[Effect of Naotaifang on microglial polarization and glial scar following cerebral ischemia reperfusion injury].

This study aims to investigate the effect of Naotaifang(NTF) on the proteins associated with microglial polarization and glial scar in the rat model of cerebral ischemia reperfusion injury(CIRI). The CIRI model was established by middle cerebral artery occlusion/reperfusion. The 48 successfully modeled rats were randomized into model 7 d, model 14 d, NTF 7 d, and NTF 14 d groups(n=12). In addition, 12 SD rats were selected as the sham group. The NTF group was administrated with NTF suspension at 27 g·kg~(-1)·d~(-1) by gavage, and the sham, model 7 d, and model 14 d groups were administrated with the same volume of normal saline every day by gavage for 7 and 14 days, respectively. After the intervention, Longa score was evaluated. The infarct volume was measured by 2,3,5-triphenyl-2H-tetrazolium chloride(TTC) staining. Morris water maze and open field tests were carried out to evaluate the spatial learning, memory, cognitive function, and anxiety degree of rats. Hematoxylin-eosin(HE) staining was employed to observe the morphological structure and damage of the brain tissue. The immunofluorescence assay was employed to measure the expression of glial fibrillary acidic protein(GFAP) and glial scar. Western blot was employed to determine the protein levels of GFAP, neurocan, phosphacan, CD206, arginase-1(Arg-1), interleukin(IL)-1ß, IL-6, and IL-4. Compared with the sham, model 7 d and model 14 d groups showed cerebral infarction of different degrees, severe pathological injury of cerebral cortex and hippocampus, neurological impairment, reduced spatial learning and memory, cognitive dysfunction, severe anxiety, astrocyte hyperplasia, thickening penumbra glial scar, and up-regulated protein levels of IL-1ß, IL-6, GFAP, neurocan, phosphacan, CD206, and Arg-1(P<0.01). Compared with the model group, NTF 7 d and NTF 14 d groups improved spatial learning, memory, and cognitive function, reduced anxiety, improved nerve function, reduced cerebral infarction volume, reduced astrocyte hyperplasia, thinned penumbra glial scar, down-regulated the protein levels of GFAP, neurocan, phosphacan, IL-6, and IL-1ß, and up-regulated the protein levels of IL-4, CD206, and Arg-1(P<0.05 or P<0.01). NTF exerts a neuroprotective effect on CIRI by inducing the M2 polarization of microglia, inhibiting inflammatory response, and reducing the formation of glial scar.

No-reflow after recanalization in ischemic stroke: From pathomechanisms to therapeutic strategies.

Endovascular reperfusion therapy is the primary strategy for acute ischemic stroke. No-reflow is a common phenomenon, which is defined as the failure of microcirculatory reperfusion despite clot removal by thrombolysis or mechanical embolization. It has been reported that up to 25% of ischemic strokes suffer from no-reflow, which strongly contributes to an increased risk of poor clinical outcomes. No-reflow is associated with functional and structural alterations of cerebrovascular microcirculation, and the injury to the microcirculation seriously hinders the neural functional recovery following macrovascular reperfusion. Accumulated evidence indicates that pathology of no-reflow is linked to adhesion, aggregation, and rolling of blood components along the endothelium, capillary stagnation with neutrophils, astrocytes end-feet, and endothelial cell edema, pericyte contraction, and vasoconstriction. Prevention or treatment strategies aim to alleviate or reverse these pathological changes, including targeted therapies such as cilostazol, adhesion molecule blocking antibodies, peroxisome proliferator-activated receptors (PPARs) activator, adenosine, pericyte regulators, as well as adjunctive therapies, such as extracorporeal counterpulsation, ischemic preconditioning, and alternative or complementary therapies. Herein, we provide an overview of pathomechanisms, predictive factors, diagnosis, and intervention strategies for no-reflow, and attempt to convey a new perspective on the clinical management of no-reflow post-ischemic stroke.

Mitochondrial dysfunctions induce PANoptosis and ferroptosis in cerebral ischemia/reperfusion injury: from pathology to therapeutic potential.

