RESUMO
BACKGROUND: Existing evidence suggests that the composition of the gut microbiota is associated with neuropathic pain (NP), but the mechanistic link is elusive. Peroxisome proliferator-activated receptor α (PPARα) has been shown to be a pharmacological target for the treatment of metabolic disorders, and its expression is also involved in inflammatory regulation. The aim of this study was to investigate the important modulatory effects of PPARα on gut microbiota and spinal cord metabolites in mice subjected to chronic constriction injury. METHODS: We analyzed fecal microbiota and spinal cord metabolic alterations in mice from the sham, CCI, GW7647 (PPARα agonist) and GW6471 (PPARα antagonist) groups by 16â¯S rRNA amplicon sequencing and untargeted metabolomics analysis. On this basis, the intestinal microbiota and metabolites that were significantly altered between treatment groups were analyzed in a combined multiomics analysis. We also investigated the effect of PPARα on the polarization fractionation of spinal microglia. RESULTS: PPARα agonist significantly reduce paw withdrawal threshold and paw withdrawal thermal latency, while PPARα antagonist significantly increase paw withdrawal threshold and paw withdrawal thermal latency. 16â¯S rRNA gene sequencing showed that intraperitoneal injection of GW7647 or GW6471 significantly altered the abundance, homogeneity and composition of the gut microbiome. Analysis of the spinal cord metabolome showed that the levels of spinal cord metabolites were shifted after exposure to GW7647 or GW6471. Alterations in the composition of gut microbiota were significantly associated with the abundance of various spinal cord metabolites. The abundance of Licheniformes showed a significant positive correlation with nicotinamide, benzimidazole, eicosanoids, and pyridine abundance. Immunofluorescence results showed that intraperitoneal injection of GW7647 or GW6471 altered microglial activation and polarization levels. CONCLUSION: Our study shows that PPARα can promote M2-type microglia polarization, as well as alter gut microbiota and metabolites in CCI mice. This study enhances our understanding of the mechanism of PPARα in the treatment of neuropathic pain.
Assuntos
Microbioma Gastrointestinal , Metabolômica , Neuralgia , PPAR alfa , RNA Ribossômico 16S , Medula Espinal , Animais , Masculino , Camundongos , Fezes/microbiologia , Microbioma Gastrointestinal/efeitos dos fármacos , Microbioma Gastrointestinal/fisiologia , Camundongos Endogâmicos C57BL , Microglia/metabolismo , Microglia/efeitos dos fármacos , Neuralgia/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/microbiologia , Oxazóis , PPAR alfa/metabolismo , RNA Ribossômico 16S/genética , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Tirosina/análogos & derivadosRESUMO
Neuropathic pain (NP) is usually treated with analgesics and symptomatic therapy with poor efficacy and numerous side effects, highlighting the urgent need for effective treatment strategies. Recent studies have reported an important role for peroxisome proliferator-activated receptor alpha (PPARα) in regulating metabolism as well as inflammatory responses. Through pain behavioral assessment, we found that activation of PPARα prevented chronic constriction injury (CCI)-induced mechanical allodynia and thermal hyperalgesia. In addition, PPARα ameliorated inflammatory cell infiltration at the injury site and decreased microglial activation, NOD-like receptor protein 3 (NLRP3) inflammasome production, and spinal dendritic spine density, as well as improved serum and spinal cord metabolic levels in mice. Administration of PPARα antagonists eliminates the analgesic effect of PPARα agonists. PPARα relieves NP by inhibiting neuroinflammation and functional synaptic plasticity as well as modulating metabolic mechanisms, suggesting that PPARα may be a potential molecular target for NP alleviation. However, the effects of PPARα on neuroinflammation and synaptic plasticity should be further explored.
Assuntos
Camundongos Endogâmicos C57BL , Neuralgia , PPAR alfa , Medula Espinal , Animais , PPAR alfa/metabolismo , Neuralgia/tratamento farmacológico , Neuralgia/metabolismo , Masculino , Camundongos , Medula Espinal/metabolismo , Medula Espinal/efeitos dos fármacos , Hiperalgesia/tratamento farmacológico , Hiperalgesia/metabolismo , Metabolômica , Microglia/efeitos dos fármacos , Microglia/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/metabolismo , Proteína 3 que Contém Domínio de Pirina da Família NLR/antagonistas & inibidores , Doenças Neuroinflamatórias/tratamento farmacológico , Doenças Neuroinflamatórias/metabolismo , Espinhas Dendríticas/efeitos dos fármacos , Espinhas Dendríticas/metabolismo , Espinhas Dendríticas/patologia , Inflamassomos/metabolismo , Inflamassomos/efeitos dos fármacosRESUMO
BACKGROUND: Pyruvate dehydrogenase complex (PDHC) deficiency is a common neurodegenerative disease associated with abnormal mitochondrial energy metabolism. The diagnosis of PDHC is difficult because of the lack of a rapid, accurate, and cost-effective clinical diagnostic method. METHODS: A 4-year-old boy was preliminarily diagnosed with putative Leigh syndrome based on the clinical presentation. PDHC activity in peripheral blood leukocytes and a corresponding gene analysis were subsequently undertaken. Sodium pyruvate 1-13 C was used for the analysis of PDHC activity in peripheral leukocytes. The genes encoding PDHC were then scanned for mutations. RESULTS: The results showed that the corresponding PDHC activity was dramatically decreased to 10.5 nmol/h/mg protein as compared with that of healthy controls (124.6 ± 7.1 nmol/h/mg). The ratio of PDHC to citrate synthase was 2.1% (control: 425.3 ± 27.1). The mutation analysis led to the identification of a missense mutation, NM_000284.4:g214C>T, in exon 3 of PDHC. CONCLUSION: The peripheral blood leukocyte PDHC activity assay may provide a practical enzymatic diagnostic method for PDHC-related mitochondrial diseases.
