Abstract:Aim To study the regulatory effect of acupuncture pretreatment on ferroptosis of nerve cells in rats with ischemic stroke. Methods Sixty SD rats were randomly divided into sham middle cerebral artery occlusion (MCAO) group, MCAO group, and acupuncture+MCAO group. In the acupuncture+MCAO group, the acupuncture points of DU26, PC6, and SP6, were selected for acupuncture pretreatment, once a day for a total of 5 days. After pretreatment, MCAO or sham MCAO models were prepared. The Zausinger sextintegral method was used to score the neurological function of rats, and the infarct volume of brain tissue was calculated by TTC staining. Electron microscopy was used to observe the pathomorphological changes of brain tissue. The iron content was detected by colorimetric method, the content of malondialdehyde (MDA) and glutathione (GSH) was determined by ELISA, and the expression of glutathione peroxidase 4 (GPX4) was detected by immunofluorescence. Results Compared with the sham MCAO group, the MCAO group had a decrease in neurological function scores, a significant increase in infarct volume, a decrease in the number of mitochondria under electron microscopy, a rupture and vacuolization of the inner mitochondrial cristae, an increase in the contents of iron and MDA in brain tissue, and a decrease in GSH content and GPX4 expression. Compared with the MCAO group, the acupuncture+MCAO group had an increase in neurological function scores, a decrease in infarct volume, a large number of mitochondria under electron microscopy, a clear structure, a decrease or disordered arrangement of some mitochondrial crest structures, a decrease in the contents of iron and MDA in brain tissue, and an increase in GSH content and GPX4 expression. Conclusion Acupuncture pretreatment can alleviate neurological damage in rats, and its mechanism may be related to regulating iron, GSH and MDA contents in brain tissue, and GPX4 expression, improving cell antioxidant capacity and inhibiting nerve cell ferroptosis.

Abstract:Aim Ischemic stroke (IS) is caused by acute ischemia of cerebral blood vessels, leading to brain tissue damage and neuronal apoptosis. The pathogenesis is complex, involving multiple cell death modes such as pyroptosis, ferroptosis and disulfide death. Disulfide death is a newly discovered form of death that helps to explore the pathological mechanisms of various diseases from a new perspective. The aim of this study is to discover and validate the differential expression of disulfide death-related genes in blood samples of ischemic patients and their association with immune regulation. Methods The relevant datasets of clinical patients (GSE16561 and GSE37587) were obtained through online big data. Differentially expressed genes related to disulfide death were identified, and gene enrichment analysis was conducted to further explore the potential mechanisms. Subsequently, immune cell infiltration was analyzed to investigate the dysregulation of immune cells in the context of IS. Finally, the accuracy of key genes was verified through ROC curves, column charts, calibration curves, and decision curves, and a disease prediction model was constructed to predict the risk of stroke. Results Based on this dataset, significant differential expression of 9 genes related to disulfide death was identified. Independent external validation was conducted using the microarray dataset GSE58294. Single item comparisons were performed on these differentially expressed genes in blood samples from 69 IS patients and 23 normal individuals.The results showed that the trends of LRPPRC, MYH9, NDUFA11, PRDX1 and RPN1, the 5 differentially expressed genes, were consistent. Immune infiltration analysis found that differentially expressed genes such as TLN1, MYH9, PRDX1, LRPPRC, NDUFA11 were also strongly correlated with CD8＋T cells, activated NK cells, macrophages, and neutrophils in IS patients. Functional enrichment analysis emphasized the important role of pathways such as focal adhesion, platelet aggregation, and activation in the occurrence and development of diseases. By using a column chart model for risk prediction, it was shown that the accuracy of these differentially expressed genes was good, and the ROC curve AUC value of the optimized combination of disulfide death-related genes could reach 0.844. Further validation through an external dataset (GSE58294) revealed that the ROC curve AUC value optimized for disulfide death-related genes reached 0.989, which had good clinical guidance significance for the risk of IS. Conclusions This study confirmed the existence of 5 disulfide death-related genes in IS patients through a dataset, including upregulation of MYH9 and downregulation of LRPPRC, NDUFA11, PRDX1 and RPN1. These gene alterations are suggested to influence IS disease progression and prognosis through immune inflammation and bleeding risk.

