Erythrocyte-derived extracellular vesicles aggravate inflammation by promoting the proinflammatory macrophage phenotype through TLR4-MyD88-NF-κB-MAPK pathway.

Transfusion of stored erythrocytes is associated with the increased risk of morbidity and mortality in critical infections, but the mechanism is incompletely understood. Previous studies have suggested that RBC-derived extracellular vesicles (EVs) may be potential risk factors for the occurrence of transfusion-related immunomodulation. The purpose of our study was to evaluate the effects of RBC-derived EVs under inflammatory conditions and explore the underlying mechanisms. In vivo, the activity of EVs was evaluated in cecal ligation and puncture (CLP)-induced sepsis. Our results showed that EVs significantly aggravated the inflammatory response to sepsis in serum and lung tissue by promoting the production of the proinflammatory factors tumor necrosis factor-α (TNF-α）-interleukin-6(IL-6), and interleukin-1ß (IL-1ß) and reduced the survival rate of septic mice in vivo. Importantly, adoptive transfer of EVs-pretreated bone marrow-derived macrophages (BMDMs) obviously aggravated systemic proinflammatory factors in mice after CLP surgery. In vitro, the proinflammatory properties of EVs were shown to elevate TNF-α, IL-6, and IL-1ß levels in lipopolysaccharide (LPS)-stimulated BMDMs. Moreover, EVs promoted LPS-induced macrophage polarization into a proinflammatory phenotype. The underlying mechanism might involve EV-mediated up-regulation of TLR4-MyD88-NF-κB-MAPK activity to favor macrophage cytokine production.

Impact of Increased Hemoglobin on Spontaneous Intracerebral Hemorrhage.

BACKGROUND: Studies of the impact of increased hemoglobin on spontaneous intracerebral hemorrhage (ICH) are limited. The present study aimed to explore the effect of increased hemoglobin on ICH. METHODS: A retrospective single-center study using medical records from a database processed by univariate and multivariate analyses was performed in the People's Hospital of Tibet Autonomous Region in Lhasa, Tibet, China. RESULTS: The mean hemoglobin level in 211 patients with ICH was 165.03 ± 34.12 g/l, and a median hematoma volume was 18.5 ml. Eighty-eight (41.7%) patients had large hematomas (supratentorial hematoma ≥ 30 ml; infratentorial hematoma ≥ 10 ml). No differences in ICH risk factors between the groups with different hemoglobin levels were detected. Increased hemoglobin was independently associated with large hematomas [odds ratio (OR) 1.013, P = 0.023]. Increased hemoglobin was independently associated with ICH with subarachnoid hemorrhage (OR 1.014, P = 0.016), which was more pronounced in men (OR 1.027, P = 0.002). Increased hemoglobin was independently associated with basal ganglia hemorrhage and lobar hemorrhage in men (OR 0.986, P = 0.022; OR 1.013, P = 0.044, respectively) but not in women (P > 0.1). CONCLUSIONS: Increased hemoglobin was independently associated with large hemorrhage volume. Increased hemoglobin was independently associated with lobar hemorrhage in men and ICH with subarachnoid hemorrhage, which was more pronounced in men. Additional studies are needed to confirm our findings and explore potential mechanisms.

Cerebral large artery occlusion in chronic graft-versus-host disease: A case report.

RATIONALE: Cerebral large artery occlusion in chronic central nervous system graft-versus-host disease after allogeneic hematopoietic stem cell transplantation (allo-HSCT) was very scarce. We described a young patient with bilateral white matter lesions and symptomatic internal carotid artery occlusion after allo-HSCT with the history of aplastic anemia. PATIENT CONCERNS: A 17-year-old girl with the history of aplastic anemia developed recurrent headache and sudden hemiplegia of right limbs 2 years after allo-HSCT. DIAGNOSES: She was diagnosed with skin chronic graft-versus-host disease 19 months after allo-HSCT. Brain magnetic resonance imaging showed bilateral subcortical white matter abnormal signals and hyperintensity of left fronto-parietal lobe on diffusion weighted imaging and corresponding hypointense apparent diffusion coefficients indicating acute infarction. CT angiography revealed thrombosis in left internal carotid artery. Carotid plaque high-resolution magnetic resonance imaging showed annular enhancement of vascular wall revealing signs of vasculitis. INTERVENTIONS: Intravenous immunoglobulin, methylprednisolone, and anticoagulant therapy were used to treat the patient. OUTCOMES: The patient's symptoms gradually resolved and she could walk with assistance after 3 weeks before returned home. LESSONS: Chronic graft-versus-host disease-associated vasculitis could involve cerebral large vessels which warrants further study.