Ischemic stroke (IS) accounts for more than 80% of the total stroke, which represents the leading cause of mortality and disability worldwide. Cerebral ischemia/reperfusion injury (CI/RI) is a cascade of pathophysiological events following the restoration of blood flow and reoxygenation, which not only directly damages brain tissue, but also enhances a series of pathological signaling cascades, contributing to inflammation, further aggravate the damage of brain tissue. Paradoxically, there are still no effective methods to prevent CI/RI, since the detailed underlying mechanisms remain vague. Mitochondrial dysfunctions, which are characterized by mitochondrial oxidative stress, Ca2+ overload, iron dyshomeostasis, mitochondrial DNA (mtDNA) defects and mitochondrial quality control (MQC) disruption, are closely relevant to the pathological process of CI/RI. There is increasing evidence that mitochondrial dysfunctions play vital roles in the regulation of programmed cell deaths (PCDs) such as ferroptosis and PANoptosis, a newly proposed conception of cell deaths characterized by a unique form of innate immune inflammatory cell death that regulated by multifaceted PANoptosome complexes. In the present review, we highlight the mechanisms underlying mitochondrial dysfunctions and how this key event contributes to inflammatory response as well as cell death modes during CI/RI. Neuroprotective agents targeting mitochondrial dysfunctions may serve as a promising treatment strategy to alleviate serious secondary brain injuries. A comprehensive insight into mitochondrial dysfunctions-mediated PCDs can help provide more effective strategies to guide therapies of CI/RI in IS.

A systematic review of the research progress of non-coding RNA in neuroinflammation and immune regulation in cerebral infarction/ischemia-reperfusion injury.

Cerebral infarction/ischemia-reperfusion injury is currently the disease with the highest mortality and disability rate of cardiovascular disease. Current studies have shown that nerve cells die of ischemia several hours after ischemic stroke, which activates the innate immune response in the brain, promotes the production of neurotoxic substances such as inflammatory cytokines, chemokines, reactive oxygen species and - nitrogen oxide, and mediates the destruction of blood-brain barrier and the occurrence of a series of inflammatory cascade reactions. Meanwhile, the expression of adhesion molecules in cerebral vascular endothelial cells increased, and immune inflammatory cells such as polymorphonuclear neutrophils, lymphocytes and mononuclear macrophages passed through vascular endothelial cells and entered the brain tissue. These cells recognize antigens exposed by the central nervous system in the brain, activate adaptive immune responses, and further mediate secondary neuronal damage, aggravating neurological deficits. In order to reduce the above-mentioned damage, the body induces peripheral immunosuppressive responses through negative feedback, which increases the incidence of post-stroke infection. This process is accompanied by changes in the immune status of the ischemic brain tissue in local and systemic systems. A growing number of studies implicate noncoding RNAs (ncRNAs) as novel epigenetic regulatory elements in the dysfunction of various cell subsets in the neurovascular unit after cerebral infarction/ischemia-reperfusion injury. In particular, recent studies have revealed advances in ncRNA biology that greatly expand the understanding of epigenetic regulation of immune responses and inflammation after cerebral infarction/ischemia-reperfusion injury. Identification of aberrant expression patterns and associated biological effects of ncRNAs in patients revealed their potential as novel biomarkers and therapeutic targets for cerebral infarction/ischemia-reperfusion injury. Therefore, this review systematically presents recent studies on the involvement of ncRNAs in cerebral infarction/ischemia-reperfusion injury and neuroimmune inflammatory cascades, and elucidates the functions and mechanisms of cerebral infarction/ischemia-reperfusion-related ncRNAs, providing new opportunities for the discovery of disease biomarkers and targeted therapy. Furthermore, this review introduces clustered regularly interspaced short palindromic repeats (CRISPR)-Display as a possible transformative tool for studying lncRNAs. In the future, ncRNA is expected to be used as a target for diagnosing cerebral infarction/ischemia-reperfusion injury, judging its prognosis and treatment, thereby significantly improving the prognosis of patients.

Uncovering the Neuroprotective Effect of Hedysarum multijugum Maxim-Chuanxiong Rhizoma Compound on Cerebral Infarction through Quantitative Proteomics.

