Study on the anti-mitochondrial apoptosis mechanism of Erigeron breviscapus injection based on UPLC-Q-TOF-MS metabolomics and molecular docking in rats with cerebral ischemia-reperfusion injury.

ETHNOPHARMACOLOGICAL RELEVANCE: Erigeron breviscapus is a common medicine of eight ethnic minorities, including Miao, Naxi, and Yi. As early as the Ming Dynasty (AD 1368-1644), Lanmao's Materia Medica of Southern Yunnan (AD 1436) recorded that the medicine is used for the treatment of "Zuo tan you huan." In modern pharmacological research, Erigeron breviscapus injection is the most commonly used preparation in the treatment of ischemic stroke caused by acute cerebral infarction, but its mechanism of action in the treatment of ischemic stroke is not well understood. AIM OF THE STUDY: In this study, a metabonomics study based on ultraperformance liquid chromatography-quadrupole time-of-flight tandem mass spectrometry (UPLC-QTOF-MS) was used in investigating the effect of a traditional Chinese medicine preparation Erigeron breviscapus injection on the rat model of focal cerebral ischemia-reperfusion and the affinity of its main components with the targets of mitochondrial apoptotic pathways. MATERIALS AND METHODS: This study used molecular docking technology to verify the effective binding ability of main effective components of Erigeron breviscapus injection to target proteins related to mitochondrial apoptosis pathway. This study developed a metabonomics method based on the ultra-performance liquid chromatography combined with quadrupole time-of-flight tandem mass spectrometry (UPLC Q-TOF MS) to evaluate the efficacy and study the mechanism of traditional Chinese medicine preparation. With pattern recognition analysis (principal component analysis and partial least squares-discriminate analysis) of urinary metabolites, a clear separation of focal cerebral ischemia-reperfusion model group and healthy control group was achieved. RESULTS: Erigeron breviscapus injection can significantly reduce the area of cerebral infarction, improve tissue morphological lesion in rats, and can increase the number of Nissl bodies. It may be a promoting factor by inhibiting hippocampal nerve cell apoptosis and Bax protein expression and by exerting effects against ischemia reperfusion after the induction of apoptosis. Thus, it plays a role in brain protection. Moreover, it can considerably promote the recovery of neurological deficiency signs in advance. Meanwhile, Erigeron breviscapus decreased malondialdehyde content and T-NOS activity. Its curative effect from strong to weak order: low dose > high dose > medium dose. The representative components of Erigeron breviscapus have good affinity with the active sites of mitochondrial apoptosis-related proteins. Metabolomics found that the potential biomarkers regulated by breviscapine are kynurequinolinic acid, succinylornithine, and leucine proline. It is speculated that it may participate in TRP-kynurequinolinic acid and succinylornithine-urea cycle-NO metabolic pathways. CONCLUSIONS: This paper revealed the potential biomarkers and metabolic pathways regulated by Erigeron breviscapus. It was speculated that the mechanism is related to its inhibition of mitochondrion-mediated apoptosis. Erigeron breviscapus could restore the metabolic profiles of the model animals to normal animal levels. The mechanism may be related to the potential biomarkers of quinolinic acid, succinylornithine, and leucine proline and the metabolic pathways involved. However, the exact mechanism by which Erigeron breviscapus inhibits mitochondrion-mediated apoptosis remains to be further explored.

Ulinastatin affects focal cerebral ischemia-reperfusion injury via SOCS1-mediated JAK2/STAT3 signalling pathway.

Cerebral ischemia results in loss of cerebral blood flow, which contributes to neuronal damage, neurocognitive impairment, as well as learning and memory difficulties. Although reperfusion is necessary to restore the blood supply to the brain, it also leads to several detrimental effects on the brain. The purpose of this study was to assess the effects of ulinastatin (UTI) on preventing focal cerebral ischemia/reperfusion-induced injury (FCIRI). First, a rat model of FCIRI was established and treated with UTI. The effects of UTI on FCIRI in rats were evaluated using Morris water maze assay, triphenyl tetrazolium chloride staining, TUNEL, western blot assay, and enzyme-linked immunosorbent assay analysis. UTI was found to improve the learning memory ability, reduce infarction area, inhibit apoptosis and decrease inflammation in FCIRI rats. Messenger RNA microarray analysis of hippocampal tissues revealed that suppressor of cytokine signalling-1 (SOCS1) was the downstream target of UTI in FCIRI. SOCS1 depletion impaired the protective effect of UTI on FCIRI in rats. SOCS1 blocked the activation of the JAK2/STAT3 pathway. JAK2 inhibitor caused the JAK2/STAT3 pathway deficit, hence reversing the effect of sh-SOCS1 on FCIRI in rats. Taken together, our results demonstrate that UTI alleviated FCIRI in rats, which was, to some extent, related to SOCS1-mediated JAK2/STAT3 pathway.

