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1.
Acta Pharmacol Sin ; 2024 Sep 03.
Artigo em Inglês | MEDLINE | ID: mdl-39227736

RESUMO

Emerging evidence shows that psychological stress promotes the progression of Parkinson's disease (PD) and the onset of dyskinesia in non-PD individuals, highlighting a potential avenue for therapeutic intervention. We previously reported that chronic restraint-induced psychological stress precipitated the onset of parkinsonism in 10-month-old transgenic mice expressing mutant human α-synuclein (αSyn) (hαSyn A53T). We refer to these as chronic stress-genetic susceptibility (CSGS) PD model mice. In this study we investigated whether ginsenoside Rg1, a principal compound in ginseng notable for soothing the mind, could alleviate PD deterioration induced by psychological stress. Ten-month-old transgenic hαSyn A53T mice were subjected to 4 weeks' restraint stress to simulate chronic stress conditions that worsen PD, meanwhile the mice were treated with Rg1 (40 mg· kg-1 ·d-1, i.g.), and followed by functional magnetic resonance imaging (fMRI) and a variety of neurobehavioral tests. We showed that treatment with Rg1 significantly alleviated both motor and non-motor symptoms associated with PD. Functional MRI revealed that Rg1 treatment enhanced connectivity between brain regions implicated in PD, and in vivo multi-channel electrophysiological assay showed improvements in dyskinesia-related electrical activity. In addition, Rg1 treatment significantly attenuated the degeneration of dopaminergic neurons and reduced the pathological aggregation of αSyn in the striatum and SNc. We revealed that Rg1 treatment selectively reduced the level of the stress-sensitive protein RTP801 in SNc under chronic stress conditions, without impacting the acute stress response. HPLC-MS/MS analysis coupled with site-directed mutation showed that Rg1 promoted the ubiquitination and subsequent degradation of RTP801 at residues K188 and K218, a process mediated by the Parkin RING2 domain. Utilizing αSyn A53T+; RTP801-/- mice, we confirmed the critical role of RTP801 in stress-aggravated PD and its necessity for Rg1's protective effects. Moreover, Rg1 alleviated obstacles in αSyn autophagic degradation by ameliorating the RTP801-TXNIP-mediated deficiency of ATP13A2. Collectively, our results suggest that ginsenoside Rg1 holds promise as a therapeutic choice for treating PD-sensitive individuals who especially experience high levels of stress and self-imposed expectations.

2.
Acta Pharmacol Sin ; 2024 Aug 07.
Artigo em Inglês | MEDLINE | ID: mdl-39112771

RESUMO

Esculetin (ESC) is a coumarin-derived phytochemical prevalent in traditional Chinese medicine that exhibits anti-acute ischemic stroke activities. Our previous studies demonstrate that CKLF1 is a potential anti-stroke target for coumarin-derived compound. In this study we investigated whether CKLF1 was involved in the neuroprotective effects of ESC against photothrombotic stroke in mice. The mice were treated with ESC (20, 40 or 80 mg·kg-1·d-1, i.g.) for two weeks. The therapeutic effect of ESC was assessed using MRI, neurological function evaluation, and a range of behavioral tests on D1, 3, 7 and 14 of ESC administration. We showed that oral administration of ESC dose-dependently reduced the cerebral infarction volume within one week after stroke, improved behavioral performance, and alleviated neuropathological damage within two weeks. Functional MRI revealed that ESC significantly enhanced the abnormal low-frequency fluctuation (ALFF) value of the motor cortex and promoted functional connectivity between the supplementary motor area (SMA) and multiple brain regions. We demonstrated that ESC significantly reduced the protein levels of CKLF1 and CCR5, as well as the CKLF1/CCR5 protein complex in the peri-infarcted area. We showed that ESC (0.1-10 µM) dose-dependently blocked CKLF1-induced chemotactic movement of neutrophils in the Transwell assay, reducing the interaction of CKLF1/CCR5 on the surface of neutrophils, thereby reducing neutrophil infiltration, and decreasing the expression of ICAM-1, VCAM-1 and MMP-9 in the peri-infarct tissue. Knockout of CKLF1 reduced brain infarction volume and motor dysfunction after stroke but also negated the anti-stroke efficacy and neutrophil infiltration of ESC. These results suggest that the efficacy of ESC in promoting post-stroke neural repair depends on its inhibition on CKLF1-mediated neutrophil infiltration, which offering novel perspectives for elucidating the therapeutic properties of coumarins.