Predictors and Prognosis of Symptomatic Intracranial Hemorrhage in Acute Ischemic Stroke Patients Without Thrombolysis: Analysis of Data From the Chinese Acute Ischemic Stroke Treatment Outcome Registry.

Background and Purpose: There is limited information on symptomatic intracranial hemorrhage (sICH) in stroke patients without thrombolysis. This study aimed to evaluate the risk factors of sICH and the association between sICH and the prognosis at 3 and 12 months in acute ischemic stroke patients without thrombolysis. Methods: Data originated from the Chinese Acute Ischemic Stroke Treatment Outcome Registry. Univariate analysis and multivariate logistic regression were used to screen the risk factors of sICH. Multivariable logistic regression models were used to assess the association of sICH with poor outcome and all-cause mortality. Results: Totally, 9,484 patients were included, of which 69 (0.73%) had sICH. Atrial fibrillation (odds ratio [OR], 3.682; 95% confidence interval [CI], 1.945-6.971; p < 0.001), history of tumors (OR, 2.956; 95% CI, 1.115-7.593; p = 0.024), and the National Institutes of Health Stroke Scale (NIHSS) score on admission ([6-15: OR, 2.344; 95% CI, 1.365-4.024; p = 0.002] [>15: OR, 4.731; 95% CI, 1.648-13.583; p = 0.004]) were independently associated with sICH. After adjustment of the confounders, patients with sICH had a higher risk of poor outcome (OR, 1.983; 95% CI, 1.117-3.521; p = 0.018) at 3 months and that of all-cause mortality at 3 (OR, 6.135; 95% CI, 2.328-16.169; p < 0.001) and 12 months (OR, 3.720; 95% CI, 1.513-9.148; p = 0.004). Conclusion: sICH occurred in 0.73% of acute ischemic stroke patients without thrombolysis and was associated with a worse prognosis at 3 and 12 months. Atrial fibrillation, history of tumors, and NIHSS score at admission were independent risk factors of sICH.

Myasthenia gravis associated with renal cell carcinoma: a paraneoplastic syndrome or just a coincidence.

BACKGROUND: Myasthenia gravis (MG) can occur as a paraneoplastic phenomenon associated with thymoma. The association of MG with renal cell carcinoma (RCC) is not clear. Herein, we describe six cases of MG associated with RCC. METHODS: There were 283 patients diagnosed with MG admitted to our hospital from 2014 to 2019. Among them, 6 patients also had RCC. None of them had immune checkpoint inhibitor therapies. We performed a retrospective clinical data collection and follow-up studies of these 6 patients. RESULTS: These 6 patients with an average MG onset age of 61.3 ± 13.3 years, were all positive for anti-acetylcholine receptor antibodies. MG symptoms appeared after RCC resection in 3 cases. RCC was discovered after the onset of MG in 2 cases, and synchronously with MG in 1 case. After nephrectomy, the MG symptoms showed a stable complete remission in 1 case. Among them, four patients met the diagnostic criteria of possible paraneoplastic neurological syndromes. CONCLUSIONS: Except for thymoma, patients with MG should pay attention to other tumors including RCC. MG may be a paraneoplastic syndrome of RCC, and further studies are needed to elucidate the relationship.

