Background:This study investigated the role of polydatin in regulating macrophage-epithelial cell(EC)interactions during asthma.An asthma model was induced in BALB/c mice using ovalbumin(20μg).Methods:The therapeutic...Background:This study investigated the role of polydatin in regulating macrophage-epithelial cell(EC)interactions during asthma.An asthma model was induced in BALB/c mice using ovalbumin(20μg).Methods:The therapeutic effects of polydatin(20 and 40 mg/kg)were evaluated in this asthmatic mouse model.To assess the underlying mechanisms,Bronchial Epithelium Adenovirus 12-SV402B(BEAS-2B)cells were cocultured with Tohoku Hospital for Pediatrics-1(THP-1)macrophages,in which toll-like receptor 4(TLR4)was either overexpressed or knocked down,and subsequently stimulated with lipopoly-saccharide(LPS)and ATP.THP-1 cells underwent a 1-h pretreatment with polydatin(50 and 100μmol/L),Class Lipid Inhibitor-095(CLI-095,TLR4 inhibitor,1μg/mL),or A438079(P2X7R antagonist,10μmol/L)prior to LPS/ATP challenge.Results:Findings from Western blotting,enzyme-linked immunosorbent assay,flow cytometry,real-time polymerase chain reaction,and immunofluorescence assays demonstrated that modulating TLR4 expression significantly altered interleukin-1β(IL-1β)secretion from THP-1 macrophages and mitochondrial reactive oxygen species(mtROS)production in BEAS-2B ECs.In the mouse asthma model,polydatin significantly alleviated airway inflammation,oxidative stress,and apoptosis,likely by interfering with TLR4/P2X7R-mediated signaling and suppressing the activation of the NOD-like receptor protein inflammasome.Additionally,polydatin significantly reduced IL-1βand IL-18 levels and inhibited the infiltration of macrophages and eosinophils.Correspondingly,polydatin significantly attenuated TLR4/P2X7R signaling in THP-1 cells stimulated with ATP and LPS,thereby reducing IL-1βand IL-18 secretion,calcium influx,mtROS production,and apoptosis in BEAS-2B ECs.Conclusions:Polydatin is a promising therapeutic candidate for asthma,possibly by targeting macrophage-epithelium cross-talk via the TLR4/P2X7R axis.Future formulations as capsules or sprays may effectively alleviate airway inflammation and remodeling.展开更多
Objective:Breast cancer is the most common malignancy in women and is characterized by a high recurrence rate that severely impacts patient survival.Regulatory T cells(Tregs)in the tumor microenvironment(TME)promote i...Objective:Breast cancer is the most common malignancy in women and is characterized by a high recurrence rate that severely impacts patient survival.Regulatory T cells(Tregs)in the tumor microenvironment(TME)promote immune evasion and metastasis,increasing recurrence risk.This study determined how the epigenetic regulators,DNMT3A and METTL7A,modulate Treg infiltration via the DDR1/STAT3/CXCL5 axis and influence breast cancer recurrence and prognosis.Methods:RNA sequencing(RNA-seq)was used to identify differentially expressed genes(DEGs),followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.Machine learning algorithms,including least absolute shrinkage and selection operator(LASSO),supported vector machine-recursive feature elimination(SVM-RFE)and ElasticNet identified DDR1 as a key gene.Validation included RT-qPCR,western blot,MSP,MeRIP-qPCR,and Co-IP to assess epigenetic regulation.Functional assays(CCK-8,Transwell,and Treg differentiation/chemotaxis)and xenograft models evaluated the role of DDR1 in tumor progression and recurrence.Results:DNMT3A upregulated DDR1 via DNA methylation,while METTL7A enhanced DDR1 mRNA stability via m6A modification.Co-regulation activated the DDR1/STAT3/CXCL5 axis,which boosted cancer cell proliferation,migration,and invasion.CXCL5 secretion increased Treg infiltration and accelerated tumor growth in vivo.DDR1 silencing reversed these effects,confirming that DDR1 has a pivotal role in breast cancer recurrence.Conclusion:DNMT3A and METTL7A were shown to cooperatively regulate DDR1 via DNA/m6A methylation,which drives Tregmediated immune suppression and recurrence.This study provided novel insights and therapeutic targets for breast cancer prognosis and treatment.展开更多