3.
J Med Genet ; 60(2): 144-153, 2023 02.
Artigo em Inglês | MEDLINE | ID: mdl-35387802

RESUMO

BACKGROUND: The genetic causes for most male infertility due to severe oligoasthenoteratozoospermia (OAT) remain unclear. OBJECTIVE: To identify the genetic cause of male infertility characterised by OAT. METHODS: Variant screening was performed by whole-exome sequencing from 325 infertile patients with OAT and 392 fertile individuals. In silico and in vitro analyses were performed to evaluate the impacts of candidate disease-causing variants. A knockout mouse model was generated to confirm the candidate disease-causing gene, and intracytoplasmic sperm injection (ICSI) was used to evaluate the efficiency of clinical treatment. RESULTS: We identified biallelic CFAP61 variants (NM_015585.4: c.1654C>T (p.R552C) and c.2911G>A (p.D971N), c.144-2A>G and c.1666G>A (p.G556R)) in two (0.62%) of the 325 OAT-affected men. In silico bioinformatics analysis predicted that all four variants were deleterious, and in vitro functional analysis confirmed the deleterious effects of the mutants. Notably, H&E staining and electron microscopy analyses of the spermatozoa revealed multiple morphological abnormalities of sperm flagella, the absence of central pair microtubules and mitochondrial sheath malformation in sperm flagella from man with CFAP61 variants. Further immunofluorescence assays revealed markedly reduced CFAP61 staining in the sperm flagella. In addition, Cfap61-deficient mice showed the OAT phenotype, suggesting that loss of function of CFAP61 was the cause of OAT. Two individuals accepted ICSI therapy using their own ejaculated sperm, and one of them succeeded in fathering a healthy baby. CONCLUSIONS: Our findings indicate that CFAP61 is essential for spermatogenesis and that biallelic CFAP61 variants lead to male infertility in humans and mice with OAT.


Assuntos
Anormalidades Múltiplas , Astenozoospermia , Infertilidade Masculina , Oligospermia , Humanos , Masculino , Animais , Camundongos , Infertilidade Masculina/genética , Oligospermia/genética , Astenozoospermia/genética , Sêmen , Espermatozoides , Anormalidades Múltiplas/genética
4.
J Sci Food Agric ; 104(10): 5735-5750, 2024 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-38441287

RESUMO

BACKGROUND: During the brewing of soy sauce, the conversion of multiple substances is driven by various microorganisms and their secreted enzyme systems. Soy sauce mash is an important source of enzyme systems during moromi fermentation, but the changes of enzyme systems in soy sauce mash during moromi fermentation are poorly understood. In order to explore the predominant enzyme systems existing during moromi fermentation and to explain the characteristics of the enzyme system changes, an enzymatic activities assay and 4D-label-free proteomics analysis were conducted on soy sauce mash at different stages of fermentation. RESULTS: The activities of hydrolytic enzymes in soy sauce mash decreased continuously throughout the fermentation process, while most of the characteristic physicochemical substances in soy sauce mash supernatant had already accumulated at the early stage of fermentation. Four hydrolytic enzymes were found to be positively correlated with important physicochemical indexes by principal component analysis and Pearson correlation analysis. The proteomics analysis revealed three highly upregulated enzymes and two enzymes that were present in important metabolic pathways throughout the fermentation process. Furthermore, it was found that Aspergillus oryzae was able to accumulate various nutrients in the soy sauce mash by downregulating most of its metabolic pathways. CONCLUSION: Enzymes present with excellent properties during the moromi fermentation period could be obtained from these results. Meanwhile, the characterization of the metabolic pathways of microorganisms during the moromi fermentation period was revealed. The results provide a basis for more scientific and purposeful improvement of moromi fermentation in the future. © 2024 Society of Chemical Industry.


Assuntos
Fermentação , Proteômica , Alimentos de Soja , Alimentos de Soja/análise , Alimentos de Soja/microbiologia , Proteínas Fúngicas/metabolismo , Aspergillus oryzae/metabolismo , Aspergillus oryzae/enzimologia
5.
J Neuroinflammation ; 20(1): 97, 2023 Apr 25.
Artigo em Inglês | MEDLINE | ID: mdl-37098609

RESUMO

Ischemic stroke is characterized by the presence of reactive microglia. However, its precise involvement in stroke etiology is still unknown. We used metabolic profiling and showed that chemokine like factor 1 (CKLF1) causes acute microglial inflammation and metabolic reprogramming from oxidative phosphorylation to glycolysis, which was reliant on the AMP-activated protein kinase (AMPK)-mammalian target of rapamycin (mTOR)-hypoxia inducible factor 1α (HIF-1α) signaling pathway. Once activated, microglia enter a chronic tolerant state as a result of widespread energy metabolism abnormalities, which reduces immunological responses, including cytokine release and phagocytosis. Metabolically dysfunctional microglia were also found in mice using genome-wide RNA sequencing after chronic administration of CKLF1, and there was a decrease in the inflammatory response. Finally, we showed that the loss of CKLF1 reversed the defective immune response of microglia, as indicated by the maintenance its phagocytosis to neutrophils, thereby mitigating the long-term outcomes of ischemic stroke. Overall, CKLF1 plays a crucial role in the relationship between microglial metabolic status and immune function in stroke, which prepares a potential therapeutic strategy for ischemic stroke.


