Sulforaphane attenuates microglia-mediated neuronal damage by down-regulating the ROS/autophagy/NLRP3 signal axis in fibrillar Aß-activated microglia.

The neuroinflammatory hypothesis of Alzheimer's disease (AD) posits that amyloid-beta (Aß) phagocytosis along with subsequent lysosomal damage and NLRP3 inflammasome activation plays important roles in Aß-induced microglia activation and microglia-induced neurotoxicity. Sulforaphane (SFN) has neuroprotective effects for AD. However, whether SFN can inhibit its cytotoxic autophagy and NLRP3 inflammasome activation in microglia remain unknown. In this study, results showed SFN played an indirect, protective role on neurons via a series of impacts on Aß-activated microglia, including inhibition of autophagy initiation as well as autophagic lysosomal membrane permeability and subsequent NLRP3/caspase-1 inflammasomes activation. M1 phenotype polarization was also inhibited. Our results demonstrated that SFN could inhibit the cytostatic autophagy-induced NLRP3 signaling pathway in Aß-activated microglia by decreasing reactive oxygen species (ROS) production. These results provide novel insight into the potential role of SFN in AD therapy.

Sulforaphane Upregulates Cultured Mouse Astrocytic Aquaporin-4 Expression through p38 MAPK Pathway.

Protein misfolding and/or aggregation are common pathological features associated with a number of neurodegenerative diseases, including Alzheimer's disease (AD) and Parkinson disease (PD). Abnormal protein aggregation may be caused by misfolding of the protein and/or dysfunction of the protein clearance system. Recent studies have demonstrated that the specific water channel protein, aquaporin-4 (AQP4), plays a role in the pathogenesis of neurodegenerative diseases involving protein clearance system. In this study, we aimed to investigate the role of sulforaphane (SFN) in the upregulation of AQP4 expression, along with its underlying mechanism using cultured mouse astrocytes as a model system. At low concentrations, SFN was found to increase cell proliferation and result in the activation of astrocytes. However, high SFN concentrations were found to suppress cell proliferation of astrocytes. In addition, our study found that a 1 µM concentration of SFN resulted in the upregulation of AQP4 expression and p38 MAPK phosphorylation in cultured mouse astrocytes. Moreover, we demonstrated that the upregulation of AQP4 expression was significantly attenuated when cells were pretreated with SB203580, a p38 MAPK inhibitor. In conclusion, our findings from this study revealed that SFN exerts hormesis effect on cultured mouse astrocytes and can upregulate astrocytic AQP4 expression by targeting the p38 MAPK pathway.

Hemispheric Difference of Regional Brain Function Exists in Patients With Acute Stroke in Different Cerebral Hemispheres: A Resting-State fMRI Study.

OBJECTIVE: To explore the different compensatory mechanisms of brain function between the patients with brain dysfunction after acute ischemic stroke (AIS) in the dominant hemisphere and the non-dominant hemisphere based on Resting-state Functional Magnetic Resonance Imaging (Rs-fMRI). METHODS: In this trial, 15 healthy subjects (HS) were used as blank controls. In total, 30 hemiplegic patients with middle cerebral artery acute infarction of different dominant hemispheres were divided into the dominant hemisphere group (DH) and the non-dominant hemisphere group (NDH), scanned by a 3.0 T MRI scanner, to obtain the amplitude of low-frequency fluctuations (ALFF) and regional homogeneity (ReHo) and compare the differences. RESULTS: Compared with the HS, increased ALFF values in the brain areas, such as the bilateral midbrain, were observed in DH. Meanwhile decreased ReHo values in the brain areas, such as the right postcentral gyrus (BA3), were also observed. Enhanced ALFF values in the brain areas, such as the left BA6, and enhanced ReHo values in the brain areas, such as the left precuneus, were observed in the NDH. The ALFF and ReHo values of the right BA9 and precentral gyrus were both increased. Compared with DH, the NDH group showed lower ALFF values in the left supplementary motor area and lower ReHo values in the right BA10. CONCLUSION: After acute infarction in the middle cerebral artery of the dominant hemisphere, a compensation mechanism is triggered in brain areas of the ipsilateral cortex regulating motor-related pathways, while some brain areas related to cognition, sensation, and motor in the contralateral cortex are suppressed, and the connection with the peripheral brain regions is weakened. After acute infarction in the middle cerebral artery of the non-dominant hemisphere, compensatory activation appears in motor control-related brain areas of the dominant hemisphere. After acute middle cerebral artery infarction in the dominant hemisphere, compared with the non-dominant hemisphere, functional specificity in the bilateral supplementary motor area weakens. After acute middle cerebral artery infarction in different hemispheres, there are hemispheric differences in the compensatory mechanism of brain function.

