Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the c...Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.展开更多
BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer...BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.展开更多
BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in ...BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.展开更多
Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and af...Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.展开更多
Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneou...Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.展开更多
Objective:To determine the effect of Zanthoxylum piperitum extracet(ZPE)on apoptosis and analyze anticancer substances in ZPE,changes in proteins related to apoptosis,and pathological changes in tumors in mouse.Method...Objective:To determine the effect of Zanthoxylum piperitum extracet(ZPE)on apoptosis and analyze anticancer substances in ZPE,changes in proteins related to apoptosis,and pathological changes in tumors in mouse.Methods:Fifteen 4-week-old female BALB/c nu/nu mice were divided into 3 groups depending on ZPE dose,with 5 in each group.AGS gastric carcinoma cells(1 x 10^(6) cells/200 jxL)were subcutaneously injected into the flank of each mouse.One week after the injection of AGS cells,ZPE was administered to the skin tissue[10 or 50 mg/(kg-d)]in the low-and high-dose groups,respectively for 20 days.Control animals were injected with vehicle only.After 3 weeks,the tumor was extracted and carried out for immunohistochemistry,the tendency of apoptosis and p53 in the body was checked using TdT-mediated dUTP nick-end labeling(TUNEL)assay.For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,annexin V dead cell staining,cell cycle arrest and Western blotting,AGS gastric carcinoma cells were incubated with various concentrations of ZPE for 24 h.Cell survival rates were analyzed by MTT assays.Apoptosis was analyzed using annexin V dead cell staining and cell cycle arrest and measured using Muse cell analyzer.Results:High performance liquid chromatography(HPLC)analysis showed that ZPE contained organic sulfur compounds such as alliin and S-allylcysteine.MTT assay results revealed that ZPE(10-85»xg/mL)could effectively inhibit the growth of AGS gastric cancer cells at higher concentrations(P<0.05,P<0.01).The annexin V&dead cell staining assay and cell cycle arrest assay confirmed a dose-dependent increase in the apoptosis rate and G!phase in ZPE(10-70 jig/mL)groups.ZPE decreased the expression of anti-apoptotic proteins(p-Akt,p-MDM2,Bcl-2),while increased pro-apoptotic proteins(cleaved PARP,p53,pro-Caspase 3,Bax).TUNEL assays revealed an increase in cell apoptosis.Immunohistochemistry staining confirmed the involvement of p53.Conclusion:ZPE decreases AGS cell proliferation and induces apoptosis by inhibiting Akt and MDM2 expression.展开更多
Type 1 diabetes mellitus(T1DM)-induced cognitive dysfunction is common,but its underlying mechanisms are still poorly understood.In this study,we found that knockout of conventional protein kinase C(cPKC)γsignificant...Type 1 diabetes mellitus(T1DM)-induced cognitive dysfunction is common,but its underlying mechanisms are still poorly understood.In this study,we found that knockout of conventional protein kinase C(cPKC)γsignificantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles,but did not affect the activities of GSK-3βand PP2A in the hippocampal neurons of T1DM mice.cPKCγdeficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice.Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγdeficiency in T1DM mice.Moreover,cPKCγdeficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro.The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor.In conclusion,these results indicated that cPKCγpromotes autophagy through the AMPK/mTOR signaling pathway,thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.展开更多
Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of ...Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer.In this work,we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer.Methods:The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform.The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform.The target genes of liver cancer were predicted using GeneCards database.The intersecting genes were then analyzed with Venn diagrams.Kyoto Encyclopedia of Genes and Genomes and Database for Annotation,Visualization,and Integrated Discovery were used to analyze the pathway.Then,cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills.The living dead cell staining method was used to observe the survival of cells.HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method,and then the mitochondrial damage was also detected by flow cytometry.The expression of target genes was detected by quantitative real-time polymerase chain reaction.Results:A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer‑related targets.We obtained 1,899 disease targets and 297 transcriptome targets from the database.Six drug-disease intersecting genes,CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3 were obtained.They are enrichment in apoptosis,PI3K-AKT signaling pathway,MAPK signaling pathway,pathways in cancer and p53 signaling pathway.Besides,it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group.And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment.Xihuang pill also induced the mitochondrial membrane potential damage.Compared with the control group,the expression level of CCNB1 and BIRC5 was induced,while the expression level of IGF2 was reduced after Xihuang pill treatment.Conclusion:Xihuang pill may act on six proteins(CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3)and cover multiple pathways to form a therapeutic network to treat liver cancer.展开更多
Due to its difficulty in early diagnosis and lack of sensitivity to chemotherapy and radiotherapy,renal cell carcinoma(RCC)remains to be a frequent cause of cancer-related death.Here,we probed into new targets for its ...Due to its difficulty in early diagnosis and lack of sensitivity to chemotherapy and radiotherapy,renal cell carcinoma(RCC)remains to be a frequent cause of cancer-related death.Here,we probed into new targets for its early diagnosis and treatment for RCC.microRNA(miRNA)data of M2-EVs and RCC were searched on the Gene Expression Omnibus database,followed by the prediction of the potential downstream target.Expression of target genes was measured via RT-qPCR and Western blot,respectively.M2 macrophage was obtained viaflow cytometry with M2-EVs extracted.The binding ability of miR-342-3p to NEDD4L and to CEP55 ubiquitination was studied with their roles in the physical abilities of RCC cells assayed.Subcutaneous tumor-bearing mouse models and lung metastasis models were prepared to observe in vivo role of target genes.M2-EVs induced RCC growth and metastasis.miR-342-3p showed high expression in both M2-EVs and RCC cells.M2-EVs carrying miR-342-3p promoted RCC cell abilities to proliferate,invade and migrate.In RCC cells,M2-EV-derived miR-342-3p could specifically bind to NEDD4L and consequently elevate CEP55 protein expression via suppressing NEDD4L,thereby exerting tumor-promoting effects.CEP55 could be degraded by ubiquitination under the function of NEDD4L,and miR-342-3p delivered by M2-EVs facilitated the RCC occurrence and development by activating the PI3K/AKT/mTOR signaling pathway.In conclusion,M2-EVs promote RCC growth and metastasis by delivering miR-342-3p to suppress NEDD4L and subsequently inhibit CEP55 ubiquitination and degradation via activation of the PI3K/AKT/mTOR signaling pathway,strongly driving the proliferative,migratory and invasive of RCC cells.展开更多
Objective:To explore the effect of acupotomy intervention on autophagy of chondrocytes in rabbits with knee osteoarthritis(KOA),and to determine the possible mechanisms of acupotomy to alleviate cartilage degeneration...Objective:To explore the effect of acupotomy intervention on autophagy of chondrocytes in rabbits with knee osteoarthritis(KOA),and to determine the possible mechanisms of acupotomy to alleviate cartilage degeneration.Methods:The modified Videman method was used to construct a KOA rabbit model.After modeling,40 rabbits were randomly divided into 4 groups by a random number table:control;KOA(model);KOA+acupotomy(acupotomy),and KOA+sham acupotomy(sham),10 in each group.After a 3-week treatment course,the knee joint activity was determined by the modified Lequesne MG index.Hematoxylin-eosin staining staining was used to examine the morphological changes of chondrocytes.Autophagy of chondrocytes was observed by transmission electron microscopy.The surface morphology of cartilage tissue was observed by scanning electron microscope.The mRNA and protein levels of AMP kinase/mammalian target of rapamycin/Unc-51(AMPK/mTOR/ULK1)signal pathway key proteins,autophagy-related factor Beclin-1 and microtubule-associated protein 1A/1B light chain 3(LC3)in rabbit knee cartilage were assessed by real-time fluorescence quantitative polymerase chain reaction and Western blot,respectively.Results:The modified Lequesne MG score of acupotomy group was significantly lower than that of model group(P<0.05).Pathological results showed that chondrocyte autophagy decreased and cartilage surface was rough in the model group,which recovered after acupotomy treatment.The mRNA expressions of AMPK,ULK1,Beclin-1 and the protein levels of p-AMPK,p-ULK1,Beclin-1,and LC3Ⅱ/LC3Ⅰwere decreased in the model group,while the mRNA and protein expressions of mTOR were increased(P<0.01).However,acupotomy treatment reversed these abnormal changes(P<0.05).Conclusions:Acupotomy could effectively up-regulate the expressions of AMPK,ULK1 and Beclin1,reduce the expression of mTOR,promote autophagy,and alleviate joint degeneration.Acupotomy is a promising complementary and alternative therapy for KOA.展开更多