Assuntos
AVC Isquêmico , Acidente Vascular Cerebral , Animais , Camundongos , Citocinas/metabolismo , Tolerância Imunológica , AVC Isquêmico/metabolismo , Mamíferos/metabolismo , Microglia/metabolismo , Acidente Vascular Cerebral/metabolismo
6.
Brief Bioinform ; 22(4)2021 07 20.
Artigo em Inglês | MEDLINE | ID: mdl-33381797

RESUMO

In drug discovery, one of the most important tasks is to find novel and biologically active molecules. Given that only a tip of iceberg of drugs was founded in nearly one-century's experimental exploration, it shows great significance to use in silico methods to expand chemical database and profile drug-target linkages. In this study, a web server named ChemGenerator was proposed to generate novel activates for specific targets based on users' input. The ChemGenerator relies on an autoencoder-based algorithm of Recurrent Neural Networks with Long Short-Term Memory by training of 7 million of molecular Simplified Molecular-Input Line-Entry System as the basic model, and further develops target guided generation by transfer learning. As results, ChemGenerator gains lower loss (<0.01) than existing reference model (0.2~0.4) and shows good performance in the case of Epidermal Growth Factor Receptor. Meanwhile, ChemGenerator is now freely accessible to the public by http://smiles.tcmobile.org. In proportion to endless molecular enumeration and time-consuming expensive experiments, this work demonstrates an efficient alternative way for the first virtual screening in drug discovery.


Assuntos
Bases de Dados de Compostos Químicos , Descoberta de Drogas , Internet , Redes Neurais de Computação , Software , Ligantes
7.
Acta Pharmacol Sin ; 44(10): 1935-1947, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37198412

RESUMO

Chemokine receptor 5 (CCR5) is one of the main co-receptors of HIV-1, and has been found to be a potential therapeutic target for stroke. Maraviroc is a classic CCR5 antagonist, which is undergoing clinical trials against stroke. As maraviroc shows poor blood-brain barrier (BBB) permeability, it is of interest to find novel CCR5 antagonists suitable for neurological medication. In this study we characterized the therapeutic potential of a novel CCR5 antagonist A14 in treating ischemic stroke mice. A14 was discovered in screening millions compounds in the Chemdiv library based on the molecular docking diagram of CCR5 and maraviroc. We found that A14 dose-dependently inhibited the CCR5 activity with an IC50 value of 4.29 µM. Pharmacodynamic studies showed that A14 treatment exerted protective effects against neuronal ischemic injury both in vitro and vivo. In a SH-SY5Y cell line overexpressing CCR5, A14 (0.1, 1 µM) significantly alleviated OGD/R-induced cell injury. We found that the expression of CCR5 and its ligand CKLF1 was significantly upregulated during both acute and recovery period in focal cortical stroke mice; oral administration of A14 (20 mg·kg-1·d-1, for 1 week) produced sustained protective effect against motor impairment. A14 treatment had earlier onset time, lower onset dosage and much better BBB permeability compared to maraviroc. MRI analysis also showed that A14 treatment significantly reduced the infarction volume after 1 week of treatment. We further revealed that A14 treatment blocked the protein-protein interaction between CCR5 and CKLF1, increasing the activity of CREB signaling pathway in neurons, thereby improving axonal sprouting and synaptic density after stroke. In addition, A14 treatment remarkably inhibited the reactive proliferation of glial cells after stroke and reduced the infiltration of peripheral immune cells. These results demonstrate that A14 is a promising novel CCR5 antagonist for promoting neuronal repair after ischemic stroke. A14 blocked the protein-protein interaction between CKLF1 and CCR5 after stroke by binding with CCR5 stably, improved the infarct area and promoted motor recovery through reversing the CREB/pCREB signaling which was inhibited by activated CCR5 Gαi pathway, and benefited to the dendritic spines and axons sprouting.


Assuntos
Antagonistas dos Receptores CCR5 , AVC Isquêmico , Neuroblastoma , Acidente Vascular Cerebral , Animais , Humanos , Camundongos , AVC Isquêmico/tratamento farmacológico , Maraviroc/uso terapêutico , Maraviroc/farmacologia , Simulação de Acoplamento Molecular , Receptores CCR5/metabolismo , Acidente Vascular Cerebral/tratamento farmacológico , Antagonistas dos Receptores CCR5/química , Antagonistas dos Receptores CCR5/farmacologia
8.
J Med Genet ; 59(10): 1010-1016, 2022 10.
Artigo em Inglês | MEDLINE | ID: mdl-35121647

RESUMO

BACKGROUND: Numerous variants of uncertain significance (VUSs) have been identified by whole exome sequencing in clinical practice. However, VUSs are not currently considered medically actionable. OBJECTIVE: To assess the splicing patterns of 49 VUSs in 48 families identified clinically to improve genetic counselling and family planning. METHODS: Forty-nine participants with 49 VUSs were recruited from the Reproductive and Genetic Hospital of CITIC-Xiangya. Bioinformatic analysis was performed to preliminarily predict the splicing effects of these VUSs. RT-PCR and minigene analysis were used to assess the splicing patterns of the VUSs. According to the results obtained, couples opted for different methods of reproductive interventions to conceive a child, including prenatal diagnosis and preimplantation genetic testing (PGT). RESULTS: Eleven variants were found to alter pre-mRNA splicing and one variant caused nonsense-mediated mRNA decay, which resulted in the reclassification of these VUSs as likely pathogenic. One couple chose to undergo in vitro fertilisation with PGT treatment; a healthy embryo was transferred and the pregnancy is ongoing. Three couples opted for natural pregnancy with prenatal diagnosis. One couple terminated the pregnancy because the fetus was affected by short-rib thoracic dysplasia and harboured the related variant. The infants of the other two couples were born and were healthy at their last recorded follow-up. CONCLUSION: RNA splicing analysis is an important method to assess the impact of sequence variants on splicing in clinical practice and can contribute to the reclassification of a significant proportion of VUSs. RNA splicing analysis should be considered for genetic disease diagnostics.