Abstract:Aim To investigate the effect of butorphanol on neuronal pyroptosis in ischemic stroke rats and its role in protein kinase A (PKA)/cyclic adenosine phosphate (cAMP) response element binding protein (CREB). Methods A rat model of ischemic stroke was established using SD rats. All rats were divided into control group, model group, butorphanol low-dose group (butorphanol L group), butorphanol high-dose group (butorphanol H group) and butorphanol+PKA inhibitor (H-89) group. Neurological function was scored, TTC staining was used to detect cerebral infarction volume, and pathological characteristics of brain tissue were detected by HE staining. Neuronal pyroptosis was observed by uranium lead double staining in hippocampus. The expression of inflammassome NOD-like receptor thermal protein domain associated protein 3(NLRP3) and Caspase-1 were detected by immunofluorescence, and the contents of interleukin-1β (IL-1β), IL-18 and cAMP were detected by enzyme linked immunosorbent assay (ELISA). The expression levels of phosphorylated PKA (p-PKA), PKA, p-CREB and CREB protein were detected by Western blot. Results Compared with the control group, the brain space of rats in the model group was enlarged, the neuron cell membrane was defective, the nucleus and nuclear membrane were sunken and constricted, and the nerve function scores, cerebral infarction volume, NLRP3, Caspase-1, IL-1β and IL-18 levels were increased; the expression levels of cAMP, p-PKA/PKA and p-CREB/CREB protein were decreased (P＜0.05). Compared with the model group, the brain structure of butorphanol L and H groups was more complete, the neuronal cell structure was improved, the neural function scores, cerebral infarction volume, NLRP3, Caspase-1, IL-1β and IL-18 levels were decreased, and the protein expression levels of cAMP, p-PKA/PKA and p-CREB/CREB were increased (P＜0.05). Compared with butorphanol H group, butorphanol+H-89 group increased vacuolar degeneration of brain tissue, abnormal neuronal structure, neural function scores, cerebral infarction volume, NLRP3, Caspase-1, IL-1β and IL-18 levels; and the expression levels of cAMP, p-PKA/PKA and p-CREB/CREB protein were decreased (P＜0.05). Conclusion Butorphanol significantly inhibits neuronal pyroptosis in ischemic stroke rats, which may be related to the activation of PKA/CREB signaling pathway.

Abstract:Aim To investigate the factors influencing carotid artery calcification in patients with ischemic stroke and to construct a predictive model for columnar plots to provide reference for clinical formulation of prevention and control measures. Methods A total of 500 patients with ischemic stroke were randomly divided into modeling group (350 cases) and verification group (150 cases) according to a ratio of 7∶3, and the incidence of carotid artery calcification was analyzed. LASSO-Logistic regression equation was used to analyze the influencing factors of carotid artery calcification, and the prediction model of carotid artery calcification risk was built. Receiver operating characteristic (ROC) curve and calibration curve were used to analyze the nomogram to predict model differentiation and accuracy. Decision curve analysis (DCA) was drawn to evaluate the validity of the prediction model. Results In the modeling group, compared with those without carotid artery calcification, the age of those with carotid artery calcification increased by 17.87%, the proportion of smoking history increased by 32.69%, the level of fasting blood glucose increased by 22.47%, the level of glycated hemoglobin increased by 0.69%, and the level of low density lipoprotein cholesterol (LDLC) increased by 17.84%, the uric acid level increased by 22.42%, the high sensitivity C-reactive protein (hs-CRP) level increased by 40.31%, and the estimated glomerular filtration rate (eGFR) level decreased by 7.04%, with statistical significance (P＜0.05). In the verification group, compared with those without carotid artery calcification, the age of those with carotid artery calcification increased by 17.23%, the proportion of smoking history increased by 33.39%, the level of fasting blood glucose increased by 22.37%, the level of glycated hemoglobin increased by 0.75%, the level of LDLC increased by 17.96%, and the level of uric acid increased by 24.44%, hs-CRP level increased by 30.81%, eGFR level decreased by 6.46%, the difference was statistically significant (P＜0.05). In the modeling group and the verification group, the prediction models of carotid artery calcification were constructed based on the above factors, and the AUC for predicting carotid artery calcification was 0.953 and 0.972, respectively, which was accurate and clinically effective. Conclusion Increasing age, smoking history and increased fasting blood glucose, glycated hemoglobin, LDLC, uric acid and hs-CRP levels are independent risk factors for the occurrence of carotid artery calcification, and elevated eGFR levels are independent protective factor. The prediction model based on the above factors has certain predictive value for the occurrence of carotid artery calcification.