Assuntos
Ensaios Enzimáticos Clínicos/métodos , Leucócitos/metabolismo , Doença da Deficiência do Complexo de Piruvato Desidrogenase/diagnóstico , Complexo Piruvato Desidrogenase/metabolismo , Pré-Escolar , Testes Genéticos/métodos , Humanos , Masculino , Mutação de Sentido Incorreto , Complexo Piruvato Desidrogenase/genética , Doença da Deficiência do Complexo de Piruvato Desidrogenase/genética , Doença da Deficiência do Complexo de Piruvato Desidrogenase/metabolismoRESUMO
Cholesterol is an essential component of lysosomal membranes. In this study, we investigated the effects of membrane cholesterol on the permeability of rat liver lysosomes to K+ and H+, and the organelle stability. Through the measurements of lysosomal beta-hexosaminidase free activity, membrane potential, membrane fluidity, intra-lysosomal pH, and lysosomal proton leakage, we established that methyl-beta-cyclodextrin (MbetaCD)-produced loss of membrane cholesterol could increase the lysosomal permeability to both potassium ions and protons, and fluidize the lysosomal membranes. As a result, potassium ions entered the lysosomes through K+/H+ exchange, which produced osmotic imbalance across the membranes and osmotically destabilized the lysosomes. In addition, treatment of the lysosomes with MbetaCD caused leakage of the lysosomal protons and raised the intra-lysosomal pH. The results indicate that membrane cholesterol plays important roles in the maintenance of the lysosomal limited permeability to K+ and H+. Loss of this membrane sterol is critical for the organelle acidification and stability.
Assuntos
Colesterol/química , Colesterol/metabolismo , Membranas Intracelulares/metabolismo , Lisossomos/metabolismo , Potássio/metabolismo , Animais , Concentração de Íons de Hidrogênio , Membranas Intracelulares/efeitos dos fármacos , Íons , Lisossomos/efeitos dos fármacos , Masculino , Permeabilidade , Potássio/química , Ratos , Ratos Wistar , beta-Ciclodextrinas/farmacologiaRESUMO
The effect of solute hydrogen bonding capacity on the osmotic stability of lysosomes was examined through measurement of free enzyme activity of lysosomes after their incubation in sucrose and poly(ethylene glycol) (PEG) (1500-6000 Da molecular mass) media. Free enzyme activity of the lysosomes was less in the PEG medium than that in the sucrose medium under the same hypotonic condition. The lysosomal enzyme latency loss decreased with increasing hydrogen bonding capacity of the solute. In addition, the lysosomes lost less latency at lower incubation temperature. The results indicate that solute hydrogen bonding capacity plays an important role in the osmotic protection of an incubation medium to lysosomes.
Assuntos
Lisossomos/enzimologia , Animais , Hexosaminidases/química , Hexosaminidases/metabolismo , Ligação de Hidrogênio , Lisossomos/química , Masculino , Concentração Osmolar , Polietilenoglicóis/química , Ratos , Ratos Wistar , Sacarose/química , TemperaturaRESUMO
Lysosomal destabilization is critical for the organelle and living cells. Using methyl-beta-cyclodextrin (M beta CD) to selectively deplete lysosomal membrane cholesterol, we investigated the effect of cholesterol on the organelle osmotic stability. The results show that loss of membrane cholesterol caused changes in the lysosomal osmotic properties. The lysosomes lost the ability to resist osmotic shock and became more sensitive to osmotic stress. As a result, the lysosomes lost membrane integrity rapidly. Microscope observation showed that the lysosomes were liable to swell in the hypotonic sucrose medium. It is presumably due to an enhancement of the lysosomal permeability to water caused by the loss of membrane cholesterol. The results indicate an important role of cholesterol in the maintenance of lysosomal stability.