Assuntos
Precursores de RNA , Splicing de RNA , Feminino , Aconselhamento Genético , Testes Genéticos/métodos , Humanos , Gravidez , Diagnóstico Pré-Natal , Splicing de RNA/genética
9.
Yi Chuan ; 45(1): 88-95, 2023 Jan 20.
Artigo em Inglês | MEDLINE | ID: mdl-36927641

RESUMO

Duchenne/Becker muscular dystrophy (DMD/BMD) is one of the most common progressive muscular dystrophy diseases with X-linked recessive inheritance. It is mainly caused by the deletion, duplication and point mutation of DMD gene. In rare cases, it is also caused by the destruction of DMD gene by chromosomal structural rearrangement. Here, we report a case of Duchenne/Becker Muscular dystrophy (DMD/BMD) with typical symptoms but unknown genetic defects after MLPA and next generation sequencing tests in other hospitals. Interestingly, we find a pericentric inversion of X chromosome (Chr.X: g. [31939463-31939465del; 31939466-131765063 inv; 131765064-131765067del]) in this patient. We then use the karyotyping, FISH, long-read sequencing and Sanger sequencing technologies to characterize the chromosome rearrangement. We find that this chromosomal aberration disrupt both the DMD gene and the HS6ST2 gene. The patient present with typical DMD symptoms such as muscle weakness, but no obvious symptoms of Paganini-Miozzo syndrome. Our results suggest that the destruction of DMD gene by structural rearrangement is also one of the important causes of DMD. Therefore, we suggest to provide further genetic testing for those DMD patients with unknown genetic defects through routine genetic testing. Cost-effective karyotyping and FISH should be considered firstly to identify chromosome rearrangements. Long-read sequencing followed by Sanger sequencing could be useful to locate the precise breakpoints. The genetic diagnosis of this case made it possible for reproductive intervention in the patient's family.


Assuntos
Distrofia Muscular de Duchenne , Humanos , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/diagnóstico , Distrofina/genética , Testes Genéticos , Rearranjo Gênico/genética , Cromossomo X , Sulfotransferases/genética
10.
Zhongguo Zhong Yao Za Zhi ; 48(6): 1673-1681, 2023 Mar.
Artigo em Zh | MEDLINE | ID: mdl-37005855

RESUMO

This study employed bibliometrics tools to review the studies of traditional Chinese medicine(TCM) treatment of Alzheimer's disease(AD) in recent ten years, aiming to explore the research status, hotspots, and future trends in this field at home and abroad. The relevant literature published from January 1, 2012 to August 15, 2022 was retrieved from Web of Science and CNKI. CiteSpace 6.1R2 and VOSviewer 1.6.15 were used for the visual analysis of authors, countries, institutions, keywords, journals, etc. A total of 2 254 Chinese articles and 545 English articles were included. The annual number of articles published showed a rising trend with fluctuations. The country with the largest number of relevant articles published and the largest centrality was China. SUN Guo-jie and WANG Qi were the authors publishing the most Chinese articles and English articles, respectively. Hubei University of Chinese Medicine and Beijing University of Chinese Medicine published the most articles in Chinese and English, respectively. Journal of Ethnopharmacology and Neuroscience Letters published the articles with the highest cited frequency and the highest centrality. According to the keywords, the research on TCM treatment of AD mainly focused on the mechanism of action and treatment methods. Metabolomics, intestinal flora, oxidative stress, tau hyperphosphorylation, ß-amyloid(Aß), inflammatory cytokines, and autophagy were the focuses of the research on mechanism of action. Acupuncture, clinical effect, kidney deficiency and phlegm stasis, and dredging governor vessel to revitalize mind were the hotspots of clinical research. This research field is still in the stage of exploration and development. Exchanges and cooperation among institutions should be encouraged to carry out more high-quality basic research on TCM treatment of AD, obtain high-level evidence, and clarify the pathogenesis and prescription mechanism.


Assuntos
Terapia por Acupuntura , Doença de Alzheimer , Medicina , Humanos , Doença de Alzheimer/tratamento farmacológico , Peptídeos beta-Amiloides , Medicina Tradicional Chinesa
11.
Zhongguo Zhong Yao Za Zhi ; 48(21): 5871-5880, 2023 Nov.
Artigo em Zh | MEDLINE | ID: mdl-38114183

