reperfusion injury. Oxygen-glucose deprivation and reoxygenation stimulated primary neural stem cells to produce abundant succinate. Succinate can be converted into diethyl succinate in cells. Exogenous diethyl succinate inhibited the proliferation of mouse-derived C17.2 neural stem cells and increased the infarct volume in the rat model of cerebral ischemia/reperfusion injury. Exogenous diethyl succinate also increased the succinylation of the Rho family GTPase Cdc42 but repressed Cdc42 GTPase activity in C17.2 cells. Increasing Cdc42 succinylation by knockdown of the desuccinylase Sirt5 also inhibited Cdc42 GTPase activity in C17.2 cells. Our findings suggest that ischemic accumulation of succinate decreases Cdc42 GTPase activity by induction of Cdc42 succinylation, which inhibits the proliferation of neural stem cells and aggravates cerebral ischemia/reperfusion injury.

DNA hypomethylation promotes learning and memory recovery in a rat model of cerebral ischemia/reperfusion injury

Cerebral ischemia/reperfusion injury impairs learning and memory in patients.Studies have shown that synaptic function is involved in the formation and development of memory,and that DNA methylation plays a key role in the regulation of learning and memory.To investigate the role of DNA hypomethylation in cerebral ischemia/reperfusion injury,in this study,we established a rat model of cerebral ischemia/reperfusion injury by occlusion of the middle cerebral artery and then treated the rats with intraperitoneal 5-aza-2′-deoxycytidine,an inhibitor of DNA methylation.Our results showed that 5-aza-2′-deoxycytidine markedly improved the neurological function,and cognitive,social and spatial memory abilities,and dose-dependently increased the synaptic density and the expression of SYP and SHANK2 proteins in the hippocampus in a dose-dependent manner in rats with cerebral ischemia/reperfusion injury.The effects of 5-aza-2′-deoxycytidine were closely related to its reduction of genomic DNA methylation and DNA methylation at specific sites of the Syp and Shank2 genes in rats with cerebral ischemia/reperfusion injury.These findings suggest that inhibition of DNA methylation by 5-aza-2′-deoxycytidine promotes the recovery of learning and memory impairment in a rat model of cerebral ischemia/reperfusion injury.These results provide theoretical evidence for stroke treatment using epigenetic methods.

Upregulation of CDGSH iron sulfur domain 2 attenuates cerebral ischemia/reperfusion injury

CDGSH iron sulfur domain 2 can inhibit ferroptosis,which has been associated with cerebral ischemia/reperfusion,in individuals with head and neck cancer.Therefore,CDGSH iron sulfur domain 2 may be implicated in cerebral ischemia/reperfusion injury.To validate this hypothesis in the present study,we established mouse models of occlusion of the middle cerebral artery and HT22 cell models of oxygen-glucose deprivation and reoxygenation to mimic cerebral ischemia/reperfusion injury in vivo and in vitro,respectively.We found remarkably decreased CDGSH iron sulfur domain 2 expression in the mouse brain tissue and HT22 cells.When we used adeno-associated virus and plasmid to up-regulate CDGSH iron sulfur domain 2 expression in the brain tissue and HT22 cell models separately,mouse neurological dysfunction was greatly improved;the cerebral infarct volume was reduced;the survival rate of HT22 cells was increased;HT22 cell injury was alleviated;the expression of ferroptosis-related glutathione peroxidase 4,cystine-glutamate antiporter,and glutathione was increased;the levels of malondialdehyde,iron ions,and the expression of transferrin receptor 1 were decreased;and the expression of nuclear-factor E2-related factor 2/heme oxygenase 1 was increased.Inhibition of CDGSH iron sulfur domain 2 upregulation via the nuclear-factor E2-related factor 2 inhibitor ML385 in oxygen-glucose deprived and reoxygenated HT22 cells blocked the neuroprotective effects of CDGSH iron sulfur domain 2 up-regulation and the activation of the nuclear-factor E2-related factor 2/heme oxygenase 1 pathway.Our data indicate that the up-regulation of CDGSH iron sulfur domain 2 can attenuate cerebral ischemia/reperfusion injury,thus providing theoretical support from the perspectives of cytology and experimental zoology for the use of this protein as a therapeutic target in patients with cerebral ischemia/reperfusion injury.

