Background:Nasopharyngeal carcinoma(NPC)exhibits a significant prevalence in the southern regions of China,and paclitaxel(PTX)is frequently employed as a medication for managing advanced NPC.However,drug resistance is...Background:Nasopharyngeal carcinoma(NPC)exhibits a significant prevalence in the southern regions of China,and paclitaxel(PTX)is frequently employed as a medication for managing advanced NPC.However,drug resistance is typically accompanied by a poor prognosis.Exploring the synergistic potential of combining multiple chemotherapeutic agents may represent a promising avenue for optimizing treatment efficacy.Methods:This study investigated whether 3-Methyladenine(3-MA)could potentiated the effect of PTX and its potential molecular mechanism.Samples were divided into the following categories:Negative control(NC)with the solvent dimethyl sulfoxide(DMSO,0.5%v/v),PTX(400 nM),3-MA(4 mM),and PTX(400 nM)+3-MA(4 mM).The viability of NPC cells was assessed using both the cell counting kit-8(CCK-8)assay and the colony formation assay.Microscopic observation was performed to identify morphological cell changes.Flow cytometry was used to assess cell cycle status,mitochondrial membrane potential(MMP),and apoptotic cells.Western blotting was conducted to quantify the protein expression.Results:3-MA enhanced PTX-specific inhibition of NPC cell proliferation.PTX,either alone or in combination with 3-MA,caused cell cycle halt at the G2/M phase in the majority of NPC cells,and the combination treatment of PTX with 3-MA induced a higher rate of NPC cell death compared to PTX alone.Western blotting results revealed the combination of PTX with 3-MA heightened activation of cyclin-dependent kinase 1(CDK1),a key molecule in shifting cells from mitotic arrest to apoptosis,led to a reduction in Myeloid Cell Leukemia 1(MCL-1)expression and an increase in Poly(ADP-ribose)polymerase(PARP)cleavage.Conclusion:The concurrent administration of PTX with 3-MA effectively enhances PTX’s inhibitory impact on NPC and activates the apoptosis signal regulated by CDK1.展开更多
AIM:To investigate the role of autophagy inhibitor 3-methyladenine(3-MA)on a diabetic mice model(DM)and the potential mechanism.METHODS:Male C57BL/6J mice were randomly divided into a normal control group(NC group)and...AIM:To investigate the role of autophagy inhibitor 3-methyladenine(3-MA)on a diabetic mice model(DM)and the potential mechanism.METHODS:Male C57BL/6J mice were randomly divided into a normal control group(NC group)and an DM group.DM were induced by multiple low-dose intraperitoneal injection of streptozotocin(STZ)60 mg/kgd for 5 consecutive days.DM mice were randomly subdivided into untreated group(DM group),3-MA(10 mg/kgd by gavage)treated group(DM+3-MA group)and chloroquine(CQ;50 mg/kg by intraperitoneal injection)treated group(DM+CQ group).The fasting blood glucose(FBG)levels were recorded every week.At the end of experiment,retinal samples were collected.The expression levels of pro-apoptotic proteins cleaved caspase-3,cleaved poly ADP-ribose polymerase 1(PARP1)and Bax,anti-apoptotic protein Bcl-2,fibrosisassociated proteins Fibronectin and type 1 collagenα1 chain(COL1A1),vascular endothelial growth factor(VEGF),inflammatory factors interleukin(IL)-1βand tumor necrosis factor(TNF)-α,as well as autophagy related proteins LC3,Beclin-1 and P62 were determined by Western blotting.The oxidative stress indicators 8-hydroxydeoxyguanosine(8-OHdG)and malondialdehyde(MDA)were detected by commercial kits.RESULTS:Both 3-MA and CQ had shor t-term hypoglycemic effect on FBG and reduced the expression of VEGF and inflammatory factors IL-1βand TNF-αin DM mice.3-MA also significantly alleviated oxidative stress indicators 8-OHdG and MDA,decreased the expression of fibrosisrelated proteins Fibronectin and COL1A1,pro-apoptotic proteins cleaved caspase-3,cleaved PARP1,as well as the ratio of Bax/Bcl-2.CQ had no significant impact on the oxidative stress indicators,fibrosis,and apoptosis related proteins.The results of Western blotting for autophagy related proteins showed that the ratio of LC3 II/LC3 I and the expression of Beclin-1 in the retina of DM mice were decreased by 3-MA treatment,and the expression of P62 was further increased by CQ treatment.CONCLUSION:3-MA has anti-apoptotic and anti-fibrotic effects on the retina of DM mice,and can attenuate retinal oxidative stress,VEGF expression and the production of inflammatory factors in the retina of DM mice.The underlying mechanism of the above effects of 3-MA may be related to its inhibition of early autophagy and hypoglycemic effect.展开更多