RESUMO

This study investigated the neuroprotective effects and underlying mechanism of Liujing Toutong Tablets(LJTT) on a rat model of permanent middle cerebral artery occlusion(pMCAO). The pMCAO model was established using the suture method. Eighty-four male SPF-grade SD rats were randomly divided into a sham operation group, a model group, a nimodipine group(0.020 g·kg~(-1)), and high-, medium-, and low-dose LJTT groups(2.8, 1.4, and 0.7 g·kg~(-1)). The Longa score, adhesive removal test and laser speckle contrast imaging technique were used to evaluate the degree of neurological functional impairment and changes in local cerebral blood flow. The survival and mortality of rats in each group were recorded daily. After seven days of continuous administration following the model induction, the rats in each group were euthanized, and brain tissue and blood samples were collected for corresponding parameter measurements. Nissl staining was used to examine pathological changes in brain tissue neurons. The levels of tumor necrosis factor-alpha(TNF-α), interleukin-6(IL-6), IL-1ß, vascular endothelial growth factor(VEGF), calcitonin gene-related peptide(CGRP), beta-endorphin(ß-EP), and endogenous nitric oxide(NO) in rat serum were measured using specific assay kits. The entropy weight method was used to analyze the weights of various indicators. The protein expression levels of nuclear factor kappa-B(NF-κB), inhibitor kappaB alpha(IκBα), phosphorylated IκBα(p-IκBα), and phosphorylated inhibitor of NF-κB kinase alpha(p-IKKα) in brain tissue were determined using Western blot. Immunohistochemistry was used to detect the protein expression of chemokine-like factor 1(CKLF1) and C-C chemokine receptor 5(CCR5) in rat brain tissue. Compared with the sham operation group, the model group showed significantly higher neurological functional impairment scores, prolonged adhesive removal time, decreased cerebral blood flow, increased neuronal damage, reduced survival rate, significantly increased levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in serum, significantly decreased levels of VEGF and ß-EP, significantly increased expression levels of NF-κB p65, p-IκBα/IκBα, and p-IKKα in rat brain tissue, and significantly upregulated protein expression of CKLF1 and CCR5. Compared with the model group, the high-dose LJTT group significantly improved the neurological functional score of pMCAO rats after oral administration for 7 days. LJTT at all doses significantly reduced adhesive removal time and restored cerebral blood flow. The high-and medium-dose LJTT groups significantly improved neuronal damage. The LJTT groups at all doses showed reduced levels of TNF-α, IL-1ß, IL-6, CGRP, and NO in rat serum, increased VEGF and ß-EP levels, and significantly decreased expression levels of NF-κB p65, p-IκBα/IκBα, p-IKKα, and CCR5 protein in rat brain tissue. The entropy weight analysis revealed that CGRP and ß-EP were significantly affected during the model induction, and LJTT exhibited a strong effect in reducing the release of inflammatory factors such as TNF-α and IL-1ß. LJTT may exert a neuroprotective effect on rats with permanent cerebral ischemia by reducing neuroinflammatory damage, and its mechanism may be related to the inhibition of the NF-κB signaling pathway and the regulation of the CKLF1/CCR5 axis. Additionally, LJTT may exert certain analgesic effects by reducing CGRP and NO levels and increasing ß-EP levels.


Assuntos
Isquemia Encefálica , NF-kappa B , Ratos , Masculino , Animais , NF-kappa B/genética , NF-kappa B/metabolismo , Inibidor de NF-kappaB alfa/genética , Inibidor de NF-kappaB alfa/metabolismo , Fator A de Crescimento do Endotélio Vascular/genética , Quinase I-kappa B/metabolismo , Quinase I-kappa B/farmacologia , Fator de Necrose Tumoral alfa/genética , Fator de Necrose Tumoral alfa/farmacologia , Interleucina-6/genética , Peptídeo Relacionado com Gene de Calcitonina/farmacologia , Ratos Sprague-Dawley , Transdução de Sinais , Isquemia Encefálica/tratamento farmacológico , Comprimidos
12.
Acta Pharmacol Sin ; 43(3): 563-576, 2022 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-34103690

RESUMO

Myelin damage and abnormal remyelination processes lead to central nervous system dysfunction. Glial activation-induced microenvironment changes are characteristic features of the diseases with myelin abnormalities. We previously showed that ginsenoside Rg1, a main component of ginseng, ameliorated MPTP-mediated myelin damage in mice, but the underlying mechanisms are unclear. In this study we investigated the effects of Rg1 and mechanisms in cuprizone (CPZ)-induced demyelination mouse model. Mice were treated with CPZ solution (300 mg· kg-1· d-1, ig) for 5 weeks; from week 2, the mice received Rg1 (5, 10, and 20 mg· kg-1· d-1, ig) for 4 weeks. We showed that Rg1 administration dose-dependently alleviated bradykinesia and improved CPZ-disrupted motor coordination ability in CPZ-treated mice. Furthermore, Rg1 administration significantly decreased demyelination and axonal injury in pathological assays. We further revealed that the neuroprotective effects of Rg1 were associated with inhibiting CXCL10-mediated modulation of glial response, which was mediated by NF-κB nuclear translocation and CXCL10 promoter activation. In microglial cell line BV-2, we demonstrated that the effects of Rg1 on pro-inflammatory and migratory phenotypes of microglia were related to CXCL10, while Rg1-induced phagocytosis of microglia was not directly related to CXCL10. In CPZ-induced demyelination mouse model, injection of AAV-CXCL10 shRNA into mouse lateral ventricles 3 weeks prior CPZ treatment occluded the beneficial effects of Rg1 administration in behavioral and pathological assays. In conclusion, CXCL10 mediates the protective role of Rg1 in CPZ-induced demyelination mouse model. This study provides new insight into potential disease-modifying therapies for myelin abnormalities.