BACKGROUND Magnesium(Mg^(2+))plays a fundamental role in numerous cellular processes,including enzymatic reactions,DNA replication,oxidative stress response,and cytoskeletal dynamics.In fact,dysregulation of Mg^(2+)ho...BACKGROUND Magnesium(Mg^(2+))plays a fundamental role in numerous cellular processes,including enzymatic reactions,DNA replication,oxidative stress response,and cytoskeletal dynamics.In fact,dysregulation of Mg^(2+)homeostasis has been increasingly associated with the development and progression of cancer,particularly colorectal cancer(CRC).Transient receptor potential melastatin(TRPM)channels,especially TRPM6 and TRPM7,are essential regulators of epithelial Mg^(2+)influx.While TRPM7 promotes CRC progression,the role of TRPM6 and TRPM6/7 channels remains unclear.AIM To investigate the role of membrane-localized TRPM6 and TRPM6/7 channels in Mg^(2+)influx,spheroid(SP)formation,stemness,and migration.METHODS We used parental and SP-derived HT-29 cells at comparable passages as in vitro models.Mass spectrometry confirmed full-length sequences,phosphorylation,and methionine oxidation of TRPM6 and TRPM7.Mg^(2+)influx,total and free Mg^(2+)levels were measured by fluorescence imaging and biochemical assays.TRPM6/TRPM7 expression and markers were analyzed by western blot.Func-tional assays,including secondary SP formation and wound healing,assessed stemness and migration.Cells were treated with Mg^(2+)transport inhibitors:Co(III)hexamine,2-aminoethyl diphenylborinate(TRPM6/7 blocker),and Mesendogen(TRPM6 inhibitor).RESULTS The expression of membrane-bound TRPM6,TRPM7,and TRPM6/7 was significantly higher in SP cells than in parental cells.Mass spectrometric analysis confirmed the presence of full-length TRPM6 and TRPM7 with increased phosphorylation and oxidation in SP cells.Enhanced Mg^(2+)influx and total intracellular Mg^(2+)levels were observed in SP cells.Free ionized intracellular Mg^(2+)levels remained comparable across all experimental groups.Pharmacological inhibition of TRPM6 and TRPM6/7 significantly reduced Mg^(2+)influx,decreased total Mg^(2+)content,compromised CRC SP stability,abolished cancer stem-like properties,impaired cell migration,and downregulated pro-tumorigenic markers,including Nanog,cyclooxygenase-2,and matrix metalloproteinase-9.CONCLUSION Membrane-localized TRPM6 and TRPM6/7 channels regulate Mg^(2+)influx and promote CRC stemness,SP stability,and migration,highlighting their potential as therapeutic targets to inhibit CRC progression and metastasis.展开更多
BACKGROUND:While theα7 nicotinic acetylcholine receptor(α7 nAChR)is implicated in sepsis-associated encephalopathy(SAE),its pathophysiological contributions require further investigation.METHODS:SAE was induced in m...BACKGROUND:While theα7 nicotinic acetylcholine receptor(α7 nAChR)is implicated in sepsis-associated encephalopathy(SAE),its pathophysiological contributions require further investigation.METHODS:SAE was induced in mice via cecal ligation and puncture(CLP),and microglia were treated with lipopolysaccharide(LPS).PHA-543613(anα7 nAChR agonist)was used to activateα7 nAChR.To study the role ofα7 nAChR in mitophagy and pyroptosis,caspase-1-deficient mice and PTEN-induced kinase 1(PINK1)small interfering RNA(siRNA)were used.Cognitive function,cerebral oxygen extraction ratio(CERO2),and brain tissue oxygen pressure(PbtO2)were measured.Blood-brain barrier(BBB)integrity was evaluated via Evan’s blue staining.Mitophagy,pyroptosis,and cytokine levels were analyzed via Western blotting and immunofl uorescence.RESULTS:CLP or LPS treatment signifi cantly down-regulatedα7 nAChR protein expression in microglia.The administration of PHA-543613 to activateα7 nAChR not only restored its expression post-sepsis,but also notably decreased BBB permeability and mitigated cognitive deficits.Bothα7 nAChR activation and caspase-1 knockout effectively suppressed microglial pyroptosis.The activation ofα7 nAChR also promoted mitophagy in microglia.This led to an amelioration of brain tissue hypoxia,as shown by elevated PbtO2 and reduced CERO2 levels.The suppression of microglial pyroptosis byα7 nAChR was counteracted when mitophagy was inhibited through the siRNA-mediated silencing of PINK1.CONCLUSION:The activation ofα7 nAChR reduces pyroptosis by enhancing microglial mitophagy,thereby mitigating SAE.展开更多
OBJECTIVE:to investigate the anti-arthritic effects of lappaconitine(LA)on adjuvant-induced arthritis in Sprague-Dawley rats and its possible involvement in the regulation of M1/M2 macrophage balance through the P2X7 ...OBJECTIVE:to investigate the anti-arthritic effects of lappaconitine(LA)on adjuvant-induced arthritis in Sprague-Dawley rats and its possible involvement in the regulation of M1/M2 macrophage balance through the P2X7 receptor(P2X7r).METHODS:Rats were immunized with complete Freund's adjuvant and then intraperitoneally administered LA(2,4,or 8 mg·kg^(-1)·d^(-1))or methotrexate(0.5 mg/kg per 3 d)for 14 d.The anti-arthritic effects of LA were evaluated through arthritis index(AI)assessment,ankle