DHA attenuates Aß-induced necroptosis through the RIPK1/RIPK3 signaling pathway in THP-1 monocytes.

Monocytes play a crucial role in Alzheimer's disease (AD), and docosahexaenoic acid (DHA) has a neuroprotective effect for many neurodegenerative diseases. However, mechanisms that regulate monocyte and Aß protein interaction in AD and the effects of DHA on monocytes in the context of AD are not fully understood. The experiments were designed to further explore possible mechanisms of interaction between monocytes and Aß plaques. Another objective of this study was to investigate a potential mechanism for Aß-induced necroptosis involving the activation of MAPK and NF-kB signaling pathways in human THP-1 monocytes, as well as how these pathways might be modulated by DHA. Our findings indicate that Aß25-35 has a "Hormesis" effect on cell viability and necroptosis in THP-1 cells, and Aß25-35 influences THP-1 cells differentiation as analyzed by flow cytometry. Pretreatment of THP-1 monocytes with DHA effectively inhibited Aß-induced activation and markedly suppressed protein expression of necroptosis (RIPK1, RIPK3, MLKL) and pro-inflammatory cytokines (TNF-α, IL-1ß, IL-6). Moreover, our findings indicate that Aß25-35 activated the ERK1/2 and p38 signaling pathways, but not NF-κB/p65 signaling, while pre-treatment with DHA followed by Aß25-35 treatment suppressed only ERK1/2 signaling. Further study revealed that the expression level of RIPK3 is reduced much more during coadministration with DHA and necrostatin-1 (NEC-1) than administration alone with either of them, indicating that DHA may have additional targets. Meanwhile, this finding indicates that DHA can prevent Aß-induced necroptosis of THP-1 cells via the RIPK1/RIPK3 signaling pathway. Our results also indicate that DHA treatment restored migration of THP-1 monocytes induced by Aß25-35, and DHA treatment could be a promising new therapy for AD management.

[Analysis of GDAP1 gene mutation in a pedigree with autosomal dominant Charcot-Marie-Tooth disease].

OBJECTIVE: To investigate the molecular genetic mechanism of Charcot- Marie-Tooth (CMT) disease in a pedigree. METHODS: Genomic DNA was extracted from the peripheral blood of the family members of a pedigree with autosomal dominant CMT disease, and 65 candidate genes of the proband were screened using target exon capture and the next generation sequencing, and the suspicious genes were verified using Sanger sequencing. PolyPhen-2, PROVEAN and SIFT software were used to predict the function of the mutant genes, and PyMOL-1 software was used to simulate the mutant protein structure. RESULTS: A heterozygous missense mutation [c.371A>G (p.Y124C)] was detected in exon 3 of GDAP1 gene of the proband. This heterozygous mutation was also detected in both the proband's mother and her brother, but not in her father. Multiple sequence alignment analysis showed that tyrosine at codon 124 of GDAP1 protein was highly conserved. All the 3 prediction software predicted that the mutation was harmful. Molecular structure simulation showed a weakened interaction force between the amino acid residues at codon 124 and the surrounding amino acid residues to affect the overall stability of the protein. CONCLUSIONS: The mutation of GDAP1 gene may be related to the pathogenesis of autosomal dominant AD-CMT in this pedigree. The newly discovered c.371A>G mutation (p.Y124C) expands the mutation spectrum of GDAP1 gene, but further study is needed to clarify the underlying pathogenesis.

[Correlation of apolipoprotein AI, apolipoprotein B and their ratio with the severity of cerebral white matter lesions].