Isoastilbin inhibits neuronal apoptosis and oxidative stress in a rat model of ischemia-reperfusion injury in the brain: Involvement of SIRT1/3/6.

BACKGROUND: Isoastilbin (IAB) has been shown to have antioxidative and anti-apoptotic functions. A recent study found that IAB can reduce oxidative stress in Alzheimer's disease. However, whether the antioxidative function of IAB is also protective in other brain diseases remains unknown. OBJECTIVES: To investigate the roles and underlying mechanisms of IAB in middle cerebral artery occlusion-reperfusion (MCAO/R) in rats. MATERIAL AND METHODS: Male Wistar rats were randomly divided into 5 groups: sham group, MCAO/R group, and 3 MCAO/R groups groups administered IAB (20 mg/kg, 40 mg/kg or 80 mg/kg) once a day for 3 days. Infarction size, modified Neurological Severity Score (mNSS), oxidative stress markers, and neuronal apoptosis markers were used to assay the function of IAB. RESULTS: Compared with the MCAO/R group, administration of IAB reduced the infarction size and mNSS scores in MCAO/R rats. Isoastilbin also decreased the level of malondialdehyde (MDA) and enhanced the activity of catalase (CAT), superoxide dismutase (SOD) and glutathione peroxidase (GSH-PX). Isoastilbin treatment attenuated MCAO/R-induced neuronal apoptosis compared with the MCAO/R group, as indicated by the results of terminal deoxynucleotide transferase-mediated X-dUTP nick end (TUNEL) and western blot assays. Isoastilbin also reversed MCAO/R-induced downregulation of SIRT1/3/6 protein expression. CONCLUSIONS: These observations suggest that IAB protects against oxidative stress and neuronal apoptosis in rats following cerebral ischemia-reperfusion (I/R) injury through the upregulation of SIRT1/3/6, indicating that IAB might be a promising therapeutic agent for cerebral I/R injury.

Neuroprotection of Chikusetsu saponin V on transient focal cerebral ischemia/reperfusion and the underlying mechanism.

BACKGROUND: Oxidative stress and frequently unwanted alterations in mitochondrial structure and function are key aspects of the pathological cascade in transient focal cerebral ischemia. Chikusetsu saponin V (CHS V), a major component of saponins from Panax japonicas, can attenuate H2O2-induced oxidative stress in SH-SY5Y cells. PURPOSE: The aim of the present study was to investigate the neuroprotective effects and the possible underlying mechanism of CHS V on transient focal cerebral ischemia/reperfusion. METHODS: Mice with middle cerebral artery occlusion (MCAO) and cultured cortical neurons exposed to oxygen glucose deprivation (OGD) were used as in vivo and in vitro models of cerebral ischemia, respectively. The neurobehavioral scores, infarction volumes, H&E staining and some antioxidant levels in the brain were evaluated. The occurrence of neuronal death was estimated. Total and mitochondrial reactive oxygen species (ROS) levels, as well as mitochondrial potential were measured using flow cytometry analysis. Mitochondrial structure and respiratory activity were also examined. Protein levels were investigated by western blotting and immunohistochemistry. RESULTS: CHS V effectively attenuated cerebral ischemia/reperfusion (CI/R) injury, including improving neurological deficits, shrinking infarct volume and reducing the number of apoptotic cells. Furthermore, CHS V treatment remarkably increased antioxidant levels and reduced ROS levels and mitochondrial damage by enhancing the expression and deacetylation of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) by activating AMPK and SIRT-1, respectively. CONCLUSION: Our data demonstrated that CHS V prevented CI/R injury by suppressing oxidative stress and mitochondrial damage through the modulation of PGC-1α with AMPK and SIRT-1.

