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@#Abstract: Objective To explore the correlation between the levels of silent information regulator 1 (SIRT1) and forkhead box protein O3 (FOXO3) in peripheral blood mononuclear cells of patients with active pulmonary tuberculosis (APTB) and macrophage-related cytokines-inducible nitric oxide synthase (iNOS) and arginase-1 (Arg-1). Methods A total of 64 APTB patients who were treated in Yubei Hospital, the First Affiliated Hospital of Chongqing Medical University from January 2020 to December 2021 were gathered as the APTB group, 59 people with latent tuberculosis infection (LTBI) were gathered as the LTBI group, and 62 healthy people were gathered as the control group. Quantitative real-time PCR (qPCR) method was performed to measure the levels of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells. The enzyme-linked immunosorbent assay (ELISA) was performed to measure serum iNOS and Arg-1 levels; ROC curve was used to analyze the value of SIRT1 mRNA and FOXO3 mRNA levels in the differential diagnosis of LTBI and APTB; Pearson correlation was performed to analyze the correlation of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells of APTB patients with serum iNOS and Arg-1 levels. Results The levels of SIRT1 mRNA, FOXO3 mRNA and serum iNOS in peripheral blood mononuclear cells decreased in control group, LTBI group and APTB group, and the level of serum Arg-1 increased in turn (P<0.05). The AUCs of SIRT1 mRNA and FOXO3 mRNA in differential diagnosis of LTBI and APTB were 0.876 and 0.887, respectively, the sensitivity was 71.2% and 76.3%, and the specificity was 96.9% and 90.6% respectively. The levels of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells of APTB patients were positively correlated (r=0.500, P<0.05), and they were positively correlated with serum iNOS and negatively correlated with serum Arg-1 (P<0.05). The SIRT1 mRNA, FOXO3 mRNA and serum iNOS in peripheral blood mononuclear cells of APTB patients after 6 months of treatment were higher than those before treatment, and serum Arg-1 was lower than before treatment (P<0.05). Conclusions The levels of SIRT1 mRNA and FOXO3 mRNA in peripheral blood mononuclear cells of APTB patients are low, and they are positively correlated with macrophage-related cytokine iNOS and negatively correlated with Arg-1.
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Background: An interdisciplinary research of public health, biomedical and pharmaceutical sciences is neededfor integrating qualitative and quantitative researches undertaken. It hence requires focus on public beneficencefor non-communicable diseases. Purpose: To study anticancer activities of soil samples of Central India andits stability for applied public health use. Material and Methods: Screening on Actinomycetes isolates obtainedfrom rural and urban farm soils illustrating arginase production was conducted from equated soil samples ofgeo-representative localities and adjoining areas of Bhopal, India. Enrichment Technique (CDSEA) was usedfor detection of extracellular production of L-arginase and their anticancer activities using MTT 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide assay along with characterization and probioticproperties of selected isolate. Results: L-Arginase activity quantified by ornithine (21.06-117.92 U/mg) wasfound in isolates BRD-21, KAR-73, BHA-162, BAR-199, ARH-210, HAB-228. Urea release (15.88 – 59.79 U/mg protein) depicted L- arginase activity in crude enzyme samples. It shows noticeable anticancer activity.Morphological and biochemical characterization of these isolates revealed metabolic diversity. Isolate KAR 73produced collagenase (specific activity 57.8 U/mg), L-asparaginase (specific activity 116 U/mg) and L-arginasewith tolerance to higher temperature (45°C) and salt concentration (2-8% w/v). Equal concentrations ofcrude L- arginase from these isolates inhibited growth and proliferation of colorectal adenocarcinoma celllines (19.99%-38.65%) under in-vitro conditions. Conclusion: Arginine depletion through arginase activity isevidenced for potential effectiveness in cancer treatment especially adenocarcinomas and squamous cellcarcinoma. It is useful for wider public health purposes
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Objective: To provide a method for preparing a specific polyclonal antibody of ARG. Methods: Firstly, the technology of polymerase chain reaction(PCR)was adopted to amplify the AbArg gene encoding arginase in Agaricus bisporus. Then the recombinant plasmid pET-28a(+)-Arg was constructed and transformed into E. coli BL21 for the expression of fusion protein. Afterwards, the purified protein was used to immunize New Zealand white rabbits, and the serum samples were collected. Results: The prokaryotic expression plasmid pET-28a(+)-Arg was constructed and the results of SDS-PAGE analysis showed that the fusion protein existing in form of inclusion body was successfully induced and expressed. After purification, the fusion protein with high purity (above 90%)was obtained. The immunity serum titer was 51 200 and Western blotting results showed that the polyclonal antibody could specifically recognize ARG protein in A. bisporus. Conclusion: Polyclonal antibody anti-ARG was prepared successfully, which lays a foundation for the further study on the mechanism of arginine metabolism.