Connexin43 promotes angiogenesis through activating the HIF-1α/VEGF signaling pathway under chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion, a major vascular contributor to vascular cognitive impairment and dementia, can exacerbate small vessel pathology. Connexin43, the most abundant gap junction protein in brain tissue, has been found to be critically involved in the pathological changes of vascular cognitive impairment and dementia caused by chronic cerebral hypoperfusion. However, the precise mechanisms underpinning its role are unclear. We established a mouse model via bilateral common carotid arteries stenosis on connexin43 heterozygous male mice and demonstrated that connexin43 improves brain blood flow recovery by mediating reparative angiogenesis under chronic cerebral hypoperfusion, which subsequently reduces the characteristic pathologies of vascular cognitive impairment and dementia including white matter lesions and irreversible neuronal injury. We additionally found that connexin43 mediates hypoxia inducible factor-1α expression and then activates the PKA signaling pathway to regulate vascular endothelial growth factor-induced angiogenesis. All the above findings were replicated in bEnd.3 cells treated with 375 µM CoCl2in vitro. These results suggest that connexin 43 could be instrumental in developing potential therapies for vascular cognitive impairment and dementia caused by chronic cerebral hypoperfusion.

Dural arteriovenous fistula, a rare cause of rapidly progressive dementia in a patient with bilateral thalamic lesions: a case report.

There are many causes of bilateral thalamic lesions, but few cases of dural arteriovenous fistula (DAVF) associated with such lesions have been reported previously. Here, we describe an adult man with reversible rapid progressive dementia (RPD) in whom bilateral thalamic lesions were caused by a DAVF that had six supply arteries and drained into both the venous sinus and cortical veins. A 53-year-old man presented with memory decline and abnormal behavior. Head computed tomography (CT) revealed insignificant low density in the bilateral thalami and high density in the right occipital lobe. Brain magnetic resonance imaging showed hyperintensities in the thalami on T2-weighted images. Magnetic resonance venogram revealed no sign of the straight sinus, but multiple tortuous vessels in the cistern of the vein of Galen. Digital subtraction angiography revealed DAVFs near the tentorium cerebelli draining into the vein of Galen, which caused the vasogenic oedema of the thalami. The patient was then treated by transarterial embolization of the feeders. He gradually recovered after the surgery. RPD with bithalamic lesions caused by DAVF is rare but reversible. Therefore, the early recognition and intervention of DAVFs is crucial for the good prognosis of patients so that fistulas can be embolized in time.

The Association between Neprilysin gene polymorphisms and Alzheimer's disease in Tibetan population.

OBJECTIVES: Alzheimer's disease (AD) is a well-known neurodegenerative disease, of which the hallmark is the disposition of ß-amyloid (Aß) in the form of plaque in the brain. Neprilysin (NEP) is the major enzyme to degrade Aß and prevent accumulation of Aß. The present study was undertaken to elucidate the correlation between the NEP gene polymorphisms and AD in Chinese Tibetan population. METHODS: Ninety-nine sporadic AD Tibetan patients and 113 healthy Tibetan controls were enrolled in this study. The genotype frequencies and allele frequencies of multiple NEP gene loci were analyzed using the case-control association analysis. RESULTS: No significant correlation was found between polymorphisms of NEP gene loci (rs9829757, rs1816558, rs6776185, rs3736187, rs701109, rs989692) and the occurrence of AD in Tibetan population. However, allele C of NEP gene locus (rs701109) and allele T of gene locus (rs3736187) were possible risk factors of male AD patients in Tibetan population. CONCLUSIONS: NEP gene loci (rs701109, rs989692, rs9829757, rs3736187, rs1816558, rs6776185) were polymorphic in Tibetan population. No difference was found between these loci but for that male gender combined with allele C of NEP gene locus (rs701109) and T of gene locus (rs3736187) might be risk factors for AD in Tibet.

Characteristics of acute ischemic stroke in hospitalized patients in Tibet: a retrospective comparative study.