EFFECTS OF ELECTROACUPUNCTURE ON EXPRESSION OF INTERCELLULAR ADHESION MOLECULE-1 IN THE RAT OF LOCAL CEREBRAL ISCHEMIA-REPERFUSION

Objective To investigate effects of electroacupuncture （EA） on expression of intercellular adhesion molecule-1 （ICAM-1） in the rat of local cerebral ischemia-reperfusion. Methods Eighty SD rats were randomly divided into a normal control group, a sham operation group, a model group and an EA treatment group, 20 rats in each group. The thread-obstruction method was used for preparation of ischemia-reperfusion model. Zea-Longa rating criteria were used for evaluation of nervous function disorder; Immunohistochemical SABC method was used for detection of ICAM-1 expression in the microvascular endothelial cell of the ischemic brain region, and ELISA method for the soluble ICAM-1 （slCAM-1） content in peripheral blood. Re. suits After cerebral ischemia-reperfusion, both ICAM-1 expression level in the microvascular endethelium cell of the ischemic brain region and slCAM-1 content in the peripheral blood significantly increased in the model group as compared with the normal group and the sham operation group （P〈0.01）; After EA treatment, the ICAM-1 expression level in the microvascular endothelial cell of the ischemic brain region and slCAM-1 content in the peripheral blood were significantly down-regulated in the EA treatment group as com- pared with the model group （P〈 0.05）. Conclusion After cerebral ischemia-reperfusion, the microvascular endothelial cell of the ischemic brain region releases ICAM-1, which induces inflammatory injury of cerebral tissues; EA treatment can decease the expression of ICAM-1, so as to prevent the brain from the injury.

Atorvastatin protects against cerebral ischemia/ reperfusion injury through anti-inflammatory and antioxidant effects

In addition to its lipid-lowering effect, atorvastatin exerts anti-inflammatory and antioxidant effects as well. In this study, we hypothesized that atorvastatin could protect against cerebral isch-emia/reperfusion injury. The middle cerebral artery ischemia/reperfusion model was established, and atorvastatin, 6.5 mg/kg, was administered by gavage. We found that, after cerebral ischemia/ reperfusion injury, levels of the inflammation-related factors E-selectin and myeloperoxidase were upregulated, the oxidative stress-related marker malondialdehyde was increased, and super- oxide dismutase activity was decreased in the ischemic cerebral cortex. Atorvastatin pretreatment significantly inhibited these changes. Our findings indicate that atorvastatin protects against ce-rebral ischemia/reperfusion injury through anti-inflammatory and antioxidant effects.

Puerarin partly counteracts the inflammatory response after cerebral ischemia/reperfusion via activating the cholinergic anti-inflammatory pathway

Puerarin, a major isoflavonoid derived from the Chinese medical herb radix puerariae （Gegen）, has been reported to inhibit neuronal apoptosis and play an anti-inflammatory role in focal cerebral ischemia model rats. Recent findings regarding stroke pathophysiology have recognized that anti-inflammation is an important target for the treatment of ischemic stroke. The cholinergic anti-inflammatory pathway is a highly robust neural-immune mechanism for inflammation control. This study was to investigate whether activating the cholinergic anti-inflammatory pathway can be involved in the mechanism of inhibiting the inflammatory response during puerarin-induced cerebral ischemia/reperfusion in rats. Results showed that puerarin pretreatment （intravenous injection） re- duced the ischemic infarct volume, improved neurological deficit after cerebral ischemia/reperfusion and decreased the levels of interleukin-1β, interleukin-6 and tumor necrosis factor-a in brain tissue. Pretreatment with puerarin （intravenous injection） attenuated the inflammatory response in rats, which was accompanied by janus-activated kinase 2 （JAK2） and signal transducers and activators of transcription 3 （STAT3） activation and nuclear factor kappa B （NF-KB） inhibition. These observa- tions were inhibited by the alpha7 nicotinic acetylcholine receptor （a7nAchR） antagonist a-bungarotoxin （a-BGT）. In addition, puerarin pretreatment increased the expression of a7nAchR mRNA in ischemic cerebral tissue. These data demonstrate that puerarin pretreatment strongly protects the brain against cerebral ischemia/reperfusion injury and inhibits the inflammatory re- sponse. Our results also indicated that the anti-inflammatory effect of puerarin may partly be medi- ated through the activation of the cholinergic anti-inflammatory pathway.