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Objective@#To investigate the potential effects of cytochrome P450 1A1 (CYP1A1) in regulating macrophages polarize to M2 type and explore the molecular mechanism.@*Methods@#All trials were completely randomized. ① Experiment 1: 6-8 weeks old healthy male C57BL/6J mice were collected, and primary peritoneal cells were extracted, then the cells were divided into phosphate buffered saline (PBS) group and interleukin-4 (IL-4) group. The cells in the IL-4 group were stimulated with 10 mg/L IL-4 (M2 macrophage inducer); and those in the PBS group were given with an equal amount of PBS. The mRNA expressions of intracellular M2 type polarized marker molecules including arginase-1 (Arg-1) and chitinase 3 like protein 1 (YM1) at 2, 4, 6 hours after IL-4 challenge were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The phosphorylation of tyrosine protein kinase 1/signaling transcriptional and transduced activator 6 (JAK1/STAT6) signaling pathway and protein expressions of CYP1A1 and Arg-1 at 6, 12, 24 hours after IL-4 challenge were determined by Western Blot. ② Experiment 2: RAW264.7 cells with high expression CYP1A1 (CYP1A1/RAW) and their negative control cells (NC/RAW) were cultured in vitro. The cells in logarithmic growth phase were collected, and then they were divided into PBS control group and IL-4 group. The treatment method was the same as experiment 1. The phosphorylations of intracellular JAK1/STAT6 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in different cells at 1 hour and 2 hours after IL-4 challenge were determined by Western Blot. The mRNA and protein expressions of Arg-1 in different cells at 2 hours and 4 hours after IL-4 challenge were determined by RT-qPCR and Western Blot, respectively.@*Results@#① Experiment 1: after IL-4 challenge for 2 hours, the mRNA expressions of Arg-1 and YM1 in the primary peritoneal macrophages of mice were significantly increased as compared with the PBS control group [Arg-1 mRNA (2-ΔΔCt): 1.75±0.82 vs. 1.00±0.21; YM1 mRNA (2-ΔΔCt): 2.58±0.53 vs. 1.00±0.20, both P < 0.05] which indicated that IL-4 induced macrophage to M2 type polarization successfully. Meanwhile, the mRNA expression of CYP1A1 in polarized mouse peritoneal primary macrophages was also elevated as compared with the PBS control group, and peaked at 4 hours [CYP1A1 mRNA (2-ΔΔCt): 2.25±0.69 vs. 1.00±0.17, P < 0.01]. The results indicated that CYP1A1 expression was enhanced when macrophages polarized to M2 type. Compared with the PBS control group, the bands of phosphorylated JAK1 (p-JAK1), phosphorylated STAT6 (p-STAT6), Arg-1 and CYP1A1 were enhanced in primary peritoneal macrophages of mice in the IL-4 group, and reached the peak value at 12 hours then gradually decreased. This result indicated that the phosphorylation of JAK1/STAT6 pathway was enhanced in M2 macrophages with high expression of CYP1A1, and the pathway was activated. ② Experiment 2: after IL-4 challenge for 2 hours, the expression of Arg-1 mRNA in CYP1A1/RAW cells was significantly higher than that in NC/RAW cells (2-ΔΔCt: 3.02±0.60 vs. 1.47±0.43, P < 0.05), and the protein band signal was also stronger, and both peaked at 4 hours. This indicated that CYP1A1 could promote the polarization of macrophages to M2. After IL-4 challenge for 2 hours, the expression of p-JAK1 and p-JAK3 protein bands in both cells were significantly enhanced as compared with the PBS control group, but the enhancement of p-STAT6 band in CYP1A1/RAW cells was stronger than that of NC/RAW cells, indicating that CYP1A1 promoted macrophage polarization by promoting phosphorylation of the JAK1/STAT6 pathway. In the meantime, the protein band of Akt, a downstream protein of the PI3K/Akt pathway, in CYP1A1/RAW cells was significantly lower than that of NC/RAW cells, indicating that CYP1A1 did not promote macrophage polarization through this pathway.@*Conclusions@#CYP1A1 promotes the polarization of macrophage to M2 type. The mechanism is related to promoting the phosphorylation of JAK1/STAT6 pathway.