Neuroprotective Effects of a Novel Inhibitor of c-Jun N-Terminal Kinase in the Rat Model of Transient Focal Cerebral Ischemia.

A novel specific inhibitor of c-Jun N-terminal kinase, 11H-indeno[1,2-b]quinoxalin-11-one oxime sodium salt (IQ-1S), has a high affinity to JNK3 compared to JNK1/JNK2. The aim of this work was to study the mechanisms of neuroprotective activity of IQ-1S in the models of reversible focal cerebral ischemia (FCI) in Wistar rats. The animals were administered with an intraperitoneal injection of IQ-1S (5 and 25 mg/kg) or citicoline (500 mg/kg). Administration of IQ-1S exerted a pronounced dose-dependent neuroprotective effect, not inferior to the effects of citicoline. Administration of IQ-1S at doses of 5 and 25 mg/kg reduced the infarct size by 20% and 50%, respectively, 48 h after FCI, whereas administration of citicoline reduced the infarct size by 34%. The administration of IQ-1S was associated with a faster amelioration of neurological status. Control rats showed a 2.0-fold increase in phospho-c-Jun levels in the hippocampus compared to the corresponding values in sham-operated rats 4 h after FCI. Administration of IQ-1S at a dose of 25 mg/kg reduced JNK-dependent phosphorylation of c-Jun by 20%. Our findings suggest that IQ-1S inhibits JNK enzymatic activity in the hippocampus and protects against stroke injury when administered in the therapeutic and prophylactic regimen in the rat model of FCI.

MicroRNA-193b-3p alleviates focal cerebral ischemia and reperfusion-induced injury in rats by inhibiting 5-lipoxygenase expression.

AIMS: Ischemic stroke has become one of the main causes of death worldwide. MicroRNAs (miRNAs) have been implicated in cerebral ischemia-reperfusion (I/R) injury and could serve as therapeutic targets. 5-Lipoxygenase (5-LOX) is a key enzyme in the biosynthesis of leukotrienes and has been implicated in inflammatory central nerve system disorders. The objective of this study was to explore the neuroprotective effects of miR-193b-3p against focal cerebral I/R injury in rats by regulating 5-LOX expression. METHODS AND MATERIALS: Adult male Sprague-Dawley rats were subjected to transient middle cerebral artery occlusion and reperfusion injury. The level of miR-193b-3p expression was observed in the rat cortical peri-infarct region after focal cerebral I/R injury. Bioinformatics analysis was used to predict the binding sites of miR-193b-3p, and a dual-luciferase reporter gene assay was applied to verify the potential interaction between 5-LOX mRNA and miR-193b-3p. Then, rats were injected with a miR-193b-3p agomir (modified and enhanced mimic) or antagomir (modified and enhanced inhibitor) in the right lateral ventricle of the brain. Neurological deficit scores, infarct volumes, neuron damage and 5-LOX enzymatic activity and expression were measured. In an in vitro experiment, cultured PC12 cells were exposed to oxygen-glucose deprivation and reperfusion (OGD/R). OGD/R-induced cells were treated with a miR-193b-3p mimic or inhibitor and 5-LOX siRNA. Cell viability, lactate dehydrogenase release, apoptosis rate and 5-LOX expression were evaluated. RESULTS: The level of miR-193b-3p expression was increased in the cortical peri-infarct region of rats with cerebral focal I/R injury. The results of the dual-luciferase reporter gene assay showed that a miR-193b-3p binding site was located in the 3' untranslated region (3'UTR) of 5-LOX mRNA. Neurological deficit scores, infarct volumes and neuronal injury were alleviated by miR-193b-3p agomir treatment but aggravated by miR-193b-3p antagomir. Furthermore, leukotriene B4, cysteinyl-leukotrienes and 5-LOX expression in the cortical peri-infarct region of rats with focal cerebral I/R injury were also downregulated by miR-193b-3p agomir treatment but upregulated by miR-193b-3p antagomir. In PC12 cells, miR-193b-3p mimic significantly decreased OGD/R-induced cell death and reduced lactate dehydrogenase release and 5-LOX expression. In contrast, miR-193b-3p inhibitor exacerbated OGD/R-induced injury in PC12 cells. Additionally, the in vitro effects of miR-193b-3p inhibitor on OGD/R-induced cell injury were partially reversed by 5-LOX siRNA treatment. CONCLUSION: MiR-193b-3p has a potentially neuroprotective effect on focal cerebral I/R-induced injury by inhibiting 5-LOX expression.