Objective: To uncover the neuroprotective effect of Hedysarum multijugum Maxim-Chuanxiong Rhizoma compound (Huangqi-Chuanxiong Compound (HCC)) on cerebral infarction (CI) through quantitative proteomics. Method: CI model was established by the modified Zea Longa intracavitary suture blocking method. After modeling, the rats were given intragastric administration for 7 days, once a day. After the 7-day intervention, the neurological function score was performed, the brain tissue was pathologically observed, and the total serum protein was extracted. Then, these proteins were analyzed by LC-MS/MS to identify the differentially expressed proteins (DEPs) in the HCC/CI group and CI/sham operation group. Finally, bioinformatics analysis was used to analyze DEPs, including gene ontology (GO) analysis, pathway analysis, and protein interaction analysis. ELISA and Western blotting were used to verify the proteomics results. Result: The neurological function scores of the HCC group were lower than those of the CI group. HE staining showed that the pathological results of the HCC group were improved. A total of 1340 proteins were identified by LC-MS/MS, of which 1138 proteins contain quantitative information. There are 122 DEPs in the CI/sham operation group and 25 DEPs in the HCC/CI group with fold change >1.3 or <0.77 and FDR<0.05. The 12 upregulated proteins in HCC/CI group include Protein Actn2, Kelch-like protein 41, Alpha-1, 4 glucan phosphorylase, Protein Lrtm2, Dystrophin, Galectin-1, and C4b-binding protein beta chain. The 13 downregulated proteins include Alpha-2 antiplasmin, Arachidonate 15-lipoxygenase, Carbonic anhydrase 2, Complement factor I, angiotensinogen, catalase, Protein LOC103691744, and Anionic trypsin-1. The bioinformatics analysis showed that HCC may treat CI through regulating cell-substrate adhesion and regulation, reactive oxygen species metabolic process, angiotensin response (cellular response to angiotensin), positive regulation of the occurrence of nerves and neurons (positive regulation of neurogenesis), inflammatory response, response to hypoxia (response to hypoxia, response to decreased oxygen levels), and cellular calcium homeostasis (cellular calcium ion homeostasis). The results of ELISA and Western blot also showed that, compared with model group, the angiotensinogen and catalase in HCC group were decreased (P < 0.05), which is consistent with the findings of proteomics. Conclusion: The therapeutic mechanism of HCC in the treatment of CI may involve fibrinolysis, cell-matrix adhesion, inflammation, hypoxia, and oxidative stress.

Exploring the Mechanism of Edaravone for Oxidative Stress in Rats with Cerebral Infarction Based on Quantitative Proteomics Technology.

OBJECTIVE: To explore the mechanism of edaravone in the treatment of oxidative stress in rats with cerebral infarction based on quantitative proteomics technology. METHOD: The modified Zea Longa intracavitary suture blocking method was utilized to make rat CI model. After modeling, the rat was intragastrically given edaravone for 7 days, once a day. After the 7-day intervention, the total proteins of serum were extracted. After proteomics analysis, the differentially expressed proteins are analyzed by bioinformatics. Then chemoinformatics methods were used to explore the biomolecular network of edaravone intervention in CI. RESULT: The neurological scores and pathological changes of rats were improved after the intervention of edaravone. Proteomics analysis showed that in the model/sham operation group, 90 proteins in comparison group were upregulated, and 26 proteins were downregulated. In the edaravone/model group, 21 proteins were upregulated, and 41 proteins were downregulated. Bioinformatics analysis and chemoinformatics analysis also show that edaravone is related to platelet activation and aggregation, oxidative stress, intercellular adhesion, glycolysis and gluconeogenesis, iron metabolism, hypoxia, inflammatory chemokines, their mediated signal transduction, and so on. CONCLUSION: The therapeutic mechanism of edaravone in the treatment of CI may involve platelet activation and aggregation, oxidative stress, intercellular adhesion, glycolysis and gluconeogenesis, iron metabolism, hypoxia, and so on. This study revealed the serum protein profile of edaravone in the treatment of cerebral infarction rats through serum TMT proteomics and discovered the relevant mechanism of edaravone regulating iron metabolism in cerebral infarction, which provides new ideas for the study of edaravone intervention in cerebral infarction and also provides reference information for future research on the mechanism of edaravone intervention in iron metabolism-related diseases.

Extract of Naotaifang, a compound Chinese herbal medicine, protects neuron ferroptosis induced by acute cerebral ischemia in rats.