基金supported by the National Natural Science Foundation of China,No.81202625the Open Fund of Key Laboratory of Cardiovascular and Cerebrovascular Diseases Translational Medicine,China Three Gorges University,China,No.2016xnxg101
文摘Puerarin suppresses autophagy to alleviate cerebral ischemia/reperfusion injury, and accumulating evidence indicates that the AMPKm TOR signaling pathway regulates the activation of the autophagy pathway through the coordinated phosphorylation of ULK1. In this study, we investigated the mechanisms underlying the neuroprotective effect of puerarin and its role in modulating autophagy via the AMPK-m TOR-ULK1 signaling pathway in the rat middle cerebral artery occlusion model of cerebral ischemia/reperfusion injury. Rats were intraperitoneally injected with puerarin, 50 or 100 mg/kg, daily for 7 days. Then, 30 minutes after the final administration, rats were subjected to transient middle cerebral artery occlusion for 90 minutes. Then, after 24 hours of reperfusion, the Longa score and infarct volume were evaluated in each group. Autophagosome formation was observed by transmission electron microscopy. LC3, Beclin-1 p62, AMPK, m TOR and ULK1 protein expression levels were examined by immunofluorescence and western blot assay. Puerarin substantially reduced the Longa score and infarct volume, and it lessened autophagosome formation in the hippocampal CA1 area following cerebral ischemia/reperfusion injury in a dose-dependent manner. Pretreatment with puerarin（50 or 100 mg/kg） reduced Beclin-1 expression and the LC3-II/LC3-I ratio, as well as p-AMPK and p S317-ULK1 levels. In comparison, it increased p62 expression. Furthermore, puerarin at 100 mg/kg dramatically increased the levels of p-m TOR and p S757-ULK1 in the hippocampus on the ischemic side. Our findings suggest that puerarin alleviates autophagy by activating the APMK-m TOR-ULK1 signaling pathway. Thus, puerarin might have therapeutic potential for treating cerebral ischemia/reperfusion injury.
基金Supported by the National Natural Science Funds of China,No.81974448Guangdong Medical Research Foundation,No.B2019126Shenzhen Science and Technology Innovation Commission,No.JCYJ20210324135005013.
文摘BACKGROUND Altered miR-188-3p expression has been observed in various human cancers.AIM To investigate the miR-188-3p expression,its roles,and underlying molecular events in gastric cancer.METHODS Fifty gastric cancer and paired normal tissues were collected to analyze miR-188-3p and CBL expression.Normal and gastric cancer cells were used to manipulate miR-188-3p and CBL expression through different assays.The relationship between miR-188-3p and CBL was predicted bioinformatically and confirmed using a luciferase gene reporter assay.A Kaplan-Meier analysis was used to associate miR-188-3p or CBL expression with patient survival.A nude mouse tumor cell xenograft assay was used to confirm the in vitro data.RESULTS MiR-188-3p was found to be lower in the plasma of gastric cancer patients,tissues,and cell lines compared to their healthy counterparts.It was associated with overall survival of gastric cancer patients(P<0.001),tumor differentiation(P<0.001),lymph node metastasis(P=0.033),tumor node metastasis stage(I/II vs III/IV,P=0.024),and American Joint Committee on Cancer stage(I/II vs III/IV,P=0.03).Transfection with miR-188-3p mimics reduced tumor cell growth and invasion while inducing apoptosis and autophagy.CBL was identified as a direct target of miR-188-3p,with its expression antagonizing the effects of miR-188-3p on gastric cancer(GC)cell proliferation by inducing tumor cell apoptosis and autophagy through the inactivation of the Akt/mTOR signaling pathway.The in vivo data confirmed antitumor activity via CBL downregulation in gastric cancer.CONCLUSION The current data provides ex vivo,in vitro,and in vivo evidence that miR-188-3p acts as a tumor suppressor gene or possesses antitumor activity in GC.