diameter measurement,and histopathological staining analysis.The analgesic effect of LA on arthritis was measured using mechanical withdrawal threshold testing and gait scoring.The impacts of LA on macrophage polarization,the expression of pro-/anti-inflammatory cytokines and P2X7r were analyzed using quantitative real-time polymerase chain reaction,enzyme-linked immunosorbent assay,and Western blotting.RESULTS:LA treatment significantly reduced AI scores,paw swelling,joint destruction,and inflammatory cell infiltration,and alleviated arthritis pain.Additionally,LA promoted a balanced M1/M2 ratio by increasing the m RNA expression level of M2 marker arginase 1 and decreasing those of M1 markers inducible nitric oxide synthase and interleukin(IL)-1βin synovial tissues.Furthermore,LA lowered the levels of three M1-related cytokines,namely tumor necrosis factor-α,IL-1βand IL-18,and raised the level of the M2-related cytokine IL-10.Further research showed that treatment with LA inhibited the expression of P2X7r.CONCLUSION:Our findings indicate that the notable therapeutic and analgesic effects of LA on AIA rats are exerted through balancing the M1/M2 ratio,probably via P2X7r.展开更多
A deficiency ofγδT cells has been described in Crohn's disease(CD).AIM To analyze the gene expression of interleukin 7(IL-7)and its receptors in the tissues of patients with CD.METHODS We studied the peripheral ...A deficiency ofγδT cells has been described in Crohn's disease(CD).AIM To analyze the gene expression of interleukin 7(IL-7)and its receptors in the tissues of patients with CD.METHODS We studied the peripheral blood of 80 patients with CD,comparing them with a group of 80 healthy subjects.The number and apoptosis ofαβandγδT cells in peripheral blood and the proportion ofαβandγδT cells in the intestinal tissues of patients with CD(n=25)were studied.The gene and protein expression of IL-7,IL-2 receptor subunitγ[cluster of differentiation 132(CD132)],receptorα(CD127),and caspase-3 in tissues was analyzed by quantitative PCR.Serum IL-7 levels were also analyzed.RESULTS In patients with CD,a decreased number ofγδT cells and an increase in the apoptosis of CD56+αβandγδT cells in peripheral blood was observed(P<0.0001 and P<0.01)respectively,and there was an inverse correlation among T subsets and their apoptosis.In addition,IL-7 gene expression and IL-7 protein in the tissues of these patients were increased.The titers of caspase-3 in tissues were low vs control group(P>0.01).The percentage of CD8+γδT cells decreased in tissues(P<0.01),and was directly related to IL-7 levels in peripheral blood.The expression of IL-2 receptor subunitγ(CD132)was greatly decreased in the tissues of patients with CD(P<0.05).CONCLUSION There may be a cause-effect relationship between the lower gene expression of the IL-2 receptor subunitγ(CD132)in tissues of patients with CD andγδT cells immunodeficiency.展开更多
BACKGROUND Diabetic cognitive dysfunction(DCD)is one of the chronic complications of diabetes,but its mechanism is currently unknown.Studies have shown that mitochondrial fission mediated by calcium overload is an imp...BACKGROUND Diabetic cognitive dysfunction(DCD)is one of the chronic complications of diabetes,but its mechanism is currently unknown.Studies have shown that mitochondrial fission mediated by calcium overload is an important mechanism of DCD.Blocking calcium overload and restoring calcium homeostasis are key steps in treatment.Transient receptor potential melastatin 7(TRPM7)is a novel player in causing calcium overload.Our previous studies have shown that genetic silencing of TRPM7 in type 1 diabetic rats leads to significant improvements in cognitive function,but the specific mechanism remains unclear.Troxerutin,extracted from the flowers of Sophora japonica,is one of the derivatives of rutin and has been shown to have neuroprotective effects.However,its association with TRPM7 remains unclear.AIM To use animal and cellular models,we investigated whether TRPM7 mediated mitochondrial fission by upregulation of calcineurin(CaN)/dynamin-related protein 1(Drp1)ser637 in DCD,and whether Troxerutin improved DCD by inhibiting TRPM7-mediated mitochondrial division.METHODS In this study,we used db/db mice and hippocampal neuronal cell lines(HT22)treated with high-concentration glucose as our study subjects.We evaluated cognitive function using Morris water maze,novel object recognition tasks,and Nesting tests.We observed mitochondrial morphology using transmission electron microscopy and measured mitochondrial energy metabolism indicators using a spectrophotometer.We