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Objective:To investigate the effect of LRRK2G2019S mutation on activation of microglia after iron deprivation and its mechanism.Methods:(1) Microglia were differentiated from human induced pluripotent stem cells (IPSC) with the help of hematopoietic progenitor cells (HPC) and identified by immunofluorescent staining, and α-synuclein (α-syn) A53T mutant protein was obtained by protein purification technology. (2) Microglia were divided into control group, α-syn group, α-syn+ deferoxamine (DFO) group; phosphate buffer solution (PBS), 1 μmol/L purified α-syn A53T mutant protein, 1 μmol/L purified α-syn A53T mutant protein+30 mmol/L DFO were given respectively for 24 h. Fe 2+ concentration was detected by colorimetry, Rab35 protein expression was detected by Western blotting, intracellular reactive oxygen species (ROS) level was detected by flow cytometry, and interleukin-6 ( IL-6), tumor necrosis factor-α ( TNF-α) and transforming growth factor-β ( TGF-β) mRNA expressions were detected by real time-PCR (RT-PCR); microglia culture supernatant (MCS) in the 3 groups were transfered to SH-SY5Y cells, and SH-SY5Y cell apoptosis was detected by flow cytometry. (3) Bidirectional DNA sequencing was used to detect leucine rich repeat kinase 2 ( LRRK2) gene mutations in microglia treated with 1 μmol/L purified α-syn A53T mutant protein. Microglia were divided into control group, α-syn group and α-syn+GSK3357679A group, and treated with corresponding drugs for 24 h, respectively (LRRK2 inhibitor GSK3357679A concentration: 10 nmol/L), and LRRK2 protein expression was detected by Western blotting; microglia were divided into control group, α-syn group, α-syn+GSK3357679A, and α-syn+GSK3357679A+DFO group, and treated with corresponding drugs for 24 h, Rab35 protein expression was detected by Western blotting, intracellular ROS level was detected by flow cytometry, and IL-6, TNF-α and TGF-β mRNA expressions were detected by RT-PCR. (4) Microglia were divided into control group, α-syn group, α-syn+rapamycin (RAPA) group, and treated with corresponding drugs for 24 h (concentration of autophagy inducer RAPA: 50 nmol/L); protein expressions of Rab35, P62 and microtubule-associated protein light chain 3 II (LC3II) were detected by Western blotting; intracellular ROS level was detected by flow cytometry, and IL-6, TNF-α and TGF-β mRNA expressions were detected by RT-PCR. (5) Microglia were divided into control group, α-syn group, and α-syn+Rab35 group, and treated with corresponding drugs for 24 h (concentration of Rab35 overexpressed plasmids: 1 μg/mL); Rab35, P62, and LC3II protein expressions were detected by Western blotting; ROS level was detected by flow cytometry, and IL-6, TNF-α and TGF-β mRNA expressions were detected by RT-PCR. Results:(1) Immunofluorescent staining showed negative neuronal nuclei (NeuN) expression and positive ionized calcium-binding adapter molecule 1 (Iba1) expression in microglia, and high LRRK2 expression; PcDNA3.1-SNCA-A53T expression plasmid was constructed and α-syn A53T mutant protein was purified. (2) The Fe 2+ concentration in α-syn group was significantly higher than that in control group, and the Fe 2+ concentration in α-syn+DFO group was significantly lower than that in α-syn group ( P<0.05); the Rab35 protein and TGF-β mRNA expressions in control group, α-syn group and α-syn+DFO group were decreased successively, while the IL-6 and TNF-α mRNA expressions were increased successively, with significant differences ( P<0.05); ROS level and SH-SY5Y cell apoptosis rate in control group, α-syn group, α-syn+DFO group were increased successively. (3) Bidirectional DNA sequencing showed that the LRRK2G2019S mutation in microglia was the most obvious after α-syn A53T mutant protein stimulation; compared with the control group, the α-syn group had significantly increased LRRK2 protein expression, while the α-syn+GSK3357679A group had significantly decreased LRRK2 protein expression compared with α-syn group ( P<0.05); compared