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1.
Proc Natl Acad Sci U S A ; 121(16): e2401313121, 2024 Apr 16.
Artículo en Inglés | MEDLINE | ID: mdl-38602916

RESUMEN

All forms of life are presumed to synthesize arginine from citrulline via a two-step pathway consisting of argininosuccinate synthetase and argininosuccinate lyase using citrulline, adenosine 5'-triphosphate (ATP), and aspartate as substrates. Conversion of arginine to citrulline predominantly proceeds via hydrolysis. Here, from the hyperthermophilic archaeon Thermococcus kodakarensis, we identified an enzyme which we designate "arginine synthetase". In arginine synthesis, the enzyme converts citrulline, ATP, and free ammonia to arginine, adenosine 5'-diphosphate (ADP), and phosphate. In the reverse direction, arginine synthetase conserves the energy of arginine deimination and generates ATP from ADP and phosphate while releasing ammonia. The equilibrium constant of this reaction at pH 7.0 is [Cit][ATP][NH3]/[Arg][ADP][Pi] = 10.1 ± 0.7 at 80 °C, corresponding to a ΔG°' of -6.8 ± 0.2 kJ mol-1. Growth of the gene disruption strain was compared to the host strain in medium composed of amino acids. The results suggested that arginine synthetase is necessary in providing ornithine, the precursor for proline biosynthesis, as well as in generating ATP. Growth in medium supplemented with citrulline indicated that arginine synthetase can function in the direction of arginine synthesis. The enzyme is widespread in nature, including bacteria and eukaryotes, and catalyzes a long-overlooked energy-conserving reaction in microbial amino acid metabolism. Along with ornithine transcarbamoylase and carbamate kinase, the pathway identified here is designated the arginine synthetase pathway.


Asunto(s)
Arginina , Ligasas , Arginina/metabolismo , Citrulina/metabolismo , Amoníaco , Ornitina/genética , Adenosina Trifosfato/metabolismo , Fosfatos , Adenosina , Catálisis
2.
Stem Cell Res Ther ; 15(1): 102, 2024 Apr 08.
Artículo en Inglés | MEDLINE | ID: mdl-38589967

RESUMEN

BACKGROUND: Premature ovarian insufficiency (POI) is a major cause of infertility. In this study, we aimed to investigate the effects of the combination of bone marrow mesenchymal stem cells (BMSCs) and moxibustion (BMSCs-MOX) on POI and evaluate the underlying mechanisms. METHODS: A POI rat model was established by injecting different doses of cyclophosphamide (Cy). The modeling of POI and the effects of the treatments were assessed by evaluating estrous cycle, serum hormone levels, ovarian weight, ovarian index, and ovarian histopathological analysis. The effects of moxibustion on BMSCs migration were evaluated by tracking DiR-labeled BMSCs and analyzing the expression of chemokines stromal cell-derived factor 1 (Sdf1) and chemokine receptor type 4 (Cxcr4). Mitochondrial function and mitophagy were assessed by measuring the levels of reactive oxygen species (ROS), mitochondrial membrane potential (MMP), ATP, and the mitophagy markers (Drp1, Pink1, and Parkin). Furthermore, the mitophagy inhibitor Mdivi-1 and the mitophagy activator CCCP were used to confirm the role of mitophagy in Cy-induced ovarian injury and the underlying mechanism of combination therapy. RESULTS: A suitable rat model of POI was established using Cy injection. Compared to moxibustion or BMSCs transplantation alone, BMSCs-MOX showed improved outcomes, such as reduced estrous cycle disorders, improved ovarian weight and index, normalized serum hormone levels, increased ovarian reserve, and reduced follicle atresia. Moxibustion enhanced Sdf1 and Cxcr4 expression, promoting BMSCs migration. BMSCs-MOX reduced ROS levels; upregulated MMP and ATP levels in ovarian granulosa cells (GCs); and downregulated Drp1, Pink1, and Parkin expression in ovarian tissues. Mdivi-1 significantly mitigated mitochondrial dysfunction in ovarian GCs and improved ovarian function. CCCP inhibited the ability of BMSCs-MOX treatment to regulate mitophagy and ameliorate Cy-induced ovarian injury. CONCLUSIONS: Moxibustion enhanced the migration and homing of BMSCs following transplantation and improves their ability to repair ovarian damage. The combination of BMSCs and moxibustion effectively reduced the excessive activation of mitophagy, which helped prevent mitochondrial damage, ultimately improving ovarian function. These findings provide a novel approach for the treatment of pathological ovarian aging and offer new insights into enhancing the efficacy of stem cell therapy for POI patients.


Asunto(s)
Trasplante de Células Madre Mesenquimatosas , Células Madre Mesenquimatosas , Moxibustión , Insuficiencia Ovárica Primaria , Humanos , Femenino , Ratas , Animales , Mitofagia , Especies Reactivas de Oxígeno/metabolismo , Carbonil Cianuro m-Clorofenil Hidrazona/efectos adversos , Carbonil Cianuro m-Clorofenil Hidrazona/metabolismo , Insuficiencia Ovárica Primaria/inducido químicamente , Insuficiencia Ovárica Primaria/terapia , Insuficiencia Ovárica Primaria/patología , Ciclofosfamida/efectos adversos , Células Madre Mesenquimatosas/metabolismo , Mitocondrias/metabolismo , Ubiquitina-Proteína Ligasas/metabolismo , Proteínas Quinasas/metabolismo , Hormonas/efectos adversos , Hormonas/metabolismo , Adenosina Trifosfato/metabolismo
3.
Biochem J ; 481(8): 587-599, 2024 Apr 24.
Artículo en Inglés | MEDLINE | ID: mdl-38592738

RESUMEN

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting α-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2- (above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2ß2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP.