Automated label-free detection of injured neuron with deep learning by two-photon microscopy.

Stroke is a significant cause of morbidity and long-term disability globally. Detection of injured neuron is a prerequisite for defining the degree of focal ischemic brain injury, which can be used to guide further therapy. Here, we demonstrate the capability of two-photon microscopy (TPM) to label-freely identify injured neurons on unstained thin section and fresh tissue of rat cerebral ischemia-reperfusion model, revealing definite diagnostic features compared with conventional staining images. Moreover, a deep learning model based on convolutional neural network is developed to automatically detect the location of injured neurons on TPM images. We then apply deep learning-assisted TPM to evaluate the ischemic regions based on tissue edema, two-photon excited fluorescence signal intensity, as well as neuronal injury, presenting a novel manner for identifying the infarct core, peri-infarct area, and remote area. These results propose an automated and label-free method that could provide supplementary information to augment the diagnostic accuracy, as well as hold the potential to be used as an intravital diagnostic tool for evaluating the effectiveness of drug interventions and predicting potential therapeutics.

Light Sheet Microscopy Using FITC-Albumin Followed by Immunohistochemistry of the Same Rehydrated Brains Reveals Ischemic Brain Injury and Early Microvascular Remodeling.

Until recently, the visualization of cerebral microvessels was hampered by the fact that only short segments of vessels could be evaluated in brain sections by histochemistry. These limitations have been overcome by light sheet microscopy, which allows the 3D analysis of microvasculature in cleared brains. A major limitation of light sheet microscopy is that antibodies do not sufficiently penetrate cleared brains. We herein describe a technique of reverse clearing and rehydration, which after microvascular network analysis allows brain sectioning and immunohistochemistry employing a broad set of antibodies. Performing light sheet microscopy on brains of mice exposed to intraluminal middle cerebral artery occlusion (MCAO), we show that in the early phase of microvascular remodeling branching point density was markedly reduced, more strongly than microvascular length. Brain infarcts in light sheet microscopy were sharply demarcated by their autofluorescence signal, closely corresponding to brain infarcts revealed by Nissl staining. Neuronal survival, leukocyte infiltration, and astrocytic reactivity could be evaluated by immunohistochemistry in rehydrated brains, as shown in direct comparisons with non-cleared brains. Immunohistochemistry revealed microthrombi in ischemic microvessels that were likely responsible for the marked branching point loss. The balance between microvascular thrombosis and remodeling warrants further studies at later time-points after stroke.

Effect of Panax notoginseng Saponins on Focal Cerebral Ischemia-Reperfusion in Rat Models: A Meta-Analysis.

With the increase of the aging population, the high mortality and disability rates caused by ischemic stroke are some of the major problems facing the world, and they dramatically burden the society. Panax notoginseng (Burk) F. H. Chen, a traditional Chinese medicine, is commonly used for promoting blood circulation and removing blood stasis, and its main bioactive components are Panax notoginseng saponins (PNS). Therefore, we performed a meta-analysis on focal cerebral ischemia-reperfusion animal models established with middle cerebral artery occlusion (MCAO) surgery to evaluate the therapeutic effect of PNS. We systematically searched the reports of PNS in MCAO animal experiments in seven databases. We assessed the study quality using two literature quality evaluation criteria; evaluated the efficacy of PNS treatment based on the outcomes of the neurological deficit score (NDS), cerebral infarct volume (CIV), and biochemical indicators via a random/fixed-effects model; and performed a subgroup analysis utilizing ischemia duration, drug dosage, intervention time, and administration duration. We also compared the efficacy of PNS with positive control drugs or combination treatment. As a result, we selected 14 eligible studies from the 3,581 searched publications based on the predefined exclusion-inclusion criteria. PNS were significantly associated with reduced NDS, reduced CIV, and inhibited release of the inflammatory factors IL-1ß and TNF-α in the focal MCAO rat models. The PNS combination therapy outperformed the PNS alone. In addition, ischemia time, drug dosage, intervention time, and administration duration in the rat models all had significant effects on the efficacy of PNS. Although more high-quality studies are needed to further determine the clinical efficacy and guiding parameters of PNS, our results also confirmed that PNS significantly relieves the focal cerebral ischemia-reperfusion in rat models. In the animal trials, it was suggested that an early intervention had significant efficacy with PNS alone or PNS combination treatment at a dosage lower than 25 mg/kg or 100-150 mg/kg for 4 days or longer. These findings further guide the therapeutic strategy for clinical cerebral ischemic stroke.