基金Supported by the National Natural Science Foundation of China,No.81702777Natural Science Foundation of Guangdong Province,No.2015A030310053
文摘BACKGROUND Growth arrest-specific gene 2(GAS2)plays a role in modulating in reversible growth arrest cell cycle,apoptosis,and cell survival.GAS2 protein is universally expressed in most normal tissues,particularly in the liver,but is depleted in some tumor tissues.However,the functional mechanisms of GAS2 in hepatocellular carcinoma(HCC)are not fully defined.AIM To investigate the function and mechanism of GAS2 in HCC.METHODS GAS2 expression in clinic liver and HCC specimens was analyzed by real-time PCR and western blotting.Cell proliferation was analyzed by counting,MTS,and colony formation assays.Cell cycle analysis was performed by flow cytometry.Cell apoptosis was investigated by Annexin V apoptosis assay and western blotting.RESULTS GAS2 protein expression was lower in HCC than in normal tissues.Overexpression of GAS2 inhibited the proliferation of HCC cells with wide-type p53,while knockdown of GAS2 promoted the proliferation of hepatocytes(P<0.05).Furthermore,GAS2 overexpression impeded the G1-to-S cell cycle transition and arrested more G1 cells,particularly the elevation of sub G1(P<0.01).Apoptosis induced by GAS2 was dependent on p53,which was increased by etoposide addition.The expression of p53 and apoptosis markers was further enhanced when GAS2 was upregulated,but became diminished upon downregulation of GAS2.In the clinic specimen,GAS2 was downregulated in more than 60%of HCCs.The average fold changes of GAS2 expression in tumor tissues were significantly lower than those in paired non-tumor tissues(P<0.05).CONCLUSION GAS2 plays a vital role in HCC cell proliferation and apoptosis,possibly by regulating the cell cycle and p53-dependent apoptosis pathway.
基金supported by American Diabetes Association,American Heart Association,NIH NIEHS,NIH NIA,NIH NINDS,and NIH ARRA
文摘Throughout the globe,diabetes mellitus（DM） is increasing in incidence with limited therapies presently available to prevent or resolve the significant complications of this disorder.DM impacts multiple organs and affects all components of the central and peripheral nervous systems that can range from dementia to diabetic neuropathy.The mechanistic target of rapamycin（m TOR） is a promising agent for the development of novel regenerative strategies for the treatment of DM.m TOR and its related signaling pathways impact multiple metabolic parameters that include cellular metabolic homeostasis,insulin resistance,insulin secretion,stem cell proliferation and differentiation,pancreatic β-cell function,and programmed cell death with apoptosis and autophagy.m TOR is central element for the protein complexes m TOR Complex 1（m TORC1） and m TOR Complex 2（m TORC2） and is a critical component for a number of signaling pathways that involve phosphoinositide 3-kinase（PI 3-K）,protein kinase B（Akt）,AMP activated protein kinase（AMPK）,silent mating type information regulation 2 homolog 1（Saccharomyces cerevisiae）（SIRT1）,Wnt1 inducible signaling pathway protein 1（WISP1）,and growth factors.As a result,m TOR represents an exciting target to offer new clinical avenues for the treatment of DM and the complications of this disease.Future studies directed to elucidate the delicate balance m TOR holds over cellular metabolism and the impact of its broad signaling pathways should foster the translation of these targets into effective clinical regimens for DM.