BACKGROUND： Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and...BACKGROUND： Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and excitotoxicity, and in patients with Alzheimer＇s disease and critical illness. The role of autophagy in developmental epilepsy, however, is unknown. The present study was to investigate the effects of recurrent neonatal seizure, in the presence and absence of autophagy inhibitor 3-methyladenine （3-MA）, on the acute phase gene expression of ZnTs, LC3 and Beclin-1 in rat cerebral cortex and the interaction among them.METHODS： Thirty-six Sprague-Dawley neonatal rats at postnatal day 6（P6） were randomly divided into three groups： a recurrent-seizures group （RS, n=12）, a 3-MA treated-seizure group （3-MA group, each rat pretreated with 3-methyladenine before seizures, 100nmol/μl/day, i.p., n=12） and a control group （n=12）. At 1.5 and 6 hours after the last seizures, the mRNA levels of ZnT1-ZnT3, microtubule-associated protein 1A/1B light chain 3 （LC3） and beclin-1 were detected using the real-time RT-PCR method. The LC3 protein level was examined by Western blotting.RESULTS： The levels of LC3, beclin-1 and ZnT-2 transcripts in the RS group elevated signi？ cantly at 1.5 and 6 hours after the last seizures compared with those in the control and 3-MA groups. At the interval of 1.5 hours, the mRNA level of ZnT-1 increased signi？ cantly after the last seizure compared with that in the control group. There was no signi？ cant difference in the transcript levels of ZnT-3 among the three groups. Linear correlation analysis showed that the expression of the ？ ve genes in the control group exhibited a signi？ cant inter-relationship. In the 3-MA group, however, the inter-relationship was only found between beclin-1 and ZnT-1. In the RS group, the inter-relationship was not observed.CONCLUSIONS： The autophagy/lysosomal pathway is immediately activated along with the elevated expression of ZnT1 and ZnT2 in the cerebral cortex after recurrent seizures. 3-MA is involved in the regulation of the autophagy/lysosomal pathway and ZnTs by down-regulating the expression of LC3 and beclin-1.展开更多
Background Mammalian target of rapamycin （mTOR） is involved in a caspase independent form of programmed cell death called autophagy. The aim of this research was to investigate the effects of rapamycin and 3-methyla...Background Mammalian target of rapamycin （mTOR） is involved in a caspase independent form of programmed cell death called autophagy. The aim of this research was to investigate the effects of rapamycin and 3-methyladenine （3-MA） on autophagy, proliferation, apoptosis, and cell-cycle parameters of rat bone marrow-derived endothelial progenitor cells （EPCs）. Methods Mononuclear cells isolated from rat bone marrow were treated with rapamycin （0.01, 0.1, 1, or 10 pg/L） or 3-MA （1.25, 2.5, 5, or 10 mmol/L） for 24 hours. Expression of the autophagy marker protein LC3-11 was analyzed by Western blotting. Apoptosis and cell-cycle progression were analyzed by flow cytometry. Cell proliferation was measured using the MTT assay. Results Rapamycin treatment of EPCs induced apoptosis and autophagy and inhibited proliferation and cell-cycle progression in a dose-dependent manner. Treatment with 5 mmol/L 3-MA promoted cell proliferation; in contrast, treatment with 10 mmol/L 3-MA promoted apoptosis and induced S-phase arrest. Conclusions Rapamycin treatment of EPCs induced apoptosis and autophagy. Low concentrations of 3-MA had no significant effect on the proliferation and apoptosis of EPCs; The 5 mmol/L group promoted cell proliferation, but had no effect on the apoptosis; the 10 mmol/L group inhibited the proliferation and promoted apoptosis through the cell cycle.展开更多
Objective To investigate the effects of 3-methyladenineon airway inflammation,airway hyperresponsivenessand mucus secretion in asthmatic mice,and to explore itsmechanism. Methods C57BL /6J female mice were randomlydiv...Objective To investigate the effects of 3-methyladenineon airway inflammation,airway hyperresponsivenessand mucus secretion in asthmatic mice,and to explore itsmechanism. Methods C57BL /6J female mice were randomlydivided into normal control group ( PBS),OVAgroup(OVA),OVA with 3-methyladenine group (OVA +3-MA),and OVA with 4-phenylbutyrate group (OVA +4-PBA). OVA group,OVA + 3-MA group and OVA + 4-PBA groups were all sensitized and challenged with OVAto establish asthmatic models,while PBS group was givenPBS as a control. At 2 h before challenge,OVA + 3-MAgroup was intraperitoneally injected with 3-methyladenine,and OVA + 4-PBA group was intraperitoneally injectedwith 4-phenylbutyrate. Airway hyperresponsiveness,eosinophils,and pathological changes of pulmonarytissue (hematoxylin-eosin,HE staining) were measuredto confirm the establishment of asthmatic models.展开更多
基金supported by the Science and Technology Innovation Program of Hunan Province(Grant Numbers:2021SK1014 and 2022WZ1027)the Colleges and Universities of Hunan Province(Grant Number:HNJG 20200440)+1 种基金the Scientific Research Fund of Hunan Provincial Education Department(Grant Number:21B0411)the Scientific Research Project of Changsha Central Hospital(Number:YNKY202201).