Abstract:Aim To investigate the effect of dendrobium officinale polysaccharide (DOP) on Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) signaling pathway in brain tissue of ischemic stroke rats. Methods The rat model of middle cerebral artery occlusion was established by thread embolism. SD rats were randomly divided into model group, DOP low dose (25 μg/g) group, DOP medium dose (50 μg/g) group, DOP high dose (100 μg/g) group and edaravone group, with 12 rats in each group; Another 12 SD rats were set as sham operation group. After drug intervention, the neurological deficit of rats in each group was scored by Longa grading method. The cerebral infarction in rats was detected by triphenyltetrazolium chloride staining. Hematoxylin-eosin staining was used to observe the pathological changes of hippocampal nerve tissue in rats. Enzyme-linked immunosorbent assay was used to detect the levels of inflammatory cytokines interferon-γ (IFN-γ), cyclooxygenase-2 (COX-2) and interleukin-6 (IL-6) in rat brain. Western blot was used to detect the expression level of JAK/STAT3 signaling pathway related proteins in rat brain. Results Compared with the sham operation group, the hippocampal neurons in the model group were shriveled, smaller, degenerated and necrotic, damaged in structure and disordered in arrangement, and the number of them decreased obviously. The whole hippocampal nerve tissue showed obvious pathological damage, and the neurological deficit score, cerebral infarction area, levels of IFN-γ, COX-2 and IL-6 in brain tissue, p-JAK/JAK, p-STAT3/STAT3 increased obviously (P＜0.05). Compared with the model group, the pathological damage of hippocampal nerve tissue in the drug treatment group was reduced, the neurological deficit score, cerebral infarction area, the levels of IFN-γ, COX-2 and IL-6 in brain tissue, p-JAK/JAK, p-STAT3/STAT3 were all decreased, and there was a dose-dependent relationship among DOP groups (P＜0.05). Compared with edaravone group, there was no significant change in all indexes of rats in DOP high dose group (P＞0.05). Conclusion DOP can inhibit the activation of JAK/STAT3 signaling pathway in ischemic stroke rats, reduce the synthesis and secretion of inflammatory cytokines, relieve brain inflammation and repair nerve function.

