BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is ...BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.展开更多
Natural products have long been a crucial source of,or provided inspiration for new agrochemical discovery.Naturally occurring 18β-glycyrrhetinic acid shows broad-spectrum bioactivities and is a potential skeleton fo...Natural products have long been a crucial source of,or provided inspiration for new agrochemical discovery.Naturally occurring 18β-glycyrrhetinic acid shows broad-spectrum bioactivities and is a potential skeleton for novel drug discovery.To extend the utility of 18β-glycyrrhetinic acid for agricultural uses,a series of novel 18β-glycyrrhetinic acid amide derivatives were prepared and evaluated for their antibacterial potency.Notably,compound 5k showed good antibacterial activity in vitro against Xanthomonas oryzae pv.oryzae(Xoo,EC50=3.64 mg L–1),and excellent protective activity(54.68%)against Xoo in vivo.Compound 5k induced excessive production and accumulation of reactive oxygen species in the tested pathogens,resulting in damaging the bacterial cell envelope.More interestingly,compound 5k could increase the activities of plant defense enzymes including catalase,superoxide dismutase,peroxidase,and phenylalanine ammonia lyase.Taken together,these enjoyable results suggested that designed compounds derived from 18β-glycyrrhetinic acid showed potential for controlling intractable plant bacterial diseases by disturbing the balance of the phytopathogen’s redox system and activating the plant defense system.展开更多
BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GR...BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.展开更多
Objective:To study the effects of 18β-glycyrrhetinic acid (GA) on proliferation inhibition, apop totic induction, and the relationship between GA-induced apoptosis and intracellular Ca2+ concentration in human breast...Objective:To study the effects of 18β-glycyrrhetinic acid (GA) on proliferation inhibition, apop totic induction, and the relationship between GA-induced apoptosis and intracellular Ca2+ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 μmol/L to 250 μmol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 μmol/L GA for 24 h, intracellular Ca2+ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 μmol/L to 250 μmol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose dependent fashion. IC50 of the proliferation inhibition was 234.33 μmol/L. Treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca2+ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18β-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca2+ level may be depended on apoptosis induced by GA in MCF-7 cells.展开更多
BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options.Cell-based therapies using mesenchymal stem cells(MSCs)emerged as an alternative approach to suppor...BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options.Cell-based therapies using mesenchymal stem cells(MSCs)emerged as an alternative approach to support hepatic regeneration.In vitro preconditioning strategies have been employed to strengthen the regenerative and differentiation potential of MSCs towards hepatic lineage.Chemical compounds of the triterpene class;glycyrrhizic acid(GA)and 18β-glycyrrhetinic acid(GT)possess diverse therapeutic properties including hepatoprotection and anti-fibrosis characteristics.They are capable of modulating several signaling pathways that are crucial in hepatic regeneration.Preconditioning with hepato-protective triterpenes may stimulate MSC fate transition towards hepatocytes.AIM To explore the effect of GA and GT on hepatic differentiation of human umbilical cord-MSCs(hUC-MSCs).METHODS hUC-MSCs were isolated and characterized phenotypically by flow cytometry and immunocytochemistry for the expression of MSC-associated surface molecules.Isolated cells were treated with GA,GT,and their combination for 24 h and then analyzed at three time points;day 7,14,and 21.qRT-PCR was performed for the expression of hepatic genes.Expression of hepatic proteins was analyzed by immunocytochemistry at day 21.Periodic acid Schiff staining was performed to determine the functional ability of treated cells.RESULTS The fusiform-shaped morphology of MSCs in the treatment groups in comparison with the untreated control,eventually progressed towards the polygonal morphology of hepatocytes with the passage of time.The temporal transcriptional profile of preconditioned MSCs displayed significant expression of hepatic genes with increasing time of differentiation.Preconditioned cells showed positive expression of hepatocyte-specific proteins.The results were further corroborated by positive periodic acid Schiff staining,indicating the presence of glycogen in their cytoplasm.Moreover,bi-nucleated cells,which is the typical feature of hepatocytes,were also seen in the preconditioned cells.CONCLUSION Preconditioning with glycyrrhizic acid,18β-glycyrrhetinic acid and their combination,successfully differentiates hUC-MSCs into hepatic-like cells.These MSCs may serve as a better therapeutic option for degenerative liver diseases in future.展开更多