BACKGROUND: Numerous studies on acute ischemic stroke (AIS) have been conducted at low-altitude regions, and the related findings have been used to guide clinical management. However, corresponding studies at high altitude are few. This study aimed to analyse the clinical characteristics of AIS patients at high-altitude regions through a hospital-based comparative study between Tibet and Beijing. METHODS: This study included the diagnoses of AIS patients from People's Hospital of Tibet Autonomous Region (PHOTAR) and Peking University First Hospital (PUFH) between 1 January 2014 and 31 December 2017, where data including patient demographics, treatment time, onset season, risk factors, infarction location, laboratory data, image examination results, treatments, and AIS subtype were collected and compared. Continuous and categorical variables were analysed with a two-sample t-test or Wilcoxon rank sum test and chi-square test, respectively. Significant risk factors were examined with binary logistic regression analysis. RESULTS: In total, 236 and 1021 inpatients from PHOTAR and PUFH were included, respectively. The PHOTAR patients were younger than the PUFH patients (P < 0.001). Young adult stroke, erythrocytosis, and hyperhomocysteinemia were more frequent in PHOTAR patients (all P < 0.001). Other vascular risk factors, including hypertension, diabetes mellitus, hyperlipidaemia, smoking and alcohol consumption history, were less prevalent in PHOTAR patients than in PUFH patients. The rate of intravenous thrombolysis and the rate of within intravenous thrombolysis window time were also lower in PHOTAR patients (both P < 0.001). The PHOTAR group also tended to have anterior circulation infarction. Erythrocytosis and hyperhomocysteinemia were independent risk factors in PHOTAR, and young adults accounted for a larger proportion of stroke cases. CONCLUSION: In Tibet, AIS patients were relatively younger, and anterior circulation infarctions were more common. Erythrocytosis and hyperhomocysteinemia may contribute to these differences. Here, young adult stroke also accounted for a higher proportion, and this may be associated with erythrocytosis. Our findings present the first hospital-based comparative study in Tibet and may contribute to policies for stroke prevention in this region.

Cerebrospinal fluid CD20 positive B-cell expansion in a case of anti-NMDAR encephalitis.

Anti-N-methyl-d-aspartate receptor (NMDAR) encephalitis is a potentially fatal autoimmune encephalitis with a strong B-cell response. We measured the proportion of CD20 positive and CD19 positive B-cells in the CSF from a case of severe anti-NMDAR encephalitis. The proportion of CD20 positive B-cells in the CSF was 15.0%, higher than CD19 positive B cells (10.1%), and higher than that seen in non-inflammatory neurological disorders (<1%). After the treatments of steroids, intravenous immunoglobulin and rituximab to eliminate B-cells, she recovered. These findings further support the use of rituximab that targets CD20 positive B-cells in anti-NMDAR encephalitis.

Plasma lipidomic analysis of sphingolipids in patients with large artery atherosclerosis cerebrovascular disease and cerebral small vessel disease.

BACKGROUND: Sphingolipids mainly consist of ceramides (Cer), sphingomyelins (SM) and glycosphingolipids. Sphingolipids are related with coronary heart disease and metabolic disease, but there're few studies about cerebrovascular disease. The purpose was to detect sphingolipids in plasma of patients with large artery atherosclerosis (LAA) cerebrovascular disease and cerebral small vessel disease (CSVD) to explore the similarities and differences of pathogenesis of the two subtypes. METHODS: 20 patients with LAA cerebrovascular disease, 20 patients with age-related CSVD, 10 patients with Fabry disease and 14 controls were enrolled from October 2017 to January 2019. Ultra-high performance liquid chromatography-quadruple-time-of-flight mass spectrometry/mass spectrometry was used to determine sphingolipids. Univariate combined with multivariate analysis was used for comparison. Receiver operating characteristic curves were used to determine sensitivities and specificities. RESULTS: 276 sphingolipids were detected, including 39 Cer, 3 ceramide phosphates, 72 glycosphingolipids and 162 SM. (1) Cer (d36:3), Cer (d34:2), Cer (d38:6), Cer (d36:4) and Cer (d16:0/18:1) were increased in LAA; SM (d34:1), Cer (d34:2), Cer (d36:4), Cer (d16:0/18:1), Cer (d38:6), Cer (d36:3) and Cer (d32:0) were increased in age-related CSVD. (2) Cer (d36:4) and SM (d34:1) were increased in age-related CSVD compared with LAA. (3) Total trihexosyl ceramides were increased in Fabry group compared with control (P<0.05); SM (d34:1) was increased in Fabry group. CONCLUSIONS: Ceramides are increased in both LAA and age-related CSVD, which may be related to similar risk factors and pathophysiological process of arteriosclerosis; SM is increased in both age-related CSVD and Fabry disease, suggesting that increased SM may be associated with CSVD. Glycosphingolipids, trihexosylceramides in particular, are increased in Fabry disease.