文摘Background:Nasopharyngeal carcinoma(NPC)exhibits a significant prevalence in the southern regions of China,and paclitaxel(PTX)is frequently employed as a medication for managing advanced NPC.However,drug resistance is typically accompanied by a poor prognosis.Exploring the synergistic potential of combining multiple chemotherapeutic agents may represent a promising avenue for optimizing treatment efficacy.Methods:This study investigated whether 3-Methyladenine(3-MA)could potentiated the effect of PTX and its potential molecular mechanism.Samples were divided into the following categories:Negative control(NC)with the solvent dimethyl sulfoxide(DMSO,0.5%v/v),PTX(400 nM),3-MA(4 mM),and PTX(400 nM)+3-MA(4 mM).The viability of NPC cells was assessed using both the cell counting kit-8(CCK-8)assay and the colony formation assay.Microscopic observation was performed to identify morphological cell changes.Flow cytometry was used to assess cell cycle status,mitochondrial membrane potential(MMP),and apoptotic cells.Western blotting was conducted to quantify the protein expression.Results:3-MA enhanced PTX-specific inhibition of NPC cell proliferation.PTX,either alone or in combination with 3-MA,caused cell cycle halt at the G2/M phase in the majority of NPC cells,and the combination treatment of PTX with 3-MA induced a higher rate of NPC cell death compared to PTX alone.Western blotting results revealed the combination of PTX with 3-MA heightened activation of cyclin-dependent kinase 1(CDK1),a key molecule in shifting cells from mitotic arrest to apoptosis,led to a reduction in Myeloid Cell Leukemia 1(MCL-1)expression and an increase in Poly(ADP-ribose)polymerase(PARP)cleavage.Conclusion:The concurrent administration of PTX with 3-MA effectively enhances PTX’s inhibitory impact on NPC and activates the apoptosis signal regulated by CDK1.
基金Supported by the National Natural Science Foundation of China(No.82270893)Joint Fund of Health Committee of Hubei Province(No.WJ2019-16).
文摘AIM:To investigate the role of autophagy inhibitor 3-methyladenine(3-MA)on a diabetic mice model(DM)and the potential mechanism.METHODS:Male C57BL/6J mice were randomly divided into a normal control group(NC group)and an DM group.DM were induced by multiple low-dose intraperitoneal injection of streptozotocin(STZ)60 mg/kgd for 5 consecutive days.DM mice were randomly subdivided into untreated group(DM group),3-MA(10 mg/kgd by gavage)treated group(DM+3-MA group)and chloroquine(CQ;50 mg/kg by intraperitoneal injection)treated group(DM+CQ group).The fasting blood glucose(FBG)levels were recorded every week.At the end of experiment,retinal samples were collected.The expression levels of pro-apoptotic proteins cleaved caspase-3,cleaved poly ADP-ribose polymerase 1(PARP1)and Bax,anti-apoptotic protein Bcl-2,fibrosisassociated proteins Fibronectin and type 1 collagenα1 chain(COL1A1),vascular endothelial growth factor(VEGF),inflammatory factors interleukin(IL)-1βand tumor necrosis factor(TNF)-α,as well as autophagy related proteins LC3,Beclin-1 and P62 were determined by Western blotting.The oxidative stress indicators 8-hydroxydeoxyguanosine(8-OHdG)and malondialdehyde(MDA)were detected by commercial kits.RESULTS:Both 3-MA and CQ had shor t-term hypoglycemic effect on FBG and reduced the expression of VEGF and inflammatory factors IL-1βand TNF-αin DM mice.3-MA also significantly alleviated oxidative stress indicators 8-OHdG and MDA,decreased the expression of fibrosisrelated proteins Fibronectin and COL1A1,pro-apoptotic proteins cleaved caspase-3,cleaved PARP1,as well as the ratio of Bax/Bcl-2.CQ had no significant impact on the oxidative stress indicators,fibrosis,and apoptosis related proteins.The results of Western blotting for autophagy related proteins showed that the ratio of LC3 II/LC3 I and the expression of Beclin-1 in the retina of DM mice were decreased by 3-MA treatment,and the expression of P62 was further increased by CQ treatment.CONCLUSION:3-MA has anti-apoptotic and anti-fibrotic effects on the retina of DM mice,and can attenuate retinal oxidative stress,VEGF expression and the production of inflammatory factors in the retina of DM mice.The underlying mechanism of the above effects of 3-MA may be related to its inhibition of early autophagy and hypoglycemic effect.