文摘Objective:To observe the impact of activation and inhibition of mitogen activated protein kinases(MAPK)/extracellular signalregulated protein kinase(ERK)signaling pathway on the proliferation and apoptosis of cutaneous squamous cell carcinoma(SCC).cells and investigate the interaction mechanism between MAPK/ERK signaling pathway and tumor suppressor gene P53 in SCC.Methods:Human A431 cells were cultured and divided into MAPK/ERK inhibition groups with low-,medium-and highconcentration of inhibitors(PD98059+DMSO),MAPK/ERK activation groups with low-,medium-and high-concentration of stimuli(IGF+PBS)and blank control group(DMSO).The cell proliferation in vitro was detected by MTT assay,with the cell apoptosis detected by flow cytometry(FCM)and the protein expression of P-ERK and P53 detected by western blot in each group.Results:The A431 cell proliferation was inhibited by different concentrations of PD98059 with a clear concentration-effect and time-effect relationship(p<.05);and the cell proliferation was promoted by the different concentrations of IGF with a clear concentration-effect and time-effect relationship(p<.05).The FCM results showed a significant increase in the apoptosis rate of A431 cells which were treated with PD98059,with a clear concentration-effect relationship(p<.05);while the apoptosis rate was decreased significantly after A431 cells were treated with IGF,also with a concentration-effect relationship(p<.05).The western blot results showed that the expression of P-ERK protein was decreased but the expression of P53 was increased after A431 cells were treated with PD98059.With the concentration of PD98059 going up,the decrease in P-ERK and the increase in P53 were more significant(p<.05);while the expression of P-ERK protein was increased but the expression of P53 was decreased after A431 cells were treated with IGF.With the concentration of IGF going up,the increase in P-ERK and the decrease in P53 were more significant(p<.05).According to Pearson correlation analysis,the expression of P53 was negatively correlated to that of P-ERK(p<.05).Conclusions:After MAPK/ERK signaling pathway was activated by IGF in A431 cells,the expression of pro-apoptotic factor P53 was decreased with the ability of cell proliferation enhanced and the ability of apoptosis reduced.However,after the inhibition of MAPK/ERK signaling pathway,the expression of pro-apoptotic factor P53 was increased with the ability of cell proliferation reduced and the ability of apoptosis increased.
文摘Objective:To determine the effect of Zanthoxylum piperitum extracet(ZPE)on apoptosis and analyze anticancer substances in ZPE,changes in proteins related to apoptosis,and pathological changes in tumors in mouse.Methods:Fifteen 4-week-old female BALB/c nu/nu mice were divided into 3 groups depending on ZPE dose,with 5 in each group.AGS gastric carcinoma cells(1 x 10^(6) cells/200 jxL)were subcutaneously injected into the flank of each mouse.One week after the injection of AGS cells,ZPE was administered to the skin tissue[10 or 50 mg/(kg-d)]in the low-and high-dose groups,respectively for 20 days.Control animals were injected with vehicle only.After 3 weeks,the tumor was extracted and carried out for immunohistochemistry,the tendency of apoptosis and p53 in the body was checked using TdT-mediated dUTP nick-end labeling(TUNEL)assay.For 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT)assay,annexin V dead cell staining,cell cycle arrest and Western blotting,AGS gastric carcinoma cells were incubated with various concentrations of ZPE for 24 h.Cell survival rates were analyzed by MTT assays.Apoptosis was analyzed using annexin V dead cell staining and cell cycle arrest and measured using Muse cell analyzer.Results:High performance liquid chromatography(HPLC)analysis showed that ZPE contained organic sulfur compounds such as alliin and S-allylcysteine.MTT assay results revealed that ZPE(10-85»xg/mL)could effectively inhibit the growth of AGS gastric cancer cells at higher concentrations(P<0.05,P<0.01).The annexin V&dead cell staining assay and cell cycle arrest assay confirmed a dose-dependent increase in the apoptosis rate and G!phase in ZPE(10-70 jig/mL)groups.ZPE decreased the expression of anti-apoptotic proteins(p-Akt,p-MDM2,Bcl-2),while increased pro-apoptotic proteins(cleaved PARP,p53,pro-Caspase 3,Bax).TUNEL assays revealed an increase in cell apoptosis.Immunohistochemistry staining confirmed the involvement of p53.Conclusion:ZPE decreases AGS cell proliferation and induces apoptosis by inhibiting Akt and MDM2 expression.