Cerebellar Neurocytoma with Excellent Response to Radiotherapy.

BACKGROUND: Extraventricular neurocytoma (EVN) is a rare neurocytoma occurring in the brain parenchyma outside the ventricular system that shares similar biological behaviors and histopathologic characteristics with central neurocytoma. Reports of EVN localized in the brainstem and cerebellum are relatively uncommon. In addition, few cases with radiotherapy as the only treatment have been reported and their outcomes were unclear. CASE DESCRIPTION: We report a case of pathologically confirmed EVN of the brainstem and cerebellum in a 43-year-old male who presented with unprovoked nausea and dizziness. The patient received radiotherapy only and showed a favorable outcome during the 2-year follow-up period. CONCLUSIONS: These results suggest that patients with EVN who are treated with radiotherapy without surgery may have a favorable prognosis.

The protective effect of polyethylene glycol-conjugated urokinase nanogels in rat models of ischemic stroke when administrated outside the usual time window.

pH-sensitive polyethylene glycol-conjugated urokinase nanogels (PEG-UK) is a new form of urokinase (UK) nanogels that could release UK at certain pH values. In our former study, we demonstrated that the pH value in the infarcted brain significantly declined to the level that could trigger the delivery of UK from PEG-UK. Thrombolysis is recommended as the first choice for ischemic stroke within the time window. However, it is common for the patients to miss the thrombolysis time window, which is one of the major causes of bad prognosis from ischemic stroke. It remains promising for seeking therapeutic approaches for ischemic stroke by investigating potential protective reagents delivered out of the usually thrombolysis time window. In this study, the protective effect of administration of PEG-UK outside the usual time window and the underlying mechanisms were investigated. PEG-UK was administrated 2 h and a half after ischemic stroke Delayed administration of PEG-UK significantly ameliorated the severity of neurological deficits of permanent middle cerebral occlusion (pMCAO) rats and reduced the infiltration of inflammatory cells and the concentration of interleukin 1ß (IL-1ß) and tumor necrosis factor-α (TNF-α) in the brain tissues. The content of water and the leakage of Evans Blue (EB) in the PEG-UK group were also decreased. Maintenance of the expression of platelet-derived growth factor-C (PDGF-C) and inhibition of the upregulation of metalloproteinase proteins, low-density lipoprotein receptor-related protein (LRP), nuclear factor κB (NF-κB) p65 and cyclooxygenase-2 (Cox-2) were observed through western blotting and realtime PCR in the PEG-UK group. Besides, delayed administration of PEG-UK attenuated the up regulation of Caspase8 and Caspase9 and the cleavage of Caspase3 and poly (ADP-ribose) polymerase 1 (PARP1) in ischemic lesion sites. Moreover, PEG-UK treatment also inhibited the upregulation and phosphorylation of N-methyl-D-aspartic acid receptors (NMDARs), which has been revealed to play a vital role in mediating excito-neurotoxicity in ischemic stroke. In conclusion, through the inhibition of LRP/NF-κB/Cox-2 pathway, the Caspase cascade and activation of NMDARs, administration of PEG-UK outside the usual time window could still exert protective effects in pMCAO rats through the maintenance of the integrity of BBB and the inhibition of apoptosis and excito-neurotoxicity.