Asunto(s)
Proteínas Quinasas Activadas por AMP , Adenosina Difosfato , Adenosina Monofosfato , Adenosina Difosfato/metabolismo , Adenosina Monofosfato/metabolismo , Humanos , Regulación Alostérica , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/química , Ligandos , Fosforilación , Adenosina Trifosfato/metabolismo , Activación Enzimática , Unión Proteica
4.
J Mater Chem B ; 12(19): 4629-4641, 2024 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-38666407

RESUMEN

Enlightened by the great success of the drug repurposing strategy in the pharmaceutical industry, in the current study, material repurposing is proposed where the performance of carbonyl iron powder (CIP), a nutritional intervention agent of iron supplement approved by the US FDA for iron deficiency anemia in clinic, was explored in anti-cancer treatment. Besides the abnormal iron metabolic characteristics of tumors, serving as potential targets for CIP-based cancer therapy under the repurposing paradigm, the efficacy of CIP as a catalyst in the Fenton reaction, activator for dihydroartemisinin (DHA), thus increasing the chemo-sensitivity of tumors, as well as a potent agent for NIR-II photothermal therapy (PTT) was fully evaluated in an injectable alginate hydrogel form. The CIP-ALG gel caused a rapid temperature rise in the tumor site under NIR-II laser irradiation, leading to complete ablation in the primary tumor. Further, this photothermal-ablation led to the significant release of ATP, and in the bilateral tumor model, both primary tumor ablation and inhibition of secondary tumor were observed simultaneously under the synergistic tumor treatment of nutritional-photothermal therapy (NT/PTT). Thus, material repurposing was confirmed by our pioneering trial and CIP-ALG-meditated NT/PTT/immunotherapy provides a new choice for safe and efficient tumor therapy.


Asunto(s)
Adenosina Trifosfato , Antineoplásicos , Rayos Infrarrojos , Animales , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/química , Ratones , Antineoplásicos/farmacología , Antineoplásicos/química , Inmunoterapia , Reposicionamiento de Medicamentos , Humanos , Rayos Láser , Terapia Fototérmica , Ratones Endogámicos BALB C , Proliferación Celular/efectos de los fármacos , Línea Celular Tumoral , Alginatos/química , Femenino , Hidrogeles/química , Hidrogeles/farmacología , Ensayos de Selección de Medicamentos Antitumorales , Tamaño de la Partícula , Artemisininas/química , Artemisininas/farmacología
5.
Sci Rep ; 14(1): 6348, 2024 03 15.
Artículo en Inglés | MEDLINE | ID: mdl-38491051

RESUMEN

Hepatocellular carcinoma (HCC) progression is associated with dysfunctional mitochondria and bioenergetics impairment. However, no data about the relationship between mitochondrial supercomplexes (hmwSC) formation and ATP production rates in HCC are available. Our group has developed an adenosine derivative, IFC-305, which improves mitochondrial function, and it has been proposed as a therapeutic candidate for HCC. We aimed to determine the role of IFC-305 on both mitochondrial structure and bioenergetics in a sequential cirrhosis-HCC model in rats. Our results showed that IFC-305 administration decreased the number and size of liver tumors, reduced the expression of tumoral markers, and reestablished the typical architecture of the hepatic parenchyma. The livers of treated rats showed a reduction of mitochondria number, recovery of the mtDNA/nDNA ratio, and mitochondrial length. Also, IFC-305 increased cardiolipin and phosphatidylcholine levels and promoted hmwSC reorganization with changes in the expression levels of hmwSC assembly-related genes. IFC-305 in HCC modified the expression of several genes encoding elements of electron transport chain complexes and increased the ATP levels by recovering the complex I, III, and V activity. We propose that IFC-305 restores the mitochondrial bioenergetics in HCC by normalizing the quantity, morphology, and function of mitochondria, possibly as part of its hepatic restorative effect.


Asunto(s)
Carcinoma Hepatocelular , Neoplasias Hepáticas , Ratas , Animales , Carcinoma Hepatocelular/inducido químicamente , Carcinoma Hepatocelular/tratamiento farmacológico , Carcinoma Hepatocelular/metabolismo , Dietilnitrosamina/toxicidad , Neoplasias Hepáticas/inducido químicamente , Neoplasias Hepáticas/tratamiento farmacológico , Neoplasias Hepáticas/metabolismo , Mitocondrias/metabolismo , Adenosina/metabolismo , Metabolismo Energético , Adenosina Trifosfato/metabolismo
6.
Phytomedicine ; 126: 155421, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38430819

RESUMEN

BACKGROUND: The presence of plasmid-mediated resistance-nodulation-division (RND) efflux pump gene cluster tmexCD1-toprJ1 and its related variants has been associated with heightened resistance to tigecycline, thus diminishing its effectiveness. In this study, we explored the potential of gramine, a naturally occurring indole alkaloid, as an innovative adjuvant to enhance the treatment of infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters. METHODS: The synergistic potential of gramine in combination with antibiotics against both planktonic and drug-tolerant multidrug-resistant Enterobacterales was evaluated using the checkerboard microbroth dilution technique and time-killing curve analyses. Afterwards, the proton motive force (PMF) of cell membrane, the function of efflux pump and the activity of antioxidant system were determined by fluorescence assay and RT-PCR. The intracellular accumulation of tigecycline was evaluated by HPLC-MS/MS. The respiration rate, bacterial ATP level and the NAD+/NADH ratio were investigated to reveal the metabolism state. Finally, the safety of gramine was assessed through hemolytic activity and cytotoxicity assays. Two animal infection models were used to evaluate the in vivo synergistic effect. RESULTS: Gramine significantly potentiated tigecycline and ciprofloxacin activity against tmexCD1-toprJ1 and its variants-positive pathogens. Importantly, the synergistic activity was also observed against bacteria in special physiological states such as biofilms and persister cells. The mechanism study showed that gramine possesses the capability to augment tigecycline accumulation within cells by disrupting the proton motive force (PMF) and inhibiting the efflux pump functionality. In addition, the bacterial respiration rate, intracellular ATP level and tricarboxylic acid cycle (TCA) were promoted under the treatment of gramine. Notably, gramine effectively restored tigecycline activity in multiple animal infection models infected by tmexCD1-toprJ1 positive K. pneumoniae (RGF105-1). CONCLUSION: This study provides the first evidence of gramine's therapeutic potential as a novel tigecycline adjuvant for treating infections caused by K. pneumoniae carrying tmexCD-toprJ-like gene clusters.