基金This work was supported by the Beijing Natural Science Foundation(7192016 and 7222064)the Scientific Research Common Program of Beijing Municipal Commission of Education(KM201910025029)the National Natural Science Foundation of China(82071539 and 31972911).
文摘Type 1 diabetes mellitus(T1DM)-induced cognitive dysfunction is common,but its underlying mechanisms are still poorly understood.In this study,we found that knockout of conventional protein kinase C(cPKC)γsignificantly increased the phosphorylation of Tau at Ser214 and neurofibrillary tangles,but did not affect the activities of GSK-3βand PP2A in the hippocampal neurons of T1DM mice.cPKCγdeficiency significantly decreased the level of autophagy in the hippocampal neurons of T1DM mice.Activation of autophagy greatly alleviated the cognitive impairment induced by cPKCγdeficiency in T1DM mice.Moreover,cPKCγdeficiency reduced the AMPK phosphorylation levels and increased the phosphorylation levels of mTOR in vivo and in vitro.The high glucose-induced Tau phosphorylation at Ser214 was further increased by the autophagy inhibitor and was significantly decreased by an mTOR inhibitor.In conclusion,these results indicated that cPKCγpromotes autophagy through the AMPK/mTOR signaling pathway,thus reducing the level of phosphorylated Tau at Ser214 and neurofibrillary tangles.
文摘Background:Xihuang pill is a kind of traditional Chinese medicine,which has been widely used in the treatment of kinds of cancer.However,there is still a lack of systematic understanding of the molecular mechanism of Xihuang pill in the treatment of liver cancer.In this work,we aim to explore the molecular mechanism of Xihuang pill in treating liver cancer.Methods:The functional components in Xihuang pill were collected from Traditional Chinese Medicine Database and Analysis Platform.The target genes of these components were also collected using Traditional Chinese Medicine Database and Analysis Platform.The target genes of liver cancer were predicted using GeneCards database.The intersecting genes were then analyzed with Venn diagrams.Kyoto Encyclopedia of Genes and Genomes and Database for Annotation,Visualization,and Integrated Discovery were used to analyze the pathway.Then,cell counting kit-8 was used to measure the half-maximal inhibitory concentration of Xihuang pills.The living dead cell staining method was used to observe the survival of cells.HepG2 cell apoptosis was tested by flow cytometry with fluorescein isothiocyanate/propidium iodide double staining method,and then the mitochondrial damage was also detected by flow cytometry.The expression of target genes was detected by quantitative real-time polymerase chain reaction.Results:A total of 130 compounds and 198 genes were identified as potential active ingredients and putative liver cancer‑related targets.We obtained 1,899 disease targets and 297 transcriptome targets from the database.Six drug-disease intersecting genes,CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3 were obtained.They are enrichment in apoptosis,PI3K-AKT signaling pathway,MAPK signaling pathway,pathways in cancer and p53 signaling pathway.Besides,it was found that the apoptosis rate of the HepG2 cells in Xihuang pill treated group was significantly higher than that of the control group.And the apoptosis rate gradually increased in a dose dependent manner of Xihuang pill treatment.Xihuang pill also induced the mitochondrial membrane potential damage.Compared with the control group,the expression level of CCNB1 and BIRC5 was induced,while the expression level of IGF2 was reduced after Xihuang pill treatment.Conclusion:Xihuang pill may act on six proteins(CCNB1,BIRC5,TOP2A,ESR1,IGF2 and IGFBP3)and cover multiple pathways to form a therapeutic network to treat liver cancer.
基金supported by the Science and Technology Department of Sichuan Province(2015SZ0117,2019YJ0701,and 2021YJ0239).