OBJECTIVE: Our previous research showed that Naotaifang (a compound traditional Chinese herbal medicine) extract (NTE) has clinically beneficial effects on neurological improvement of patients with acute cerebral ischemia. In this study, we investigated whether NTE protected acute brain injury in rats and whether its effects on ferroptosis could be linked to the dysfunction of glutathione peroxidase 4 (GPX4) and iron metabolism. METHODS: We established an acute brain injury model of middle cerebral artery occlusion (MCAO) in rats, in which we could observe the accumulation of iron in neurons, as detected by Perl's staining. Using assay kits, we measured expression levels of ferroptosis biomarkers, such as iron, glutathione (GSH), reactive oxygen species (ROS) and malonaldehyde (MDA); further the expression levels of transferrin receptor 1 (TFR1), divalent metal transporter 1 (DMT1), solute carrier family 7 member 11 (SLC7A11) and GPX4 were determined using immunohistochemical analysis, real-time quantitative polymerase chain reaction and Western blot assays. RESULTS: We found that treatment with NTE reduced the expression levels of TFR1 and DMT1, reduced ROS, MDA and iron accumulation and reduced neurobehavioral scores, relative to untreated MCAO rats. Treatment with NTE increased the expression levels of SLC7A11, GPX4 and GSH, and the number of Nissl bodies in the MCAO rats. CONCLUSION: Taken together, our data suggest that acute cerebral ischemia induces neuronal ferroptosis and the effects of treating MCAO rats with NTE involved inhibition of ferroptosis through the TFR1/DMT1 and SCL7A11/GPX4 pathways.

[Analysis on medication rules for treatment of dementia by ancient physicians based on data mining methods].

To analyze the medication features and regularity of prescriptions of Chinese medicine in treating patients with dementia based on ancient medical records. In the article, we retrieved the ancient medical records related to the treatment of dementia (from the Han Dynasty to the late Qing period) in Chinese Medical Classics, Chinese Ancient Medical Books and digital library, and then set up a medical records normalized database. The medication features and prescription rules for dementia were analyzed by frequency statistics and association rules (Apriori algorithm, improved mutual information algorithm and complex system entropy clustering). Finally, a total of 156 prescriptions were selected, involving 123 Chinese herbs, with a total frequency of 11 747 for the herbs, and 8 core prescriptions were mined. After the association rules between the frequency and prescriptions for the treatment of dementia were determined, we found that the most commonly used herbs included Fuling (Poria), Yuanzhi (Polygalae Radix), Renshen (Ginseng Radix et Rhizoma), Shichangpu (Acori Tatarinowii Rhizoma), Gancao (Glycyrrhizae Radix et Rhizoma), Danggui (Angelicae Sinensis Radix), Maidong (Ophiopogonis Radix), Baizhu (Bletillae Rhizoma), Dihuang (Rehmanniae Radix) and Ganjiang (Zingiberis Rhizoma); the frequently-used drugs compatibility was mainly for tonifying Qi-blood, regulating Yin and Yang and inducing resuscitation. The drugs were mainly of warm nature and sweet (mild) flavor, and the channel tropism of drugs mainly distributed to the heart, liver, spleen and kidney. The core prescriptions were composed of Renshen (Ginseng Radix et Rhizoma), Fuling (Poria), Yuanzhi (Polygalae Radix), Shichangpu (Acori Tatarinowii Rhizoma), and Baizhu(Bletillae Rhizoma). In conclusion, high frequency herbs and core prescriptions reflect the prescriptions by ancient physicians mainly focus on Qi-replenishing, spleen-invigorating and heart-nourishing, but also reflect the prescription rules of nourishing Yin, enriching blood, eliminating phlegm and warming Yang for the treatment of dementia. The medication features and prescription rules for the treatment of dementia obtained by association rules are useful to guide the clinical practice of Chinese medicine in treatment of dementia.

[Corrigendum] Naotaifang extract treatment results in increased ferroportin expression in the hippocampus of rats subjected to cerebral ischemia.