BACKGROUND Gastric carcinoma(GC)is a common gastrointestinal malignancy worldwide.Based on the cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore...BACKGROUND Gastric carcinoma(GC)is a common gastrointestinal malignancy worldwide.Based on the cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the mechanism by which 18β-glycyrrhetinic acid(18β-GRA)regulates mitochondrial ribosomal protein L35(MRPL35)related signal proteins to inhibit the proliferation of GC cells.METHODS Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823.The apoptosis and cell cycle were assessed by flow cytometry.Cell invasion and migration were evaluated by Transwell assay,and cell scratch test was used to detect cell migration.Furthermore,a tumor model was established by hypodermic injection of 2.5×106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect MRPL35 expression in the engrafted tumors in mice.We used the term tandem mass tag(TMT)labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins(DEPs)extracted from GC cells and control cells after 18β-GRA intervention.A detailed bioinformatics analysis of these DEPs was performed,including Gene Ontology annotation and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis,and so on.Moreover,STRING database(https://string-db.org/)was used to predict proteinprotein interaction(PPI)relationships and Western blot was used to detect the expression of proteins of interest in GC cells.RESULTS The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose-and timedependent manner.It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase.The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose,and the migration and invasiveness of GC cells were inhibited.The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice,and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice.Using TMT technology,609 DEPs,among which 335 were up-regulated and 274 were down-regulated,were identified in 18β-GRA intervention compared with control.We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways,such as cellular processes,biological regulation,and TP53 signaling pathway.Notably,after the drug intervention,MRPL35 expression was significantly down-regulated(P=0.000247),TP53 expression was up-regulated(P=0.02676),and BCL2L1 was down-regulated(P=0.01699).Combined with the Retrieval of Interacting Genes/Proteins database,we analyzed the relationship between MRPL35,TP53,and BCL2L1 signaling proteins,and we found that COPS5,BAX,and BAD proteins can form a PPI network with MRPL35,TP53,and BCL2L1.Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells,MRPL35,TP53,and BCL2L1 showed dose-dependent up/down-regulation,and the expression of COPS5,BAX,and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION 18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35,COPS5,TP53,BCL2L1,BAX,and BAD.展开更多
Glycyrrhiza uralensis Fisch has been used to treat the symptoms of organ fever, food poisoning, typhoid fever, sore throat, cough due to lung heat, children's diseases and so on since the ancient times. It is a so...Glycyrrhiza uralensis Fisch has been used to treat the symptoms of organ fever, food poisoning, typhoid fever, sore throat, cough due to lung heat, children's diseases and so on since the ancient times. It is a solid Chinese herbal medicine which is good for the health. In recent years, it has been found that licorice extract also has excellent anti-cancer effect. The 18β glycyrrhetinic acid, obtained by hydrolysis of precursor glycyrrhizic acid, is a relatively efficient anti-cancer ingredient. 18β glycyrrhetinic acid can exert anti-cancer effects by inhibiting cancer cell proliferation, inducing apoptosis of cancer cells, inhibiting invasion and metastasis of cancer cells, preventing oxidation, inhibiting neovascularization, inhibiting lymphangiogenesis, and regulating hormone secretion. This paper reviewed the advances in research of the anticancer mechanism of 18β glycyrrhetinic acid, to provide a theoretical basis for future research.展开更多
Cisplatin （CP） , a highly effective and widely used chemotherapeutic agent, has a major limitation for its nephrotoxicity. We recently identified a novel strategy for attenuating its nephrotoxicity in chemotherapy b...Cisplatin （CP） , a highly effective and widely used chemotherapeutic agent, has a major limitation for its nephrotoxicity. We recently identified a novel strategy for attenuating its nephrotoxicity in chemotherapy by an ef- fective adjuvant via epigenetic modification through targeting Histone deacetylase 2 （HDAC2）. Glycyrrhizic acid （GA） ,a major active component of Licorice, was described here for its new application. Molecular docking and Surface Plasmon resonance （SPR） assay firstly reported that 18βGA, GA metabolite in vivo, could directly bind to HDAC2 and prevent HDAC2 activation. The effects and mechanisms of GA and its major metabolite 18βGA were assessed in CP-induced acute kidney injury （AKI） in C57BL/6 mice, and in CP-treated HK-2 and mTEC cells lines. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） and flow cytometry （FCM） results confirmed that GA and 18βGA could inhibit apoptosis of renal tubular epithelial cells induced by CP in vivo and in vitro. Western blot and immunofluorescence results demonstrated that the expression of bone morphogenetic protein- 7 （BMP-7） , a protective molecule in renal inflammation, was clearly induced by 18βGA in AKI models while siR- NA BMP-7 could reduce the inhibitory effect of 18βGA on apoptosis. Results of current study indicated that 18βGA inhibited apoptosis of renal tubular epithelial cells via enhancing level of BMP-7 epigenetically through targeting HDAC2, therefore protecting against CP-induced AKI. These available evidence, which led to an improved under- standing of molecular recognition, suggested that 18βGA could serve as a potential clinical adjuvant in chemothera-展开更多
The in vitro antibacterial activities of 18β-glycyrrhetinic acid alone or combined with first-line antituberculosis drugs including isoniazid（INH）,rifampicin（RFP） and streptomycin（SM） against Mycobacterium tube...The in vitro antibacterial activities of 18β-glycyrrhetinic acid alone or combined with first-line antituberculosis drugs including isoniazid（INH）,rifampicin（RFP） and streptomycin（SM） against Mycobacterium tuberculosis were detected using MABA method.The minimum inhibitory concentrations（MICs） of18β-glycyrrhetinic acid against M.tuberculosis H37Rv（ATCC 27294） and M.bovis（ATCC 19210） were 50 and 100 μg/m L,respectively.The MICs of two clinical drug-susceptible isolates and six drug-resistant isolates were 25-50 and 100-200 μg/m L,respectively.As 18β-glycyrrhetinic acid combined with INH,RFP and SM,they exhibited synergistic effects against six drug-resistant isolates,and MICs decreased significantly：MIC of INH decreased by 2-32 folds（FICIs 0.125-0.375）;MIC of RFP decreased by 4-8 folds（FICIs 0.240-0.490）;MIC of SM decreased by 4-16 folds（FICIs 0.165-0.460）.Traditional medicine monomer had low cytotoxicity on normal cell BHK-21 and could restraint SMMC fission.The results showed that 18β-glycyrrhetinic acid combined with anti-TB drugs（INH,RFP and SM） had good antibacterial activity against M.tuberculosis.These findings indicated that 18β-glycyrrhetinic acid might serve as the potential therapeutic compound for future development of anti-TB drugs.展开更多
基金Supported by the Ningxia Natural Science Foundation,No.2022AAC03144.
文摘BACKGROUND Gastric cancer(GC)is a common gastrointestinal malignancy worldwide.Based on cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the molecular mechanism of 18β-glycyrrhetinic acid(18β-GRA)regulating the miR-345-5p/TGM2 signaling pathway to inhibit the proliferation of GC cells.METHODS CCK-8 assay was used to determine the effect of 18β-GRA on the survival rate of GES-1 cells and AGS and HGC-27 cells.Cell cycle and apoptosis were detected by flow cytometry,cell migration was detected by a wound healing assay,the effect of 18β-GRA on subcutaneous tumor growth in BALB/c nude mice was investigated,and the cell autophagy level was determined by MDC staining.TMT proteomic analysis was used to detect the differentially expressed autophagy-related proteins in GC cells after 18β-GRA intervention,and then the protein-protein interaction was predicted using STRING(https://string-db.org/).MicroRNAs(miRNAs)transcriptome analysis was used to detect the miRNA differential expression profile,and use miRBase(https://www.mirbase/)and TargetScan(https://www.targetscan.org/)to predict the miRNA and complementary binding sites.Quantitative real-time polymerase chain reaction was used to detect the expression level of miRNA in 18β-GRA treated cells,and western blot was used to detect the expression of autophagy related proteins.Finally,the effect of miR-345-5p on GC cells was verified by mir-345-5p overexpression.RESULTS 18β-GRA could inhibit GC cells viability,promote cell apoptosis,block cell cycle,reduce cell wound healing ability,and inhibit the GC cells growth in vivo.MDC staining results showed that 18β-GRA could promote autophagy in GC cells.By TMT proteomic analysis and miRNAs transcriptome analysis,it was concluded that 18β-GRA could down-regulate TGM2 expression and up-regulate miR-345-5p expression in GC cells.Subsequently,we verified that TGM2 is the target of miR-345-5p,and that overexpression of miR-345-5p significantly inhibited the protein expression level of TGM2.Western blot showed that the expression of autophagy-related proteins of TGM2 and p62 was significantly reduced,and LC3II,ULK1 and AMPK expression was significantly increased in GC cells treated with 18β-GRA.Overexpression of miR-345-5p not only inhibited the expression of TGM2,but also inhibited the proliferation of GC cells by promoting cell apoptosis and arresting cell cycle.CONCLUSION 18β-GRA inhibits the proliferation of GC cells and promotes autophagy by regulating the miR-345-5p/TGM2 signaling pathway.