also detected mRNA and protein expression of TRPM7,CaN,p-Drp1^(ser637),caspase-3,B-cell lymphoma 2 associated X protein,and B-cell lymphoma 2 using quantitative real-time polymerase chain reaction,western blotting,and immunofluorescence.RESULTS In the db/db diabetic mice with cognitive dysfunction,as well as in hippocampal neurons exposed to high-concentration glucose,TRPM7 and CaN expression were upregulated,phosphorylated Drp1^(ser637)expression was downregulated,and mitochondrial fission was increased.By modulating(inhibiting or overexpressing)TRPM7,it was further validated that TRPM7 activates the CaN/Drp1^(ser637)pathway,resulting in an increase in mitochondrial fission and neuronal cell apoptosis.Troxerutin downregulated TRPM7/CaN/Drp1^(ser637),reduced mitochondrial fission,and improved DCD.CONCLUSION TRPM7 promotes mitochondrial fission via the CaN/Drp1^(ser637)pathway.Troxerutin improves mitochondrial function and reduces neuronal damage by inhibiting this pathway,suggesting TRPM7 as a potential therapeutic target for DCD.展开更多
Crohn’s disease(CD)is a chronic inflammatory disorder characterized by dysregulated immune responses and significant disruption of intestinal immunity.A recent case-control study by Andreu-Ballester et al revealed de...Crohn’s disease(CD)is a chronic inflammatory disorder characterized by dysregulated immune responses and significant disruption of intestinal immunity.A recent case-control study by Andreu-Ballester et al revealed decreased expression of interleukin(IL)-2 receptor subunitγ(CD132)in CD tissues,a finding that has profound implications for understanding immune dysregulation in CD.CD132,an essential component of the IL-7/IL-2 signaling axis,is critical forγδT cell survival and function,which are pivotal for maintaining gut integrity and modulating inflammation.Here,we propose that reduced CD132 expression represents a key mechanism underlyingγδT cell deficiencies in CD,contributing to impaired immune surveillance and exacerbated inflammation.This hypothesis integrates emerging evidence from cytokine signaling and immunopathology in CD,offering new insights into its pathogenesis.These findings highlight the therapeutic potential of targeting the IL-7/IL-2 axis to restore immune homeostasis in CD,presenting a novel avenue for future research and intervention.展开更多
The P2X7 receptor mRNA and proteins in guinea-pig dorsal root ganglia （DRG） were studied by using RT-PCR and immunohistochemistry. The co-localization of P2X7 receptor with four cytochemical markers, the neurofilame...The P2X7 receptor mRNA and proteins in guinea-pig dorsal root ganglia （DRG） were studied by using RT-PCR and immunohistochemistry. The co-localization of P2X7 receptor with four cytochemical markers, the neurofilament protein NF200, S100, substance P and isolectin t34 （IB4） binding glyco-conjugates, were also examined. It was found that P2X7 receptor immunoreactivity （P2X7 R-IR） was present mostly in large-and medium-sized DRG neurons （62%±9% and 36%±6% respectively in all P2X7 R-IR neurons）. All the P2X7 R-IR neurons were also NF200 and S100 immunopositive. However, in a small number of NF200 or S100 immunopositive neurons no P2XTR-IR was detectable. All the IB4-positive or substance P-immunopositive neurons had no P2X7 R-IR. These results demonstrate that P2X7 receptors are expressed in a large subpopulation of DRG neurons and they may play a role in the transduction of specific peripheral sensory signals.展开更多
AIM To determine if activation of the ATP-gated P2X7 receptor channel induces phosphatidylserine(PS)exposure in erythrocytes from multiple dog breeds.METHODS Peripheral blood was collected from 25 dogs representing 13...AIM To determine if activation of the ATP-gated P2X7 receptor channel induces phosphatidylserine(PS)exposure in erythrocytes from multiple dog breeds.METHODS Peripheral blood was collected from 25 dogs representing 13 pedigrees and seven crossbreeds.ATP-induced PS exposure on canine erythrocytes in vitro was assessed using a flow cytometric Annexin V binding assay.RESULTS ATP induced PS exposure in erythrocytes from all dogs studied.ATP caused PS exposure in a concentrationdependent manner with an EC50 value of 395μmol/L.The non-P2X7 agonists,ADP or AMP,did not cause PS exposure.The P2X7 antagonist,AZ10606120,but not the P2X1 antagonist,NF449,blocked ATP-induced PS exposure.CONCLUSION The results indicate that ATP induces PS exposure in erythrocytes from various dog breeds and that this process is mediated by P2X7 activation.展开更多