with the control group, the α-syn group had significantly decreased Rab35 protein and TGF-β mRNA expressions, and statistically increased IL-6 and TNF-α mRNA expressions ( P<0.05); compared with α-syn group, the α-syn+GSK3357679A group had significantly increased Rab35 protein and TGF-β mRNA expressions, and statistically decreased IL-6 and TNF-α mRNA expressions ( P<0.05); compared with α-syn+GSK3357679A group, α-syn+GSK3357679A+DFO group had significantly increased IL-6 and TNF-α mRNA expressions, and significantly decreased Rab35 protein and TGF-β mRNA expressions ( P<0.05). The α-syn group had higher ROS level than the control group, the α-syn+GSK3357679A group had lower ROS level than the α-syn group, and the α-syn+GSK3357679A+DFO group had higher ROS level than the α-syn+GSK3357679A group. (4) Compared with the control group, the α-syn group had significantly decreased Rab35 and LC3II protein, and TGF-β mRNA expressions, and significantly increased P62 protein, IL-6 and TNF-α mRNA expressions ( P<0.05); compared with α-syn group, the α-syn+RAPA group had significantly increased Rab35 and LC3II protein, and TGF-β mRNA expressions, and significantly decreased P62 protein, and IL-6 and TNF-α mRNA expressions ( P<0.05); the α-syn group had higher ROS level than the control group and α-syn+RAPA group. (5) Compared with the control group, the α-syn group had significantly decreased Rab35 and LC3II protein, and TGF-β mRNA expressions, and statistically increased P62 protein, and IL-6 and TNF-α mRNA expressions ( P<0.05); compared with the α-syn group, the α-syn+Rab35 group had significantly increased Rab35 and LC3II protein, and TGF-β mRNA expressions, and significantly decreased P62 protein, and IL-6 and TNF-α mRNA expressions ( P<0.05). The α-syn group had higher ROS level than the control group and α-syn+Rab35 group. Conclusion:LRRK2G2019S can induce neuroinflammation by inhibiting Rab35-related autophagy under iron deprivation, and Rab35 is expected to be a key factor in intervening neuroinflammation.
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@#Background & Objective: Endovascular treatment is the widely accepted treatment for patients with anterior circulation stroke within 6 hours of onset of stroke. We aimed to evaluate the advantages of endovascular treatment compared to standard medical treatment in treating patients with anterior circulation stroke beyond the 6-hour therapeutic window. Methods: We reviewed the literature concerning endovascular treatment versus medical treatment beyond the 6-hour therapeutic window. Using random-effects meta-analysis, we evaluated the following outcomes: modified Rankin scale in the three-month follow-up [excellent outcome (mRS≤1), functional independence (mRS≤2), moderate outcome(mRS≤3)], recanalization rate at 24 hours, mortality at 90 days or in-hospital, symptomatic intracranial hemorrhage, parenchymal hematoma type 2 and hemorrhagic infarction 1. Results: Four studies including 642 patients were evaluated. Endovascular treatment was associated with higher odds of excellent outcome (OR 2.55; 95% CI 1.48 to 4.41,), functional independence (OR 3.64; 95% CI 2.43 to 5.45), moderate outcome (OR 2.70; 95% CI 1.95-3.74) and recanalization rate at 24 hours (OR 8.81; 95%CI 2.81 to 27.69) compared to MT. No difference in the rates of mortality, symptomatic intracranial hemorrhage, parenchymal hematoma type 2 or hemorrhagic infarction 1 was found between the 2 groups. Studies using strict perfusion imaging inclusion selection showed better moderate outcome in comparison to the studies without perfusion imaging inclusion selection (P <0.012). Conclusion: Our study highlights the superiority of endovascular treatment over standard medical treatment alone for treating patients with anterior circulation stroke beyond 6 hours since stroke onset, although more studies are required for further investigation. Standard of strict selection for eligible patients before endovascular treatment should be based on DAWN or DEFFUSE 3 inclusion criteria.