Mol Med Rep 11: [Related article:] 4047­4052, 2015; DOI: 10.3892/mmr.2015.3309 Following the publication of this article, an interested reader drew to our attention anomalies associated with the presentation of Figs. 1 and 4. The image selected for Fig. 1A, the data pertaining to '2 h', was inadvertently selected for Fig. 4A, the image labeled 'Sham'. Additionally, in Fig. 4, the same source image had inadvertently been used to provide the images for the 'Low dose group' and 'Model' panels (although the view presented differed in these panels). On re-examining our data, we realized that these errors had occurred during the compilation of Fig. 4, and that the images were correctly selected for Fig. 1. An amended version of Fig. 4 is presented below, featuring images which correctly show the data for the 'Low dose', 'Sham' and 'Model' groups. The immunohistochemical results suggested that, following treatment with 27 g/kg naotaifang extract, the expression of Fpn increased significantly compared with the other treatment doses (P<0.05), whereas significant changes were not observed among the other groups (P>0.05). The errors made in the selection of certain images for Fig. 4A did not affect the overall conclusions reported in the present study. We sincerely apologize for this mistake, and thank the reader of our article who drew this matter to our attention. Furthermore, we regret any inconvenience this mistake has caused.

Naotaifang extract treatment results in increased ferroportin expression in the hippocampus of rats subjected to cerebral ischemia.

The expression of Ferroportin (Fpn) was examined at different time points in rats following focal cerebral ischemia treated with or without the traditional Chinese medicine Naotaifang. Initially, rats were randomly divided into 2, 6, 12, 24 and 72 h groups following middle cerebral artery occlusion (MCAO) and the mRNA and protein level of Fpn was detected by immunohistochemistry and reverse transcription polymerase chain reaction (RT­PCR) at the above time points. Secondly, the rats were randomly divided into five groups as follows: Sham surgery group, model group, low­dose group (3 g/kg NTE), medium dose group (9 g/kg NTE) and the high­dose group (27 g/kg NTE). After 3 days of corresponding therapy by intragastric administration once a day, the regional cerebral ischemia model was reproduced by the MCAO suture method. On the third day, the neurological behavior of the rats was analyzed by neurobehavioral assessment. Fpn in the hippocampal CA2 region was measured by immunohistochemistry and the mRNA level of Fpn was detected by RT­PCR. Expression of Fpn in the hippocampal CA2 region reached a peak 12 h after surgery (P<0.05, compared with the model group). The high­dose group (27 g/kg NTE) exhibited a lower neurological behavior score (P<0.05) and a higher level of expression of Fpn at the mRNA and protein level compared with the sham surgery group and model group (P<0.05). Dysregulation of intracellular iron balance is possibly a new mechanism underlying cerebral ischemia. NTE can protect the neuronal population in the hippocampal CA2 region by adjusting the expression of Fpn to balance iron levels following cerebral ischemia.

[Regulation of naotai recipe on the expression of HIF-lα/VEGF signaling pathway in cerebral ischemia/reperfusion rats].

OBJECTIVE: To observe the therapeutic angiogenesis effect of naotai recipe (NR) on local ischemia/reperfusion (I/R) injury of rats by observing signaling pathway of hypoxia-inducible factor-lα (HIF-1α) and vascular endothelial growth factor (VEGF). METHODS: Totally 120 Sprague-Dawley (SD) rats were randomly divided into 4 groups, namely, the normal control group (n =12), the sham-operation group (n =12), the I/R model group (n =48), and the NR group (n =48). Cerebral I/R injury models were established using thread suture method. Rats in the I/R model group and the NR group were sub-divided into 4 sub-groups according to the 1st, 3rd, 5th, and 7th I/R day (n =12). The phenomenon of neovasculization was observed by immunofluorescence staining. The protein and mRNA expression levels of HIF-la, VEGF-A, and VEGFR II receptor were detected by RT-PCR. RESULTS: There were a large amount of labels for neovasculization in the ischemic area of the NR group. Double-immunofluorescence labeling [vWF (red) and BrdU (green)] was observed in the NR group. Compared with the model group, the HIF-1α protein expression was obviously enhanced on the 1 st day of I/R (P <0.01), and the VEGF protein expression started to enhance on the 3rd day in the NR group (P <0.01). The VEGFR protein expression level was the highest in the NR group on the 5th day of I/R (P <0.01). The protein expression of VEGF and HIF-1α started to decrease on the 7th day of I/R. CONCLUSION: NR could strengthen angiogenesis after I/R by elevating the expression of HIF-lα and activating HIF-lα/VEGF signaling pathway.