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This study aimed to investigate the relationship between coagulating cold and blood stasis syndrome and glycolysis, and observe the intervention effect of Liangfang Wenjing Decoction(LFWJD) on the expression of key glycolytic enzymes in the uterus and ovaries of rats with coagulating cold and blood stasis. The rat model of coagulating cold and blood stasis syndrome was established by ice-water bath. After modeling, the quantitative scoring of symptoms were performed, and according to the scoring results, the rats were randomly divided into a model group and LFWJD low-, medium-and high-dose groups(4.7, 9.4, 18.8 g·kg~(-1)·d~(-1)), with 10 in each group. Another 10 rats were selected as the blank group. After 4 weeks of continuous administration by gavage, the quantitative scoring of symptoms was repeated. Laser speckle flowgraphy was used to detect the changes of microcirculation in the ears and uterus of rats in each group. Hematoxylin-eosin(HE) staining was used to observe the pathological morphology of uterus and ovaries of rats in each group. The mRNA and protein expressions of pyruvate dehydrogenase kinase 1(PDK1), hexokinase 2(HK2) and lactate dehydrogenase A(LDHA) in the uterus and ovaries of rats were examined by real-time quantitative polymerase chain reaction(RT-qPCR) and Western blot, respectively. The rats in the model group showed signs of coagulating cold and blood stasis syndrome, such as curl-up, less movement, thickened veins under the tongue, and reduced blood perfusion in the microcirculation of the ears and uterus, and HE staining revealed a thinning of the endometrium with disorganized arrangement of epithelial cells and a decrease in the number of ovarian follicles. Compared with the model group, the treatment groups had alleviated coagulating cold and blood stasis, which was manifested as red tongue, reduced nail swelling, no blood stasis at the tail end as well as increased blood perfusion of the microcirculation in the ears and uterus(P<0.05 or P<0.01). Among the groups, the LFWJD medium-and high-dose groups had the most significant improvement in coagulating cold and blood stasis, with neatly arranged columnar epithelial cells in uterus, and the number of ovarian follicles was higher than that in the model group, especially mature follicles. The mRNA and protein expressions of PDK1, HK2, LDHA in uterus and ovaries were up-regulated in the model group(P<0.05 or P<0.01), while down-regulated in LFWJD medium-and high-dose groups(P<0.05 or P<0.01). The LFWJD low-dose group presented a decrease in the mRNA expressions of PDK1, HK2 and LDHA in uterus and ovaries as well as in the protein expressions of HK2 and LDHA in uterus and HK2 and PDK1 in ovaries(P<0.05 or P<0.01). The therapeutic mechanism of LFWJD against coagulating cold and blood stasis syndrome is related to the down-regulation of key glycolytic enzymes PDK1, HK2 and LDHA, and the inhibition of glycolytic activities in uterus and ovaries.
Subject(s)
Female , Animals , Rats , Ovary , Uterus , Ovarian Follicle , Lactate Dehydrogenase 5 , GlycolysisABSTRACT
La medición de glucosa en caninos es un procedimiento habitual en la clínica diaria, actualmente este valor se puede obtener mediante dispositivos portátiles y pruebas laboratoriales. Se realizó esta investigación con el fin de aportar mayor conocimiento sobre la importancia de la medición de glucosa, ya que en los últimos años ha perdido valor entre las pruebas hematológicas a considerar debido a que solo se relaciona con determinadas patologías como la diabetes u otras enfermedades metabólicas. El presente trabajo tiene como objetivo comparar los valores de glucosa en caninos obtenidos mediante un glucómetro portátil de uso humano (Accu-chek® Active, Roche Diagnostic, Mannheim, Alemania); veterinario (aLcose® Vet Glu, jjPlus Corporation, New Taipei, Taiwán) y la prueba estándar de laboratorio, esto nos indicará la fiabilidad de los resultados obtenidos mediante estos métodos. Se realizó la toma de muestras de sangre de 50 caninos clínicamente sanos, de los cuales se obtuvo el resultado de glucemia mediante estos tres métodos. Los resultados de nuestra investigación evidenciaron que las tres formas de evaluación de la glucosa sanguínea en perros brindaban resultados estadísticamente diferentes (p < 0.05). Se obtuvo valores de glucosa diferentes entre los tres métodos de medición, teniendo como promedios finales 84.14 mg/dL, 101.12 mg/dL y 91.12 mg/dL correspondientes al glucómetro portátil de uso humano, veterinario y a la prueba estándar de laboratorio respectivamente. En conclusión, los glucómetros portátiles de uso humano subestiman los valores reales de glucosa, mientras que los de uso veterinario lo sobreestiman, comparados con la prueba estándar de laboratorio.