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Objective To compare the effect of repeated transcranial magnetic stimulation (rTMS) at different frequencies acting on left dorsolateral prefrontal cortex (LPFC) on arousal for coma patients after brain stem injury.Methods Ninety-nine patients with coma resulted from brain stem injury,admitted to our hospital from February 1,2015 to April 10,2018,were chosen in our study.Among them,30 patients weren't treated with rTMS (control group),33 patients were treated with 10 Hz rTMS (10 Hz rTMS treatment group),and 36 patients were treated with 20 Hz rTMS (20 Hz rTMS treatment group);treatments lasted for 20 d.All patients received routine coma arousal treatment.Glasgow coma scale (GCS) scores,electroencephalogram (EEG) grading,brainstem auditory evoked potential (BAEP) grading and incidence of adverse reactions were compared among the three groups before and after treatment.Results Before treatment,there were no significant differences in GCS total scores,language response scores,motor response scores,eye opening reaction scores,EEG grading and BAEP grading among the three groups (P＞0.05).The total GCS scores,and scores of language response,motor response and open eye response of patients in the 20 Hz rTMS treatment group and 10 Hz rTMS treatment group after treatment were significantly higher than those in the control group (P＜0.05);the total GCS scores and motor response scores of patients in the 20 Hz rTMS treatment group were significantly higher than those in 10 Hz rTMS treatment group after treatment (P＜0.05).After treatment,patients in the control group,10 Hz rTMS treatment group,and 20 Hz rTMS treatment group showed statistically significant differences in EEG grading and BAEP grading (P＜0.05).The incidence of adverse reactions in the 20 Hz rTMS group (19.4％) was statistically higher than that in the 10 Hz rTMS group (3.0％,P＜0.05).Conclusion High-frequency rTMS has an awakening effect on patients with coma resulted from brain stem injury,and the coma awakening effect of 20 Hz rTMS is partially better than that of 10 Hz rTMS,but it has the risk of increasing side effects such as epilepsy and scalp bum.
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Objective To analyze the influence of general anesthesia (GA) on bilateral subthalamic nucleus deep brain stimulation (STN-DBS) in treating Parkinson's disease (PD) through microelectrode recording (MER),and discuss the differences between different modes of anesthesia.Methods A retrospective analysis was performed on clinical data of 31 PD patients accepted bilateral STN-DBS in our hospital from June 2015 to June 2017.Nine patients accepted surgery under GA (A group):4 patients were treated with intravenous anesthesia (A1 group),and 5 patients were treated with inhalation anesthesia (A2 group);22 patients accepted surgery under local anesthesia LA group.MER indexes,including STN discharge frequency,STN recorded length,and maximum target error,and short-term (6 months) efficacy were recorded.A linear regression analysis was performed to find possible influence factors on discharge frequency and improving rate of UPDRS scores.Results The discharge frequencies of B group,A1 group and A2 group were 51.42 Hz±6.28 Hz,35.79 Hz±7.02 Hz and 43.18 Hz±5.87 Hz,respectively,with significant differences (F=12.181,P=0.000);as compared with that in the B group,the discharge frequencies of A1 group and A2 group were significantly lower (P＜0.05).The STN recorded lengths of B group,A1 group and A2 group were 5.48 mm±0.33 mm,5.06 mm±0.15 mm and 5.22 mam±0.16 mm,respectively,with significant differences (F=4.115,P=0.027);as compared with that in the B group,the recorded lengths of A1 group and A2 group were significantly shorter (P＜0.05).A1 group had the maximum target error,but no significant differences were noted among the 3 groups (P＞ 0.05).Six months after the surgery,the UPDRS-Ⅲ scores and Schwab-England scores of A group and B group were decreased and daily levodopa equivalent (LEDD) was decreased.As compared with B group,A group had significantly better improvement in Hoehn & Yahr grading (P＜0.05).Disease durations were positively correlated with discharge frequency (r=0.539,P=0.002);age and improving rate of UPDRS scores were negatively correlated (r=-0.572,P=-0.001);preoperative LEDD and improving rate of UPDRS scores were positively correlated (r=0.725,P=-0.000).Conclusions Bilateral STN-DBS performed under GA in PD enjoys good efficacy,which shows no obvious difference as compared with that under LA.Inhalation anesthesia had less influence on electrophysiology than intravenous anesthesia.