OBJECTIVE: To investigate the correlation of apolipoprotein AI (ApoAI), ApoB, ApoB/ApoAI and the severity of brain white matter lesions (WML). METHODS: A total of 648 patients with WML confirmed by brain magnetic resonance imaging (MRI) were divided into mild WML group (n=386) and moderate to severe WML group (n=262) according to evaluations with the Fazekas scale. The demographic data, blood biochemical parameters and the levels of ApoAI, ApoB and ApoB/AI ratio were compared between the two groups to identify the risk factors of moderate to severe WML. RESULTS: Univariate analysis showed that age, gender, hypertension, diabetes, coronary heart disease, previous stroke, homocysteine, HDL-C, ApoAI, and ApoB/AI ratio all differed significantly between the two groups (P < 0.05), but ApoB levels were similar between them (P > 0.05). Multivariate logistic regression analysis revealed that with ApoAI and ApoB/AI ratio as the continuous variables, after adjustment for the compounding factors, ApoB/AI ratio was an independent risk factor (OR=11.456, 95% CI: 3.622-36.229, P < 0.001) and ApoAI was an independent protective factor for moderate to severe WML (OR=0.068, 95% CI: 0.018-0.262, P < 0.001). With the upper quartiles of ApoAI level (1.38 g/L) and ApoB/AI ratio (0.58) as their respective cutoff values, patients with a high ApoAI level and a low ApoB/AI ratio were found to have the lowest incidence of moderate to severe WML (P < 0.001). CONCLUSIONS: An increased ApoB/AI ratio is an independent risk factor and an increased ApoAI level is an independent protective factor for moderate to severe WML.

Sulforaphane attenuates microglia-mediated neuronal necroptosis through down-regulation of MAPK/NF-κB signaling pathways in LPS-activated BV-2 microglia.

Sulforaphane (SFN), a natural dietary isothiocyanate in cruciferous vegetables such as broccoli and cabbage, has very strong anti-inflammatory activity. Activation of microglia leads to overexpression of a series of pro-inflammatory mediators, which play a vital role in neuronal damage. SFN may have neuroprotective effects in different neurodegenerative diseases related to inflammation. However, the mechanisms underlying SFN's protection of neurons against microglia-mediated neuronal damage are not fully understood. Here, we investigated how SFN attenuated microglia-mediated neuronal damage. Our results showed that SFN could not directly protect the viability of neurons following pro-inflammatory mediators, but increased the viability of BV-2 microglia and down-regulated the mRNA and protein levels of pro-inflammatory mediators including TNF-α, IL-1ß, IL-6 and iNOS in a concentration-dependent manner in BV-2 cells. SFN also significantly blocked the phosphorylation of MAPKs (p38, JNK, and ERK1/2) and NF-κB p65, both by itself and with MAPK inhibitors (SB203580, SP 600125, and U0126) or an NF-κB inhibitor (PDTC). The expression of pro-inflammatory proteins was also blocked by SFN with or without inhibitors. Further, SFN indirectly increased the viability and maintained the morphology of neurons, and the protein expression of RIPK3 and MLKL was significantly suppressed by SFN in neuronal necroptosis through p38, JNK, and NF-κB p65 but not ERK1/2 signaling pathways. Together, our results demonstrate that SFN attenuates LPS-induced pro-inflammatory responses through down-regulation of MAPK/NF-κB signaling pathway in BV-2 microglia and thus indirectly suppresses microglia-mediated neuronal damage.

[Expression of HDAC9 in different brain regions in mice with cerebral ischemic stroke].

OBJECTIVE: To investigate the expression and the subcellular localization of HDAC9 in different brain regions of mice after cerebral ischemic injury and explore the association between HDAC9 and ischemic stroke. METHODS: Twenty-one male C57BL/6 mice were randomly divided into sham-operated group (n=9) and operated group (n=12). In the latter group, the mice with Zea-Longa neurological deficit scores of 2 or 3 following middle cerebral artery occlusion (MCAO) were assigned into MCAO group (n=9). Immunofluorescence was performed to investigate the subcellular localization of HDAC9 in the brain tissues on day 3 after MCAO. Western blotting and qRT-PCR were used to analyze the expression of HDAC9 in different regions of the brain. Results Immunofluorescence showed more intense HDAC9 expressions in the brain tissues around the infarct focus, and in the cells surrounding the infarct, HDAC9 expression was obviously increased in the cytoplasm and reduced in the cell nuclei. Compared with the other brain regions, the ipsilesional cortex with MCAO showed more abundant HDAC9 expressions at both the mRNA and protein levels (P<0.05). CONCLUSION: HDAC9 may be closely related to cerebral ischemic injury and participate in the pathophysiological process of ischemic stroke.

Non-enhanced MR lymphography of the thoracic duct: improved visualization following ingestion of a high fat meal-initial experience.