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To investigate the potential effects of cytochrome P450 1A1 (CYP1A1) in regulating macrophages polarize to M2 type and explore the molecular mechanism. Methods All trials were completely randomized. ① Experiment 1: 6-8 weeks old healthy male C57BL/6J mice were collected, and primary peritoneal cells were extracted, then the cells were divided into phosphate buffered saline (PBS) group and interleukin-4 (IL-4) group. The cells in the IL-4 group were stimulated with 10 mg/L IL-4 (M2 macrophage inducer); and those in the PBS group were given with an equal amount of PBS. The mRNA expressions of intracellular M2 type polarized marker molecules including arginase-1 (Arg-1) and chitinase 3 like protein 1 (YM1) at 2, 4, 6 hours after IL-4 challenge were determined by quantitative reverse transcription-polymerase chain reaction (RT-qPCR). The phosphorylation of tyrosine protein kinase 1/signaling transcriptional and transduced activator 6 (JAK1/STAT6) signaling pathway and protein expressions of CYP1A1 and Arg-1 at 6, 12, 24 hours after IL-4 challenge were determined by Western Blot. ② Experiment 2: RAW264.7 cells with high expression CYP1A1 (CYP1A1/RAW) and their negative control cells (NC/RAW) were cultured in vitro. The cells in logarithmic growth phase were collected, and then they were divided into PBS control group and IL-4 group. The treatment method was the same as experiment 1. The phosphorylations of intracellular JAK1/STAT6 and phosphatidylinositol 3-kinase/protein kinase B (PI3K/Akt) pathways in different cells at 1 hour and 2 hours after IL-4 challenge were determined by Western Blot. The mRNA and protein expressions of Arg-1 in different cells at 2 hours and 4 hours after IL-4 challenge were determined by RT-qPCR and Western Blot, respectively. Results ① Experiment 1: after IL-4 challenge for 2 hours, the mRNA expressions of Arg-1 and YM1 in the primary peritoneal macrophages of mice were significantly increased as compared with the PBS control group [Arg-1 mRNA (2-ΔΔCt): 1.75±0.82 vs. 1.00±0.21; YM1 mRNA (2-ΔΔCt): 2.58±0.53 vs. 1.00±0.20, both P < 0.05] which indicated that IL-4 induced macrophage to M2 type polarization successfully. Meanwhile, the mRNA expression of CYP1A1 in polarized mouse peritoneal primary macrophages was also elevated as compared with the PBS control group, and peaked at 4 hours [CYP1A1 mRNA (2-ΔΔCt): 2.25±0.69 vs. 1.00±0.17, P < 0.01]. The results indicated that CYP1A1 expression was enhanced when macrophages polarized to M2 type. Compared with the PBS control group, the bands of phosphorylated JAK1 (p-JAK1), phosphorylated STAT6 (p-STAT6), Arg-1 and CYP1A1 were enhanced in primary peritoneal macrophages of mice in the IL-4 group, and reached the peak value at 12 hours then gradually decreased. This result indicated that the phosphorylation of JAK1/STAT6 pathway was enhanced in M2 macrophages with high expression of CYP1A1, and the pathway was activated. ② Experiment 2: after IL-4 challenge for 2 hours, the expression of Arg-1 mRNA in CYP1A1/RAW cells was significantly higher than that in NC/RAW cells (2-ΔΔCt:3.02±0.60 vs. 1.47±0.43, P < 0.05), and the protein band signal was also stronger, and both peaked at 4 hours. This indicated that CYP1A1 could promote the polarization of macrophages to M2. After IL-4 challenge for 2 hours, the expression of p-JAK1 and p-JAK3 protein bands in both cells were significantly enhanced as compared with the PBS control group, but the enhancement of p-STAT6 band in CYP1A1/RAW cells was stronger than that of NC/RAW cells, indicating that CYP1A1 promoted macrophage polarization by promoting phosphorylation of the JAK1/STAT6 pathway. In the meantime, the protein band of Akt, a downstream protein of the PI3K/Akt pathway, in CYP1A1/RAW cells was significantly lower than that of NC/RAW cells, indicating that CYP1A1 did not promote macrophage polarization through this pathway. Conclusions CYP1A1 promotes the polarization of macrophage to M2 type. The mechanism is related to promoting the phosphorylation of JAK1/STAT6 pathway.
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The objective was to investigate the effect of kinsenoside (Kin) treatments on macrophage polarity and evaluate the resulting protection of chondrocytes to attenuate osteoarthritis (OA) progression. RAW264.7 macrophages were polarized to M1/M2 subtypes then administered with different concentrations of Kin. The polarization transitions were evaluated with quantitative real-time polymerase chain reaction (qRT-PCR), confocal observation and flow cytometry analysis. The mechanism of Kin repolarizing M1 macrophages was evaluated by Western blot. Further, macrophage conditioned medium (CM) and IL-1 were administered to chondrocytes. Micro-CT scanning and histological observations were conducted on anterior cruciate ligament transection (ACLT) mice with or without Kin treatment. We found that Kin repolarized M1 macrophages to the M2 phenotype. Mechanistically, Kin inhibited the phosphorylation of IB, which further reduced the downstream phosphorylation of P65 in nuclear factor-B (NF-B) signaling. Moreover, Kin inhibited mitogen-activated protein kinases (MAPK) signaling molecules p-JNK, p-ERK and p-P38. Additionally, Kin attenuated macrophage CM and IL-1-induced chondrocyte damage. , Kin reduced the infiltration of M1 macrophages, promoted M2 macrophages in the synovium, inhibited subchondral bone destruction and reduced articular cartilage damage induced by ACLT. All the results indicated that Kin is an effective therapeutic candidate for OA treatment.