目的从结直肠癌症患者血浆中分离微量来源于肿瘤细胞的游离DNA,检测甲基化EphA7作为肿瘤标志。方法提取血浆中微量游离DNA,并以亚硫酸氢钠修饰DNA。根据修饰后的DNA序列,设计EphA7甲基化特异性引物和探针,利用Real Time PCR仪扩增甲基化...目的从结直肠癌症患者血浆中分离微量来源于肿瘤细胞的游离DNA,检测甲基化EphA7作为肿瘤标志。方法提取血浆中微量游离DNA,并以亚硫酸氢钠修饰DNA。根据修饰后的DNA序列,设计EphA7甲基化特异性引物和探针,利用Real Time PCR仪扩增甲基化EphA7基因。结果从大肠癌患者血浆中检出微量甲基化EphA7基因。结论应用荧光标记TaqMan探针RealTime PCR可以检测出结直肠癌患者血浆中微量游离DNA中甲基化EphA7基因,血浆甲基化EphA7可能作为一种新的肿瘤标志物。展开更多
基金National Natural Science Foundation of China,Grant/Award Number:82260007Jilin Province Health Commission,Grant/Award Number:2024A062+1 种基金Jilin Provincial Department of Education,Grant/Award Number:JJKH20240698KJJilin Province Science and Technology Department,Grant/Award Number:20240404025ZP and 20240602100RC。
文摘Background:This study investigated the role of polydatin in regulating macrophage-epithelial cell(EC)interactions during asthma.An asthma model was induced in BALB/c mice using ovalbumin(20μg).Methods:The therapeutic effects of polydatin(20 and 40 mg/kg)were evaluated in this asthmatic mouse model.To assess the underlying mechanisms,Bronchial Epithelium Adenovirus 12-SV402B(BEAS-2B)cells were cocultured with Tohoku Hospital for Pediatrics-1(THP-1)macrophages,in which toll-like receptor 4(TLR4)was either overexpressed or knocked down,and subsequently stimulated with lipopoly-saccharide(LPS)and ATP.THP-1 cells underwent a 1-h pretreatment with polydatin(50 and 100μmol/L),Class Lipid Inhibitor-095(CLI-095,TLR4 inhibitor,1μg/mL),or A438079(P2X7R antagonist,10μmol/L)prior to LPS/ATP challenge.Results:Findings from Western blotting,enzyme-linked immunosorbent assay,flow cytometry,real-time polymerase chain reaction,and immunofluorescence assays demonstrated that modulating TLR4 expression significantly altered interleukin-1β(IL-1β)secretion from THP-1 macrophages and mitochondrial reactive oxygen species(mtROS)production in BEAS-2B ECs.In the mouse asthma model,polydatin significantly alleviated airway inflammation,oxidative stress,and apoptosis,likely by interfering with TLR4/P2X7R-mediated signaling and suppressing the activation of the NOD-like receptor protein inflammasome.Additionally,polydatin significantly reduced IL-1βand IL-18 levels and inhibited the infiltration of macrophages and eosinophils.Correspondingly,polydatin significantly attenuated TLR4/P2X7R signaling in THP-1 cells stimulated with ATP and LPS,thereby reducing IL-1βand IL-18 secretion,calcium influx,mtROS production,and apoptosis in BEAS-2B ECs.Conclusions:Polydatin is a promising therapeutic candidate for asthma,possibly by targeting macrophage-epithelium cross-talk via the TLR4/P2X7R axis.Future formulations as capsules or sprays may effectively alleviate airway inflammation and remodeling.
基金supported by the National Natural Science Foundation of China(Grant No.82060479)Key Research and Development Program of Ningxia Hui Autonomous Region(Grant No.2021BEG03062)Ningxia Natural Science Fund Key Project(Grant No.2024AAC02080).
文摘Objective:Breast cancer is the most common malignancy in women and is characterized by a high recurrence rate that severely impacts patient survival.Regulatory T cells(Tregs)in the tumor microenvironment(TME)promote immune evasion and metastasis,increasing recurrence risk.This study determined how the epigenetic regulators,DNMT3A and METTL7A,modulate Treg infiltration via the DDR1/STAT3/CXCL5 axis and influence breast cancer recurrence and prognosis.Methods:RNA sequencing(RNA-seq)was used to identify differentially expressed genes(DEGs),followed by Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes(KEGG)enrichment.Machine learning algorithms,including least absolute shrinkage and selection operator(LASSO),supported vector machine-recursive feature elimination(SVM-RFE)and ElasticNet identified DDR1 as a key gene.Validation included RT-qPCR,western blot,MSP,MeRIP-qPCR,and Co-IP to assess epigenetic regulation.Functional assays(CCK-8,Transwell,and Treg differentiation/chemotaxis)and xenograft models evaluated the role of DDR1 in tumor progression and recurrence.Results:DNMT3A upregulated DDR1 via DNA methylation,while METTL7A enhanced DDR1 mRNA stability via m6A modification.Co-regulation activated the DDR1/STAT3/CXCL5 axis,which boosted cancer cell proliferation,migration,and invasion.CXCL5 secretion increased Treg infiltration and accelerated tumor growth in vivo.DDR1 silencing reversed these effects,confirming that DDR1 has a pivotal role in breast cancer recurrence.Conclusion:DNMT3A and METTL7A were shown to cooperatively regulate DDR1 via DNA/m6A methylation,which drives Tregmediated immune suppression and recurrence.This study provided novel insights and therapeutic targets for breast cancer prognosis and treatment.