The Role of T Follicular Helper Cells and T Follicular Regulatory Cells in the Pathogenesis of Autoimmune Hemolytic Anemia.

Autoimmune hemolytic anemia (AIHA) is an acquired autoimmune disease mediated by antibodies against the patient's red blood cells. However, the underlying mechanisms for antibody production are not fully understood. Previous studies of etiology and pathogenesis of AIHA mainly focus on autoreactive B cells that have escaped tolerance mechanisms. Few studies have reported the function of TFH and TFR cells in the process of AIHA. The present study aimed to explore the potential mechanism of TFH and TFR cells in the pathogenesis of AIHA. With the model of murine AIHA, increased ratios of TFH:TFR, elevated serum IL-21 and IL-6 levels, and upregulated Bcl-6 and c-Maf expression were reported. Also, adoptive transfer of purified CD4+CXCR5+CD25- T cells from immunized mice promoted the induction of autoantibody in the AIHA mouse model. Altogether, our data demonstrate the important role of TFH cells for control and induction of AIHA. In the light of the key contributions of TFH cells to the immune response in AIHA, strategies aimed at inhibiting the TFH development or function should be emphasized.

Cilostazol ameliorates ischemia/reperfusion-induced tight junction disruption in brain endothelial cells by inhibiting endoplasmic reticulum stress.

Endoplasmic reticulum (ER) stress is essential for brain ischemia/reperfusion (I/R) injury. However, whether it contributes to I/R-induced blood-brain barrier (BBB) injury remains unclear. cilostazol exerts protective effects toward I/R-induced BBB injury, with unclear mechanisms. This study explored the potential role of ER stress in I/R-induced endothelial cell damage and determined whether the therapeutic potential of cilostazol, with respect to I/R-induced endothelial cell damage, is related to inhibition of ER stress. We found that exposing brain endothelial cells (bEnd.3) to oxygen-glucose deprivation/reoxygenation (OGD/R) significantly activated ER stress and diminished the barrier function of cell monolayers; treatment with the ER stress inhibitor 4-phenylbutyric acid (4-PBA) or cilostazol prevented OGD/R-induced ER stress and preserved barrier function. Furthermore, OGD/R induced the expression and secretion of matrix metalloproteinase-9 and nuclear translocation of phosphorylated NF-κB. These changes were partially reversed by 4-PBA or cilostazol treatment. In vivo, 4-PBA or cilostazol significantly attenuated I/R-induced ER stress and ameliorated Evans blue leakage and tight junction loss. These results demonstrate that I/R-induced ER stress participates in BBB disruption. Targeting ER stress could be a useful strategy to protect the BBB from ischemic stroke, and cilostazol is a promising therapeutic agent for this process.-Nan, D., Jin, H., Deng, J., Yu, W., Liu, R., Sun, W., Huang, Y. Cilostazol ameliorates ischemia/reperfusion-induced tight junction disruption in brain endothelial cells by inhibiting endoplasmic reticulum stress.

Neuromyelitis optica spectrum disorder coincident with renal clear cell carcinoma: A case report.

RATIONALE: Detection of aquaporin-4 (AQP4) antibody in cerebrospinal fluid (CSF) was not suggested for the diagnosis of neuromyelitis opica spectrum disorders (NMOSD). However, some patients with NMOSD have only AQP4 antibody positive in CSF but not in serum with unknown cause. Besides, it is rarely reported that NMOSD complicated with renal clear cell carcinoma. So, the relationship between AQP4-Ab, NMOSD and malignant tumors warrants an investigation. PATIENTS CONCERNS: A 31-year-old female presented in our hospital with chief complaints of urinary retention and weakness in bilateral lower extremities for more than 10 days. DIAGNOSES: The patient was diagnosed as NMOSD by neuroimaging and laboratory examination, with AQP4 antibody positive only in CSF. Besides, asymptomatic clear cell carcinoma was also found in left kidney. INTERVENTIONS: The patient underwent 2-month immunosuppressive therapy for NMOSD at first, including intravenous administration of immunoglobulin (IVIG) and methylprednisone, with oral drugs of predisone and tacrolimus. After that, Partial nephrectomy of left kidney was performed. OUTCOMES: The patient demonstrated almost complete remission for NMOSD after immunosuppressive therapy, and the renal tumor was cured by partial nephrectomy. LESSON: This case indicates that neuromyelitis optica (NMO)-IgG positive only in CSF could have potential association with the etiology of NMOSD, and renal clear cell carcinoma could be found complicated with NMOSD coincidently. Besides, it is necessary to examine NMO-IgG in CSF for patients suspicious with NMOSD, even when the serum test is negative, especially for those with complicated malignant tumors.