Asunto(s)
Infecciones por Klebsiella , Klebsiella pneumoniae , Animales , Tigeciclina/metabolismo , Tigeciclina/farmacología , Tigeciclina/uso terapéutico , Klebsiella pneumoniae/genética , Klebsiella pneumoniae/metabolismo , Minociclina/farmacología , Minociclina/metabolismo , Minociclina/uso terapéutico , Espectrometría de Masas en Tándem , Infecciones por Klebsiella/tratamiento farmacológico , Infecciones por Klebsiella/microbiología , Farmacorresistencia Bacteriana , Antibacterianos/farmacología , Alcaloides Indólicos/farmacología , Adenosina Trifosfato/metabolismo , Pruebas de Sensibilidad Microbiana
7.
Biosci Biotechnol Biochem ; 88(5): 529-537, 2024 Apr 22.
Artículo en Inglés | MEDLINE | ID: mdl-38509025

RESUMEN

Four ethanol fractionated crude extracts (EFCEs [A-D]) purified from the leaves of Cinnamomum macrostemon Hayata were screened for antioxidative effects and mitochondrial function in HaCaT cells. The higher cell viability indicated that EFCE C was mildly toxic. Under the treatment of 50 ng/mL EFCE C, the hydrogen peroxide (H2O2)-induced cytosolic and mitochondrial reactive oxygen species levels were reduced as well as the H2O2-impaired cell viability, mitochondrial membrane potential (MMP), ATP production, and mitochondrial mass. The conversion of globular mitochondria to tubular mitochondria is coincident with EFCE C-restored mitochondrial function. The mitophagy activator rapamycin showed similar effects to EFCE C in recovering the H2O2-impaired cell viability, MMP, ATP production, mitochondrial mass, and also mitophagic proteins such as PINK1, Parkin, LC3 II, and biogenesis protein PGC-1α. We thereby propose the application of EFCE C in the prevention of oxidative stress in skin cells.


Asunto(s)
Supervivencia Celular , Cinnamomum , Peróxido de Hidrógeno , Queratinocitos , Potencial de la Membrana Mitocondrial , Mitocondrias , Mitofagia , Estrés Oxidativo , Extractos Vegetales , Especies Reactivas de Oxígeno , Humanos , Mitofagia/efectos de los fármacos , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Queratinocitos/citología , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/farmacología , Extractos Vegetales/química , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Peróxido de Hidrógeno/metabolismo , Supervivencia Celular/efectos de los fármacos , Cinnamomum/química , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Hojas de la Planta/química , Antioxidantes/farmacología , Ubiquitina-Proteína Ligasas/metabolismo , Sirolimus/farmacología , Células HaCaT , Proteínas Quinasas/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/metabolismo , Coactivador 1-alfa del Receptor Activado por Proliferadores de Peroxisomas gamma/genética
8.
J Ethnopharmacol ; 325: 117885, 2024 May 10.
Artículo en Inglés | MEDLINE | ID: mdl-38331123

RESUMEN

ETHNOPHARMACOLOGICAL RELEVANCE: The Timosaponin BⅡ (TBⅡ) is one of the main active components of the traditional Chinese medicine Anemarrhena asphodeloides, and it is a steroidal saponin with various pharmacological activities such as anti-oxidation, anti-inflammatory and anti-apoptosis. However, its role in acute ulcerative colitis remains unexplored thus far. AIM OF THE STUDY: This study aims to investigate the protective effect of TBⅡ against dextran sulfate sodium (DSS)-induced ulcerative colitis in mice and elucidate its underlying mechanisms. METHODS: Wild-type (WT) and NLRP3 knockout (NLRP3-/-) mice were applied to evaluate the protective effect of TBⅡ in DSS-induced mice colitis. Pharmacological inhibition of NLRP3 or adenovirus-mediated NLRP3 overexpression in bone marrow-derived macrophages (BMDM) from WT mice and colonic epithelial HCoEpiC cells was used to assess the role of TBⅡ in LPS + ATP-induced cell model. RNA-seq, ELISA, western blots, immunofluorescence staining, and expression analysis by qPCR were performed to examine the alterations of colonic NLRP3 expression in DSS-induced colon tissues and LPS + ATP-induced cells, respectively. RESULTS: In mice with DSS-induced ulcerative colitis, TBⅡ treatment attenuated clinical symptoms, repaired the intestinal mucosal barrier, reduced inflammatory infiltration, and alleviated colonic inflammation. RNA-seq analysis and protein expression levels demonstrated that TBⅡ could prominently inhibit NLRP3 signaling. TBⅡ-mediated NLRP3 inhibition was associated with alleviating intestinal permeability and inflammatory response via the blockage of communication between epithelial cells and macrophages, probably in an NLRP3 inhibition mechanism. However, pharmacological inhibition of NLRP3 by MCC950 or Ad-NLRP3 mediated NLRP3 overexpression significantly impaired the TBⅡ-mediated anti-inflammatory effect. Mechanistically, TBⅡ-mediated NLRP3 inhibition may be partly associated with the suppression of NF-κB, a master pro-inflammatory factor for transcriptional regulation of NLRP3 expression in the priming step. Moreover, co-treatment TBⅡ with NF-κB inhibitor BAY11-7082 partly impaired TBⅡ-mediated NLRP3 inhibition, and consequently affected the IL-1ß mature and secretion. Importantly, TBⅡ-mediated amelioration was not further enhanced in NLPR3-/- mice. CONCLUSION: TBⅡ exerted a prominent protective effect against DSS-induced colitis via regulation of alleviation of intestinal permeability and inflammatory response via the blockage of crosstalk between epithelial cells and macrophages in an NLRP3-mediated inhibitory mechanism. These beneficial effects could make TBⅡ a promising drug for relieving colitis.