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Introduction: Salivary components can be used as biomarkers for diagnosing and monitoring oral diseases. There is evidence that one potential biomarker, arginase, is associated with the inflammatory processes of periodontal disease, and its enzymatic activity is reduced according to the improvement in the clinical parameters after treatment. Objective: The present study aimed to evaluate the salivary arginase activity in gingivitis and periodontitis patients treated with full-mouth mechanical procedures combined with the adjunctive use of essential oils or chlorhexidine mouthwash, respectively. Material and method: Twenty-six gingivitis and 16 periodontitis patients received complete periodontal examinations at the baseline and 3 months after therapy, in which the periodontal probing depth, clinical attachment loss, plaque index, and gingival index measurements were taken. At these same appointments, the salivary total protein level and salivary arginase activity were also established via spectrophotometry. Result: There were improvements in all of the clinical parameters (p < 0.05) evaluated from the baseline to 3 months in both groups. In addition, the salivary arginase activity and total protein levels were reduced after the gingivitis treatment. Conclusion: Similar to the clinical results, both therapeutic protocols positively affected the salivary arginase activity; however, further studies are necessary to clarify its potential as a salivary biomarker for periodontal monitoring.
Introdução: Componentes salivares podem ser usados como biomarcadores para diagnóstico e monitoramento de doenças orais. Há evidências de que um potencial biomarcador, arginase, está associado com os processos inflamatórios da doença periodontal, e após o tratamento sua atividade enzimática é reduzida em concordância com a melhora nos parâmetros clínicos. Objetivo: O presente estudo objetivou avaliar a atividade da arginase salivar em pacientes com gengivite e periodontite tratados com procedimentos mecânicos em estágio único combinados ao uso coadjuvante de enxaguatórios com óleos essenciais ou clorexidina, respectivamente. Material e método: Vinte e seis pacientes com gengivite e 16 pacientes com periodontite receberam exame periodontal completo antes e 3 meses após a terapia, em que mensurações de profundidade de sondagem, perda de inserção clínica, índice de placa e índice gengival foram realizadas. Nestas mesmas consultas os níveis de proteína total e a atividade de arginase salivar foram estabelecidos via espectrofotometria. Resultado: Todos os parâmetros clínicos melhoraram, em ambos os grupos, do exame inicial para o de 3 meses (p < 0,05). Adicionalmente, após tratamento para gengivite houve redução da atividade de arginase salivar e do nível de proteína total. Conclusão: Semelhante aos resultados clínicos, ambos os protocolos terapêuticos afetaram positivamente a atividade da arginase salivar; entretanto, estudos futuros são necessários para clarificar seu potencial como biomarcador salivar para o monitoramento periodontal.
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Humans , Periodontitis , Arginase , Saliva , Oils, Volatile , Chlorhexidine , Diagnosis , Gingivitis , Periodontal Diseases , Biomarkers , Mouth DiseasesABSTRACT
Arginase is a urea cycle enzyme which catalyzes the cleavage of L-arginine to L-ornithine,and urea. Arginase deficiency is inherited as an autosomal recessive genetic disorder. Hyperammonemia refers to a condition with elevated levels of ammonia in the blood, which is a product of protein degradation. The lack of the arginase enzyme results in excessive accumulation of nitrogen, in the form of ammonia (hyperammonemia) and arginine (hyperargininemia) in the blood. In the present study, the erythrocyte arginase activity is measured along with plasma ammonia concentration in the newborns and children. The study group consists of 133 subjects which are divided into two groups based on the ammonia level. Group 1 consists of subjects with normal ammonia (n=92) and Group 2 consists of subjects with high ammonia (n=41). We found a significant decrease in arginase activity in the high ammonia group compared to the normal ammonia group. A significant negative correlation between arginase and ammonia is observed in both the groups. The result of this study suggests that arginase deficiency could be the cause for hyperammonemia in these cases. Hence, we suggest that estimation of erythrocyte arginase activity can be used as a screening procedure to detect arginase deficiency in newborns, infants, and children with hyperammonemia
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Viperin is a multifunctional protein that was first identified in human primary macrophages treated with interferon-γ and in human fibroblasts infected with human cytomegalovirus. This protein plays a role as an anti-viral protein and a regulator of cell signaling pathways or cellular metabolism when induced in a variety of cells such as fibroblasts, hepatocytes and immune cells including T cells and dendritic cells. However, the role of viperin in macrophages is unknown. Here, we show that viperin is basally expressed in murine bone marrow cells including monocytes. Its expression is maintained in bone marrow monocyte-derived macrophages (BMDMs) depending on macrophage colony-stimulating factor (M-CSF) treatment but not on granulocyte-macrophage colony-stimulating factor (GM-CSF) treatment. In wild type (WT) and viperin knockout (KO) BMDMs differentiated with M-CSF or G-MCSF, there are little differences at the gene expression levels of M1 and M2 macrophage markers such as inducible nitric oxide synthase (iNOS) and arginase-1, and cytokines such as IL-6 and IL-10, indicating that viperin expression in BMDMs does not affect the basal gene expression of macrophage markers and cytokines. However, when BMDMs are completely polarized, the levels of expression of macrophage markers and secretion of cytokines in viperin KO M1 and M2 macrophages are significantly higher than those in WT M1 and M2 macrophages. The data suggest that viperin plays a role as a regulator in polarization of macrophages and secretion of M1 and M2 cytokines.