Assuntos
Quimiocina CXCL10/antagonistas & inibidores , Doenças Desmielinizantes/patologia , Ginsenosídeos/farmacologia , Animais , Cuprizona/farmacologia , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Hipocinesia/patologia , Mediadores da Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Microglia/efeitos dos fármacos , NF-kappa B/efeitos dos fármacos , Panax/química , Panax/metabolismo , Fagocitose/efeitos dos fármacos , RNA Interferente Pequeno/farmacologia
13.
Acta Pharmacol Sin ; 43(5): 1217-1230, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-34385606

RESUMO

The phenotypic transformation of microglia in the ischemic penumbra determines the outcomes of ischemic stroke. Our previous study has shown that chemokine-like-factor 1 (CKLF1) promotes M1-type polarization of microglia. In this study, we investigated the cellular source and transcriptional regulation of CKLF1, as well as the biological function of CKLF1 in ischemic penumbra of rat brain. We showed that CKLF1 was significantly up-regulated in cultured rat cortical neurons subjected to oxygen-glucose deprivation/reoxygenation (ODG/R) injury, but not in cultured rat microglia, astrocytes and oligodendrocytes. In a rat model of middle cerebral artery occlusion, we found that CKLF1 was up-regulated and co-localized with neurons in ischemic penumbra. Furthermore, the up-regulated CKLF1 was accompanied by the enhanced nuclear accumulation of NF-κB. The transcriptional activity of CKLF1 was improved by overexpression of NF-κB in HEK293T cells, whereas application of NF-κB inhibitor Bay 11-7082 (1 µM) abolished it, caused by OGD/R. By using chromatin-immunoprecipitation (ChIP) assay we demonstrated that NF-κB directly bound to the promoter of CKLF1 (at a binding site located at -249 bp to -239 bp of CKLF1 promoter region), and regulated the transcription of human CKLF1. Moreover, neuronal conditional medium collected after OGD/R injury or CKLF1-C27 (a peptide obtained from secreted CKLF1) induced the M1-type polarization of microglia, whereas the CKLF1-neutralizing antibody (αCKLF1) or NF-κB inhibitor Bay 11-7082 abolished the M1-type polarization of microglia. Specific knockout of neuronal CKLF1 in ischemic penumbra attenuated neuronal impairments and M1-type polarization of microglia caused by ischemic/reperfusion injury, evidenced by inhibited levels of M1 marker CD16/32 and increased expression of M2 marker CD206. Application of CKLF1-C27 (200 nM) promoted the phosphorylation of p38 and JNK in microglia, whereas specific depletion of neuronal CKLF1 in ischemic penumbra abolished ischemic/reperfusion-induced p38 and JNK phosphorylation. In summary, CKLF1 up-regulation in neurons regulated by NF-κB is one of the crucial mechanisms to promote M1-type polarization of microglia in ischemic penumbra.


Assuntos
Isquemia Encefálica , Acidente Vascular Cerebral , Animais , Encéfalo/metabolismo , Isquemia Encefálica/metabolismo , Quimiocinas/metabolismo , Células HEK293 , Humanos , Proteínas com Domínio MARVEL , Microglia/metabolismo , NF-kappa B/metabolismo , Neurônios/metabolismo , Ratos , Acidente Vascular Cerebral/metabolismo , Regulação para Cima
14.
J Med Genet ; 58(3): 168-172, 2021 03.
Artigo em Inglês | MEDLINE | ID: mdl-32303603

RESUMO

BACKGROUND: The genetic causes of the majority of cases of female infertility caused by premature ovarian insufficiency (POI) are unknown. OBJECTIVE: To identify the genetic causes of POI in 110 patients. METHODS: Whole-exome sequencing was performed on 110 patients with POI, and putative disease-causative variants were validated by Sanger sequencing. Bioinformatic and in vitro functional analyses were performed for functional characterisation of the identified candidate disease-causative variants. RESULTS: We identified two homozygous variants (NM_001040274: c.150_151del (p.Ser52Profs*7), c.999A>G (p.Ile333Met)) in SYCP2L in two patients, which had co-segregated with POI in these families. Bioinformatic analysis predicted that the two variants are deleterious, and in vitro functional analysis showed that mutant SYCP2L proteins exhibited mislocalisation and loss of function. CONCLUSIONS: SYCP2L is a novel gene found to be responsible for human POI. Our findings provide a potential molecular marker for POI and improve the understanding of the genetic basis of female infertility.