基金fundings provided by the National Natural Science Foundation of China(21877021 and 32160661)the Guizhou Provincial S&T Program[(2018)4007]the Program of Introducing Talents of Discipline to Universities of China(D20023,111 Program).
文摘Natural products have long been a crucial source of,or provided inspiration for new agrochemical discovery.Naturally occurring 18β-glycyrrhetinic acid shows broad-spectrum bioactivities and is a potential skeleton for novel drug discovery.To extend the utility of 18β-glycyrrhetinic acid for agricultural uses,a series of novel 18β-glycyrrhetinic acid amide derivatives were prepared and evaluated for their antibacterial potency.Notably,compound 5k showed good antibacterial activity in vitro against Xanthomonas oryzae pv.oryzae(Xoo,EC50=3.64 mg L–1),and excellent protective activity(54.68%)against Xoo in vivo.Compound 5k induced excessive production and accumulation of reactive oxygen species in the tested pathogens,resulting in damaging the bacterial cell envelope.More interestingly,compound 5k could increase the activities of plant defense enzymes including catalase,superoxide dismutase,peroxidase,and phenylalanine ammonia lyase.Taken together,these enjoyable results suggested that designed compounds derived from 18β-glycyrrhetinic acid showed potential for controlling intractable plant bacterial diseases by disturbing the balance of the phytopathogen’s redox system and activating the plant defense system.
基金Ningxia Medical University Project,No. XZ2021005Ningxia Natural Science Foundation,Nos. 2022AAC03144 and 2022AAC02039National Natural Science Foundation of China,No. 82260879
文摘BACKGROUND Gastric cancer(GC)is one of the most common cancer types worldwide,and its prevention and treatment methods have garnered much attention.As the active ingredient of licorice,18β-glycyrrhetinic acid(18β-GRA)has a variety of pharmacological effects.The aim of this study was to explore the effective target of 18β-GRA in the treatment of GC,in order to provide effective ideas for the clinical prevention and treatment of GC.AIM To investigate the mechanism of 18β-GRA in inhibiting cell proliferation and promoting autophagy flux in GC cells.METHODS Whole transcriptomic analyses were used to analyze and screen differentially expressed microRNAs(miRNAs)in GC cells after 18β-GRA intervention.Lentivirus-transfected GC cells and the Cell Counting Kit-8 were used to detect cell proliferation ability,cell colony formation ability was detected by the clone formation assay,and flow cytometry was used to detect the cell cycle and apoptosis.A nude mouse transplantation tumor model of GC cells was constructed to verify the effect of miR-328-3p overexpression on the tumorigenicity of GC cells.Tumor tissue morphology was observed by hematoxylin and eosin staining,and microtubule-associated protein light chain 3(LC3)expression was detected by immunohistochemistry.TransmiR,STRING,and miRWalk databases were used to predict the relationship between miR-328-3p and signal transducer and activator of transcription 3(STAT3)-related information.Expression of STAT3 mRNA and miR-328-3p was detected by quantitative polymerase chain reaction(qPCR)and the expression levels of STAT3,phosphorylated STAT3(p-STAT3),and LC3 were detected by western blot analysis.The targeted relationship between miR-328-3p and STAT3 was detected using the dual-luciferase reporter gene system.AGS cells were infected with monomeric red fluorescent protein-green fluorescent protein-LC3 adenovirus double label.LC3 was labeled and autophagy flow was observed under a confocal laser microscope.RESULTS The expression of miR-328-3p was significantly upregulated after 18β-GRA intervention in AGS cells(P=4.51E-06).Overexpression of miR-328-3p inhibited GC cell proliferation and colony formation ability,arrested the cell cycle in the G0/G1 phase,promoted cell apoptosis,and inhibited the growth of subcutaneous tumors in BALB/c nude mice(P<0.01).No obvious necrosis was observed in the tumor tissue in the negative control group(no drug intervention or lentivirus transfection)and vector group(the blank vector for lentivirus transfection),and more cells were loose and necrotic in the miR-328-3p group.Bioinformatics tools predicted that miR-328-3p has a targeting relationship with STAT3,and STAT3 was closely related to autophagy markers such as p62.After overexpressing miR-328-3p,the expression level of STAT3 mRNA was significantly decreased(P<0.01)and p-STAT3 was downregulated(P<0.05).The dual-luciferase reporter gene assay showed that the luciferase activity of miR-328-3p and STAT33’untranslated regions of the wild-type reporter vector group was significantly decreased(P<0.001).Overexpressed miR-328-3p combined with bafilomycin A1(Baf A1)was used to detect the expression of LC3 II.Compared