基金Supported by Burapha University,Thailand Science Research and Innovation,and National Science Research and Innovation Fund,No.53/2567.
文摘BACKGROUND Magnesium(Mg^(2+))plays a fundamental role in numerous cellular processes,including enzymatic reactions,DNA replication,oxidative stress response,and cytoskeletal dynamics.In fact,dysregulation of Mg^(2+)homeostasis has been increasingly associated with the development and progression of cancer,particularly colorectal cancer(CRC).Transient receptor potential melastatin(TRPM)channels,especially TRPM6 and TRPM7,are essential regulators of epithelial Mg^(2+)influx.While TRPM7 promotes CRC progression,the role of TRPM6 and TRPM6/7 channels remains unclear.AIM To investigate the role of membrane-localized TRPM6 and TRPM6/7 channels in Mg^(2+)influx,spheroid(SP)formation,stemness,and migration.METHODS We used parental and SP-derived HT-29 cells at comparable passages as in vitro models.Mass spectrometry confirmed full-length sequences,phosphorylation,and methionine oxidation of TRPM6 and TRPM7.Mg^(2+)influx,total and free Mg^(2+)levels were measured by fluorescence imaging and biochemical assays.TRPM6/TRPM7 expression and markers were analyzed by western blot.Func-tional assays,including secondary SP formation and wound healing,assessed stemness and migration.Cells were treated with Mg^(2+)transport inhibitors:Co(III)hexamine,2-aminoethyl diphenylborinate(TRPM6/7 blocker),and Mesendogen(TRPM6 inhibitor).RESULTS The expression of membrane-bound TRPM6,TRPM7,and TRPM6/7 was significantly higher in SP cells than in parental cells.Mass spectrometric analysis confirmed the presence of full-length TRPM6 and TRPM7 with increased phosphorylation and oxidation in SP cells.Enhanced Mg^(2+)influx and total intracellular Mg^(2+)levels were observed in SP cells.Free ionized intracellular Mg^(2+)levels remained comparable across all experimental groups.Pharmacological inhibition of TRPM6 and TRPM6/7 significantly reduced Mg^(2+)influx,decreased total Mg^(2+)content,compromised CRC SP stability,abolished cancer stem-like properties,impaired cell migration,and downregulated pro-tumorigenic markers,including Nanog,cyclooxygenase-2,and matrix metalloproteinase-9.CONCLUSION Membrane-localized TRPM6 and TRPM6/7 channels regulate Mg^(2+)influx and promote CRC stemness,SP stability,and migration,highlighting their potential as therapeutic targets to inhibit CRC progression and metastasis.
基金supported by grants from the Natural Science Foundation of Hunan Province(2022JJ70037 and 2022JJ40402)the National Natural Science Foundation of China(82472220 and 82002074)+1 种基金the Natural Science Foundation of Guangdong Province(2023A1515010267,2023A1515012665 and 2024A1515010073)the China International Medical Foundation Cerebrovascular Disease Youth Innovation Fund(Z-2016-20-2201).
文摘BACKGROUND:While theα7 nicotinic acetylcholine receptor(α7 nAChR)is implicated in sepsis-associated encephalopathy(SAE),its pathophysiological contributions require further investigation.METHODS:SAE was induced in mice via cecal ligation and puncture(CLP),and microglia were treated with lipopolysaccharide(LPS).PHA-543613(anα7 nAChR agonist)was used to activateα7 nAChR.To study the role ofα7 nAChR in mitophagy and pyroptosis,caspase-1-deficient mice and PTEN-induced kinase 1(PINK1)small interfering RNA(siRNA)were used.Cognitive function,cerebral oxygen extraction ratio(CERO2),and brain tissue oxygen pressure(PbtO2)were measured.Blood-brain barrier(BBB)integrity was evaluated via Evan’s blue staining.Mitophagy,pyroptosis,and cytokine levels were analyzed via Western blotting and immunofl uorescence.RESULTS:CLP or LPS treatment signifi cantly down-regulatedα7 nAChR protein expression in microglia.The administration of PHA-543613 to activateα7 nAChR not only restored its expression post-sepsis,but also notably decreased BBB permeability and mitigated cognitive deficits.Bothα7 nAChR activation and caspase-1 knockout effectively suppressed microglial pyroptosis.The activation ofα7 nAChR also promoted mitophagy in microglia.This led to an amelioration of brain tissue hypoxia,as shown by elevated PbtO2 and reduced CERO2 levels.The suppression of microglial pyroptosis byα7 nAChR was counteracted when mitophagy was inhibited through the siRNA-mediated silencing of PINK1.CONCLUSION:The activation ofα7 nAChR reduces pyroptosis by enhancing microglial mitophagy,thereby mitigating SAE.