Assuntos
Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Infertilidade Feminina/genética , Menopausa Precoce/genética , Insuficiência Ovariana Primária/genética , Adulto , Biologia Computacional , Feminino , Homozigoto , Humanos , Infertilidade Feminina/patologia , Mutação/genética , Linhagem , Insuficiência Ovariana Primária/epidemiologia , Insuficiência Ovariana Primária/patologia , Sequenciamento do Exoma
15.
Acta Pharmacol Sin ; 42(6): 848-860, 2021 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-33028984

RESUMO

Sustained elevation of corticosterone (CORT) is one of the common causes of aging and major depression disorder. However, the role of elevated CORT in late life depression (LLD) has not been elucidated. In this study, 18-month-old female rats were subjected to bilateral adrenalectomy or sham surgery. Their CORT levels in plasma were adjusted by CORT replacement and the rats were divided into high-level CORT (H-CORT), low-level CORT (L-CORT), and Sham group. We showed that L-CORT rats displayed attenuated depressive symptoms and memory defects in behavioral tests as compared with Sham or H-CORT rats. Furthermore, we showed that glutamatergic transmission was enhanced in L-CORT rats, evidenced by enhanced population spike amplitude (PSA) recorded from the dentate gyrus of hippocampus in vivo and increased glutamate release from hippocampal synaptosomes caused by high frequency stimulation or CORT exposure. Intracerebroventricular injection of an enzymatic glutamate scavenger system, glutamic-pyruvic transmine (GPT, 1 µM), significantly increased the PSA in Sham rats, suggesting that extracelluar accumulation of glutamate might be the culprit of impaired glutamatergic transmission, which was dependent on the uptake by Glt-1 in astrocytes. We revealed that hippocampal Glt-1 expression level in the L-CORT rats was much higher than in Sham and H-CORT rats. In a gradient neuron-astrocyte coculture, we found that the expression of Glt-1 was decreased with the increase of neural percentage, suggesting that impairment of Glt-1 might result from the high level of CORT contributed neural damage. In sham rats, administration of DHK that inhibited Glt-1 activity induced significant LLD symptoms, whereas administration of RIL that promoted glutamate uptake significantly attenuated LLD. All of these results suggest that glutamatergic transmission impairment is one of important pathogenesis in LLD induced by high level of CORT, which provide promising clues for the treatment of LLD.


Assuntos
Corticosterona/metabolismo , Depressão/metabolismo , Ácido Glutâmico/metabolismo , Transmissão Sináptica/fisiologia , Animais , Astrócitos/metabolismo , Transportador 2 de Aminoácido Excitatório/metabolismo , Feminino , Glutamina/metabolismo , Hipocampo/citologia , Hipocampo/metabolismo , Neurônios/metabolismo , Ratos Sprague-Dawley , Sinaptossomos/metabolismo
16.
Reprod Biomed Online ; 38(3): 397-406, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30691934

RESUMO

RESEARCH QUESTION: What is the genetic aetiology of three resistant ovary syndrome (ROS) pedigrees from 13 Chinese Han families with non-syndromic premature ovarian insufficiency (POI). DESIGN: The proband in each family was subjected to whole-exome sequencing. Bioinformatic and in-vitro functional analyses were performed for the functional characterization of the FSHR mutations. RESULTS: Four novel mutations, two homozygous mutations (c.419delA, c.1510C>T), and a compound heterozygous mutation (c.44G>A and deletion of exons 1 and 2) of FSHR were identified in the three non-syndromic POI-with-ROS families. Bioinformatic analysis predicted that the three novel point mutations in FSHR are deleterious and associated with POI in the three families, which was confirmed by in-vitro functional analysis, in which FSH-induced adenosine 3',5'-cyclic monophosphate production was abolished for all receptors. CONCLUSIONS: The three novel point mutations in FSHR were all functional inactivating mutations, and were the genetic aetiology of the three non-syndromic POI-with-ROS families. The first FSHR frameshift mutation is reported here, and the first missense mutation in the signal peptide-encoding region of FSHR to be associated with POI. Women affected by ROS should consider undergoing mutation screening for FSHR.


Assuntos
Mutação de Sentido Incorreto , Insuficiência Ovariana Primária/genética , Receptores do FSH/genética , Adulto , Animais , Células CHO , Cricetulus , Família , Feminino , Humanos , Linhagem
17.
Acta Pharmacol Sin ; 40(1): 13-25, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30262824

RESUMO

Ginsenoside Rg1 (Rg1), a saponin extracted from Panax ginseng, has been well documented to be effective against ischemic/reperfusion (I/R) neuronal injury. However, the underlying mechanisms remain obscure. In the present study, we investigated the roles of Nrf2 and miR-144 in the protective effects of Rg1 against I/R-induced neuronal injury. In OGD/R-treated PC12 cells, Rg1 (0.01-1 µmol/L) dose-dependently attenuated the cell injury accompanied by prolonging nuclear accumulation of Nrf2, enhancing the transcriptional activity of Nrf2, as well as promoting the expression of ARE-target genes. The activation of the Nrf2/ARE pathway by Rg1 was independent of disassociation with Keap1, but resulted from post-translational regulations. Knockdown of Nrf2 abolished all the protective changes of Rg1 in OGD/R-treated PC12 cells. Furthermore, Rg1 treatment significantly decreased the expression of miR-144, which downregulated Nrf2 production by targeting its 3'-untranlated region after OGD/R. Knockdown of Nrf2 had no effect on the expression of miR-144, suggesting that miR-144 was an upstream regulator of Nrf2. We revealed that there was a direct binding between Nrf2 and miR-144 in PC12 cells. Application of anti-miR-144 occluded the activation of the Nrf2/ARE pathway by Rg1 in OGD/R-treated PC12 cells. In tMCAO rats, administration of Rg1 (20 mg/kg) significantly alleviated ischemic injury, and activated Nrf2/ARE pathway. The protective effects of Rg1 were abolished by injecting of AAV-HIF-miR-144-shRNA into the predicted ischemic penumbra. In conclusion, our results demonstrate that Rg1 alleviates oxidative stress after I/R through inhibiting miR-144 activity and subsequently promoting the Nrf2/ARE pathway at the post-translational level.