with the vector group,the expression level of LC3 II in the overexpressed miR-328-3p group was downregulated(P<0.05),and compared with the Baf A1 group,the expression level of LC3 II in the overexpressed miR-328-3p+Baf A1 group was upregulated(P<0.01).The expression of LC3 II was detected after intervention of 18β-GRA in GC cells,and the results were consistent with the results of miR-328-3p overexpression(P<0.05).Additional studies showed that 18β-GRA promoted autophagy flow by promoting autophagosome synthesis(P<0.001).qPCR showed that the expression of STAT3 mRNA was downregulated after drug intervention(P<0.05).Western blot analysis showed that the expression levels of STAT3 and p-STAT3 were significantly downregulated after drug intervention(P<0.05).CONCLUSION 18β-GRA promotes the synthesis of autophagosomes and inhibits GC cell proliferation by regulating the miR-328-3p/STAT3 signaling pathway.
文摘Objective:To study the effects of 18β-glycyrrhetinic acid (GA) on proliferation inhibition, apop totic induction, and the relationship between GA-induced apoptosis and intracellular Ca2+ concentration in human breast carcinoma (MCF-7) cells. Methods: After MCF-7 cells were treated with GA at the concentrations from 50 μmol/L to 250 μmol/L for 24 h, cell viability of proliferation was assessed by MTT assay. After the cells were treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L GA for 24 h, the rates of cell apoptosis were examined by terminal deoxynucleotide transferase mediated dUTP nick-end-labeling method and flow cytometry with Annexin V/propidium iodide fluorescent stain. After the cells treated with 150 μmol/L GA for 24 h, intracellular Ca2+ concentration was measured by Fure-2 fluorescein load method. Results: After the cells were treated with GA at the concentrations from 100 μmol/L to 250 μmol/L, the rates of proliferative inhibition were increased significantly (P<0.05 and P<0.01) in a dose dependent fashion. IC50 of the proliferation inhibition was 234.33 μmol/L. Treated with 100 μmol/L, 150 μmol/L, and 200 μmol/L, the rates of cell apoptosis were increased significantly (P<0.01). Intracellular Ca2+ concentration after treatment with GA was higher evidently than that of control (P<0.05). Conclusion: 18β-glycyrrhetinic acid has the effects of the proliferation inhibition and the apoptotic induction on MCF-7 cells. The rise of intracellular Ca2+ level may be depended on apoptosis induced by GA in MCF-7 cells.
文摘BACKGROUND End-stage liver disease is a global health complication with high prevalence and limited treatment options.Cell-based therapies using mesenchymal stem cells(MSCs)emerged as an alternative approach to support hepatic regeneration.In vitro preconditioning strategies have been employed to strengthen the regenerative and differentiation potential of MSCs towards hepatic lineage.Chemical compounds of the triterpene class;glycyrrhizic acid(GA)and 18β-glycyrrhetinic acid(GT)possess diverse therapeutic properties including hepatoprotection and anti-fibrosis characteristics.They are capable of modulating several signaling pathways that are crucial in hepatic regeneration.Preconditioning with hepato-protective triterpenes may stimulate MSC fate transition towards hepatocytes.AIM To explore the effect of GA and GT on hepatic differentiation of human umbilical cord-MSCs(hUC-MSCs).METHODS hUC-MSCs were isolated and characterized phenotypically by flow cytometry and immunocytochemistry for the expression of MSC-associated surface molecules.Isolated cells were treated with GA,GT,and their combination for 24 h and then analyzed at three time points;day 7,14,and 21.qRT-PCR was performed for the expression of hepatic genes.Expression of hepatic proteins was analyzed by immunocytochemistry at day 21.Periodic acid Schiff staining was performed to determine the functional ability of treated cells.RESULTS The fusiform-shaped morphology of MSCs in the treatment groups in comparison with the untreated control,eventually progressed towards the polygonal morphology of hepatocytes with the passage of time.The temporal transcriptional profile of preconditioned MSCs displayed significant expression of hepatic genes with increasing time of differentiation.Preconditioned cells showed positive expression of hepatocyte-specific proteins.The results were further corroborated by positive periodic acid Schiff staining,indicating the presence of glycogen in their cytoplasm.Moreover,bi-nucleated cells,which is the typical feature of hepatocytes,were also seen in the preconditioned cells.CONCLUSION Preconditioning with glycyrrhizic acid,18β-glycyrrhetinic acid and their combination,successfully differentiates hUC-MSCs into hepatic-like cells.These MSCs may serve as a better therapeutic option for degenerative liver diseases in future.