A medição de glicose nos cães é um procedimento habitual realizado no atendimento clínico. Atualmente este valor pode ser obtido por meio de dispositivos portáteis e testes laboratoriais. Esta pesquisa foi realizada com a finalidade de destacar a importância da medição de glicose, visto que nos últimos anos esta avaliação não tem sido muito valorada entre os testes hematológicos, sendo considerada relevante apenas em relação a patologias como a diabetes e outras doenças metabólicas. O presente estudo teve como objetivo comparar os valores de glicose em cães obtidos com glicômetro portátil de uso humano; veterinário e o teste padrão de laboratório. Esta comparação poderá indicar a confiabilidade dos resultados obtidos mediante os métodos avaliados. Foi realizada a amostragem do sangue de 50 caninos clinicamente sadios os quais foram submetidos a avaliação de glicose mediante os três métodos. Os resultados de nossa investigação evidenciaram que as três formas de avaliação da glicose sanguínea têm resultados estatisticamente diferentes (p < 0,05). Os valores de glicose tiveram medias finais de 84,14 mg/dL, 101,12 mg/dL e 91,12 mg/dL para o glicômetro portátil de uso humano (Accu-chek® Active, Roche Diagnostic, Mannheim, Alemanha), veterinário (aLcose® Vet Glu, jjPlus Corporation, Nova Taipei, Taiwan) e o teste padrão de laboratório, respectivamente. Ao concluir, os glicômetros portáteis de uso humano subestimam os valores reais de glicose e os de uso veterinário os superestimam quando comparados com o teste padrão de laboratório.
The measurement of glucose in canines is a common procedure in daily clinical practice. Currently this value can be obtained by use of portable devices and laboratory tests. This research was carried out in order to provide more knowledge about the importance of glucose measurement, since in recent years it has lost value among the hematological tests to be considered because it is only related to certain pathologies such as diabetes or other metabolic diseases. The present study aimed to compare the glucose values in dogs obtained with a portable glucometer for human use, veterinarian use, and the standard laboratory test. This comparison may indicate the reliability of the results obtained through the evaluated methods. A blood sampling of 50 clinically healthy canines was taken and submitted to glucose evaluation using the three methods. Our investigation showed that the three ways of assessing blood glucose have statistically different results (p < 0.05). Glucose values had final averages of 84.14 mg/dL, 101.12 mg/dL, and 91.12 mg/dL for the portable glucometer for human use (Accu-chek® Active, Roche Diagnostic, Mannheim, Germany), veterinary (aLcose® Vet Glu, jjPlus Corporation, New Taipei, Taiwan) and the standard laboratory test, respectively. In conclusion, portable glucometers for human use underestimate the glucose values, and those for veterinary use overestimate them compared to the standard laboratory test.
Subject(s)
Animals , Dogs , Blood Chemical Analysis/veterinary , Blood Glucose/analysis , Blood Glucose Self-Monitoring/veterinary , Dogs/blood , Glucose/analysis , Glucose Tolerance Test/veterinaryABSTRACT
Objective@#To investigate the regulatory relationship of Protein Phosphatase 2 Regulatory Subunit B"Alpha ( PPP2R3A) and hexokinase 1 ( HK1) in glycolysis of hepatocellular carcinoma (HCC).@*Methods@#In HepG2 and Huh7 cells, PPP2R3A expression was silenced by small interfering RNA (siRNA) and overexpression by plasmid transfection. The PPP2R3A-related genes were searched by RNA sequencing. Glycolysis levels were measured by glucose uptake and lactate production. QRT-PCR, ELISA, western blot and immunofluorescence assay were performed to detect the changes of PPP2R3A and HK1. Cell proliferation, migration and invasion assay were used to study the roles of HK1 regulation by PPP2R3A.@*Results@#RNA sequencing data revealed that PPP2R3A siRNA significantly downregulated the expression of HK1. PPP2R3A gene overexpression promotes, while gene silencing suppresses, the level of HK1 and glycolysis in HCC cells. In HCC tissue samples, PPP2R3A and HK1 were colocalized in the cytoplasm, and their expression showed a positive correlation. HK1 inhibition abrogated the promotion of glycolysis, proliferation, migration and invasion by PPP2R3A overexpression in liver cancer cells.@*Conclusion@#Our findings showed the correlation of PPP2R3A and HK1 in the glycolysis of HCC, which reveals a new mechanism for the oncogenic roles of PPP2R3A in cancer.