Conditioned medium obtained from human amniotic mesenchymal stem cells attenuates focal cerebral ischemia/reperfusion injury in rats by targeting mTOR pathway.

Conditioned medium obtained from human amniotic mesenchymal stem cells (hAMSC-CM) was recently shown to have many antioxidant, antiapoptotic and proangiogenic growth factors. The present study was performed to investigate whether protective effects of hAMSC-CM against focal cerebral ischemia/ reperfusion (I/R) injury is associated with modulation of the mammalian target of rapamycin (mTOR) pathway. A rat model of middle cerebral artery occlusion (MCAO) was created and the animals were divided into three groups including sham, MCAO and MCAO + hAMSC-CM. Drug was administrated immediately after cerebral reperfusion (i.v). The expressions of mTOR, p-mTOR and LC3 were measured using Western blotting and real time-PCR, respectively. Apoptosis and neuronal loss were determined using TUNEL and Nissl staining, respectively. Infarct volume and the blood-brain barrier (BBB) damage were evaluated using 2,3,5-triphenyltetrazolium chloride (TTC) staining and Evans Blue (EB) uptake, respectively. Compared with sham, significant infarct volume, apoptotic cell death, and neuronal loss were found in MCAO rats that reversed by hAMSC-CM (P < 0.05). Likewise, MCAO rats exhibited increased mRNA level of light-chain 3 (LC3) and the LC3II/LC3I ratio as well as decreased expression level of p-mTOR that reversed by hAMSC-CM (P < 0.05). There were no significant differences in the expression of total mTOR among the experimental groups. In summary, our results demonstrate that hAMSC-CM gives rise to neuroprotection following ischemic stroke by restoring mTOR activity and inhibiting autophagy.

Synthesis, characterization and in vivo evaluation of honokiol bisphosphate prodrugs protects against rats' brain ischemia-reperfusion injury.

Honokiol (HK) usage is greatly restricted by its poor aqueous solubility and limited oral bioavailability. We synthesized and characterized a novel phosphate prodrug of honokiol (HKP) for in vitro and in vivo use. HKP greatly enhanced the aqueous solubility of HK (127.54 ±â€¯15.53 mg/ml) and the stability in buffer solution was sufficient for intravenous administration. The enzymatic hydrolysis of HKP to HK was extremely rapid in vitro (T1/ 2  = 8.9 ±â€¯2.11 s). Pharmacokinetics studies demonstrated that after intravenous administration of HKP (32 mg/kg), HKP was converted rapidly to HK with a time to reach the maximum plasma concentration of ∼5 min. The prodrug HKP achieved an improved T1/2 (7.97 ±â€¯1.30 h) and terminal volume of distribution (26.02 ±â€¯6.04 ml/kg) compared with direct injection of the equimolar parent drug (0.66 ±â€¯0.01 h) and (2.90 ±â€¯0.342 ml/kg), respectively. Furthermore, oral administration of HKP showed rapid and improved absorption compared with the parent drug. HKP was confirmed to maintain the bioactivity of the parent drug for ameliorating ischemia-reperfusion injury by decreasing brain infarction and improving neurologic function. Taken together, HKP is a potentially useful aqueous-soluble prodrug with improved pharmacokinetic properties which may merit further development as a potential drug candidate.

MiR-34b Protects Against Focal Cerebral Ischemia-Reperfusion (I/R) Injury in Rat by Targeting Keap1.