Assuntos
Ginsenosídeos/farmacologia , Infarto da Artéria Cerebral Média/tratamento farmacológico , MicroRNAs/genética , Fármacos Neuroprotetores/farmacologia , Traumatismo por Reperfusão/tratamento farmacológico , Transdução de Sinais/efeitos dos fármacos , Animais , Elementos de Resposta Antioxidante/genética , Linhagem Celular Tumoral , Regulação para Baixo/efeitos dos fármacos , Masculino , Fator 2 Relacionado a NF-E2/metabolismo , Ratos Sprague-Dawley , Regulação para Cima/efeitos dos fármacos
19.
Prenat Diagn ; 39(8): 603-608, 2019 07.
Artigo em Inglês | MEDLINE | ID: mdl-31069818

RESUMO

OBJECTIVE: To predict the risk of dystrophinopathy in fetal carriers of dystrophin gene (DMD) mutations. METHODS: Twenty-three pregnant women, with a total of 25 female fetuses carrying DMD mutations, were recruited. Among them, 13 pregnant women who participated in this study were only used to analyse the incidence of induced abortion after fetuses were diagnosed as dystrophinopathy carriers. Eleven fetal carriers from 10 pregnant women were tested to analyse X-chromosome inactivation (XCI) using amniocytes to assess the risk of dystrophinopathy. Follow-ups were conducted on all cases. RESULTS: Approximately one-third of fetuses were aborted before assessing the risk of dystrophinopathy. XCI analysis of amniocytes showed that 10 fetuses had random XCI patterns, and one fetus exhibited a highly skewed XCI pattern (100:0) with primary expression of the maternal X chromosome that carried the mutant allele. These 11 fetal carriers were born, and follow-up showed that the girl who showed the skewed XCI pattern as a fetus was diagnosed with Duchenne muscular dystrophy (DMD) at the age of four. The others did not present with dystrophinopathy-associated symptoms. CONCLUSIONS: XCI was significantly implicated in symptomatic female carriers of dystrophinopathies, and XCI pattern analysis of amniocytes may be useful in predicting the risk of dystrophinopathy in fetal carriers.


Assuntos
Âmnio/metabolismo , Distrofina/genética , Feto/metabolismo , Distrofia Muscular de Duchenne/diagnóstico , Inativação do Cromossomo X/fisiologia , Aborto Induzido/estatística & dados numéricos , Adulto , Âmnio/patologia , Estudos de Coortes , Feminino , Testes Genéticos , Heterozigoto , Humanos , Incidência , Recém-Nascido , Masculino , Distrofia Muscular de Duchenne/epidemiologia , Distrofia Muscular de Duchenne/genética , Distrofia Muscular de Duchenne/patologia , Mutação , Linhagem , Fenótipo , Gravidez , Diagnóstico Pré-Natal/métodos , Prognóstico , Fatores de Risco
20.
J Med Genet ; 55(3): 198-204, 2018 03.
Artigo em Inglês | MEDLINE | ID: mdl-29331980

RESUMO

BACKGROUND: The genetic causes of the majority of male and female infertility caused by human non-obstructive azoospermia (NOA) and premature ovarian insufficiency (POI) with meiotic arrest are unknown. OBJECTIVE: To identify the genetic cause of NOA and POI in two affected members from a consanguineous Chinese family. METHODS: We performed whole-exome sequencing of DNA from both affected patients. The identified candidate causative gene was further verified by Sanger sequencing for pedigree analysis in this family. In silico analysis was performed to functionally characterise the mutation, and histological analysis was performed using the biopsied testicle sample from the male patient with NOA. RESULTS: We identified a novel homozygous missense mutation (NM_007068.3: c.106G>A, p.Asp36Asn) in DMC1, which cosegregated with NOA and POI phenotypes in this family. The identified missense mutation resulted in the substitution of a conserved aspartic residue with asparaginate in the modified H3TH motif of DMC1. This substitution results in protein misfolding. Histological analysis demonstrated a lack of spermatozoa in the male patient's seminiferous tubules. Immunohistochemistry using a testis biopsy sample from the male patient showed that spermatogenesis was blocked at the zygotene stage during meiotic prophase I. CONCLUSIONS: To the best of our knowledge, this is the first report identifying DMC1 as the causative gene for human NOA and POI. Furthermore, our pedigree analysis shows an autosomal recessive mode of inheritance for NOA and POI caused by DMC1 in this family.


Assuntos
Azoospermia/genética , Proteínas de Ciclo Celular/genética , Proteínas de Ligação a DNA/genética , Insuficiência Ovariana Primária/genética , Espermatogênese/genética , Adulto , Azoospermia/patologia , Consanguinidade , Feminino , Homozigoto , Humanos , Masculino , Meiose/genética , Mutação de Sentido Incorreto , Insuficiência Ovariana Primária/patologia , Sequenciamento do Exoma , Adulto Jovem
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