Gallic acid attenuates cerebral ischemia/re-perfusion-induced blood-brain barrier injury by modifying polarization of microglia.

Microglia, the main immune effector cells in the central nervous system, play a dual role in the function/structure of the blood-brain barrier (BBB) and brain health. During and soon after a cerebral ischemic injury, microglia produce neurotrophic factors and neurotoxins that can impact on the injury itself and pathology progression. At the same time, microglia undergo polarization to M1 or M2 pro- vs. anti-inflammatory subtypes that also help drive the outcome of the injury process. Thus, agents that can mitigate cerebral ischemic injury progression, promote protective functions of microglia, and help maintain BBB and overall brain health/host neurologic function after a cerebral ischemic event would be of great use in clinical settings. Protective effects from gallic acid (GA) in cerebral ischemia/re-perfusion-induced injury to the BBB and other sites in the brain have not yet been assessed. To address this, a middle cerebral artery occlusion (MCAO) method was used to establish an experimental ischemic stroke model in mice. Mice were placed in sham operation (Sham), model (MCAO), MCAO + GA (50 mg/kg), MCAO + GA (100 mg/kg), or MCAO + GA (150 mg/kg) groups. At various times post-stroke, cerebral infarct volume and host neurological function were evaluated. In addition, qRT-PCR, Western blotting, and ELISA were used to evaluate the expression and tissue content of microglia-related factors. The results showed GA treatment protected the integrity of the BBB, significantly reduced brain edema, and helped lead to improved neurological function scores in the MCAO mice. Whether these changes were due to that GA attenuated cerebral ischemia/re-perfusion-induced activation of microglial cells overall, in part, by inhibiting their polarization to the M1 subtype, is uncertain. Taking these outcomes together, for now it is reasonable to suggest that use of GA either as a prophylactic or immediately in the event of a cerebral ischemic event/stroke could help to promote neuronal survival and allow for a more likely of host neurological function over time.

Cryptotanshinone reduces neurotoxicity induced by cerebral ischemia-reperfusion injury involving modulation of microglial polarization.

BACKGROUND: The diterpenoid cryptotanshinone (CTS) has wide biological functions, including inhibition of tumor growth, inflammation and apoptosis. The present study aimed to explore the possible effect of CTS on cerebral ischemia/reperfusion (I/R) injury and the underlying mechanisms. METHODS: Male C57BL/6J mice underwent transient middle cerebral artery occlusion (tMCAO) and murine microglia BV2 cells were challenged by Oxygen/glucose deprivation, to mimic I/R and ischemic/hypoxic and reperfusion (H/R) injury, respectively. CTS was administered 0.5 h (10 mg/kg) after the onset of MCAO or 2 h (20µM) post OGD. Infarct volume and neurological deficit were measured. Immunofluorescence, qPCR, and western blot, were performed to detect the expression of cytokines, apoptotic marker, and M1/M2 phenotype-specific genes. Flow cytometry was applied for M1/M2 subpopulation or Annexin V/PI apoptosis assessment. RESULTS: CTS significantly reduced cerebral infarct volume, neurologic deficit scores, pro-inflammatory cytokine production (IL-6, TNF-α, and IL-1ß), apoptotic protein expression (cleaved caspase-3) of mice after tMCAO challenge. Furthermore, CTS attenuated CD16+ M1-type and elevated CD206+ M2-type microglia in vivo or in vitro. CONCLUSIONS: We propose that the neuroprotective effect of CTS in the I/R or H/R context are explained modulation of microglial polarization, suggesting therapeutic potential for cerebral ischemic stroke.

Vitexin alleviates interleukin-1ß-induced inflammatory responses in chondrocytes from osteoarthritis patients: Involvement of HIF-1α pathway.