Assuntos
Colesterol/química , Membranas Intracelulares/fisiologia , Lisossomos/fisiologia , beta-Ciclodextrinas/farmacologia , Animais , Lisossomos/química , Lisossomos/efeitos dos fármacos , Masculino , Pressão Osmótica , Ratos , Ratos WistarRESUMO
A point mutation of mitochondrial DNA (mtDNA) at nucleotide position 3243 A to G (mt.3243A>G) is involved in many common diseases, including maternally inherited diabetes and deafness (MIDD) and mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS). However, the mutant level of mt.3243A>G varies both among individuals and in different organs, tissues, and even cells of single individuals. For detection of this mutation, current methods have limited universality and sensitivity and may be not adequate for a routine clinical test. Here, we develop and evaluate a rapid TaqMan-MGB quantitative real-time PCR (qPCR) method for detecting and quantifying the heteroplasmy level of mt.3243A>G in single-tube analysis. With our method, the sensitivity of detection was as low as 0.1%, but the accuracy of quantification was reliable, down to 4%. All positives could be correctly identified, and the heteroplasmy levels determined by qPCR correlated well with the results from restriction fragment length polymorphism (RFLP) and pyrosequencing assays (r = 0.921~0.973 and 0.972~0.984). In addition, we demonstrated that the urinary sediments, leukocytes, or hair follicles might be ideal templates to detect and quantify the heteroplasmy of mt.3243A>G mutation; however, they should be optimized or retreated for further accurate quantification. Our study should allow rapid and high throughput diagnostic testing and can potentially be used to clarify the association between clinical phenotype and pathogenic mitochondrial mutations derived from various tissues.
Assuntos
DNA Mitocondrial/genética , Mutação/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Taq Polimerase/metabolismo , Adolescente , Adulto , Sequência de Bases , Criança , DNA/genética , Estudos de Viabilidade , Feminino , Fluorescência , Humanos , Limite de Detecção , Masculino , Pessoa de Meia-Idade , Taxa de Mutação , Polimorfismo de Fragmento de Restrição/genética , Padrões de Referência , Análise de Regressão , Moldes GenéticosRESUMO
OBJECTIVE: We investigated the effect of ovariectomy (OVX) and 17ß-estradiol (E2) replacement on both mitochondrial and myocardial function in cTnT-Q92 transgenic mice generated by cardiac-restricted expression of a human hypertrophic cardiomyopathy (HCM) mutation. METHODS: The cTnT-Q92 mice were ovariectomized at twenty weeks of age and were treated with either placebo (OVX group) or E2 (OVX+E2 group) for twelve weeks before being sacrificed. Wild-type and cTnT-Q92 female mice receiving sham operation were used as controls. Indices of diastolic function such as mitral early (E) and late (A) inflow as well as isovolumic relaxation time (IVRT) were measured by echocardiography. A Clark-type electrode was used to detect respiratory control, and ATP levels were determined at the mitochondrial level using HPLC. Key components related to mitochondrial energy metabolism, such as peroxisome proliferator-activated receptor α (PPARα), PPARγ coactivator 1α (PGC-1α) and nuclear respiratory factor-1 (NRF-1), were also analyzed using Western blot and RT-PCR. The levels of oxidative stress markers were determined by measuring malondialdehyde (MDA) using the thiobarbituric acid assay. RESULTS: The cTnT-Q92 mice had impaired diastolic function compared with wild-type mice (E/A ratio, 1.39 ± 0.04 vs. 1.21 ± 0.01, p<0.001; IVRT, 19.17 ± 0.85 vs. 22.15 ± 1.43 ms, p=0.028). In response to ovariectomy, cardiac function further decreased compared with that observed in cTnT-Q92 mice that received the sham operation (E/A ratio, 1.15 ± 0.04 vs. 1.21 ± 0.01, p<0.001; IVRT, 28.31 ± 0.39 vs. 22.15 ± 1.43 ms, p=0.002). Myocardial energy metabolism, as determined by ATP levels (3.49 ± 0.31 vs. 5.07 ± 0.47 µmol/g, p<0.001), and the mitochondrial respiratory ratio (2.04 ± 0.10 vs. 2.63 ± 0.11, p=0.01) also decreased significantly. By contrast, myocardial concentrations of MDA increased significantly in the OVX group, and PGC-1α, PPARα and NRF-1decreased significantly. E2 supplementation significantly elevated myocardial ATP levels (4.55 ± 0.21 vs. 3.49 ± 0.31 µmol/g, p=0.003) and mitochondrial respiratory function (3.93 ± 0.05 vs. 2.63 ± 0.11, p=0.001); however, it reduced the MDA level (0.21 ± 0.02 vs. 0.36 ± 0.03 nmol/g, p<0.001), which subsequently improved diastolic function (E/A ratio, 1.35 ± 0.06 vs. 1.15 ± 0.04, p<0.001; IVRT, 18.22 ± 1.16 vs. 28.31 ± 0.39 ms, p=0.007). CONCLUSIONS: Our study has shown that 17ß-estradiol improved myocardial diastolic function, prevented myocardial energy dysregulation, and reduced myocardial oxidative stress in cTnT-Q92 mice.