Erythropoietin-derived peptide ARA290 mediates brain tissue protection through the ß-common receptor in mice with cerebral ischemic stroke.

AIM: To explore the neuroprotective effects of ARA290 and the role of ß-common receptor (ßCR) in a mouse model of middle cerebral artery occlusion (MCAO). METHODS: This study included male C57BL/6J mice that underwent MCAO and reperfusion. The neuroprotective effect of ARA290 on MCAO-induced brain injury was investigated using neurological function tests (Longa and modified neurological severity score). Cerebral infarction was examined by 2, 3, 5-triphenyl tetrazolium chloride staining, neuronal apoptosis was assessed by immunofluorescence staining, blood parameters were measured using a flow cytometry-based automated hematology analyzer, liquid chromatography with tandem mass spectrometry was used to identify the serum metabolomics signature, inflammatory cytokines and liver index were detected by commercially available kits, and the protein levels of the erythropoietin (EPO) receptor and ßCR were measured by western blot. RESULTS: ARA290 exerted a qualitatively similar neuroprotective effect after MCAO as EPO. ARA290 significantly reduced neuronal apoptosis and the level of inflammatory cytokines in the brain tissue. However, ARA290's neuroprotective effect was significantly suppressed following the injection of siRNA against ßCR. CONCLUSION: ARA290 provided a neuroprotective effect via ßCR in cerebral ischemic mice without causing erythropoiesis. This study provides novel insights into the role of ARA290 in ischemic stroke intervention.

Pathological Observation of Blood Stasis Syndrome in Non-diabetic Peripheral Neuropathies: A Retrospective Analysis Based on Nerve Biopsy.

OBJECTIVE: To investigate the pathological features of blood stasis syndrome (BSS) in non-diabetic peripheral neuropathy. METHODS: Clinical data of 31 patients with non-diabetic peripheral neuropathy who had undergone nerve biopsy during December 2004 and December 2010 in Xuanwu Hospital Capital Medical University were retrospectively analyzed. According to Chinese medicine (CM) syndrome differentiation and signs, 26 patients were blood stasis type and 5 patients were non-blood stasis type. Clinical and pathological data were compared in detail. RESULTS: Clinically, although both groups shared similar symptoms of limb numbness, weakness and sensory disturbances, the prevalence of neuralgia was much grievous in BSS group (73.1%, 26/31) compared with the non-BSS group (0%, 0/5). As for signs, dermal nutrients disturbance (84.6%, 22/26), dark or purple tongue (100.0%, 26/26), and sublingual varices (80.7%, 21/26) were more common in the BSS group than the non-BSS group (0%, 60%, 20%, respectively). The prevalence of qi deficiency cases (19/26) in the BSS group was significantly higher compared with the non-BSS group (1/5). The unique histological manifestations of BSS were axonal degeneration (16/26 vs 2/5 in non-BSS group), which was the hallmark of ischemia. Cases with BSS had prominent microangiopathy (61.5%, 16/26), manifested as epineurium vasculitis (inflammatory cell infiltrated to the vessel wall, obliteration and recanalization, vascular proliferation, extravascular hemosiderin deposition), angiotelectasis, proliferation and hyaline degeneration of endoneurium capillary. In the BSS group, impaired blood-nerve barrier was indicated by sub-perineurial edema (46.2%, 11/26) and endoneurial edema (15.4%, 4/26). The Renaut body (15.4%, 4/26) and amyloid deposition (3.8%, 1/26) found in the BSS group were absent in the non-BSS group. CONCLUSIONS: BBS was common in non-diabetic peripheral neuropathies. The nerves exhibited ischemic alteration of primary axon degeneration and secondary demyelination. The interstitial tissue revealed microcirculation impairment, blood-nerve barrier disturbance, amyloid deposition and proliferation changes. The high prevalence of qi deficiency also highlights the therapy of promotion of blood circulation and removal of blood stasis.

Huoluo Yinao decoction mitigates cognitive impairments after chronic cerebral hypoperfusion in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Huoluo Yinao decoction (HLYND) has been used to ameliorate cognitive impairment induced by chronic cerebral hypoperfusion in clinical for years. However, the exact mechanisms remain unknown. AIM OF THE STUDY: To investigate the effects and mechanisms underlying HLYND-mediated improvement in cognitive deficits associated with chronic cerebral hypoperfusion. MATERIALS AND METHODS: Thirty-six Sprague-Dawley rats were randomly allocated to three groups: sham, model, and HLYND. Daily administration of HLYND or volume-matched vehicle by gavage was initiated 1 day after bilateral carotid artery stenosis (BCAS) and continued for 42 days. The Morris water maze (MWM) test was used to assess cognitive functions from days 36-42. Via western blot and immunofluorescent staining, restoration of neuronal plasticity and remyelination of white matter were evaluated by analyzing the expression profiles of MAP-2, synaptophysin and MBP. In addition, macrophage/microglial activation was assessed by quantifying changes in Iba1, and macrophage/microglial polarization was assessed by changes in iNOS and CD16 (M1 markers), as well as Arg1 and CD206 (M2 markers). RESULTS: In the MWM test, BCAS rats showed significantly extended escape latency and reduced platform crossing times, while those in the HLYND group had shortened escape latency and increased frequency of platform crossing. In addition, rats in the model group showed decreased levels and abnormal morphological changes of MAP-2, synaptophysin and MBP, whereas HLYND administration reversed these effects. As expected, Iba1 levels were elevated in both the model and HLYND groups but rats in the model group showed increased levels of the M1 markers, iNOS and CD16, and a correspondent decrease in the M2 marker, Arg1. In contrast, in the HLYND group, iNOS and CD16 levels were suppressed, while Arg1 levels were elevated. CONCLUSIONS: Our findings demonstrate that HLYND mitigates cognitive impairment after chronic cerebral hypoperfusion in rats through mechanisms involving increased neuronal plasticity and white matter remyelination, with a subtile modulation of macrophage/microglial polarization toward the M2 phenotype.