Subject(s)
Humans , Carcinoma, Hepatocellular/pathology , Cell Line, Tumor , Cell Proliferation , Gene Expression Regulation, Neoplastic , Glycolysis , Hexokinase/metabolism , Liver Neoplasms/pathology , Protein Phosphatase 2/metabolism , RNA, Small Interfering/metabolismABSTRACT
In this study, we aimed to evaluate the toxic effects, changes in life span, and expression of various metabolism-related genes in Caenorhabditis elegans, using RNA interference (RNAi) and mutant strains, after 3-bromopyruvate (3-BrPA) treatment. C. elegans was treated with various concentrations of 3-BrPA on nematode growth medium (NGM) plates, and their survival was monitored every 24 h. The expression of genes related to metabolism was measured by the real-time fluorescent quantitative polymerase chain reaction (qPCR). Nematode survival in the presence of 3-BrPA was also studied after silencing three hexokinase (HK) genes. The average life span of C. elegans cultured on NGM with 3-BrPA was shortened to 5.7 d compared with 7.7 d in the control group. hxk-1, hxk-2, and hxk-3 were overexpressed after the treatment with 3-BrPA. After successfully interfering hxk-1, hxk-2, and hxk-3, the 50% lethal concentration (LC50) of all mutant nematodes decreased with 3-BrPA treatment for 24 h compared with that of the control. All the cyp35 genes tested were overexpressed, except cyp-35B3. The induction of cyp-35A1 expression was most obvious. The LC50 values of the mutant strains cyp-35A1, cyp-35A2, cyp-35A4, cyp-35B3, and cyp-35C1 were lower than that of the control. Thus, the toxicity of 3-BrPA is closely related to its effect on hexokinase metabolism in nematodes, and the cyp-35 family plays a key role in the metabolism of 3-BrPA.
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Objective::To study the effect of Qingzao Jiufei Tang on the expression of key limiting enzymes hexokinase 2(HK2), phosphofructokinase 2(PFK2) and pyruvate kinase M2 (PKM2), and the glucose content in Lewis mice colon cancer cells. Method::A total of 50 male C57BL/6J mice were randomly divided into model group, chemotherapy group, and high, middle and low-dose Qingzao Jiufei Tang groups, with 10 mice in each group. The lung cancer cell model was established by injecting Lewis lung cancer cells into the right axilla. The high, middle and low dose groups were administered at the doses of 11, 5.5, 2.75 g·kg-1·d-1 for 2 weeks before modeling. The drug was administered through intraperitoneal injection at a dose of 50 mg·kg-1·(2 d)-1 in the chemotherapy group. The model group was intragastrically administered with an equal volume of normal saline. After the inoculation, the drug was administered for two weeks. Two weeks later, all of the mice were put to death, and tumor tissues were collected. The mRNA expression of HK2 was detected by Real-time PCR. the protein expression of PFK2 was detected by Western blot, the PKM2 activity was detected by enzyme-linked immunosorbent assay (ELISA). Result::Compared with the model group, mRNA expressions and activity of PKM2 in lung cancer cells of treatment groups were significantly declined, and glucose content increased significantly, with significant differences from those of model group (P<0.01). The PFK2 protein expressions in lung cancer cells of treatment groups (high, medium and low-dose groups) were significantly decreased (P<0.05, P<0.01). Conclusion::Qingzao Jiufei Tang could inhibit Lewis proliferation, and decrease the glucose intake in lung cancer cells. The effect targets may be the key rate-limiting enzymes HK2, PFK2, PKM2.