Electroacupuncture preconditioning protects against focal cerebral ischemia/reperfusion injury via suppression of dynamin-related protein 1

Electroacupuncture preconditioning at acupoint Baihui （GV20） can reduce focal cerebral ischemia/reperfusion injury. However, the precise protective mechanism remains unknown. Mitochondrial fission mediated by dynamin-related protein 1 （Drp1） can trigger neuronal apoptosis following cerebral ischemia/reperfusion injury. Herein, we examined the hypothesis that electroacupuncture pretreatment can regulate Drp1, and thus inhibit mitochondrial fission to provide cerebral protection. Rat models of focal cerebral ischemia/reperfusion injury were established by middle cerebral artery occlusion at 24 hours after 5 consecutive days of preconditioning with electroacupuncture at GV20 （depth 2 mm, intensity 1 mA, frequency 2/15 Hz, for 30 minutes, once a day）. Neurological function was assessed using the Longa neurological deficit score. Pathological changes in the ischemic penumbra on the injury side were assessed by hematoxylin-eosin staining. Cellular apoptosis in the ischemic penumbra on the injury side was assessed by terminal deoxyribonucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling staining. Mitochondrial ultrastructure in the ischemic penumbra on the injury side was assessed by transmission electron microscopy. Drp1 and cytochrome c expression in the ischemic penumbra on the injury side were assessed by western blot assay. Results showed that electroacupuncture preconditioning decreased expression of total and mitochondrial Drp1, decreased expression of total and cytosolic cytochrome c, maintained mitochondrial morphology and reduced the proportion of apoptotic cells in the ischemic penumbra on the injury side, with associated improvements in neurological function. These data suggest that electroacupuncture preconditioning-induced neuronal protection involves inhibition of the expression and translocation of Drp1.

Picroside Ⅱ down-regulates matrix metalloproteinase-9 expression following cerebral ischemia/reperfusion injury in rats

Studies have shown that Picroside Ⅱ attenuates inflammatory reactions following brain ischemia through the inhibition of the TLR-4-NF-KB signal transduction pathway, and ameliorates cerebral edema through the reduction of aquaporin-4 expression. Matrix metalloproteinase-9 （MMP-9）, located downstream of the TLR-4-NF-KB signal transduction pathway, can degrade the neurovascular matrix, damage the blood-brain barrier to induce cerebral edema, and directly result in neuronal apoptosis and brain injury, Therefore, the present study further observed MMP-9 expression in the brain tissues of rats with cerebral ischemia/reperfusion injury following Picroside Ⅱ treatment. Results demonstrated that Picroside Ⅱ significantly reduced MMP-9 expression in ischemic brain tissues, as well as neuronal apoptosis and brain infarct volume, suggesting Picroside Ⅱ exhibits neuroprotection by down-regulating MMP-9 expression and inhibiting cell apoptosis.

Comparison of the anti-apoptotic effects of 15-and 35-minute suspended moxibustion after focal cerebral ischemia/reperfusion injury