文摘OBJECTIVE:to investigate the anti-arthritic effects of lappaconitine(LA)on adjuvant-induced arthritis in Sprague-Dawley rats and its possible involvement in the regulation of M1/M2 macrophage balance through the P2X7 receptor(P2X7r).METHODS:Rats were immunized with complete Freund's adjuvant and then intraperitoneally administered LA(2,4,or 8 mg·kg^(-1)·d^(-1))or methotrexate(0.5 mg/kg per 3 d)for 14 d.The anti-arthritic effects of LA were evaluated through arthritis index(AI)assessment,ankle diameter measurement,and histopathological staining analysis.The analgesic effect of LA on arthritis was measured using mechanical withdrawal threshold testing and gait scoring.The impacts of LA on macrophage polarization,the expression of pro-/anti-inflammatory cytokines and P2X7r were analyzed using quantitative real-time polymerase chain reaction,enzyme-linked immunosorbent assay,and Western blotting.RESULTS:LA treatment significantly reduced AI scores,paw swelling,joint destruction,and inflammatory cell infiltration,and alleviated arthritis pain.Additionally,LA promoted a balanced M1/M2 ratio by increasing the m RNA expression level of M2 marker arginase 1 and decreasing those of M1 markers inducible nitric oxide synthase and interleukin(IL)-1βin synovial tissues.Furthermore,LA lowered the levels of three M1-related cytokines,namely tumor necrosis factor-α,IL-1βand IL-18,and raised the level of the M2-related cytokine IL-10.Further research showed that treatment with LA inhibited the expression of P2X7r.CONCLUSION:Our findings indicate that the notable therapeutic and analgesic effects of LA on AIA rats are exerted through balancing the M1/M2 ratio,probably via P2X7r.
文摘A deficiency ofγδT cells has been described in Crohn's disease(CD).AIM To analyze the gene expression of interleukin 7(IL-7)and its receptors in the tissues of patients with CD.METHODS We studied the peripheral blood of 80 patients with CD,comparing them with a group of 80 healthy subjects.The number and apoptosis ofαβandγδT cells in peripheral blood and the proportion ofαβandγδT cells in the intestinal tissues of patients with CD(n=25)were studied.The gene and protein expression of IL-7,IL-2 receptor subunitγ[cluster of differentiation 132(CD132)],receptorα(CD127),and caspase-3 in tissues was analyzed by quantitative PCR.Serum IL-7 levels were also analyzed.RESULTS In patients with CD,a decreased number ofγδT cells and an increase in the apoptosis of CD56+αβandγδT cells in peripheral blood was observed(P<0.0001 and P<0.01)respectively,and there was an inverse correlation among T subsets and their apoptosis.In addition,IL-7 gene expression and IL-7 protein in the tissues of these patients were increased.The titers of caspase-3 in tissues were low vs control group(P>0.01).The percentage of CD8+γδT cells decreased in tissues(P<0.01),and was directly related to IL-7 levels in peripheral blood.The expression of IL-2 receptor subunitγ(CD132)was greatly decreased in the tissues of patients with CD(P<0.05).CONCLUSION There may be a cause-effect relationship between the lower gene expression of the IL-2 receptor subunitγ(CD132)in tissues of patients with CD andγδT cells immunodeficiency.
基金Supported by the Natural Science Foundation of Hebei Province,No.H2021206187 and No.H2021206452.
文摘BACKGROUND Diabetic cognitive dysfunction(DCD)is one of the chronic complications of diabetes,but its mechanism is currently unknown.Studies have shown that mitochondrial fission mediated by calcium overload is an important mechanism of DCD.Blocking calcium overload and restoring calcium homeostasis are key steps in treatment.Transient receptor potential melastatin 7(TRPM7)is a novel player in causing calcium overload.Our previous studies have shown that genetic silencing of TRPM7 in type 1 diabetic rats leads to significant improvements in cognitive function,but the specific mechanism remains unclear.Troxerutin,extracted from the flowers of Sophora japonica,is one of the derivatives of rutin and has been shown to have neuroprotective effects.However,its association with TRPM7 remains unclear.AIM To use animal and cellular models,we investigated whether TRPM7 mediated mitochondrial fission by upregulation of calcineurin(CaN)/dynamin-related protein 1(Drp1)ser637 in DCD,and whether Troxerutin improved DCD by inhibiting TRPM7-mediated mitochondrial division.METHODS In this study,we used db/db mice and hippocampal neuronal cell lines(HT22)treated with high-concentration glucose as our study subjects.We evaluated cognitive function using Morris water maze,novel object recognition tasks,and Nesting tests.We observed mitochondrial morphology using transmission electron microscopy and measured mitochondrial energy metabolism indicators using a spectrophotometer.We also detected mRNA and protein expression of TRPM7,CaN,p-Drp1^(ser637),caspase-3,B-cell lymphoma 2 associated X protein,and B-cell lymphoma 2 using quantitative real-time polymerase chain reaction,western blotting,and immunofluorescence.RESULTS In the db/db diabetic mice with cognitive dysfunction,as well as in hippocampal neurons exposed to high-concentration glucose,TRPM7 and CaN expression were upregulated,phosphorylated Drp1^(ser637)expression was downregulated,and mitochondrial fission was increased.By modulating(inhibiting or overexpressing)TRPM7,it was further validated that TRPM7 activates the CaN/Drp1^(ser637)pathway,resulting in an increase in mitochondrial fission and neuronal cell apoptosis.Troxerutin downregulated TRPM7/CaN/Drp1^(ser637),reduced mitochondrial fission,and improved DCD.CONCLUSION TRPM7 promotes mitochondrial fission via the CaN/Drp1^(ser637)pathway.Troxerutin improves mitochondrial function and reduces neuronal damage by inhibiting this pathway,suggesting TRPM7 as a potential therapeutic target for DCD.