Ischemic stroke is one of the leading causes of death and disability globally and has been regarded as a major public health problem. Understanding the mechanism of ischemia/reperfusion (I/R)-induced oxidative stress injury may provide new treatment for ischemic stroke. Kelch-like ECH-associated protein 1 (Keap1)/ NF-E2-related factor 2 (Nrf2)/ antioxidant response elements (ARE) signaling pathway has been considered to be the major cellular defense against oxidative stress. In the present study, our objective is to evaluate the molecular mechanism of miR-34b/Keap1 in modulating focal cerebral I/R induced oxidative injury. miR-34b was predicted to target the 3'-UTR of the rat Keap1. After focal cerebral I/R, miR-34b expression was downregulated in a time-dependent manner; miR-34b overexpression ameliorated I/R-induced oxidative stress injury in middle cerebral artery occlusion (MCAO) rats by reducing the infarction volume, the neurological severity scores, the levels of nitric oxide (NO) and (3-nitrotyrosine) 3-NT while increasing total (superoxide dismutases) SOD and manganese SOD (MnSOD). Through direct targeting, miR-34b could suppress the protein levels of Keap1 and increase the protein levels of Nrf2 and heme oxygenase (HO-1). Regarding the molecular mechanism, Keap1 overexpression exacerbated, while miR-34b improved H2O2-induced oxidative stress injury; the effect of miR-34b could be partially attenuated by Keap1 overexpression, suggesting that miR-34b modulated oxidative stress injury in vitro and in vivo through targeting Keap1. Taken together, we demonstrate that miR-34b protects against focal cerebral I/R-induced oxidative stress injury in MCAO rats and H2O2-induced oxidative stress injury in rat neuroblast B35 cells through targeting Keap1 and downstream Keap1/Nrf2 signaling pathway. We provided a novel mechanism of focal cerebral I/R injury from the perspective of miRNA regulation.

Neuroprotective Effects of Dibornol in Focal Cerebral Ischemia/Reperfusion in Rats.

Neuroprotective activity of 2,6-diisobornyl-4-methylphenol (Dibornol) was studied under conditions of experimental focal cerebral ischemia/reperfusion modeled by intraluminal occlusion of the left middle cerebral artery for 1 h followed by recirculation. Dibornol administered in a dose of 10 mg/kg intragastrically 24 h and 30 min before and 24 h after focal ischemia/reperfusion modeling reduced the size of the brain infarction zone by 52% (48 h after recirculation) and neurological deficit by 1.7-2.4 times in comparison with that in control animals.

The protective role of verbenalin in rat model of focal cerebral ischemia reperfusion.

To investigate the protective mechanism of verbenalin on cerebral ischemia-reperfusion injury. Middle cerebral artery occlusion in the left hemisphere was induced in rats by filament insertion, and rat model of focal cerebral ischemia-reperfusion was established. The high, medium and low dose of verbenalin groups were injected in the tail vein of corresponding drugs 10 min before reperfusion, and submitted for 22 h of reperfusion after the operation. Mortality rate was then calculated, and neurological deficits of rats were scored. The serum of rats was got to determine the S-100ß protein level, and the brain tissue was removed to determine the levels of Bax, Bcl-2, Caspase-3 and ATPase. TTC staining was performed on the brain tissue to calculate the percentage of cerebral infarct size. Changes in brain tissue morphology were observed. Rat model of focal cerebral ischemia-reperfusion was successfully replicated. In groups that have taken different doses of verbenalin, the mortality rate, neurological deficit score and the percentage of cerebral infarction size were significantly reduced, and the levels of Bax, Caspase-3, S-100ß level of the serum in the brain tissue were also significantly reduced. Increases in the levels of Bcl-2 and ATPase in brain tissue and improvement of pathological damage of hippocampus and cortex were observed. Verbenalin can inhibit the expression of apoptosis genes, promote the expression of anti-apoptosis genes, improve brain microcirculation and energy metabolism, hence reducing cerebral ischemia-reperfusion injury.

[Electroacupuncture Combined with Intracerebral Injection of VEGF Improves Neurological Dysfunction Possibly by Down-regulating Expression of Endoplasmic Reticulum Stress Related Proteins ATF 6, etc. in Cerebral Ischemia-reperfusion Injury Rats].