Autophagy inhibits C2-ceramide-mediated cell death by decreasing the reactive oxygen species levels in SH-SY5Y cells.

Ceramide has been recognized as a second messenger that regulates several intracellular processes in neuronal cells. However, its role in neuronal autophagy is not fully understood. In this study, we used a human neuroblastoma cell line (SH-SY5Y) to investigate the mechanisms underlying C2-ceramide-mediated cell death and autophagy. C2-ceramide induced caspase-3-independent cell death. In addition, C2-ceramide induced autophagy, decreased the activation of Akt and mTOR, and increased the activation of JNK and ERK1/2. However, only inhibition of ERK1/2 with PD98059 prevented C2-ceramide-induced autophagy, indicating that the ERK1/2 pathway contributes to ceramide-induced autophagy. According to the results of the flow cytometric assays, C2-ceramide-induced cell death was increased by 3-methyadenine (3-MA) and decreased by rapamycin. Furthermore, the generation of reactive oxygen species (ROS) in the cells was increased by 3-MA and decreased by rapamycin. Based on these datas, autophagy protected SH-SY5Y cells from C2-ceramide-induced cell death by decreasing ROS production. Therapeutic strategies that regulate autophagy may be used in the treatment of neurological disorders associated with ceramide-induced cell death.

Phloretin induces cell cycle arrest and apoptosis of human glioblastoma cells through the generation of reactive oxygen species.

Phloretin, a flavonoid present in various plants, has been reported to exert anticarcinogenic effects. However, the mechanism of its chemo-preventive effect on human glioblastoma cells is not fully understood. This study aimed to investigate the molecular mechanism of phloretin and its associated chemo-preventive effect in human glioblastoma cells. The results indicate that phloretin inhibited cell proliferation by inducing cell cycle arrest at the G0-G1 phase and induced apoptosis of human glioblastoma cells. Phloretin-induced cell cycle arrest was associated with increased expression of p27 and decreased expression of cdk2, cdk4, cdk6, cyclinD and cyclinE. Moreover, the PI3K/AKT/mTOR signaling cascades were suppressed by phloretin in a dose-dependent manner. In addition, phloretin triggered the mitochondrial apoptosis pathway and generated reactive oxygen species (ROS). This was accompanied by the up-regulation of Bax, Bak and c-PARP and the down-regulation of Bcl-2. The antioxidant agents N-acetyl-L-cysteine and glutathione weakened the effect of phloretin on glioblastoma cells. In conclusion, these results demonstrate that phloretin exerts potent chemo-preventive activity in human glioblastoma cells through the generation of ROS.

Treatment with the herbal medicine, naoxintong improves the protective effect of high-density lipoproteins on endothelial function in patients with type 2 diabetes.