基金Supported by Ningxia Natural Science Foundation,No.2020AAC03130Ningxia Medical University Project,No.XM2020005.
文摘BACKGROUND Gastric carcinoma(GC)is a common gastrointestinal malignancy worldwide.Based on the cancer-related mortality,the current prevention and treatment strategies for GC still show poor clinical results.Therefore,it is important to find effective drug treatment targets.AIM To explore the mechanism by which 18β-glycyrrhetinic acid(18β-GRA)regulates mitochondrial ribosomal protein L35(MRPL35)related signal proteins to inhibit the proliferation of GC cells.METHODS Cell counting kit-8 assay was used to detect the effects of 18β-GRA on the survival rate of human normal gastric mucosal cell line GES-1 and the proliferation of GC cell lines MGC80-3 and BGC-823.The apoptosis and cell cycle were assessed by flow cytometry.Cell invasion and migration were evaluated by Transwell assay,and cell scratch test was used to detect cell migration.Furthermore,a tumor model was established by hypodermic injection of 2.5×106 BGC-823 cells at the selected positions of BALB/c nude mice to determine the effect of 18β-GRA on GC cell proliferation,and quantitative reverse transcription-polymerase chain reaction(qRT-PCR)was used to detect MRPL35 expression in the engrafted tumors in mice.We used the term tandem mass tag(TMT)labeling combined with liquid chromatography–tandem mass spectrometry to screen for differentially expressed proteins(DEPs)extracted from GC cells and control cells after 18β-GRA intervention.A detailed bioinformatics analysis of these DEPs was performed,including Gene Ontology annotation and enrichment analysis,Kyoto Encyclopedia of Genes and Genomes pathway enrichment analysis,and so on.Moreover,STRING database(https://string-db.org/)was used to predict proteinprotein interaction(PPI)relationships and Western blot was used to detect the expression of proteins of interest in GC cells.RESULTS The results indicated that 18β-GRA could inhibit the proliferation of GC cells in a dose-and timedependent manner.It could induce GC cell apoptosis and arrest the cell cycle at G0/G1 phase.The proportion of cells arrested at S phase decreased with the increase of 18-GRA dose,and the migration and invasiveness of GC cells were inhibited.The results of animal experiments showed that 18β-GRA could inhibit tumor formation in BALB/c nude mice,and qRT-PCR results showed that MRPL35 expression level was significantly reduced in the engrafted tumors in mice.Using TMT technology,609 DEPs,among which 335 were up-regulated and 274 were down-regulated,were identified in 18β-GRA intervention compared with control.We found that the intervention of 18β-GRA in GC cells involved many important biological processes and signaling pathways,such as cellular processes,biological regulation,and TP53 signaling pathway.Notably,after the drug intervention,MRPL35 expression was significantly down-regulated(P=0.000247),TP53 expression was up-regulated(P=0.02676),and BCL2L1 was down-regulated(P=0.01699).Combined with the Retrieval of Interacting Genes/Proteins database,we analyzed the relationship between MRPL35,TP53,and BCL2L1 signaling proteins,and we found that COPS5,BAX,and BAD proteins can form a PPI network with MRPL35,TP53,and BCL2L1.Western blot analysis confirmed the intervention effect of 18β-GRA on GC cells,MRPL35,TP53,and BCL2L1 showed dose-dependent up/down-regulation,and the expression of COPS5,BAX,and BAD also increased/decreased with the change of 18β-GRA concentration.CONCLUSION 18β-GRA can inhibit the proliferation of GC cells by regulating MRPL35,COPS5,TP53,BCL2L1,BAX,and BAD.