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Objective: To explore the expression of hexokinase 2 (HK2) in ovarian cancer tissues and its effect on the proliferation of ovarian cancer cells. Methods: The levels of HK2 in cancer tissues of 27 patients and normal tissues of 14 patients were detected with qRT-PCR and immunohistochemical analysis. Overexpression of HK2 by transfected PLHKII-pGFPN3 plasmid and knockdown of HK2 by transfected HK2 siRNA were used in our study. Proliferation of SKOV3 cells was detected by CCK8 method. The expression of HK2 was measured by Western blot. Results: The expression of HK2 in 27 patients with ovarian cancer (92.6%, 25/27) was up-regulated compared with normal tissues (42.9%, 6/14). The expression of HK2 in the cancer tissues of patients with clinical stage III-, pathological grade G3, and distant metastasis was up-regulated significantly (P<0.05). The growth of SKOV3 cells was significantly promoted by overexpression of HK2, and the growth of SKOV3 cells was inhibited by downexpression of HK2. Conclusion: The expression of HK2 is significantly higher in ovarian cancer tissues than in normal ovarian tissues. HK2 can promote the proliferation of ovarian cancer cells.
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Objective@#To analyze the association between hexokinase 2 (HK2) gene expression and clinical pathological characteristics in renal clear cell carcinoma using database and bioinformatics methods.@*Methods@#Oncomine database was used to analyze the most significant differentially expressed genes between renal clear cell carcinoma and non-kidney tissue. The expression levels of mRNA and protein were detected by GPEIA and The Human Protein Atlas database. Correlation of HK2 expression level between clinicopathological features and prognosis of renal clear cell carcinoma was analyzed using LinkedOmics and KM-plotter databases, respectively. The STRING database was used to predict the potential protein interaction mechanism.@*Results@#The most significant difference protein in renal clear cell carcinoma, i.e. HK2, was found, which was highly expressed in renal clear cell carcinoma tissues, and positively correlated with pathological stage, T stage and N stage of renal cell carcinoma (all P<0.05). The overall survival rate of the renal clear cell carcinoma patients with high expression of HK2 was significantly lower than that of the patients with low expression (P<0.05).@*Conclusions@#High expression of HK2 gene may be associated with pathological staging, high T stage, high N stage, and poor prognosis of renal clear cell carcinoma.
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Objective@#To investigate the role of hexokinase Ⅱ in the changes of autophagic flow in cardiomyocytes of mice with ischemia-hypoxia in vitro.@*Methods@#The hearts of totally six male and female C57BL/6 mice aged from 1 to 2 days were isolated to culture primary cardiomyocytes which were used for the following experiments. (1) The cells were divided into 6 groups according to the random number table (the same grouping method below), i. e., normal control 3, 6, and 9 h groups and ischemia-hypoxia 3, 6, and 9 h groups, with 4 wells in each group. After being regularly cultured for 48 h with Dulbecco′s modified Eagle medium/nutrient mixture F12 (DMEM/F12) medium (the same regular culture condition below), the cells in normal control 3, 6, and 9 h groups were cultured with replaced fresh DMEM/F12 medium for 3, 6, and 9 h, respectively, and the cells in ischemia-hypoxia 3, 6, and 9 h groups were cultured with replaced sugar-free serum-free medium in the low-oxygen incubator with a volume fraction of 1% oxygen and a volume fraction of 5% carbon dioxide at 37 ℃ (the same hypoxic culture condition below) for 3, 6, and 9 h, respectively. Cell viability was measured by the cell counting kit 8 (CCK-8) method. (2) The cells were grouped and treated the same as those in experiment (1), with 1 well in each group. Western blotting was used to detect the protein expressions of microtubule-associated protein 1 light chain 3 Ⅰ (LC3Ⅰ), LC3Ⅱ, p62, and hexokinase Ⅱ. (3) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, and ischemia-hypoxia 9 h+ 2-deoxyglucose (2-DG) group, with 4 wells in each group. After a regular culture for 48 h, the cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h; the cells in simple ischemia-hypoxia 9 h group were replaced with sugar-free serum-free medium, and the cells in ischemia-hypoxia 9 h+ 2-DG group were replaced with sugar-free serum-free medium in which 2-DG was dissolved in a concentration of 10 mmol/L (20 μmol), and then they were cultured with hypoxia for 9 h. Cell viability was measured by CCK-8 method. (4) The