Abstract:Aim To explore the effect and molecular mechanism of procaine on hypoxia induced injury of N9 and PC12 cells. Methods N9 cells and PC12 cells were divided into control group, hypoxia group, low, medium and high dose (hypoxia＋2,6, 18 mg/L procaine) procaine group, anti-miR-con group, anti-miR-369-3p group, miR-con＋high dose procaine group (transfection miR-con＋hypoxia＋18 mg/L procaine), miR-369-3p＋high dose procaine group (transfection miR-369-3p＋hypoxia＋18 mg/L procaine). Flow cytometry was used to detect apoptosis, reagent kit was used to detect the content of reactive oxygen species (ROS) and malondialdehyde (MDA) and the activity of superoxide dismutase (SOD), ELISA was used to detect the levels of inflammatory factors such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6). RT-qPCR was used to detect the expression of miR-369-3p. Results Compared with the control group, the apoptosis rate of N9 cells and PC12 cells in the hypoxia group increased, the content of MDA and ROS increased, the activity of SOD decreased, the levels of TNF-α, IL-1β and IL-6 increased, the expression of miR-369-3p increased (P＜0.05). Compared with the hypoxia group, the apoptosis rate of N9 cells and PC12 cells in the low, medium and high dose procaine groups decreased, the content of MDA and ROS decreased, the activity of SOD increased, the levels of TNF-α, IL-1β and IL-6 decreased, the expression of miR-369-3p decreased in a concentration dependent manner (P＜0.05). Compared with the anti-miR-con group, the apoptosis rate of N9 cells and PC12 cells in the anti-miR-369-3p group decreased, the content of ROS and MDA decreased, the activity of SOD increased, the levels of TNF-α, IL-1β and IL-6 decreased (P＜0.05). Compared with miR-con＋high dose procaine group, the apoptosis rate of N9 cells and PC12 cells in miR-369-3p＋high dose procaine group increased, the expression of cleaved Caspase-3 protein increased, the content of MDA and ROS increased, the activity of SOD decreased, the levels of TNF-α, IL-1β and IL-6 increased (P＜0.05). Conclusion Procaine can reduce hypoxia induced injury to N9 and PC12 cells, and its mechanism may be related to inhibiting the expression of miR-369-3p.

Abstract:Aim To investigate the correlation between triglyceride/high density lipoprotein cholesterol ratio (TG/HDLC) and carotid plaque instability in patients with ischemic stroke (IS). Methods 594 patients with acute IS treated in the Department of Neurology of Baoding First Central Hospital from January 2019 to January 2020 were included retrospectively. All subjects underwent ultrasound examination to understand the carotid plaque. According to the ultrasound results, the subjects were divided into non-plaque group (105 cases), stable plaque group (63 cases) and unstable plaque group (426 cases). The routine biochemical indexes and blood lipid indexes were detected by automatic biochemical analyzer, and the differences of risk factors, biochemical indexes and blood lipid were compared in each group. Logistic regression analysis was used to evaluate the influencing factors of carotid plaque instability, and the odds ratio (OR) and 95% confidence interval (95%CI) were calculated. Receiver operating characteristic (ROC) curve was used to analyze the predictive value of TG/HDLC for carotid plaque instability. Results Among the 594 patients with IS, 105 had no carotid stenosis and 489 had carotid stenosis, including 439 cases of mild stenosis, 20 cases of moderate stenosis, and 30 cases of severe stenosis. The male, body mass index (BMI), smoking history, drinking history, hemoglobin, TG/HDLC in unstable plaque group were higher than those in stable plaque group, and age, HDLC were lower than that in stable plaque group (P＜0.05). Multivariate Logistic regression analysis showed that TG/HDLC was an independent risk factor for carotid plaque instability (OR 1.8,5%CI 1.027～2.551, P＝0.038). ROC curve analysis showed that the area under the curve of TG/HDLC for predicting carotid plaque instability was 0.619 (95%CI 0.542～0.696), the best cut-off value was 1.60, the sensitivity was 35.4%, and the specificity was 84.1%. Conclusion TG/HDLC is an independent risk factor for carotid plaque instability in patients with IS, and it has a certain predictive value for carotid plaque instability.

Abstract:Ischemic stroke is the most common type of stroke, and its disability rate is very high, which can lead to death in severe cases. Biomarkers can accelerate the definite diagnosis, which is very important to guide doctors to choose effective treatment methods. In recent years, the correlation between ischemic stroke and amino acid metabolism has attracted extensive attention. Studies have found that there are significant changes in amino acid metabolites and metabolic pathways after stroke, and some amino acids may be potential biomarkers. This article mainly reviews the changes of different types of amino acid metabolism in patients with ischemic stroke, in order to provide reference for exploring biomarkers and pathogenesis of ischemic stroke, so as to open up a new direction for its diagnosis, treatment and prevention.