It has been reported that vitexin has anti-inflammatory effects in osteoarthritis (OA) rats. However, the effects of vitexin on interleukins-1ß (IL-1ß)-stimulated OA patient-derived chondrocytes have not been reported. The purpose of this study was to investigate the anti-inflammatory effects of vitexin on IL-1ß-stimulated human osteoarthritis chondrocytes and to reveal the involvement of hypoxia-inducible factor 1α (HIF-1α) pathway. Enzyme-linked immunosorbent assay, quantitative real-time PCR and Western blotting assays were employed. ELISA results demonstrated that the proinflammatory cytokine levels of interleukins-6 (IL-6) and tumour necrosis factor α (TNF-α) in the serum and synovial fluid and HIF-1α level in the synovial fluid were significantly elevated in OA patients compared to normal healthy subjects. Moreover, the Western blotting results indicated that the protein expression of HIF-1α was significantly higher in the cartilage tissues of OA patients. OA patient-derived chondrocytes were stimulated by IL-1ß and treated with different concentration of vitexin for 24 hours. Vitexin showed no cytotoxicity and increased the survival of chondrocytes under IL-1ß stimulation. Vitexin suppressed IL-1ß-induced production of NO and prostaglandin E2 (PGE2 ) in chondrocytes culture. The treatment of vitexin significantly inhibited IL-1ß-induced expressions of proinflammatory cytokine levels of IL-6, TNF-α, matrix metalloproteinase (MMP)-1, MMP-3 and MMP-13. Furthermore, Western blotting results demonstrated that HIF-1α is involved in vitexin's protective effects on IL-1ß-stimulated injuries in OA patient-derived chondrocytes. Our study demonstrates that vitexin alleviates IL-1ß-induced inflammatory responses in chondrocytes from osteoarthritis patients, which may be attributed partly to the inhibition of HIF-1α pathway.

Intranasal Insulin Ameliorates Cerebral Hypometabolism, Neuronal Loss, and Astrogliosis in Streptozotocin-Induced Alzheimer's Rat Model.

Intracerebroventricular injection of streptozotocin (ICV-STZ) in rodents leads to cognitive impairments and several pathological changes like Alzheimer's disease (AD). However, there is hardly any research about the effect of ICV-STZ on regional cerebral glucose metabolism in rodents. Previous studies have demonstrated that intranasal insulin improves cognition in AD patients. However, the underlying mechanism remains elusive. Here, we treated the ICV-STZ rats with daily intranasal delivery of insulin (2 U/day) for 6 consecutive weeks, then monitored 18F-fluorodeoxyglucose (18F-FDG) uptake using a high-resolution small-animal positron emission tomography (microPET) and studied the expression of neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) using immunohistochemical staining. We observed that 18F-FDG uptake decreased significantly at the prefrontal cortex, cingulate cortex, striatum, hippocampus, and entorhinal cortex in ICV-STZ rats as compared with the control rats. Intranasal insulin restores the cerebral glucose metabolism in prefrontal and cingulate cortex and attenuates astroglia activation and neuronal loss in the hippocampus of ICV-STZ rats. These findings provide the mechanistic basis for treating AD patients with intranasal insulin.

Relationship between glycated haemoglobin concentration and erythrocyte survival in type 2 diabetes mellitus determined by a modified carbon monoxide breath test.

In clinical practice, an unexplained discordance between percentage haemoglobin A1c (HbA1c) and the progression of diabetes and its complication is observed. HbA1c is determined by the blood glucose level and the red blood cell (RBC) lifespan. Whether the RBC lifespan changes in diabetic patients remains undefined because of the lack of a convenient and accurate measurement method. In the present study, we aim to observe the RBC lifespan in type 2 diabetic patients with poor blood glucose control by an endogenous carbon monoxide (CO) measurement using a rapid and simplified CO breath test machine. The RBC lifespan, age, RBC count, haemoglobin, haematocrit, fasting blood glucose (FBG) level, HbA1c, blood lipids and the liver and kidney function were compared between 38 diabetic patients and 40 healthy individuals. Compared with the control group, in the diabetic patients, the RBC lifespan was significantly decreased by 17.52 ± 4.58 (86.08 ± 18.13 d versus 103.6 ± 22.02 d, p = 0.00). Although a univariate linear correlation analysis showed that the RBC lifespan was negatively correlated with the FBG level (r = -0.386, p = 0.000), haemoglobin A1c (r = -0.346, p = 0.002) and age (r = -0.291, p = 0.010), a stepwise multiple linear regression analysis showed that the RBC lifespan was most affected by the FBG level (t = -3.554, p = 0.001), but not by HbA1c or age, while HbA1c was most affected by the FBG level (t = 13.989, p = 0.000), but not the RBC lifespan. The RBC lifespan in diabetic patients with poor glycaemic control was reduced. The decrease in the RBC lifespan caused by hyperglycaemia was not associated with HbA1c. Thus, a decrease in the RBC lifespan will lead to an underestimation of the actual level of hyperglycaemia and the progression of disease by HbA1c in type 2 diabetic patients if we do not adjust the RBC lifespan.