Asunto(s)
Colitis Ulcerosa , Colitis , Saponinas , Animales , Ratones , Colitis Ulcerosa/inducido químicamente , Colitis Ulcerosa/tratamiento farmacológico , Colitis Ulcerosa/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , FN-kappa B/metabolismo , Lipopolisacáridos/metabolismo , Inflamasomas/metabolismo , Colitis/tratamiento farmacológico , Inflamación/tratamiento farmacológico , Inflamación/metabolismo , Antiinflamatorios/efectos adversos , Saponinas/farmacología , Saponinas/uso terapéutico , Adenosina Trifosfato/metabolismo , Sulfato de Dextran/toxicidad , Ratones Endogámicos C57BL , Colon/metabolismo
9.
Int Immunopharmacol ; 129: 111656, 2024 Mar 10.
Artículo en Inglés | MEDLINE | ID: mdl-38340422

RESUMEN

Geraniin, a chemical component of the traditional Chinese medicine geranii herba, possesses anti-inflammatory and anti-oxidative activities. However, its anti-inflammatory role in managing NLRP3 inflammasome and pyroptosis remains to be elucidated. To investigate the anti-inflammation mechanism of geraniin, LPS-primed macrophages were incubated with classical activators of NLRP3 inflammasome (such as ATP, Nigericin, or MSU crystals), and MSU crystals were injected into the ankle joints of mice to establish an acute gouty arthritis model. The propidium iodide (PI) staining results showed that geraniin could restrain cell death in the ATP- or nigericin-stimulated bone marrow-derived macrophages (BMDMs). Geraniin decreased the release of lactate dehydrogenase (LDH) and interleukin (IL)-1ß from cytoplasm to cell supernatant. Geraniin also inhibited the expression of caspase-1 p20, IL-1ß in cell supernatant and N-terminal of gasdermin D (GSDMD-NT) while blocking the oligomerization of ASC to form speck. The inhibitory effects of geraniin on caspase-1 p20, IL-1ß, GSDMD-NT, and ASC speck were not observed in NLRP3 knockout (NLRP3-/-) BMDMs. Hence, the resistance of geraniin to inflammasome and pyroptosis was contingent upon NLRP3 presence. Geraniin reduced reactive oxygen species (ROS) production and maintained mitochondrial membrane potential while preventing interaction between ASC and NLRP3 protein. Additionally, geraniin diminished MSU crystal-induced mouse ankle joint swelling and IL-1ß expression. Geraniin blocked the recruitment of neutrophils and macrophages to the synovium of joints. Our results demonstrate that geraniin prevents the assembly of ASC and NLRP3 through its antioxidant effect, thereby inhibiting inflammasome activation, pyroptosis, and IL-1ß release to provide potential insights for gouty arthritis targeted therapy.


Asunto(s)
Artritis Gotosa , Glucósidos , Taninos Hidrolizables , Inflamasomas , Ratones , Animales , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Artritis Gotosa/inducido químicamente , Piroptosis , Nigericina/farmacología , Macrófagos , Antiinflamatorios/efectos adversos , Adenosina Trifosfato/metabolismo , Caspasas/metabolismo , Interleucina-1beta/metabolismo
10.
Brain Res Bull ; 209: 110909, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38402994

RESUMEN

BACKGROUND: The efficacy of acupuncture at Zusanli (ST36) in alleviating lower-limb pain is widely acknowledged in clinical practice, while its underlying mechanism remains incompletely elucidated. Our previous research had revealed that the prompt analgesia induced by needling-ST36 was accompanied by expression alterations in certain exco-nucleotidases within the sciatic nerve. Building upon this finding, the current work focused on NTPDase1, the primary ecto-nucleotidase in the human body, which converts ATP into AMP. METHODS: A 20-min acupuncture was administered unilaterally at the ST36 on rats with acute ankle arthritis. The pain thresholds of the injured hind paws were determined. Pharmacological interference was carried out by introducing the corresponding reagents to the sciatic nerve. ATP levels around the excised nerve were measured using a luciferase-luciferin assay. Live calcium imaging, utilizing the Fura 2-related-F340/F380 ratio, was conducted on Schwann cells in excised nerves and cultured rat SCs line, RSC96 cells. RESULTS: The analgesic effect induced by needling-ST36 was impaired when preventing ATP degradation via inhibiting NTPDase1 activities with ARL67156 or Ticlopidine. Conversely, increasing NTPDase1 activities with Apyrase duplicated the acupuncture effect. Similarly, preventing the conversion of AMP to adenosine via suppression of NT5E with AMP-CP hindered the acupuncture effect. Unexpectedly, impeded ATP hydrolysis ability and diminished NTPDase1 expression were observed in the treated group. Agonism at P2Y2Rs with ATP, UTP, or INS365 resulted in anti-nociception. Contrarily, antagonism at P2Y2Rs with Suramin or AR-C 118925xx prevented acupuncture analgesia. Immunofluorescent labeling demonstrated that the treated rats expressed more P2Y2Rs that were predominant in Schwann cells. Suppression of Schwann cells by inhibiting ErbB receptors also prevented acupuncture analgesia. Finally, living imaging on the excised nerves or RSC96 cells showed that agonism at P2Y2Rs indeed led to [Ca2+]i rise. CONCLUSION: These findings strongly suggest that the analgesic mechanism of needling-ST36 on the hypersensation in the lower limb partially relies on NTPDase1 activities in the sciatic nerve. In addition to facilitating adenosine signaling in conjunction with NT5E, most importantly, NTPDase1 may provide an appropriate low-level ATP milieu for the activation of P2Y2R in the sciatic nerve, particularly in Schwann cells.