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Humans , Bone Marrow , Bone Marrow Cells , Cytokines , Cytomegalovirus , Dendritic Cells , Fibroblasts , Gene Expression , Granulocyte-Macrophage Colony-Stimulating Factor , Hepatocytes , Interleukin-10 , Interleukin-6 , Macrophage Colony-Stimulating Factor , Macrophages , Metabolism , Monocytes , Nitric Oxide Synthase Type II , T-LymphocytesABSTRACT
Endothelial dysfunction is a major risk factor for vascular complication. Nitric oxide (NO),the most important product of endothelial cells,lays a key role in regulating vasomotor as an endogenous vasodilatation factor. NO is produced by endothelial NO synthase (eNOS) on its substrate L-arginine. Arginase,which metabolizes L-arginine to urea and ornithine,competes directly with eNOS for L-arginine leading to the decrease of NO production. This paper reviews the basic role of arginase, the development of arginase inhibitors and its effects on diabetic vascular complications.
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In this study,we compared the serum levels of transforming growth factor-β1 (TGF-β1),interleukin-10 (IL-10),and arginase-1 in long-term survival kidney transplant recipients (LTSKTRs) with those in short-term survival kidney transplant recipients (STSKTRs).We then evaluated the relationship between these levels and graft function.Blood samples were collected from 50 adult LTSKTRs and 20 STSKTRs (graft survival approximately 1-3 years post-transplantation).All patients had stable kidney function.The samples were collected at our institution during the patients' follow-up examinations between March 2017 and September 2017.The plasma levels of TGF-β1,IL-10,and arginase-1 were analyzed using enzyme-linked immunosorbent assays (ELISA).The levels of TGF-β1 and arginase-1 were significantly higher in the LTSKTRs than in the STSKTRs.The time elapsed since transplantation was positively correlated with the levels of TGF-β 1 and arginase-1 in the LTSKTRs.The estimated glomerular filtration rate was positively correlated with the TGF-β1 level,and the serum creatinine level was negatively correlated with the TGF-β1 level.Higher serum levels of TGF-β1 and arginase-1 were found in LTSKTRs than in STSKTRs,and we found that TGF-β1 was positively correlated with long-term graft survival and function.Additionally,TGF-β1 and arginase-1 levels were positively correlated with the time elapsed since transplantation.On the basis of these findings,TGF-β1 and arginase-1 may play important roles in determining long-term graft survival.Thus,we propose that TGF-β1 and arginase-1 may potentially be used as predictive markers for evaluating long-term graft survival.
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In this study,we compared the serum levels of transforming growth factor-β1 (TGF-β1),interleukin-10 (IL-10),and arginase-1 in long-term survival kidney transplant recipients (LTSKTRs) with those in short-term survival kidney transplant recipients (STSKTRs).We then evaluated the relationship between these levels and graft function.Blood samples were collected from 50 adult LTSKTRs and 20 STSKTRs (graft survival approximately 1-3 years post-transplantation).All patients had stable kidney function.The samples were collected at our institution during the patients' follow-up examinations between March 2017 and September 2017.The plasma levels of TGF-β1,IL-10,and arginase-1 were analyzed using enzyme-linked immunosorbent assays (ELISA).The levels of TGF-β1 and arginase-1 were significantly higher in the LTSKTRs than in the STSKTRs.The time elapsed since transplantation was positively correlated with the levels of TGF-β 1 and arginase-1 in the LTSKTRs.The estimated glomerular filtration rate was positively correlated with the TGF-β1 level,and the serum creatinine level was negatively correlated with the TGF-β1 level.Higher serum levels of TGF-β1 and arginase-1 were found in LTSKTRs than in STSKTRs,and we found that TGF-β1 was positively correlated with long-term graft survival and function.Additionally,TGF-β1 and arginase-1 levels were positively correlated with the time elapsed since transplantation.On the basis of these findings,TGF-β1 and arginase-1 may play important roles in determining long-term graft survival.Thus,we propose that TGF-β1 and arginase-1 may potentially be used as predictive markers for evaluating long-term graft survival.