文摘BACKGROUND： Autophagy is a homeostatic process for intracellular recycling of bulk proteins and aging organelles. Increased autophagy has now been reported in experimental models of traumatic brain injury, stroke and excitotoxicity, and in patients with Alzheimer＇s disease and critical illness. The role of autophagy in developmental epilepsy, however, is unknown. The present study was to investigate the effects of recurrent neonatal seizure, in the presence and absence of autophagy inhibitor 3-methyladenine （3-MA）, on the acute phase gene expression of ZnTs, LC3 and Beclin-1 in rat cerebral cortex and the interaction among them.METHODS： Thirty-six Sprague-Dawley neonatal rats at postnatal day 6（P6） were randomly divided into three groups： a recurrent-seizures group （RS, n=12）, a 3-MA treated-seizure group （3-MA group, each rat pretreated with 3-methyladenine before seizures, 100nmol/μl/day, i.p., n=12） and a control group （n=12）. At 1.5 and 6 hours after the last seizures, the mRNA levels of ZnT1-ZnT3, microtubule-associated protein 1A/1B light chain 3 （LC3） and beclin-1 were detected using the real-time RT-PCR method. The LC3 protein level was examined by Western blotting.RESULTS： The levels of LC3, beclin-1 and ZnT-2 transcripts in the RS group elevated signi？ cantly at 1.5 and 6 hours after the last seizures compared with those in the control and 3-MA groups. At the interval of 1.5 hours, the mRNA level of ZnT-1 increased signi？ cantly after the last seizure compared with that in the control group. There was no signi？ cant difference in the transcript levels of ZnT-3 among the three groups. Linear correlation analysis showed that the expression of the ？ ve genes in the control group exhibited a signi？ cant inter-relationship. In the 3-MA group, however, the inter-relationship was only found between beclin-1 and ZnT-1. In the RS group, the inter-relationship was not observed.CONCLUSIONS： The autophagy/lysosomal pathway is immediately activated along with the elevated expression of ZnT1 and ZnT2 in the cerebral cortex after recurrent seizures. 3-MA is involved in the regulation of the autophagy/lysosomal pathway and ZnTs by down-regulating the expression of LC3 and beclin-1.
基金The work was supported by a grant from the National Natural Science Foundation of China
文摘Background Mammalian target of rapamycin （mTOR） is involved in a caspase independent form of programmed cell death called autophagy. The aim of this research was to investigate the effects of rapamycin and 3-methyladenine （3-MA） on autophagy, proliferation, apoptosis, and cell-cycle parameters of rat bone marrow-derived endothelial progenitor cells （EPCs）. Methods Mononuclear cells isolated from rat bone marrow were treated with rapamycin （0.01, 0.1, 1, or 10 pg/L） or 3-MA （1.25, 2.5, 5, or 10 mmol/L） for 24 hours. Expression of the autophagy marker protein LC3-11 was analyzed by Western blotting. Apoptosis and cell-cycle progression were analyzed by flow cytometry. Cell proliferation was measured using the MTT assay. Results Rapamycin treatment of EPCs induced apoptosis and autophagy and inhibited proliferation and cell-cycle progression in a dose-dependent manner. Treatment with 5 mmol/L 3-MA promoted cell proliferation; in contrast, treatment with 10 mmol/L 3-MA promoted apoptosis and induced S-phase arrest. Conclusions Rapamycin treatment of EPCs induced apoptosis and autophagy. Low concentrations of 3-MA had no significant effect on the proliferation and apoptosis of EPCs; The 5 mmol/L group promoted cell proliferation, but had no effect on the apoptosis; the 10 mmol/L group inhibited the proliferation and promoted apoptosis through the cell cycle.
文摘Objective To investigate the effects of 3-methyladenineon airway inflammation,airway hyperresponsivenessand mucus secretion in asthmatic mice,and to explore itsmechanism. Methods C57BL /6J female mice were randomlydivided into normal control group ( PBS),OVAgroup(OVA),OVA with 3-methyladenine group (OVA +3-MA),and OVA with 4-phenylbutyrate group (OVA +4-PBA). OVA group,OVA + 3-MA group and OVA + 4-PBA groups were all sensitized and challenged with OVAto establish asthmatic models,while PBS group was givenPBS as a control. At 2 h before challenge,OVA + 3-MAgroup was intraperitoneally injected with 3-methyladenine,and OVA + 4-PBA group was intraperitoneally injectedwith 4-phenylbutyrate. Airway hyperresponsiveness,eosinophils,and pathological changes of pulmonarytissue (hematoxylin-eosin,HE staining) were measuredto confirm the establishment of asthmatic models.