Unique MicroRNAs Signature of Lymphocyte of Yang and Yin Syndromes in Acute Ischemic Stroke Patients.

OBJECTIVE: To identify the differentially expressed microRNAs (miRNAs) profiles of yang and yin syndromes in patients with acute ischemic stroke, and to provide the molecular basis of the classification of these two syndrome types in acute ischemic stroke patients. METHODS: A microarray assay was performed to assess the expression pattern of miRNAs in the lymphocyte of acute ischemic stroke patients. Target genes for the deregulated miRNAs were predicated using the online bioinformatic algorithms and functional annotation via Kyoto encyclopedia of genes and genomes pathway analysis for miRNAs predicted targets was carried out. Based on the predicted target genes of differentially expressed miRNAs, the miRNA-gene-network and miRNA-pathway-network were constructed. RESULTS: Yang score based on tongue texture, urine, dejecta, and appearance, etc. showed that clinical symptoms were distinct between yang and yin syndromes. There were significantly higher total leukocyte number and lower total protein level in patients with yang syndrome compared with those in patients with yin syndrome (P<0.05). Comprehensive miRNA analysis identified 36 unique down-regulated miRNAs in yang syndrome group, and 20 unique down-regulated and 2 unique up-regulated miRNAs in yin syndrome group. The key regulatory miRNAs, gene, and pathways in the yang syndrome were hsa-miR-93-5p and -320b, enabled homolog, the metabolic pathways and mitogen-activated protein kinase signaling pathways, respectively, while those in the yin syndrome were hsa-miR-424-5p and -106b-5p, CNOT4, hepatitis B and pathways in cancer, respectively. CONCLUSION: These results offered insight into the molecular basis underlying the different pathogenesis of yang or yin syndrome, providing clues for the individualized therapeutic strategies of acute ischemic stroke.

AKT/GSK3ß-dependent autophagy contributes to the neuroprotection of limb remote ischemic postconditioning in the transient cerebral ischemic rat model.

BACKGROUND: Limb remote ischemic postconditioning (RIPostC) has been recognized as an applicable strategy in protecting against cerebral ischemic injury. However, the time window for application of limb RIPostC and the mechanisms behind RIPostC are still unclear. AIMS: In this study, we investigated the protective efficacy and the role of autophagy in limb RIPostC using a transient middle cerebral artery occlusion rat model. RESULTS: Limb RIPostC applied in the early phase of reperfusion reduced infarct size and improved neurological function. Autophagy levels in penumbral tissues were elevated in neurons of limb RIPostC rats, with an increase in the phosphorylation of AKT and glycogen synthase kinase 3ß (GSK3ß). Blocking the AKT/GSK3ß pathway via the AKT inhibitor LY294002 prior to limb RIPostC suppressed the RIPostC-induced autophagy and resulted in the activation of caspase-3 in RIPostC rats, suggesting a critical role for AKT/GSK3ß-dependent autophagy in reducing cell death after cerebral ischemia. CONCLUSIONS: These results aid optimization of the time window for RIPostC use and offer novel insight into, and a better understanding of, the protective mechanism of autophagy in limb RIPostC.

[Dephosphorelation of Bad and upregulation of Bcl-2 in hippocampus of rats following limbic seizure induced by kainic acid injection into amygdaloid nucleus].

The purpose of the present study was to explore the seizure-induced changes in Bad (Bcl-2-associated death protein), 14-3-3, phosphoBad, Bcl-2 and Bcl-XL expression in the rat model of focal limbic seizure. Unilateral intra-amygdaloid injection of kainic acid (KA) was made to induce seizure. Electroencephalogram (EEG) and regional cerebral flow (r-CBF) were monitored continuously. Diazepam (30 mg/kg) was administered to terminate the seizure. The apoptotic and surviving neurons in the hippocampus were observed by terminal deoxynucleotidyl transferrase-mediated dUTP nick end labeling (TUNEL) and cresyl violet staining, the expression of Bad, 14-3-3, phosphoBad, Bcl-2 and Bcl-XL were detected with immunofluorescence, Western blot and immunoprecipitation. The results showed that TUNEL-positive neurons appeared at 8 h and reached maximum at 24 h following seizure cessation within the ipsilateral CA3 subfield of the hippocampus. Seizure induced the dephosphorylation of Bad and the dissociation of Bad from its chaperone protein 14-3-3 and subsequent dimerization of Bad with Bcl-XL. The expression of phosphoBad decreased and Bcl-2 increased. There was little change in r-CBF after the seizure. These results suggest that seizure leads to a dephosphorylation of Bad and an upregulation of Bcl-2. Dephosphorylation of Bad may be injurious while the upregulation of Bcl-2 may be protective to the brain damage induced by seizures, but not related with r-CBF.