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PURPOSE: Vascular smooth muscle cell (VSMC) proliferation induced by native low-density lipoprotein (nLDL) stimulation is dependent on superoxide production from activated NADPH oxidase. The present study aimed to investigate whether the novel arginase inhibitor limonin could suppress nLDL-induced VSMC proliferation and to examine related mechanisms. MATERIALS AND METHODS: Isolated VSMCs from rat aortas were treated with nLDL, and cell proliferation was measured by WST-1 and BrdU assays. NADPH oxidase activation was evaluated by lucigenin-induced chemiluminescence, and phosphorylation of protein kinase C (PKC) βII and extracellular signal-regulated kinase (ERK) 1/2 was determined by western blot analysis. Mitochondrial reactive oxygen species (ROS) generation was assessed using MitoSOX-red, and intracellular L-arginine concentrations were determined by high-performance liquid chromatography (HPLC) in the presence or absence of limonin. RESULTS: Limonin inhibited arginase I and II activity in the uncompetitive mode, and prevented nLDL-induced VSMC proliferation in a p21Waf1/Cip1-dependent manner without affecting arginase protein levels. Limonin blocked PKCβII phosphorylation, but not ERK1/2 phosphorylation, and translocation of p47phox to the membrane was decreased, as was superoxide production in nLDL-stimulated VSMCs. Moreover, mitochondrial ROS generation was increased by nLDL stimulation and blocked by preincubation with limonin. Mitochondrial ROS production was responsible for the phosphorylation of PKCβII. HPLC analysis showed that arginase inhibition with limonin increases intracellular L-arginine concentrations, but decreases polyamine concentrations. L-Arginine treatment prevented PKCβII phosphorylation without affecting ERK1/2 phosphorylation. CONCLUSION: Increased L-arginine levels following limonin-dependent arginase inhibition prohibited NADPH oxidase activation in a PKCβII-dependent manner, and blocked nLDL-stimulated VSMC proliferation.
Subject(s)
Animals , Rats , Aorta , Arginase , Arginine , Blotting, Western , Bromodeoxyuridine , Cell Proliferation , Chromatography, High Pressure Liquid , Chromatography, Liquid , Lipoproteins , Luminescence , Membranes , Muscle, Smooth, Vascular , NADP , NADPH Oxidases , Phosphorylation , Phosphotransferases , Protein Kinase C , Reactive Oxygen Species , SuperoxidesABSTRACT
Background and purpose: Abnormal expression of arginase 2 (ARG2) in a variety of human malignant tumors was detected. Previous studies found that ARG2 significantly increased in hepatocellular carcinoma (HCC) and was related to histological grading of HCC. This study aimed to analyze the association of ARG2 expression with cell proliferation, apoptosis and prognosis in HCC. Methods: The expression levels of ARG2 mRNA in 14 samples of HCC, paracancerous liver tissues and 14 samples of normal liver were detected by reverse transcriptionpolymerase chain reaction (RT-PCR). Tissue sections from 158 HCC patients were examined immunohistochemically for protein expression of ARG2, proliferation-related proteins (Ki-67 and cyclin D1) and apoptosis-related proteins (activated caspase-3, caspase-8 and caspase-9). Immunofluorescence double labeling method was used to detect the coexpression of ARG2 and activated caspase-3, and the colocalization between ARG2 and apoptotic cells. Patients were followed up by telephone. Results: TThe expression of ARG2 mRNA was significantly increased in HCC compared with the paracancerous liver tissues and normal liver tissues (F=27.10, P＜0.01). The expression of ARG2 was positively correlated with the expression of Ki-67 and cyclin D1, respectively (r=0.247 8, P＜0.01; r=0.372 7, P＜0.01). The expression of ARG2 was positively correlated with the expression of activated caspase-3 and caspase-8, respectively (r=0.191 0, P＜0.05; r=0.180 5, P＜0.05), but not with the caspase-9 (r=0.108 9, P＞0.05). Immunofluorescence double labeling showed that ARG2 was coexpressed with the activated caspase-3 and colocalized with apoptotic cells. Kaplan-Meier survival curves showed that the median survival time was 32 months in ARG2(-) group, 18 months in ARG2(+) group and 15 months in ARG2(++) group. The log-rank test results showed that there were significant differences in median survival time between the groups, and the median survival time in ARG2(-) group was longer than that in ARG2(+) and ARG2(++) groups (χ2=12.278, P＜0.01). Conclusion: ARG2 may be involved in regulating the proliferation and apoptosis of HCC cancer cells. Detecting the expression of ARG2 in HCC tissues may indicate prognosis.
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Purpose To evaluate the expression of Arginine-1 (Arg-1) and Glypican-3 (GPC-3) in hepatocellular carcinoma (HCC) and non-hepatocellular carcinoma,as well as to summarize the related researches.Methods 156 cases of HCC,5 cases of cholangiocarcinoma,20 cases of metastatic adenocarcinoma and 18 cases of other types of tumors were studied.Immunohistochemical study for Arg-1 and GPC-3 was performed on the formalin-fixed and paraffin-embedded tumor tissues.Results The positive expression rates of Arg-1 in HCC and non-hepatocellular carcinoma were 93.6％ (146/156) and 0 (0/43),respectively,meanwhile the expression rate decreased with decreasing of differentiation (r =-0.264,P =0.001).GPC-3 expression was observed in 141 of 156 cases of HCC (90.4％) and 6 of 43 cases of non-hepatocellular carcinoma (14％),and the expression rate increased with decreasing of differentiation (r =0.179,P =0.026).The sensitivity,specificity,positive predictive value and negative predictive value of Arg-1 and GPC-3 in distinguishing HCC from non-hepatocellular carcinoma were 93.6％,100％,100％,81.1％ and 90.4％,86％,96.0％,71.2％,respectively.Conclusion Arg-1 is a sensitive and specific marker of hepatocytes.The application of Arg-1 and GPC-3 is of great significance in diagnosis of HCC and liver metastatic adenocarcinoma.