cells were grouped and treated the same as those in experiment (3), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, and p62. (5) The cells were grouped and treated the same as those in experiment (3), with 2 wells in each group. Transmission electron microscope was used to observe autophagosomes/autolysosomes in cardiomyocytes. (6) The cells were divided into normal control group, simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ hexosinase Ⅱ small interfering RNA1 (HK-ⅡsiRNA1) group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group, with 4 wells in each group. The cells in normal control group and simple ischemia-hypoxia 9 h group were regularly cultured for 48 h, and the cells in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were respectively transfected with 200 nmol/L HK-ⅡsiRNA1 and HK-ⅡsiRNA2 and then also cultured for 48 h. The cells in normal control group were cultured with replaced fresh DMEM/F12 medium for 9 h, and the cells in simple ischemia-hypoxia 9 h group, ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group, and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were cultured with replaced sugar-free serum-free medium and hypoxia for 9 h. Cell viability was measured by CCK-8 method. (7) The cells were grouped and treated the same as those in experiment (6), with 1 well in each group. Western blotting was used to detect the protein expressions of LC3Ⅰ, LC3Ⅱ, p62, and hexokinase Ⅱ. Except for experiment (5), each experiment was repeated 3 times. Data were processed with one-way analysis of variance and lest significant difference t test, and Bonferroni correction.@*Results@#(1) The viabilities of cardiomyocytes in ischemia-hypoxia 3, 6, and 9 h groups were 0.450±0.022, 0.385±0.010, and 0.335±0.015, respectively, which were significantly lower than 0.662±0.026, 0.656±0.028, and 0.661±0.021 of the corresponding normal control 3, 6, and 9 h groups, respectively (t=6.21, 9.12, 12.48, P<0.01). (2) Compared with those of corresponding normal control 3, 6, and 9 h groups, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 3, 6, and 9 h groups were significantly increased (t3 h=16.15, 10.99, 5.30, t6 h=6.79, 10.42, 9.42, t9 h=15.76, 16.51, 7.20, P<0.05 or P<0.01). (3) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.353±0.022, which was significantly lower than 0.673±0.027 of normal control group (t=9.29, P<0.01). The viability of cardiomyocytes in ischemia-hypoxia 9 h+ 2-DG group was 0.472±0.025, which was significantly higher than that of simple ischemia-hypoxia 9 h group (t=3.60, P<0.05). (4) Compared with those of normal control group, the LC3Ⅱ/Ⅰ ratio and protein expression of p62 in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=9.45, 8.40, P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰratio and protein expression of p62 in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group were significantly decreased (t=4.39, 4.74, P<0.05). (5) In cardiomyocytes of normal control group, only single autophagosome/autolysosome with bilayer membrane structure was observed. Compared with that of normal control group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of simple ischemia-hypoxia 9 h group was increased significantly. Compared with that of simple ischemia-hypoxia 9 h group, the number of autophagosome/autolysosome with bilayer membrane structure in cardiomyocytes of ischemia-hypoxia 9 h+ 2-DG group was significantly decreased. (6) The viability of cardiomyocytes in simple ischemia-hypoxia 9 h group was 0.358±0.023, which was significantly lower than 0.673±0.026 in normal control group (t=9.12, P<0.01). The viabilities of cardiomyocytes in ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were 0.487±0.027 and 0.493±0.022, respectively, which were significantly higher than the viability in simple ischemia-hypoxia 9 h group (t=3.63, 4.28, P<0.05). (7) Compared with those of normal control group, the LC3Ⅱ/Ⅰratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of simple ischemia-hypoxia 9 h group were significantly increased (t=6.08, 6.31, 4.83, P<0.05 or P<0.01). Compared with those of simple ischemia-hypoxia 9 h group, the LC3Ⅱ/Ⅰ ratio and protein expressions of p62 and hexokinase Ⅱ in cardiomyocytes of ischemia-hypoxia 9 h+ HK-ⅡsiRNA1 group and ischemia-hypoxia 9 h+ HK-ⅡsiRNA2 group were significantly decreased (t=5.10, 7.76, 15.33, 4.17, 8.42, 12.11, P<0.05 or P<0.01).@*Conclusions@#Ischemia-hypoxia upregulates the expression level of hexokinase Ⅱ protein in mouse cardiomyocytes cultured in vitro, which decreases the viability of cardiomyocytes by impairing autophagic flow. To inhibit the activity of hexokinase Ⅱ or its expression can alleviate the ischemia-hypoxia damage of cardiomyocytes.