Heat-sensitive suspended moxibustion has a neuroprotective effect against focal cerebral ischemia/reperfusion injury, but the underly- ing mechanisms remain unclear. The duration of heat-sensitive suspended moxibustion （usually from 30 minutes to 1 hour） is longer than traditional suspended moxibustion （usually 15 minutes）. However, the effects of 15- and 35-minute suspended moxibustion in rats with cerebra/ischemia/reperfusion injury are poorly understood. In this study, we performed 15- or 35-minute suspended moxibustion at acupoint Dazhui （GV14） in an adult rat model of focal cerebral ischemia/reperfusion injury. Infarct volume was evaluated with the 2,3,5-triphenyltetrazolium chloride assay. Histopathological changes and neuronal apoptosis at the injury site were assessed by hematoxy- lin-eosin staining and terminal deoxynucleotidyl transferase dUTP nick end labeling assay. Caspase-9 and caspase-3 expression at the in- jury site was detected using immunofluorescent staining. Bax and Bcl-2 expression at the injury site was assessed using western blot assay. In the 35-minute moxibustion group, infarct volume was decreased, neuronal apoptosis was reduced, caspase-9, caspase-3 and Bax expres- sion was lower, and Bcl-2 expression was increased, compared with the 15-minute moxibustion group. Our findings show that 35-minute moxibustion has a greater anti-apoptotic effect than 15-minute moxibustion after focal cerebral ischemia/reperfusion injury.

Electroacupuncture reduces apoptotic index and inhibits p38 mitogen-activated protein kinase signaling pathway in the hippocampus of rats with cerebral ischemia/reperfusion injury

Electroacupuncture attenuates cerebral hypoxia and neuronal apoptosis induced by cerebral ischemia/reperfusion injury.To further identify the involved mechanisms,we assumed that electroacupuncture used to treat cerebral ischemia/reperfusion injury was associated with the p38 mitogen-activated protein kinase（MAPK） signaling pathway.We established rat models of cerebral ischemia/reperfusion injury using the modified Zea-Longa＇s method.At 30 minutes before model establishment,p38 MAPK blocker SB20358 was injected into the left lateral ventricles.At 1.5 hours after model establishment,electroacupuncture was administered at acupoints of Chize（LU5）,Hegu（LI4）,Zusanli（ST36）,and Sanyinjiao（SP6） for 20 minutes in the affected side.Results showed that the combination of EA and SB20358 injection significantly decreased neurologic impairment scores,but no significant differences were determined among different interventional groups.Hematoxylin-eosin staining also showed reduced brain tissue injuries.Compared with the SB20358 group,the cells were regularly arranged,the structures were complete,and the number of viable neurons was higher in the SB20358 ＋ electroacupuncture group.Terminal deoxynucleotidyl transferase（Td T）-mediated d UTP nick-end labeling assay showed a decreased apoptotic index in each group,with a significant decrease in the SB20358 ＋ electroacupuncture group.Immunohistochemistry revealed reduced phosphorylated p38 expression at 3 days in the electroacupuncture group and SB20358 ＋ electroacupuncture group compared with the ischemia/reperfusion group.There was no significant difference in phosphorylated p38 expression between the ischemia/reperfusion group and SB20358 group.These findings confirmed that the electroacupuncture effects on mitigating cerebral ischemia/reperfusion injury are possibly associated with the p38 MAPK signaling pathway.A time period of 3 days could promote the repair of ischemic cerebral nerves.

13-Methyltetradecanoic acid mitigates cerebral ischemia/reperfusion injury

13-Methyltetradecanoic acid can stabilize cell membrane and have anti-inflammatory, antioxidant and anti-apoptotic effects. Previous studies mainly focused on peripheral nerve injury, but seldom on the central nervous system. We investigated whether these properties of 13-methyltetradecanoic acid have a neuroprotective effect on focal cerebral ischemia/reperfusion injury, and detected the expression of basic fibroblast growth factor and vascular endothelial growth factor. This study established rat models of middle cerebral artery occlusion/ reperfusion injury by ischemia for 2 hours and reperfusion for 24 hours. At the beginning of reperfusion, 13-methyltetradecanoic acid 10, 40 or 80 mg/kg was injected into the tail vein. Results found that various doses of 13-methyltetradecanoic acid effectively reduced infarct volume, mitigate cerebral edema, and increased the mRNA and protein expression of basic fibroblast growth factor and vascular endothe- lial growth factor at 24 hours of reperfusion. The effect was most significant in the 13-methyltetradecanoic acid 40 and 80 mg/kg groups. The findings suggest that 13-methyltetradecanoic acid can relieve focal ischemia/reperfusion injury immediately after reperfusion, stimu- late the upregulation of basic fibroblast growth factor and vascular endothelial growth factor to exert neuroprotective effects.