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Objective To explore the role of the changes in endogenous L-citrulline levels and arginases expression in erectile dysfunction of diabetic rats,and investigate the effect of L-citrulline incubation ex vivo on the erectile dysfunction.Methods Type 2 diabetic rats were induced by high fat diet plus a single intraperitoneal injection of small dose streptozotocin (30 mg/kg),followed by high fat diet for 8 weeks.The erectile function of rats was reflected by the relaxation response to acetylcholine (ACh) of corpus cavernosum detected by isometric tension measurement.Levels of endogenous nitric oxide inhibitor asymmetric dimethylarginine (ADMA) and cyclic guanosine monophosphate (cGMP) were measured by enzyme-linked immunosorbent assay (ELISA).The content of L-citrulline,nitric oxide (NO) and the activity of nitric oxide synthase (NOS) were determined with colorimetric method.Western blotting was applied to detect the protein expression of NOS,arginases and phosphodiesterase 5 (PEDS).Results The relaxation response to ACh of corpus cavernosum from diabetic rats was significantly lower than that of control rats (P ＜ 0.01),indicating the erectile dysfunction of diabetic rats.This erectile dysfunction was companied by the decreases of L-citrulline levels in serum and corpus cavernosum as well as up-regulation of arginaseⅠ & Ⅱ expression in the corpus cavernosum of diabetic rats.Incubation with L-citrulline (3 mmol/L) for 45 min ex vivo improved the relaxation function of corpus cavernosum from diabetic rats (P ＜ 0.05),while incubation with PED5 inhibitor silaenafil (3 μmoL/L) had no significant effect.Furthermore,the ADMA level was increased,while the NOS activity,cGMP and NO contents were decreased in corpus cavernosum of diabetic rats compared to control rats.Down-regulation of endothelial nitric oxide synthase (eNOS) and neuronal nitric oxide synthase (nNOS) protein expression as well as up-regulation of inducible nitric oxide synthase (iNOS) and PDE5 protein expression were also observed in the corpus cavernosum of diabetic rats.Conclusions The decreased endogenous L-citrulline levels and upregulated arginases expression was closely related to erectile dysfunction of diabetic rats,and the mechanism is associated to the up-regulation of arginase activity and decrease of NO synthesis in corpus cavernosum.L-citrulline incubation ex vivo exerted therapeutic effect on the relaxation dysfunction of corpus cavernosum from diabetic rats.
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Objective To investigate the curative effect of microwave ablation for non-small cell lung cancer (NSCLC) patients,and to analyze the serum concentration changes of vascular endothelial growth factor (VEGF),arginase-1 (Arg-1),inducible nitric oxide synthase (iNOS) before and after microwave ablation and their correlations.Methods A total of 30 cases of healthy people (control group) and 30 cases of advanced NSCLC (test group) were selected.The serum concentrations of VEGF,Arg-1 and iNOS in control group and test group (before microwave ablation,the first postoperative day,the third postoperative day and the first postoperative month) were measured by enzyme-linked immunosorbent assay (ELISA).Results The effective rate of microwave ablation for advanced NSCLC was 33.3％ (10/30),and the disease control rate was 70.0％ (21/30).The concentrations of VEGF,Arg-1 and iNOS in test group before microwave ablation were (816.56 ± 13.26)pg/ml,(5.17±0.20) ng/ml and (544.18 ± 13.93)pg/ml,which were higher than those in control group (93.43 ± 9.93) pg/ml,(1.08 ± 0.05) ng/ml and (8.08 ± 0.33) pg/ml,and the differences were statistically significant (t =239.093,P ＜ 0.001;t =110.359,P ＜ 0.001;t =210.792,P ＜ 0.001).The concentrations of VEGF were (708.41 ± 10.49) pg/ml,(592.63 ± 7.25) pg/ml and (521.91 ± 8.32) pg/ml on the first day,third day and 1 month after microwave ablation in patients with advanced NSCLC,which were significantly lower than those before treatment (all P ＜ 0.05).The homologous concentrations of Arg-1 were (5.95 ± 0.10) ng/ml,(7.02 ± 0.13) ng/ml and (7.67 ± 0.92) ng/ml,which were significantly higher than those before treatment (all P ＜ 0.05).The homologous concentrations of iNOS were (453.01 ± 9.48) pg/ml,(393.21 ± 9.42) pg/ml and (352.60 ± 8.31) pg/ml,which were significantly lower than those before treatment (all P ＜ 0.05).The expression of iNOS was positively related with VEGF in NSCLC patients before treatment (r =0.379,P =0.039),and the expression of Arg-1 was negatively related with VEGF (r =-0.556,P =0.001).However,the expression of iNOS was not associated with Arg-1 (r =-0.238,P =0.205).Conclusion Microwave ablation is effective for local therapy of NSCLC,which can directly kill cancer cells,and affect the levels of VEGF,Arg-1 and iNOS.VEGF has certain correlation with iNOS and Arg-1,but there was no correlation between iNOS and Arg-1.Microwave ablation can change the tumor microenvironment in a certain extent,and stimulate the body to produce anti-tumor immunity.