Stable cell lines of human SH-SY5Y uniformly expressing wild-type or mutant-type FERM domain containing 7 gene.

It has been reported that FERM domain containing 7 (FRMD7) may cause X-linked idiopathic congenital nystagmus (ICN). A total of >40 mutations of the FRMD7 gene have been identified, however their pathogenic role remains unclear. In the present study, enhanced green fluorescent protein-tagged wild-type (WT) and mutant (MT) FRMD7 (c. C781>G) were expressed in stably expressing human neuroblastoma SH-SY5Y cells following viral transfection and antibiotic selection. Uniform expression of the FRMD7 fusion proteins was confirmed via fluorescence microscopy and western blotting. The expression profiles of neuron-specific proteins and Rho guanine triphosphatases (GTPases) differed significantly between the wild-type and mutant cell lines. Levels of Mtap2, NF-M, nestin, GAP43 and Rac1 mRNA were significantly increased in MT-FRMD7 cells compared with controls (P<0.01). However, the expression of Rac1 protein did not differ significantly among the two cell lines. Taken together, the results of the current study suggest that MT-FRMD7 influences the expression of neuron-specific genes and Rho GTPases, which may be involved in the pathogenesis of ICN. The FRMD7 stable expression cell line may facilitate future studies investigating the role of this protein in neuronal development.

Long-term treatment with intranasal insulin ameliorates cognitive impairment, tau hyperphosphorylation, and microglial activation in a streptozotocin-induced Alzheimer's rat model.

Recent evidence reveals that aberrant brain insulin signaling plays an important role in the pathology of Alzheimer's disease (AD). Intranasal insulin administration has been reported to improve memory and attention in healthy participants and in AD patients. However, the underlying molecular mechanisms are poorly understood. Here, we treated intracerebroventricular streptozotocin-injected (ICV-STZ) rats, a commonly used animal model of sporadic AD, with daily intranasal delivery of insulin (2 U/day) for 6 consecutive weeks and then studied their cognitive function with the Morris water maze test and biochemical changes via Western blotting. We observed cognitive deficits, tau hyperphosphorylation, and neuroinflammation in the brains of ICV-STZ rats. Intranasal insulin treatment for 6 weeks significantly improved cognitive function, attenuated the level of tau hyperphosphorylation, ameliorated microglial activation, and enhanced neurogenesis in ICV-STZ rats. Additionally, our results indicate that intranasal delivery of insulin probably attenuates tau hyperphosphorylation through the down-regulation of ERK1/2 and CaMKII in the brains of ICV-STZ rats. Our findings demonstrate a beneficial effect of intranasal insulin and provide the mechanistic basis for treating AD patients with intranasal insulin.

Plasma Exosomes Spread and Cluster Around ß-Amyloid Plaques in an Animal Model of Alzheimer's Disease.