文摘Due to its difficulty in early diagnosis and lack of sensitivity to chemotherapy and radiotherapy,renal cell carcinoma(RCC)remains to be a frequent cause of cancer-related death.Here,we probed into new targets for its early diagnosis and treatment for RCC.microRNA(miRNA)data of M2-EVs and RCC were searched on the Gene Expression Omnibus database,followed by the prediction of the potential downstream target.Expression of target genes was measured via RT-qPCR and Western blot,respectively.M2 macrophage was obtained viaflow cytometry with M2-EVs extracted.The binding ability of miR-342-3p to NEDD4L and to CEP55 ubiquitination was studied with their roles in the physical abilities of RCC cells assayed.Subcutaneous tumor-bearing mouse models and lung metastasis models were prepared to observe in vivo role of target genes.M2-EVs induced RCC growth and metastasis.miR-342-3p showed high expression in both M2-EVs and RCC cells.M2-EVs carrying miR-342-3p promoted RCC cell abilities to proliferate,invade and migrate.In RCC cells,M2-EV-derived miR-342-3p could specifically bind to NEDD4L and consequently elevate CEP55 protein expression via suppressing NEDD4L,thereby exerting tumor-promoting effects.CEP55 could be degraded by ubiquitination under the function of NEDD4L,and miR-342-3p delivered by M2-EVs facilitated the RCC occurrence and development by activating the PI3K/AKT/mTOR signaling pathway.In conclusion,M2-EVs promote RCC growth and metastasis by delivering miR-342-3p to suppress NEDD4L and subsequently inhibit CEP55 ubiquitination and degradation via activation of the PI3K/AKT/mTOR signaling pathway,strongly driving the proliferative,migratory and invasive of RCC cells.
基金Supported by the National Natural Science Foundation of China(No.81774436)the Science and Technology Innovation Fund Project of Anhui University of Traditional Chinese Medicine(No.2021ZC02)the“14th Five-Year Plan”Provincial Traditional Chinese Medicine Advantage Characteristic Specialty Project[No.(2021]71)]。
文摘Objective:To explore the effect of acupotomy intervention on autophagy of chondrocytes in rabbits with knee osteoarthritis(KOA),and to determine the possible mechanisms of acupotomy to alleviate cartilage degeneration.Methods:The modified Videman method was used to construct a KOA rabbit model.After modeling,40 rabbits were randomly divided into 4 groups by a random number table:control;KOA(model);KOA+acupotomy(acupotomy),and KOA+sham acupotomy(sham),10 in each group.After a 3-week treatment course,the knee joint activity was determined by the modified Lequesne MG index.Hematoxylin-eosin staining staining was used to examine the morphological changes of chondrocytes.Autophagy of chondrocytes was observed by transmission electron microscopy.The surface morphology of cartilage tissue was observed by scanning electron microscope.The mRNA and protein levels of AMP kinase/mammalian target of rapamycin/Unc-51(AMPK/mTOR/ULK1)signal pathway key proteins,autophagy-related factor Beclin-1 and microtubule-associated protein 1A/1B light chain 3(LC3)in rabbit knee cartilage were assessed by real-time fluorescence quantitative polymerase chain reaction and Western blot,respectively.Results:The modified Lequesne MG score of acupotomy group was significantly lower than that of model group(P<0.05).Pathological results showed that chondrocyte autophagy decreased and cartilage surface was rough in the model group,which recovered after acupotomy treatment.The mRNA expressions of AMPK,ULK1,Beclin-1 and the protein levels of p-AMPK,p-ULK1,Beclin-1,and LC3Ⅱ/LC3Ⅰwere decreased in the model group,while the mRNA and protein expressions of mTOR were increased(P<0.01).However,acupotomy treatment reversed these abnormal changes(P<0.05).Conclusions:Acupotomy could effectively up-regulate the expressions of AMPK,ULK1 and Beclin1,reduce the expression of mTOR,promote autophagy,and alleviate joint degeneration.Acupotomy is a promising complementary and alternative therapy for KOA.