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Objective To investigate the role of long noncoding RNA nuclear paraspeckle assembly transcript 1 (NEA T1) in regulating the activation of microglias.Methods (1) Microglias BV2 were routinely cultured in vitro and NEA T1 mRNA expression was detected by real-time PCR (RT-PCR) after 0,0.1,0.5 and 1 μg/mL lipopolysaccharide (LPS) stimulation for 6 h;NEAT1 mRNA expression was detected by RT-PCR after 1 μg/mL LPS stimulation for 0,6,12 and 24 h.(2) NEAT1 siRNA (NEA T1-si) was transfected into BV2,and RT-PCR and Western blotting were employed to detect the LC3 mRNA and protein expressions.(3) The BV2 cells were divided into control group,NEAT1-si group,LPS group and NEAT1-si+LPS group;RT-PCR was used to detect the tumor necrosis factor-α (TNF-oα) and inducible nitric oxide synthase (iNOS) mRNA expressions;morphological changes of BV2 cells were observed under inverted microscope.(4) The BV2 cells were divided into control group,negative control+Torin-1 group and NEAT1-si+Torin-1 group;Western blotting was used to detect the LC3 protein expression and LC3,TNF-α and iNOS mRNA expressions were detected by RT-PCR.Results (1) NEA T1 was significantly up-regulated in LPS-stimulated BV2 cells in dose-and time-d ependent manners;significant differences were noted between each two groups (P＜0.05).(2) The LC3-Ⅱ mRNA expression in the NEAT1-si group was significantly decreased as compared with that in the control group (P＜0.05);LC3-Ⅱ/Ⅰ protein ratio (0.7) in the NEAT1-si group was significantly lower than that in the control group (1.03,P＜0.05).(3) As compared with those in the control group,the TNF-α and iNOS mRNA expressions in the NEAT1-si group were decreased;As compared with those in the LPS group,the TNF-oα and iNOS mRNA expressions in the NEA T1-si+LPS group were significantly decreased (P＜0.05).(4) LC3-Ⅱ/Ⅰ protein ratio in the control group,Torin-1 group and Torin-l+NEAT1-si group were 0.7,1.14 and 0.97,respectively;LC3-Ⅱ,TNF-α and iNOS mRNA expressions in the Torin-1 group were significantly higher than those in the control group (P＜0.05);LC3-Ⅱ,TNF-α and iNOS mRNA expressions in the NEAT1-si+Torin-1 group were significantly higher than those in the Torin-1 group (P＜0.05).Conclusion Knockdown of long noncoding RNA NEA T1 could attenuate microglia activation through inhibiting autophagy,and NEA T1 maybe the key molecule for the mitigation and cure of the neuroinflammation related diseases.
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Objective To investigate the role of micro RNA-124 (miR-124) in regulating activation of microglias and secretion of pro-inflammation cytokines and its potential mechanism.Methods (1) BV-2 cells were exposed to different concentration oflipopolysaccharide (LPS) for different durations;relative expression level of miR-124 was detected by real time-quantitative PCR (RT-qPCR).(2) The BV-2 cells were divided into four groups:PBS group,LPS group,LPS+ctrl-simulant group and LPS+miR-124 simulant group.Protein and mRNA expressions of inflammatory factors (tumor necrosis factor [TNF]-α and interleukin [IL] 1-β) were evaluated by RT-qPCR and ELISA.Expressions of p38α,phosphorylated (p)-p38α,ERK and p-ERK were detected by Western blotting.(3) Besides the above groups,four groups were added:LPS+VX702 group,LPS+miR-124 inhibitor group,LPS+VX702+miR-124 simulant group and LPS+VX702+miR-124 inhibitor group;pretreatment with p38α specific inhibitor VX702 was given to the BV-2 cells,the latter two groups were given miR-124 simulant or miR-124 inhibitor,and LPS was used to activate the cells;the expressions of TNF-α and IL-1β were evaluated by ELISA.Results (1) As compared with control group,miR-124 was significantly down-regulated in LPS-stimulated BV-2 cells (P＜0.05),in a dose-and time-dependent manner.(2) As compared with cells in the LPS+ctrl-simulant group,cells in the LPS+miR-124 simulant group had significantly decreased TNF-α and IL-lβ mRNA and protein expressions,and p38α and p-p38α levels (P＜0.05);the ERK and p-ERK levels showed no significant difference between the two groups (P＞0.05).(3) The TNF-α and IL-1β levels between LPS+VX702 group and LPS+VX702+miR-124 simulant group were not significantly different (P＞0.05);those between the LPS+VX702+miR-124 inhibitor group and the LPS+VX702 group were not significantly different (P＞0.05).Conclusion The miR-124 is down-regulated in LPS-activated BV-2 cells and miR-124 could suppress the secretion of pro-inflammatory mediators by targeting to p38α.