ABSTRACT
Objective To investigate the effect of arginase (Arg) inhibitor N-ω-Hydroxy-L norArginine (nor-NOHA) on high glucose cultured rhesus macaque retinal vascular endothelial cell line (RF/6A) in vitro.Methods The RF/6A cells were divided into the following 4 groups:normal control group (5.0 mmol/L of glucose,group A),high glucose group (25.0 mmol/L,group B),high glucose with 125 mg/L nor-NOHA group (group C),and high glucose with 1％ DMSO group (group D).The proliferation,migration ability and angiogenic ability of RF/6A cells were measured by Methyl thiazolyl tetrazolium (MTT),transwell chamber and tube assay respectively.The express of Arg Ⅰ,eNOS,iNOS mRNA of RF/6A cells were measured by real-time polymerase chain reaction (RT-PCR),Enzyme-linked immuno sorbent assay (ELISA) was used to detect the expression of NO and interleukine (IL)-1b of RF/6A cells.Results The proliferation,migration,and tube formation ability of group A (t=2.367,5.633,7.045;P＜0.05) and group C (t=5.260,6.952,8.875;P＜0.05)were significantly higher than group B.RT-PCR results showed the Arg Ⅰ and iNOS expression in group B was higher than that in group A (t=6.836,3.342;P＜0.05) and group C (t=4.904,7.192;P＜0.05).The eNOS expression in group B was lower than that in group A and group C (t=4.165,6.594;P＜0.05).ELISA results showed NO expression in group B was lower than that in group A and group C (t=4.925,5.368;P＜0.05).IL-1b expression in group B was higher than that in group A and group C (t=5.032,7.792;P＜0.05).Conclusions Nor-NOHA has a protective effect on cultured RF/6A cells in vitro and can enhance its proliferation,migration and tube formation.The mechanism may be inhibiting the oxidative stress by balancing the expression of Arg/NOS.
ABSTRACT
Objective To investigate the curative effect of microwave ablation for non-small cell lung cancer (NSCLC) patients,and to analyze the serum concentration changes of vascular endothelial growth factor (VEGF),arginase-1 (Arg-1),inducible nitric oxide synthase (iNOS) before and after microwave ablation and their correlations.Methods A total of 30 cases of healthy people (control group) and 30 cases of advanced NSCLC (test group) were selected.The serum concentrations of VEGF,Arg-1 and iNOS in control group and test group (before microwave ablation,the first postoperative day,the third postoperative day and the first postoperative month) were measured by enzyme-linked immunosorbent assay (ELISA).Results The effective rate of microwave ablation for advanced NSCLC was 33.3％ (10/30),and the disease control rate was 70.0％ (21/30).The concentrations of VEGF,Arg-1 and iNOS in test group before microwave ablation were (816.56 ± 13.26)pg/ml,(5.17±0.20) ng/ml and (544.18 ± 13.93)pg/ml,which were higher than those in control group (93.43 ± 9.93) pg/ml,(1.08 ± 0.05) ng/ml and (8.08 ± 0.33) pg/ml,and the differences were statistically significant (t =239.093,P ＜ 0.001;t =110.359,P ＜ 0.001;t =210.792,P ＜ 0.001).The concentrations of VEGF were (708.41 ± 10.49) pg/ml,(592.63 ± 7.25) pg/ml and (521.91 ± 8.32) pg/ml on the first day,third day and 1 month after microwave ablation in patients with advanced NSCLC,which were significantly lower than those before treatment (all P ＜ 0.05).The homologous concentrations of Arg-1 were (5.95 ± 0.10) ng/ml,(7.02 ± 0.13) ng/ml and (7.67 ± 0.92) ng/ml,which were significantly higher than those before treatment (all P ＜ 0.05).The homologous concentrations of iNOS were (453.01 ± 9.48) pg/ml,(393.21 ± 9.42) pg/ml and (352.60 ± 8.31) pg/ml,which were significantly lower than those before treatment (all P ＜ 0.05).The expression of iNOS was positively related with VEGF in NSCLC patients before treatment (r =0.379,P =0.039),and the expression of Arg-1 was negatively related with VEGF (r =-0.556,P =0.001).However,the expression of iNOS was not associated with Arg-1 (r =-0.238,P =0.205).Conclusion Microwave ablation is effective for local therapy of NSCLC,which can directly kill cancer cells,and affect the levels of VEGF,Arg-1 and iNOS.VEGF has certain correlation with iNOS and Arg-1,but there was no correlation between iNOS and Arg-1.Microwave ablation can change the tumor microenvironment in a certain extent,and stimulate the body to produce anti-tumor immunity.