Asunto(s)
Analgesia por Acupuntura , Terapia por Acupuntura , Antígenos CD , Artritis , Ratas , Humanos , Animales , Apirasa , Tobillo , Dolor , Nervio Ciático/metabolismo , Adenosina Trifosfato/metabolismo , Analgésicos , Adenosina Monofosfato , Adenosina , Puntos de Acupuntura
11.
Anim Reprod Sci ; 263: 107437, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38395011

RESUMEN

Short-term sperm storage is a straightforward and cost-effective method of managing logistics in large scale fish hatchery operations but may result in decline in sperm quality. For effective artificial reproduction of fish, use of an appropriate additive to optimize sperm storage conditions is essential. In this study, it was investigated the effect of purified seminal plasma transferrin (Tf) at 10 µg/ml on relevant parameters in common carp Cyprinus carpio sperm during short-term storage. We compared sperm motility and curvilinear velocity, adenosine triphosphate (ATP) content and DNA fragmentation of fresh spermatozoa to that stored for 24, 48, 72, and 144 h with or without Tf. The percentage of motile cells and the curvilinear velocity of spermatozoa in stored samples for 72 h with transferrin supplementation were greater compared to samples with no added protein. The ATP content in samples without added transferrin was reduced (P < 0.05) after 72 h of storage, in contrast to the levels observed in transferrin-supplemented sperm. A time-dependent increase in DNA fragmentation was observed. Significantly lower DNA damage, expressed as percent tail DNA (10.99 ±â€¯1.28) and olive tail moment (0.54 ±â€¯0.12), was recorded in Tf-supplemented samples stored for 48 h compared to that with no Tf. Hence, it is concluded that the beneficial effects of transferrin on common carp sperm could serve as an additional tool for developing and enhancing short-term sperm preservation procedures commonly used in aquaculture.


Asunto(s)
Carpas , Preservación de Semen , Masculino , Animales , Semen/metabolismo , Transferrina/farmacología , Adenosina Trifosfato/metabolismo , Motilidad Espermática , Espermatozoides , Preservación de Semen/veterinaria , Preservación de Semen/métodos , ADN/metabolismo
12.
J Nutr Biochem ; 127: 109602, 2024 May.
Artículo en Inglés | MEDLINE | ID: mdl-38373509

RESUMEN

This study evaluated the effect of vitamin D3 (VIT D3) supplementation on the enzymatic activities and density of ectonucleoside triphosphate diphosphohydrolase (E-NTPDase), ecto-5-nucleotidase (E-5'-NT), adenosine deaminase (ADA), as well as the density of P2 × 7R, P2Y12R, A1R, A2AR receptors, IL-1ß, and oxidative parameters in type 2 diabetic rats. Forty male Wistar rats were fed a high carbohydrate-high fat diet (HCHFD) and received an intraperitoneal injection containing a single dose of streptozotocin (STZ, 35 mg/kg). Animals were divided into four groups: 1) control; 2) control/VIT D3 12 µg/kg; 3) diabetic; and 4) diabetic/VIT D3 12 µg/kg. Results show that VIT D3 reduced blood glucose, ATP hydrolysis, ADA activity, P2Y12R density (platelets), as well as ATP, ADP, and AMP hydrolysis and ADA activity (synaptosomes). Moreover, VIT D3 increased insulin levels and AMP hydrolysis (platelets) and improved antioxidant defense. Therefore, we suggest that VIT D3 treatment modulates hyperglycemia-induced changes via purinergic enzymes and receptor expression, consequently attenuating insulin homeostasis dysregulation in the diabetic state.


Asunto(s)
Diabetes Mellitus Experimental , Diabetes Mellitus Tipo 2 , Insulinas , Ratas , Masculino , Animales , Ratas Wistar , Colecalciferol/farmacología , Diabetes Mellitus Experimental/tratamiento farmacológico , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Dieta Alta en Grasa/efectos adversos , Vitaminas , Adenosina Monofosfato/metabolismo , Adenosina Trifosfato/metabolismo
13.
Phytother Res ; 38(4): 1990-2006, 2024 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-38372204