PURPOSE: The objective of this study was to compare the clarity of magnetic resonance lymphography (MRL) images of the thoracic duct (TD) obtained from subjects following an overnight fast with those obtained from the same subjects after ingestion of a high fat meal. METHODS: Nineteen healthy volunteers were included in this study. TD images were acquired on a 3·0T MRI system with the imaging sequence of magnetic resonance cholangiopancreatography. TD MRL images were obtained from subjects following an overnight fast and from the same subjects 3-4 h after ingestion of a high fat meal. Images were displayed in maximum intensity projection format and degree of visualization was evaluated using a scoring system. RESULTS: The mean TD score obtained following an overnight fast was significantly lower than the mean TD score obtained 3-4 h after ingestion of a high fat meal (P<0·05). CONCLUSION: The clarity of TD MRL images is improved if subjects have ingested a high fat meal 3-4 h prior to examination.

The Efficacy of Trastuzumab in Animal Models of Breast Cancer: A Systematic Review and Meta-Analysis.

BACKGROUND: Breast cancer is the most frequent cancers and is the second leading cause of cancer death among women. Trastuzumab is an effective treatment, the first monoclonal antibody directed against the human epidermal growth factor receptor 2 (HER2). To inform the development of other effective treatments we report summary estimates of efficacy of trastuzumab on survival and tumour volume in animal models of breast cancer. METHODS: We searched PubMed and EMBASE systematically to identify publications testing trastuzumab in animal models of breast cancer. Data describing tumour volume, median survival and animal features were extracted and we assessed quality using a 12-item checklist. We analysed the impact of study design and quality and evidence for publication bias. RESULTS: We included data from 83 studies reporting 169 experiments using 2076 mice. Trastuzumab treatment caused a substantial reduction in tumour growth, with tumours in treated animals growing to 32.6% of the volume of tumours in control animals (95%CI 27.8%-38.2%). Median survival was prolonged by a factor of 1.45 (1.30-1.62). Many study design and quality features accounted for between-study heterogeneity and we found evidence suggesting publication bias. CONCLUSION: We have found trastuzumab to be effective in animal breast cancer models across a range of experimental circumstances. However the presence of publication bias and a low prevalence of measures to reduce bias provide a focus for future improvements in preclinical breast cancer research.

Aquaporin-4 and Alzheimer's Disease.

Alzheimer's disease (AD) is the most common form of dementia. Although the pathogenesis of AD remains unclear, AD is thought to result from an imbalance in the production and clearance of amyloid-ß protein (Aß). Aquaporin-4 (AQP4) is the major aquaporin in the mammalian brain, is mostly expressed on astrocytic endfeet, and functions as a water transporter. However, the distribution and expression of AQP4 are altered in both AD clinical populations and animal models. Recent studies have revealed that AQP4 is important to the clearance of Aß in brain via lymphatic clearance, transcytotic delivery, and glial degradation, as well as to the synaptic function. Thus, AQP4 likely plays an important role in the pathogenesis of AD. Further studies would provide new targets for prevention, ultimately leading to improved treatment options for AD.

Key regions of the cerebral network are altered after electroacupuncture at the Baihui (GV20) and Yintang acupuncture points in healthy volunteers: an analysis based on resting fcMRI.

OBJECTIVE: To identify the key cerebral functional region affected by acupuncture point needling by examining cerebral networks using functional connectivity MRI (fcMRI) and analysing changes in the key regions of these brain networks at different time points after needle removal. METHODS: Twelve healthy volunteers received 30 min of electroacupuncture (EA) at the Baihui (GV20) and Yintang acupuncture points and then underwent two fMRI scans, one each at 5 and 15 min after needle removal. Related brain networks were analysed centred at different 'seeds', centres which functionally connect the other cerebral regions in an organised network, such as the anterior frontal lobe, anterior cingulate gyrus, parahippocampal gyrus, amygdala, hypothalamus, head of the caudate nucleus and anterior lobe of the cerebellum. Networks were analysed based on the resting cerebral functional connection, and the differences in the activities of the brain networks between the two time points were compared. RESULTS: At 5 min after needle removal, 12 brain functional regions were involved in organising the network centred at the caudate nucleus 'seed.' This number was greater than the number of related brain networks centred at the other 'seeds'. At 15 min after needle removal, 15 and 14 brain functional regions were involved in organised networks centred at the parahippocampal and hypothalamus 'seeds', respectively; these numbers were greater than the numbers of other related brain networks centred at the other 'seeds'. CONCLUSIONS: A brain network composed of a large number of cerebral functional regions was found after EA at GV20 and Yintang in healthy volunteers. The key brain 'seed' supporting the largest brain network changed between 5 and 15 min after needle removal.