基金Supported by the Multigrain Production and Processing Characteristic Discipline Construction Projectthe Postdoctoral Scientific Research Foundation of Heilongjiang Province of China(LBH-Q13132)National key research and Development Plan"Regulation and Activity Retention Technology and Application of Coarse Cereals Active Components in Processing"(2017YFD0401203)
文摘Glycyrrhiza uralensis Fisch has been used to treat the symptoms of organ fever, food poisoning, typhoid fever, sore throat, cough due to lung heat, children's diseases and so on since the ancient times. It is a solid Chinese herbal medicine which is good for the health. In recent years, it has been found that licorice extract also has excellent anti-cancer effect. The 18β glycyrrhetinic acid, obtained by hydrolysis of precursor glycyrrhizic acid, is a relatively efficient anti-cancer ingredient. 18β glycyrrhetinic acid can exert anti-cancer effects by inhibiting cancer cell proliferation, inducing apoptosis of cancer cells, inhibiting invasion and metastasis of cancer cells, preventing oxidation, inhibiting neovascularization, inhibiting lymphangiogenesis, and regulating hormone secretion. This paper reviewed the advances in research of the anticancer mechanism of 18β glycyrrhetinic acid, to provide a theoretical basis for future research.
文摘Cisplatin （CP） , a highly effective and widely used chemotherapeutic agent, has a major limitation for its nephrotoxicity. We recently identified a novel strategy for attenuating its nephrotoxicity in chemotherapy by an ef- fective adjuvant via epigenetic modification through targeting Histone deacetylase 2 （HDAC2）. Glycyrrhizic acid （GA） ,a major active component of Licorice, was described here for its new application. Molecular docking and Surface Plasmon resonance （SPR） assay firstly reported that 18βGA, GA metabolite in vivo, could directly bind to HDAC2 and prevent HDAC2 activation. The effects and mechanisms of GA and its major metabolite 18βGA were assessed in CP-induced acute kidney injury （AKI） in C57BL/6 mice, and in CP-treated HK-2 and mTEC cells lines. Terminal deoxynucleotidyl transferase dUTP nick end labeling （TUNEL） and flow cytometry （FCM） results confirmed that GA and 18βGA could inhibit apoptosis of renal tubular epithelial cells induced by CP in vivo and in vitro. Western blot and immunofluorescence results demonstrated that the expression of bone morphogenetic protein- 7 （BMP-7） , a protective molecule in renal inflammation, was clearly induced by 18βGA in AKI models while siR- NA BMP-7 could reduce the inhibitory effect of 18βGA on apoptosis. Results of current study indicated that 18βGA inhibited apoptosis of renal tubular epithelial cells via enhancing level of BMP-7 epigenetically through targeting HDAC2, therefore protecting against CP-induced AKI. These available evidence, which led to an improved under- standing of molecular recognition, suggested that 18βGA could serve as a potential clinical adjuvant in chemothera-
基金Supported by Scientific Research Project at Universities of Inner Mongolia Autonomous Region(NJZY14332)
文摘The in vitro antibacterial activities of 18β-glycyrrhetinic acid alone or combined with first-line antituberculosis drugs including isoniazid（INH）,rifampicin（RFP） and streptomycin（SM） against Mycobacterium tuberculosis were detected using MABA method.The minimum inhibitory concentrations（MICs） of18β-glycyrrhetinic acid against M.tuberculosis H37Rv（ATCC 27294） and M.bovis（ATCC 19210） were 50 and 100 μg/m L,respectively.The MICs of two clinical drug-susceptible isolates and six drug-resistant isolates were 25-50 and 100-200 μg/m L,respectively.As 18β-glycyrrhetinic acid combined with INH,RFP and SM,they exhibited synergistic effects against six drug-resistant isolates,and MICs decreased significantly：MIC of INH decreased by 2-32 folds（FICIs 0.125-0.375）;MIC of RFP decreased by 4-8 folds（FICIs 0.240-0.490）;MIC of SM decreased by 4-16 folds（FICIs 0.165-0.460）.Traditional medicine monomer had low cytotoxicity on normal cell BHK-21 and could restraint SMMC fission.The results showed that 18β-glycyrrhetinic acid combined with anti-TB drugs（INH,RFP and SM） had good antibacterial activity against M.tuberculosis.These findings indicated that 18β-glycyrrhetinic acid might serve as the potential therapeutic compound for future development of anti-TB drugs.