The protective effect of high­density lipoprotein (HDL) on endothelial function is impaired in patients with type 2 diabetes mellitus (T2DM), which may result in atherosclerotic complications. Naoxintong (NXT) is a compound preparation that includes Radix Astragali, Angelicae sinensis, Radix Paeoniae Rubra and Ligusticum wallichii. It is widely administered in China to prevent atherosclerotic complications. In the present study, NXT was administered to 69 patients with T2DM. HDLs were isolated from patient blood samples prior to and following the intervention. In vitro endothelial functions of HDL, including proliferation, migration, angiogenesis, and anti­apoptosis were investigated by bromodeoxyuridine, wound healing, Transwell and Matrigel tube formation assays on human umbilical vein endothelial cells (HUVECs). The results from the present study demonstrated that HUVECs treated with HDL isolated from diabetic patients following NXT therapy exhibited increased proliferative effects (10­27%; P<0.05), and improved migration ability (15­35%; P<0.05), anti­apoptotic function (23­34%; P<0.05) and angiogenesis (30­54%; P<0.001). Furthermore, the phosphorylation levels of Akt (26­36%; P<0.01) and extracellular signal­regulated kinase (16­80%; P<0.01) were increased following NXT therapy. The present in vitro study demonstrates that the protective effect of HDL on endothelial function is markedly impaired in diabetic patients who tend to develop atherosclerosis, and the impaired function may be partly abrogated by NXT.

pH gradient difference around ischemic brain tissue can serve as a trigger for delivering polyethylene glycol-conjugated urokinase nanogels.

BACKGROUND AND PURPOSE: pH-sensitive polyethylene glycol-conjugated urokinase nanogels (PEG-UKs) were previously reported to be a new form of UK nanogels that could release UK at certain pH values. In this study, we evaluated the effect of PEG-UK targeted to ischemic tissue with microcirculation failure in rat model of ischemic stroke and investigated the possible mechanisms of action. METHODS: Surgeries were performed to induce persistent middle cerebral artery (MCA) occlusion in adult Sprague-Dawley rats. The pH distribution in the brain was mapped 1h after ischemia using a needle-type pH micro sensor. The release curve of active UK from PEG-UK was also mapped by a continuous measurement of the peripheral blood. The thrombolytic effects of PEG-UK, when it was administrated 1h after occlusion, including dynamic changes in the D-dimer level, neurological deficits and infarction volume, were observed. Next, the possible mechanisms underlying these effects were explored, including the BBB integrity and the extent of apoptosis and neurotoxicity. Additionally, the long-term effects of PEG-UK during the four weeks after treatment were evaluated using the dynamic changes in the body weights and clinical scores and the numbers of deaths and hemorrhagic transformations (HTs). To evaluate the systemic side effects of PEG-UK, the fluctuations of cytokines in the liver and kidney were evaluated. RESULTS: On average, MCA occlusion for 1h induced an approximately 0.49 decline in the pH value (from 7.12 to 6.73), and the lowest value was 6.32 in the predominantly affected region around the cortex. PEG suspended the release of UK from PEG-UK into the circulation. When it was administrated 1h after occlusion, PEG-UK treatment clearly reduced the severity of neurological deficits in the acute phase (P=0.001). The relative infarct volume also decreased significantly in PEG-UK rats (P<0.001). As to the integrity of BBB, the EB leakage in the PEG-UK group was reduced (P=0.001). Maintenance of the expression of TIMP-1 (P=0.032) and claudin5 (P<0.001) and inhibition of MMP9 upregulation (P<0.001) were observed through both immunohistochemistry and Western blot in the PEG-UK group. Moreover, the expression of both NMDAR1 (P<0.001) and Caspase9 (P=0.013) in PEG-UK-treated rats was reduced. As to the long-term prognosis, the rats in PEG-UK group recovered faster and better, and the numbers of deaths and HTs were not increased. No significant fluctuation in IL-1ß and TNF-α was found in the PEG-UK-treated rats during the four post-treatment weeks. When PEG-UK was administrated 2.5h after occlusion, no clearly better outcomes were observed; however, the number of HTs was not increased. CONCLUSIONS: Treatment with PEG-UK decreased the severity of ischemic stroke by improving ischemic brain tissue and protecting the BBB and by inhibiting apoptosis and decreasing neurotoxicity. PEG-UK could further inhibit HT through its BBB protection effect. The administration of PEG-UK also improved the long-term prognosis and had no obvious systemic side effects in rats. Our data provide new insights into the thrombolytic treatment of ischemic stroke.