Abstract:Aim To investigate the relationship between estimated glomerular filtration rate (EGFR), serum uric acid (SUA), fibrinogen (FIB) and cerebral hemorrhage transformation and clinical outcome after thrombolysis in ischemic stroke. Methods 158 patients with ischemic stroke treated with recombinant tissue plasminogen activator (rt-PA) intravenous thrombolysis were selected, including 121 cases without cerebral hemorrhage transformation and 37 cases with cerebral hemorrhage transformation. Logistic regression equation was used to analyze the influencing factors of cerebral hemorrhage transformation after intravenous thrombolysis with rt-PA. The levels of eGFR, SUA and FIB were compared in patients with different early neurological function. The predictive values of eGFR, SUA and FIB for early neurological deterioration (END) were evaluated. The cumulative survival rates of patients with different levels of eGFR, SUA and FIB were compared. Results After intravenous thrombolysis with rt-PA for 2 h and 24 h, the levels of eGFR, SUA and FIB in patients with cerebral hemorrhage transformation were lower than those without cerebral hemorrhage transformation (P＜0.05). Logistic regression analysis showed that age, baseline NIHSS score, baseline diastolic blood pressure, large-area cerebral infarction, levels of eGFR, SUA and FIB at 2 h and 24 h after thrombolysis were the influencing factors of cerebral hemorrhage transformation after rt-PA intravenous thrombolysis (P＜0.05). eGFR, SUA and FIB in END patients were lower than those in non-END patients at 2 h and 24 h after thrombolysis (P＜0.05). ROC curve analysis showed that the area under curve for the joint predicting END by 24 h eGFR, 24 h SUA and 24 h FIB was 0.809, which was greater than any single index, and its sensitivity and specificity were 80.95% and 74.14% respectively. The results of survival analysis showed that the cumulative survival rate of high-level eGFR, SUA and FIB group was higher than that of low-level group 24 h after thrombolysis (P＜0.05). Conclusion eGFR, SUA, FIB are closely related to the prognosis of ischemic stroke. Monitoring the above indicators is helpful for the diagnosis of cerebral hemorrhage transformation and END prediction.

Abstract:Aim To investigate the relationship between plasma homocysteine levels and acute ischemic stroke in young people in Xinjiang. Methods A total of 186 young patients with acute ischemic stroke who were hospitalized in the Department of Neurology, the First Affiliated Hospital of Xinjiang Medical University from January 2019 to December 2020 were selected as the observation group. A total of 180 patients with non-cardiovascular and cerebrovascular diseases who were hospitalized during the same period were randomly selected as the control group. The patients in the observation group were divided into mild neurological impairment group, moderate neurological impairment group and severe neurological impairment group according to the National Institutes of Health Stroke Scale score at admission. According to the carotid intima-media thickness, they were divided into normal intima group, intimal thickening group, plaque formation group and carotid artery stenosis group. According to the modified Rankin scale score at 14 days, the patients were divided into a short-term good prognosis group and a short-term poor prognosis group. The clinical data and laboratory results of each group were compared. Risk factors for ischemic stroke, carotid atherosclerosis, and short-term poor prognosis were explored in young adults. Results The blood homocysteine level was significantly higher in young patients with acute ischemic stroke than that in the control group (P＜0.05). The risk of acute ischemic stroke in young people increased with the level of homocysteine, and the two groups of patients with moderate and severe neurological impairment had higher blood homocysteine levels than those with mild neurological impairment patients (P＜0.05). Homocysteine was a risk factor for carotid atherosclerosis (OR=34.4,5%CI:8.184～142.876) and short-term poor prognosis (OR=388.7,5%CI:50.271～3 003.108) in young patients with acute ischemic stroke. The area under the curve of homocysteine for predicting the short-term poor prognosis of acute ischemic stroke in young people was 0.712 (95%CI:0.616～0.807), and the area under the curve when it was combined with leukocytes was 0.831 (95%CI:0.745～0.916). Conclusions High plasma homocysteine level is a risk factor for acute ischemic stroke, carotid atherosclerosis and short-term poor prognosis in young people in Xinjiang. It has a certain predictive value for the short-term poor prognosis of young patients with acute ischemic stroke.