RESUMEN

Osteoarthritis (OA) is characterized by an imbalance between M1 and M2 polarized synovial macrophages. Quercetin has shown protective effects against OA by altering M1/M2-polarized macrophages, but the underlying mechanisms remain unclear. In this study, rat chondrocytes were treated with 10 ng/mL of IL-1ß. To create M1-polarized macrophages in vitro, rat bone marrow-derived macrophages (rBMDMs) were treated with 100 ng/mL LPS. To mimic OA conditions observed in vivo, a co-culture system of chondrocytes and macrophages was established. ATP release assays, immunofluorescence assays, Fluo-4 AM staining, Transwell assays, ELISA assays, and flow cytometry were performed. Male adult Sprague-Dawley (SD) rats were used to create an OA model. Histological analyses, including H&E, and safranin O-fast green staining were performed. Our data showed a quercetin-mediated suppression of calcium ion influx and ATP release, with concurrent downregulation of TRPV1 and P2X7 in the chondrocytes treated with IL-1ß. Activation of TRPV1 abolished the quercetin-mediated effects on calcium ion influx and ATP release in chondrocytes treated with IL-1ß. In the co-culture system, overexpression of P2X7 in macrophages attenuated the quercetin-mediated effects on M1 polarization, migration, and inflammation. Either P2X7 or NLRP3 knockdown attenuated IL-1ß-induced M1/M2 polarization, migration, and inflammation. Moreover, overexpression of TRPV1 reduced the quercetin-mediated suppressive effects on OA by promoting M1/M2-polarized macrophages in vivo. Collectively, our data showed that quercetin-induced suppression of TRPV1 leads to a delay in OA progression by shifting the macrophage polarization from M1 to M2 subtypes via modulation of the P2X7/NLRP3 pathway.


Asunto(s)
Osteoartritis , Quercetina , Animales , Masculino , Ratas , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Inflamación/metabolismo , Macrófagos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Osteoartritis/tratamiento farmacológico , Quercetina/farmacología , Ratas Sprague-Dawley , Transducción de Señal
14.
Theriogenology ; 216: 185-195, 2024 Mar 01.
Artículo en Inglés | MEDLINE | ID: mdl-38194754

RESUMEN

Hypothermic liquid storage at 4-5 °C has emerged as a novel approach for preserving boar semen, offering innovative possibilities for semen preservation. However, this method also presents challenges, including cold shock and excessive reactive oxygen species (ROS) production. Therefore, reducing oxidative damage induced by low temperatures becomes essential while supplementing appropriate protectants. In this study, we investigated the efficacy of Bovine Serum Albumin (BSA) compared to Polyvinylpyrrolidone (PVP) and Skim Milk Powder (SMP) in maintaining boar sperm motility and progressive motility using computer-assisted sperm analysis (CASA). Among the tested concentrations, 4 g/L of BSA exhibited the best protective effect. Subsequently, we supplemented different concentrations of l-cysteine (LC) and N-acetyl-l-cysteine (NAC) as additives in the presence of BSA as a protectant. Our results demonstrated that 1 mmol/L of LC and 0.5 mmol/L of NAC exhibited superior protection of sperm quality compared to other concentrations. Furthermore, the 1 mmol/L LC and 0.5 mmol/L NAC groups showed significantly improved plasma membrane integrity and acrosome integrity compared to the control group. These groups also exhibited enhanced antioxidant capacity, evidenced by increased mitochondrial membrane potential (MMP), ATP production, total superoxide dismutase (T-SOD) activity, total antioxidant capacity (T-AOC), glutathione (GSH), glutathione peroxidase (GSH-PX), and GPX-4 levels. Additionally, they demonstrated decreased reactive oxygen species (ROS) and malondialdehyde (MDA) levels, as well as reduced oxidized glutathione (GSSG) and glutathione reductase (GR) levels. Furthermore, LC and NAC treatment enhanced AMP-activated protein kinase (AMPK) phosphorylation. However, inhibiting AMPK using compound C did not inhibit the protective effects of LC and NAC on low-temperature preserved boar sperm. These findings suggest that 4 g/L BSA can serve as an effective protectant for hypothermic liquid storage of boar semen. Additionally, LC and NAC supplementation reduces oxidative damage by enhancing antioxidant capacity rather than through AMPK-mediated ATP supplementation. These results contribute to advancing the application of LC and NAC in hypothermic liquid storage of boar semen.


Asunto(s)
Preservación de Semen , Semen , Masculino , Porcinos , Animales , Acetilcisteína/metabolismo , Acetilcisteína/farmacología , Especies Reactivas de Oxígeno/metabolismo , Albúmina Sérica Bovina/farmacología , Albúmina Sérica Bovina/metabolismo , Antioxidantes/farmacología , Antioxidantes/metabolismo , Proteínas Quinasas Activadas por AMP/metabolismo , Motilidad Espermática , Espermatozoides/fisiología , Análisis de Semen/veterinaria , Glutatión/metabolismo , Adenosina Trifosfato/metabolismo , Preservación de Semen/veterinaria , Preservación de Semen/métodos
15.
J Control Release ; 367: 1-12, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38244844

RESUMEN

Immunogenic cell death (ICD) is associated with the release of damage-associated molecular patterns, including ATP, to promote an effective immune cycle against tumors. However, tumors have evolved an effective strategy for degrading extracellular immunostimulatory ATP via the ATP-adenosine axis, allowing the sequential action of the ectonucleotidases CD39 to degrade accumulated immunostimulatory ATP into pleiotropic immunosuppressive adenosine. Here, an ingenious dissolving microneedle patch (DMNs) is designed for the intralesional delivery of CD39 inhibitor (sodium polyoxotungstate, POM-1) and ICD inducer (IR780) co-encapsulated solid lipid nanoparticles (P/I SLNs) for antitumor therapy. Upon insertion into the tumor site, IR780 induces ICD modalities with the release of damage-associated molecular patterns from endogenous tissues, which activates the antitumor immune cycle. Simultaneously, POM-1 promotes the liberation of immunostimulatory ATP and lowers the level of immunosuppressive extracellular adenosine, which supported immune control of tumors via recruiting CD39-expressing immune cells. In vivo antitumor studies prove that this platform can effectively eliminate mice melanoma (tumor growth inhibitory rate of 96.5%) and colorectal adenocarcinoma (tumor growth inhibitory rate of 93.5%). Our results shed light on the immunological aspects of combinatorial phototherapy and ATP-adenosine regulation, which will broaden the scope of synergistic antitumor immunotherapy.