Exosomes, a type of extracellular vesicle, have been shown to be involved in many disorders, including Alzheimer's disease (AD). Exosomes may contribute to the spread of misfolded proteins such as amyloid-ß (Aß) and α-synuclein. However, the specific diffusion process of exosomes and their final destination in brain are still unclear. In the present study, we isolated exosomes from peripheral plasma and injected them into the hippocampus of an AD mouse model, and investigated exosome diffusion. We found that injected exosomes can spread from the dentate gyrus (DG) to other regions of hippocampus and to the cortex. Exosomes targeted microglia preferentially; this phenomenon is stable and is not affected by age. In AD mice, microglia take up lower levels of exosomes. More interestingly, plasma exosomes cluster around the Aß plaques and are engulfed by activated microglia nearby. Our data indicate that exosomes can diffuse throughout the brain and may play a role in the dynamics of amyloid deposition in AD through microglia.

A novel tyrosine hydroxylase variant in a group of Chinese patients with dopa-responsive dystonia.

Dopa-responsive dystonia (DRD) comprises a heterogeneous group of movement disorders. A limited number of studies of Chinese patients with DRD have been reported. In the present study, we investigated the clinical and genetic features of 12 Chinese DRD families. Point mutation analysis of the GTP-cyclohydrolase I (GCH1), tyrosine hydroxylase (TH) and sepiapterin reductase (SPR) genes was conducted by direct sequencing. In addition, multiplex ligation-dependent probe amplification targeting GCH1 and TH was performed in "mutation-free" patients. Three reported mutations (IVS2-2A>G, c.293C>T, c.550C>T) were detected in GCH1, whereas two compound heterozygous variants were identified in TH, one of which was novel (c.1083C>A). Furthermore, this novel variant was not detected in any of the 250 ethnicity-matched, healthy controls. No exon deletions or duplicate mutations in the two genes were found in patients with DRD. No mutation in SPR was found. In addition, one patient with the IVS2-2A>G mutation in GCH1 showed signs of Parkinsonism. In conclusion, we here identified a novel heterozygous variant in TH (c.1083C>A). It is important to perform routine screening of GCH1 and TH for patients with DRD. While for patients with Parkinsonism, GCH1 mutation analysis should be performed after screening of genes like PARKIN, PARK7 (DJ-1) and PINK1.

Intranasal insulin alleviates cognitive deficits and amyloid pathology in young adult APPswe/PS1dE9 mice.

Brain insulin signaling deficits contribute to multiple pathological features of Alzheimer's disease (AD). Although intranasal insulin has shown efficacy in patients with AD, the underlying mechanisms remain largely unillustrated. Here, we demonstrate that intranasal insulin improves cognitive deficits, ameliorates defective brain insulin signaling, and strongly reduces ß-amyloid (Aß) production and plaque formation after 6 weeks of treatment in 4.5-month-old APPswe/PS1dE9 (APP/PS1) mice. Furthermore, c-Jun N-terminal kinase activation, which plays a pivotal role in insulin resistance and AD pathologies, is significantly inhibited. The alleviation of amyloid pathology by intranasal insulin results mainly from enhanced nonamyloidogenic processing and compromised amyloidogenic processing of amyloid precursor protein (APP), and from a reduction in apolipoprotein E protein which is involved in Aß metabolism. In addition, intranasal insulin effectively promotes hippocampal neurogenesis in APP/PS1 mice. This study, exploring the mechanisms underlying the beneficial effects of intranasal insulin on Aß pathologies in vivo for the first time, highlights important preclinical evidence that intranasal insulin is potentially an effective therapeutic method for the prevention and treatment of AD.

Association of CD33 and MS4A cluster variants with Alzheimer's disease in East Asian populations.