OBJECTIVE: To observe the effect of electroacupuncture (EA) and EA combined with intracerebral injection of vascular endothelial growth factor (VEGF) on endoplasmic reticulum stress (ERS) related proteins and genes as activating transcription factor (ATF 6), inositol requiring enzyme-1 (IRE 1), CCAAT/enhancer binding protein homologous protein (CHOP), X box-binding protein-1 (XBP 1) of cerebral ischemia reperfusion injury (CIRI) rats, so as to study its repair effect for CIRI. METHODS: Forty male SD rats were equally and randomly divided into 5 groups: sham operation, model, EA, VEGF and EA+VEGF groups (n=8). The CIRI model was established by occlusion of the middle cerebral artery (MCAO) with thread embolism method. For rats of the sham operation group, the right common carotid artery was isolated without MCAO. EA (2 Hz/100 Hz, 1-3 mA) was applied to "Baihui" (GV 20), left "Quchi" (LI 11) and left "Zusanli" (ST 36) for 30 min, once a day for 14 days. For rats of the VEGF and EA+VEGF groups, 10 µL VEGF (0.025 µg/µL) was injected into the lateral ventricle 24 h after successful modeling. The rats' neurological function was assessed by using the modified neurological severity score (mNSS), and the histopathological changes of cerebral tissue were observed by Nissl staining method. The expression levels of ERS related proteins and genes ATF 6, IRE 1, XBP 1 and CHOP were determined by western blot (WB) and fluorescent quantitative PCR, separately. RESULTS: After modeling, the level of mNSS was significantly higher in the model group than in the sham operation group (P<0.05), and the number of Nissl bodies was markedly lower in the model group than in the sham operation group (P<0.05). Following the treatment, the mNSS was significantly lower in the EA, VEGF and EA+VEGF groups than in the model group (P<0.05), and the numbers of Nissl bodies were obviously higher in the EA, VEGF and EA+VEGF groups than in the model group (P<0.05), suggesting an improvement of neurological dysfunction and a repair of the injured cerebral tissue after the treatment. The levels of CIRI-induced increase of mNSS and CIRI-induced decrease of the number of Nissl bodies in the EA+VEGF group were respectively remarkably lower or higher than those of the simple EA and simple VEGF groups (P<0.05). WB and PCR showed that the expression levels of ATF 6, IRE 1, XBP1 and CHOP proteins and genes were notably higher in the model group than in the sham operation group (P<0.05), and considerably lower in the EA, VEGF and EA+VEGF groups than in the model group (P<0.05). Comparison among the three treatment groups showed that after the treatment, the expression levels of ATF 6, IRE 1, XBP1 and CHOP proteins and genes were obviously lower in the EA+VEGF group than in the EA and VEGF groups (P<0.05). CONCLUSION: EA and EA plus intracerebral microinjection of VEGF can improve neurological function and promote cerebral tissue repair in CIRI rats, which is associated with their effects in down-regulating the expression of ERS related proteins ATF 6, IRE 1, XBP1 and CHOP. The effect of EA+VEGF is superior to that of simple EA and simple VEGF.

Microglia TREM2 is required for electroacupuncture to attenuate neuroinflammation in focal cerebral ischemia/reperfusion rats.

Neuroinflammation plays a critical role in ischemic stroke pathology and could be a promising target in ischemic stroke. Triggering receptor expressed on myeloid cells 2 (TREM2) is a microglia-specific receptor in the CNS that is involved in regulating neuroinflammation in cerebral ischemia. However, the role of TREM2 in ischemic stroke is controversial. Electroacupuncture (EA) is an effective therapy for alleviating stroke-induced neuroinflammation. Here, we found that ischemic stroke induced an increased microglial TREM2 expression, and EA treatment can further promote microglial TREM2 expression following cerebral ischemia. TREM2 overexpression was observed to play a neuroprotective role by improving the neurobehavioral deficit and reducing the cerebral infarct volume 72 h after reperfusion, whereas TREM2 silencing had the opposite effects. Moreover, the effects of EA on improving stroke outcome and suppressing neuroinflammation in the brain were reversed by TREM2 silencing. Finally, TREM2 silencing also suppressed the ability of EA to regulate the PI3K/Akt and NF-κB signaling pathways. Altogether, the results show that TREM2 could be a potential target in EA treatment for attenuating inflammatory injury following cerebral ischemia/reperfusion.

Protective effects of Progranulin against focal cerebral ischemia-reperfusion injury in rats by suppressing endoplasmic reticulum stress and NF-κB activation in reactive astrocytes.