Asunto(s)
Adenosina , Neoplasias , Animales , Ratones , Fototerapia/métodos , Neoplasias/terapia , Adenosina Trifosfato/metabolismo , Inmunoterapia , Línea Celular Tumoral
16.
Int J Med Sci ; 21(2): 369-375, 2024.
Artículo en Inglés | MEDLINE | ID: mdl-38169534

RESUMEN

Heart failure is a condition where reduced levels of adenosine triphosphate (ATP) affect energy supply in myocardial cells. Nicotinamide adenine dinucleotide (NAD+) plays a crucial role as a coenzyme for electron transfer in energy metabolism. Decreased NAD+ levels in myocardial cells lead to inadequate ATP production and increased susceptibility to heart failure. Researchers are exploring ways to increase NAD+ levels to alleviate heart failure. Targets such as sirtuin2 (sirt2), sirtuin3 (sirt3), Poly (ADP-ribose) polymerase (PARP), and diastolic regulatory proteins are being investigated. NAD+ supplementation has shown promise, even in heart failure with preserved ejection fraction (HFpEF). By focusing on NAD+ as a central component of energy metabolism, it is possible to improve myocardial activity, heart function, and address energy deficiency in heart failure.


Asunto(s)
Insuficiencia Cardíaca , Humanos , NAD/metabolismo , Volumen Sistólico , Metabolismo Energético , Poli(ADP-Ribosa) Polimerasas/metabolismo , Adenosina Trifosfato/metabolismo
17.
Mol Neurodegener ; 19(1): 13, 2024 Jan 29.
Artículo en Inglés | MEDLINE | ID: mdl-38282024

RESUMEN

BACKGROUND: Bioenergetic maladaptations and axonopathy are often found in the early stages of neurodegeneration. Nicotinamide adenine dinucleotide (NAD), an essential cofactor for energy metabolism, is mainly synthesized by Nicotinamide mononucleotide adenylyl transferase 2 (NMNAT2) in CNS neurons. NMNAT2 mRNA levels are reduced in the brains of Alzheimer's, Parkinson's, and Huntington's disease. Here we addressed whether NMNAT2 is required for axonal health of cortical glutamatergic neurons, whose long-projecting axons are often vulnerable in neurodegenerative conditions. We also tested if NMNAT2 maintains axonal health by ensuring axonal ATP levels for axonal transport, critical for axonal function. METHODS: We generated mouse and cultured neuron models to determine the impact of NMNAT2 loss from cortical glutamatergic neurons on axonal transport, energetic metabolism, and morphological integrity. In addition, we determined if exogenous NAD supplementation or inhibiting a NAD hydrolase, sterile alpha and TIR motif-containing protein 1 (SARM1), prevented axonal deficits caused by NMNAT2 loss. This study used a combination of techniques, including genetics, molecular biology, immunohistochemistry, biochemistry, fluorescent time-lapse imaging, live imaging with optical sensors, and anti-sense oligos. RESULTS: We provide in vivo evidence that NMNAT2 in glutamatergic neurons is required for axonal survival. Using in vivo and in vitro studies, we demonstrate that NMNAT2 maintains the NAD-redox potential to provide "on-board" ATP via glycolysis to vesicular cargos in distal axons. Exogenous NAD+ supplementation to NMNAT2 KO neurons restores glycolysis and resumes fast axonal transport. Finally, we demonstrate both in vitro and in vivo that reducing the activity of SARM1, an NAD degradation enzyme, can reduce axonal transport deficits and suppress axon degeneration in NMNAT2 KO neurons. CONCLUSION: NMNAT2 ensures axonal health by maintaining NAD redox potential in distal axons to ensure efficient vesicular glycolysis required for fast axonal transport.


Asunto(s)
Transporte Axonal , NAD , Nicotinamida-Nucleótido Adenililtransferasa , Animales , Ratones , Adenosina Trifosfato/metabolismo , Proteínas del Dominio Armadillo/metabolismo , Axones/metabolismo , Proteínas del Citoesqueleto/metabolismo , Glucólisis , Homeostasis , NAD/metabolismo , Nicotinamida-Nucleótido Adenililtransferasa/metabolismo
18.
Antimicrob Agents Chemother ; 68(2): e0093723, 2024 Feb 07.
Artículo en Inglés | MEDLINE | ID: mdl-38169282

RESUMEN

Entering a dormant state is a prevailing mechanism used by bacterial cells to transiently evade antibiotic attacks and become persisters. The dynamic progression of bacterial dormancy depths driven by protein aggregation has been found to be critical for antibiotic persistence in recent years. However, our current understanding of the endogenous genes that affects dormancy depth remains limited. Here, we discovered a novel role of phage shock protein A (pspA) gene in modulating bacterial dormancy depth. Deletion of pspA of Escherichia coli resulted in increased bacterial dormancy depths and prolonged lag times for resuscitation during the stationary phase. ∆pspA exhibited a higher persister ratio compared to the wild type when challenged with various antibiotics. Microscopic images revealed that ∆pspA showed accelerated formation of protein aggresomes, which were collections of endogenous protein aggregates. Time-lapse imaging established the positive correlation between protein aggregation and antibiotic persistence of ∆pspA at the single-cell level. To investigate the molecular mechanism underlying accelerated protein aggregation, we performed transcriptome profiling and found the increased abundance of chaperons and a general metabolic slowdown in the absence of pspA. Consistent with the transcriptomic results, the ∆pspA strain showed a decreased cellular ATP level, which could be rescued by glucose supplementation. Then, we verified that replenishment of cellular ATP levels by adding glucose could inhibit protein aggregation and reduce persister formation in ∆pspA. This study highlights the novel role of pspA in maintaining proteostasis, regulating dormancy depth, and affecting antibiotic persistence during stationary phase.