CD33 and MS4A cluster variants have been identified to modulate the risk of Alzheimer's disease (AD) in several recent genome-wide association studies (GWAS) in Caucasians. In the present study, we first conducted a case-control study to investigate the CD33 single nucleotide polymorphisms (SNPs) rs3865444 and rs3826656 and the MS4A cluster SNPs rs610932 and rs670139 in a cohort from eastern China that comprised 126 late-onset Alzheimer's disease (LOAD) patients and 129 healthy controls. The results revealed that the frequency of rs3826656 major (G) allele carriers was higher among the LOAD patients than among the controls [P=0.005; odds ratio (OR), 1.760; 95% confidence interval (CI), 1.185-2.615]. In apolipoprotein E (APOE) ε4 allele carriers, the G allele of the SNP rs3865444 was found to be associated with an increased risk of LOAD (P=0.002; OR, 3.391; 95% CI, 1.512-7.605). Next, we re-evaluated the association between these variants and LOAD by conducting a meta-analysis using data from studies of East Asian populations, including the present case-control study, and confirmed that rs3826656 increased the risk of LOAD. In addition, we identified a significant association between rs610932 and LOAD (P=0.035; OR, 0.79; 95% CI, 0.63-0.98). Note that heterogeneity should be considered during the interpretation of these results; significant heterogeneity was identified among studies on rs3865444, even in a subgroup analysis based on stratification of studies by the country of origin. In summary, our results suggest that CD33 and MS4A cluster variants are associated with LOAD susceptibility in East Asian populations.

FERM domain containing protein 7 interacts with the Rho GDP dissociation inhibitor and specifically activates Rac1 signaling.

The FERM domain containing protein 7 gene (FRMD7) associated with the X-linked disorder idiopathic congenital nystagmus (ICN) is involved in the regulation of neurite elongation during neuronal development. Members of the Rho family of small G-proteins (Rho GTPases) are key regulators of the actin cytoskeleton and are implicated in the control of neuronal morphology. The Rho GDP dissociation inhibitor alpha, RhoGDIα, the main regulator of Rho GTPases, can form a complex with the GDP-bound form of Rho GTPases and inhibit their activation. Here, we demonstrate that the full length of the mouse FRMD7, rather than the N-terminus or the C-terminus alone, directly interacts with RhoGDIα and specifically initiates Rac1 signaling in mouse neuroblastoma cell line (neuro-2a). Moreover, we show that wild-type human FRMD7 protein is able to activate Rac1 signaling by interacting with RhoGDIα and releasing Rac1 from Rac1-RhoGDIα complex. However, two missense mutations (c.781C>G and c.886G>C) of human FRMD7 proteins weaken the ability to interact with RhoGDIα and release less Rac1, that induce the activation of Rac1 to a lesser degree; while an additional mutant, c.1003C>T, which results in a C-terminal truncated protein, almost fails to interact with RhoGDIα and to activate Rac1 signaling. Collectively, these results suggest that FRMD7 interacts with one of the Rho GTPase regulators, RhoGDIα, and activates the Rho subfamily member Rac1, which regulates reorganization of actin filaments and controls neuronal outgrowth. We predict that human mutant FRMD7 thus influences Rac1 signaling activation, which can lead to abnormal neuronal outgrowth and cause the X-linked ICN.

[The effect of smoking on resting energy expenditure in patients with early diabetic kidney disease].

OBJECTIVE: To study the effect of smoking on resting energy expenditure (REE) and the relationships among REE, smoking, inflammation and oxidative stress in patients with diabetic kidney disease. METHODS: A case control study of 31 smokers and 40 non-smokers with early stage of diabetic kidney disease (stage III) were performed to evaluate the chronic effect of smoking on REE. REE/fat free mass (FFM), biomarkers of oxidative stress malondialdehyde (MDA), superoxide dismutase (SOD) and inflammation high-sensitivity C-reactive protein (hs-CRP), adiponectin, TNFα were also measured in these subjects. Data were analyzed by Pearson correlation analysis. RESULTS: Compared with non-smokers, REE/FFM in smokers group was significantly increased by 15.96% (P = 0.001). Pearson analysis showed that smoking was significantly correlated with REE/FFM (t = 0.395, P = 0.001). There were significantly different between smokers and non-smokers in MDA, SOD and hs-CRP (P < 0.05). But no difference between two groups in adiponectin and TNFα (P > 0.05). No significant relationships between REE/FFM and MDA, SOD, hs-CRP, adiponectin, TNFα was found (P > 0.05). CONCLUSION: Chronic smoking can lead to increased REE, arouse oxidative stress and inflammatory in patients with early stage of diabetic kidney disease. However, there is no relationship between increased REE due to smoking and oxidative stress and inflammatory.