The aim of this study is to explore the effect progranulin (PGRN) has on endoplasmic reticulum (ER) stress and the NF-κB activation in reactive astrocytes found in rat models with focal cerebral ischemia-reperfusion (I/R) injury. Sprague-Dawley (SD) rats were grouped into the sham, I/R, PGRN-high dose, PGRN-low dose, and negative control (NC) groups. TTC staining was applied in order to detect the cerebral infarction volume, a TUNEL assay to detect the apoptosis rate of neurons, an ELISA to measure MDA, SOD, iNOS, LDH, TNF-α, IL-1ß, IL-6, and IL-10 levels, and both RT-qPCR and western blotting methods in order to detect PGRN, GFAP, GRP78, CHOP, and NF-κB p65 expressions. The astrocytes (AST) cells were then assigned into the normal, I/R, negative control (NC), PGRN-high dose, and PGRN-low dose groups. After completing the transfection process, the proliferative capacity of AST cells was detected by use of the CCK-8 assay. Both the in vivo and in vitro results, in comparison with the I/R and the NC groups, the PGRN-high dose and PGRN-low dose groups both presented a decrease in cerebral infarction volume, apoptosis rate of neurons, MDA, LDH, iNOS, TNF-α, IL-1ß, IL-6 levels, and GFAP, GRP78, CHOP, NF-κB p65 expressions, and an increase in SOD, IL-10, and PGRN levels as well as cell proliferation depending on dosage. Based on our results, we came to the confirmation that PGRN can reduce neuronal apoptosis by mitigating ER stress in the reactive astrocytes as well as downregulating the inflammatory levels by suppressing the NF-κB signaling 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.

The effect of focal cerebral ischemia-reperfusion injury on TLR4 and NF-κB signaling pathway.

The present study analyzed the change of Toll-like receptor 4 (TLR4) and nuclear factor-κB (NF-κB) expression in focal cerebral ischemia-reperfusion injury model. A sample of 36 Sprague-Dawley rats were randomly selected and divided into sham operation group (group S), control group (group C) and Chrysanthemum ester group (NF-κB inhibitor, group CE), each group consisted of 12 rats. The rat model of focal cerebral ischemia-reperfusion was established. The physiological indexes and neurological severity score of rats was recorded by a double-blind method. The cerebral infarction area was evaluated by triphenyltetrazole oxide (TTC) staining on brain slices. Apoptosis was evaluated by TUNEL staining. Semi-quantitative PCR and western blot analysis was used to measure the expression of TLR4 and NF-κB. The neurological severity score of rats in group C and CE were found to be significantly lower than group S (P<0.01). The TTC staining results showed that group C and CE had different levels of cerebral infarction but the area of infarction in group CE was significantly lower than group C (P<0.01). In addition, the number of TUNEL positive cells in group CE was significantly lower than group C (P<0.01). Semi-quantitative PCR and westernblot analysis results showed that the expression of NF-κB and TLR4 of group S was significantly lower than that of group C and group CE (P<0.01), the relative expression of NF-κB and TLR4 of group CE was significantly lower than that of group C (P<0.01). Moreover, the expression of NF-κB p65/p50 of group CE and group C was significantly higher than that of group S (P<0.01). This study concludes that the focal cerebral ischemia-reperfusion injury in rats can cause brain damage and cell apoptosis. This effect might be associated to the increased expression of NF-κB and TLR4, and the activation of TLR4/NF-κB signaling pathway.

Sesamol attenuates oxidative stress, apoptosis and inflammation in focal cerebral ischemia/reperfusion injury.

The aim of the present study was to evaluate the therapeutic potential of sesamol treatment on focal ischemia/reperfusion (I/R) injury in the rat brain. The results demonstrated that pretreatment with sesamol seven days prior to focal cerebral I/R injury had significant positive effects, including improvements in neurological deficits (P<0.05), and a reduction in malondialdehyde content and elevation of antioxidant levels (superoxide dismutase, glutathione and glutatione peroxidase; both P<0.05). Furthermore, levels of B cell lymphoma-2 (Bcl-2)-associated X protein and caspase-3 were significantly downregulated, whereas the level of Bcl-2 was effectively increased. Conversely, the mRNA expression of proinflammatory cytokines were significantly reduced in focal cerebral I/R injury rats upon sesamol intervention. Therefore, the beneficial effects of sesamol on cerebral I/R injury may be due to the reduction of oxidative stress, inhibition of apoptosis and inflammation. The findings of the present study suggest that sesamol supplementation may serve as potent adjuvant in the treatment of focal cerebral ischemia/reperfusion injury due to its neuroprotective effects.