Asunto(s)
Antibacterianos , Agregado de Proteínas , Antibacterianos/farmacología , Escherichia coli/genética , Adenosina Trifosfato/metabolismo , Glucosa/metabolismo
19.
Microbiol Spectr ; 12(3): e0255223, 2024 Mar 05.
Artículo en Inglés | MEDLINE | ID: mdl-38270436

RESUMEN

Obesity and overweight among companion animals are significant concerns, paralleling the issues observed in human populations. Recent research has highlighted the potential benefits of various probiotics in addressing weight-related changes, obesity, and associated pathologies. In this study, we delved into the beneficial probiotic mechanisms in high-fat-induced obese canines, revealing that Enterococcus faecium IDCC 2102 (IDCC 2102) and Bifidobacterium lactis IDCC 4301 (IDCC 4301) have the capacity to mitigate the increase in body weight and lipid accumulation in obese canines subjected to a high-fat diet and hyperlipidemic Caenorhabditis elegans (C. elegans) strain VS29. Both IDCC 2102 and IDCC 4301 demonstrated the ability to reduce systemic inflammation and hormonal disruptions induced by obesity. Notably, these probiotics induced modifications in the microbiota by promoting lactic acid bacteria, including Lactobacillaceae, Ruminococcaceae, and S24-7, with concomitant activation of pyruvate metabolism. IDCC 4301, through the generation of bacterial short-chain fatty acids and carboxylic acids, facilitated glycolysis and contributed to ATP synthesis. Meanwhile, IDCC 2102 produced bacterial metabolites such as acetic acid and butyric acid, exhibiting a particular ability to stimulate dopamine synthesis in a canine model. This stimulation led to the restoration of eating behavior and improvements in glucose and insulin tolerance. In summary, we propose novel probiotics for the treatment of obese animals based on the modifications induced by IDCC 2102 and IDCC 4301. These probiotics enhanced systemic energy utilization in response to high caloric intake, thereby preventing lipid accumulation and restoring stability to the fecal microbiota. Consequently, this intervention resulted in a reduction in systemic inflammation caused by the high-fat diet.IMPORTANCEProbiotic supplementation affected commensal bacterial proliferation, and administering probiotics increased glycolysis and activated pyruvate metabolism in the body, which is related to propanate metabolism as a result of pyruvate metabolism activation boosting bacterial fatty acid production via dopamine and carboxylic acid specialized pathways, hence contributing to increased ATP synthesis and energy metabolism activity.


Asunto(s)
Microbioma Gastrointestinal , Probióticos , Humanos , Perros , Animales , Caenorhabditis elegans/metabolismo , Dopamina , Obesidad/terapia , Obesidad/veterinaria , Obesidad/metabolismo , Pérdida de Peso , Ácidos Grasos Volátiles , Metabolismo Energético , Inflamación , Piruvatos , Adenosina Trifosfato/metabolismo
20.
Fish Shellfish Immunol ; 146: 109387, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38272331

RESUMEN

Acetyl-CoA carboxylase (ACC) plays a regulatory role in both fatty acid synthesis and oxidation, controlling the process of lipid deposition in the liver. Given that existing studies have shown a close relationship between low phosphorus (P) and hepatic lipid deposition, this study was conducted to investigate whether ACC plays a crucial role in this relationship. Zebrafish liver cell line (ZFL) was incubated under low P medium (LP, P concentration: 0.77 mg/L) or adequate P medium (AP, P concentration: 35 mg/L) for 240 h. The results showed that, compared with AP-treated cells, LP-treated cells displayed elevated lipid accumulation, and reduced fatty acid ß-oxidation, ATP content, and mitochondrial mass. Furthermore, transcriptomics analysis revealed that LP-treated cells significantly increased lipid synthesis (Acetyl-CoA carboxylases (acc), Stearyl coenzyme A dehydrogenase (scd)) but decreased fatty acid ß-oxidation (Carnitine palmitoyltransferase I (cptI)) and (AMP-activated protein kinase (ampk)) mRNA levels compared to AP-treated cells. The phosphorylation of AMPK and ACC, and the protein expression of CPTI were significantly decreased in LP-treated cells compared with those in AP-treated cells. After 240 h of LP treatment, PF-05175157 (an ACC inhibitor) was supplemented in the LP treatment for an additional 12 h. PF-05175157-treated cells showed higher phosphorylation of ACC, higher protein expression of CPTI, and lower protein expression of FASN, lower TG content, enhanced fatty acid ß-oxidation, increased ATP content, and mitochondrial mass compared with LP-treated cells. PF-05175157 also relieved the LP-induced oxidative stress and inflammatory response. Overall, these findings suggest that ACC is a promising target for treating LP-induced elevation of lipid deposition in ZFL, and can alleviate oxidative stress and inflammatory response.


Asunto(s)
Acetil-CoA Carboxilasa , Pez Cebra , Animales , Pez Cebra/metabolismo , Acetil-CoA Carboxilasa/genética , Acetil-CoA Carboxilasa/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Hígado/metabolismo , Estrés Oxidativo , Ácidos Grasos/metabolismo , Fósforo , Lípidos , Adenosina Trifosfato/metabolismo
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