文摘Crohn’s disease(CD)is a chronic inflammatory disorder characterized by dysregulated immune responses and significant disruption of intestinal immunity.A recent case-control study by Andreu-Ballester et al revealed decreased expression of interleukin(IL)-2 receptor subunitγ(CD132)in CD tissues,a finding that has profound implications for understanding immune dysregulation in CD.CD132,an essential component of the IL-7/IL-2 signaling axis,is critical forγδT cell survival and function,which are pivotal for maintaining gut integrity and modulating inflammation.Here,we propose that reduced CD132 expression represents a key mechanism underlyingγδT cell deficiencies in CD,contributing to impaired immune surveillance and exacerbated inflammation.This hypothesis integrates emerging evidence from cytokine signaling and immunopathology in CD,offering new insights into its pathogenesis.These findings highlight the therapeutic potential of targeting the IL-7/IL-2 axis to restore immune homeostasis in CD,presenting a novel avenue for future research and intervention.
文摘The P2X7 receptor mRNA and proteins in guinea-pig dorsal root ganglia （DRG） were studied by using RT-PCR and immunohistochemistry. The co-localization of P2X7 receptor with four cytochemical markers, the neurofilament protein NF200, S100, substance P and isolectin t34 （IB4） binding glyco-conjugates, were also examined. It was found that P2X7 receptor immunoreactivity （P2X7 R-IR） was present mostly in large-and medium-sized DRG neurons （62%±9% and 36%±6% respectively in all P2X7 R-IR neurons）. All the P2X7 R-IR neurons were also NF200 and S100 immunopositive. However, in a small number of NF200 or S100 immunopositive neurons no P2XTR-IR was detectable. All the IB4-positive or substance P-immunopositive neurons had no P2X7 R-IR. These results demonstrate that P2X7 receptors are expressed in a large subpopulation of DRG neurons and they may play a role in the transduction of specific peripheral sensory signals.
基金The Centre for Medical and Molecular Bioscience(University of Wollongong)the American Kennel Club Canine Health Foundation
文摘AIM To determine if activation of the ATP-gated P2X7 receptor channel induces phosphatidylserine(PS)exposure in erythrocytes from multiple dog breeds.METHODS Peripheral blood was collected from 25 dogs representing 13 pedigrees and seven crossbreeds.ATP-induced PS exposure on canine erythrocytes in vitro was assessed using a flow cytometric Annexin V binding assay.RESULTS ATP induced PS exposure in erythrocytes from all dogs studied.ATP caused PS exposure in a concentrationdependent manner with an EC50 value of 395μmol/L.The non-P2X7 agonists,ADP or AMP,did not cause PS exposure.The P2X7 antagonist,AZ10606120,but not the P2X1 antagonist,NF449,blocked ATP-induced PS exposure.CONCLUSION The results indicate that ATP induces PS exposure in erythrocytes from various dog breeds and that this process is mediated by P2X7 activation.
文摘目的从结直肠癌症患者血浆中分离微量来源于肿瘤细胞的游离DNA,检测甲基化EphA7作为肿瘤标志。方法提取血浆中微量游离DNA,并以亚硫酸氢钠修饰DNA。根据修饰后的DNA序列,设计EphA7甲基化特异性引物和探针,利用Real Time PCR仪扩增甲基化EphA7基因。结果从大肠癌患者血浆中检出微量甲基化EphA7基因。结论应用荧光标记TaqMan探针RealTime PCR可以检测出结直肠癌患者血浆中微量游离DNA中甲基化EphA7基因,血浆甲基化EphA7可能作为一种新的肿瘤标志物。