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Objective To explore the expressions ofmiR-124* and laminin-8 in different grades of human gliomas,and analyze the potential regulatory relationship between them.Methods Thirty human glioma specimens of different stages and two healthy brain tissues,collected in our hospital from January 2011 to December 2012,were used in our study; Western blotting was performed to detect the laminin-8 and miR-124* protein expressions; plasmid vectors carried psiCHECKTM-2-wild-type (WT) laminin β1 chain 3 'untranslated region (3'UTR) and psiCHECKTM-2-mutant (MUT) laminin β1 chain 3'UTR were established,and they were,respectively,co-transfected with the miR-124* mimics,negative control ofmiRNA mimics,miR-124* inhibitors,negative control ofmiRNA inhibitors into the U87 cells;besides,cotransfection with psiCHECKTM-2 into U87 cells was used as blank control group; the fluorescence intensity of each group was determined by dual-luciferase assay 48 h after the cotransfection.Dual-luciferase assay was developed to verify the regulatory effects ofmiR-124* on laminin-8.The nu/nu nude mice were given subcutaneous injection of U87 cells to establish giloma animal models; the expression of β1 chain-containing laminin-8 protein was detected in gliomas by Western blotting; the expression of vascular endothelial cell marker CD31 was observed by immunohistochemical staining.Results Western blotting showed high expressions of laminin-8 protein in high-grade gliomas (WHO grade Ⅲ/ⅣV),but low expressions in low-grade gliomas (WHO grade Ⅰ/Ⅱ); real-time fluorescent quantitative PCR showed that the expression of miR-124* in high-grade gliomas was significantly lower than that in low-grade gliomas.Dual-luciferase assay confirmed that fluorescence intensity of U87 cells carried psiCHECKTM-2-WT laminin β1 chain 3'UTR and miR-124* mimics (2.13±0.25) was significantly weaker than that of cells carried psiCHECKTM-2-WT laminin β1 chain 3'UTR and negative control of miRNA mimics (2.71±0.08,P＜0.05); that of those carried psiCHECKTM-2-MUT laminin β1 chain 3'UTR and miR-124* inhibitors (3.18 ±0.22) was significantly stronger than those carried,psiCHECKTM-2-MUT laminin β1 chain 3'UTR and negative control of miRNA inhibitors (2.70±0.17,P＜0.05).The animal model results indicated the laminin-8 and CD31 expressions in miR-124* treatment group were obviously lower than those in control group.Conclusion MiR-124*,which can affect the malignant degrees of human gliomas through playing its negative regulation role in laminin-8 expression,maybe a target gene for the diagnosis and therapy of gliomas.
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SUMMARY Constitutional full trisomy 21 is a common disorder in which abnormal spermatogenesis has been previously described. However, constitutional mosaic trisomy 21 in an otherwise normal but infertile male has not been explored. We report a case with low level mosaic trisomy 21 in a non-syndrome but azoospermic patient. We also propose that the patient's azoospermia may be related to the constitutional mosaic trisomy 21 and thus resulting in a late onset of testicular failure.
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SUMMARY Therapy-related acute myeloid leukemia (tAML) is one of the two forms of secondary acute myeloid leukemia, with one derived from de novo myelodysplastic syndrome (MDS) and the other from exposure to environmental or therapeutic agents such as radiation and toxins. There has been a marked increase in the number of incidences of therapy-related acute myeloid leukemia. It has become a distinctive disease because of its etiology and genetic tumorigenesis. The majority of tAML resulting from the use of cytotoxic agents can be divided into two groups based on the drugs administered to the patient. The first group includes the use of alkylating agents, and the second group includes agents that bind to the enzyme DNA-topoisomerase Ⅱ. Due to the unfavorable outcome of the disease and the need for prompt intensive treatment, a timely accurate diagnosis of tAML is critical to patient care. Cytogenetic study can detect abnormalities most commonly associated with tAML and thus providing important diagnostic information. However, utilizing cytogenetic analysis alone cannot guarantee prompt and accurate results. In this study, an interesting case with therapy-related myelodysplastic syndrome and acute myeloid leukemia (tMDS/tAML) will be presented. A laboratory diagnostic strategy for tAML laboratory diagnosis will also be proposed.