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Objective To analyze the association between hexokinase 2 (HK2) gene expression and clinical pathological characteristics in renal clear cell carcinoma using database and bioinformatics methods. Methods Oncomine database was used to analyze the most significant differentially expressed genes between renal clear cell carcinoma and non-kidney tissue. The expression levels of mRNA and protein were detected by GPEIA and The Human Protein Atlas database. Correlation of HK2 expression level between clinicopathological features and prognosis of renal clear cell carcinoma was analyzed using LinkedOmics and KM-plotter databases, respectively. The STRING database was used to predict the potential protein interaction mechanism. Results The most significant difference protein in renal clear cell carcinoma, i.e. HK2, was found, which was highly expressed in renal clear cell carcinoma tissues, and positively correlated with pathological stage, T stage and N stage of renal cell carcinoma (all P<0.05). The overall survival rate of the renal clear cell carcinoma patients with high expression of HK2 was significantly lower than that of the patients with low expression (P<0.05). Conclusions High expression of HK2 gene may be associated with pathological staging, high T stage, high N stage, and poor prognosis of renal clear cell carcinoma.
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Objective To evaluate the effect of sildenafil on the expression of hexokinase-2 (HK-2) in lung tissues in a rat model of pulmonary arterial hypertension. Methods Thirty-two male Sprague-Dawley rats, aged 2 months, weighing 200-220 g, were allocated into 4 groups with 8 rats in each group using a random number table: control group ( group C), pulmonary arterial hypertension group ( group PAH), low-dose sildenafil group (group S1 ) and high-dose sildenafil group (group S2 ). The model of pul-monary arterial hypertension was established through combining left pneumonectomy with subcutaneous in-jection of 60 mg∕kg monocrotaline. Two percent sildenafil 30 and 50 mg∕kg were administered by intragastric gavage once a day for 3 consecutive weeks starting from 5 weeks after pneumonectomy in S1 and S2 groups, respectively. The chest was opened after the end of administration for measurement of mean pulmonary arte-rial pressure (mPAP) and right ventricular systolic pressure (RVSP). The hearts and lungs were excised for determination of the percentage of the thickness of tunica media of pulmonary arterioles, size of right ventricular cardiomyocytes, α-smooth muscle actin (α-SMA) expression (by immunohistochemistry) and expression of hypoxia-inducible factor-1α (HIF-1α) and HK-2 ( by Western blot). Results Compared with group C, the mPAP, RVSP and percentage of MT were significantly increased, the size of right ven-tricular cardiomyocytes was enlarged, and the expression of HIF-1α, HK-2 and α-SMA was up-regulated in PAH and S1 groups, and the RVSP and percentage of MT were significantly increased, the size of right ventricular cardiomyocytes was enlarged, and the expression of HIF-1α, HK-2 and α-SMA was up-regula-ted (P<0. 05), and no significant change was found in mPAP in group S2 (P>0. 05). Compared with group PAH, the percentage of MT was significantly decreased, the size of right ventricular cardiomyocytes was decreased, and the expression of HIF-1α, HK-2 and α-SMA was down-regulated in group S1 , and the mPAP, RVSP and percentage of MT were significantly decreased, the size of right ventricular cardiomyo-cytes was decreased, and the expression of HIF-1α, HK-2 and α-SMA was down-regulated in group S2 (P<0. 05). Conclusion The mechanism by which sildenafil inhibits proliferation of pulmonary vascular smooth muscle cells is related to inhibiting the expression of HK-2 in lung tissues in a rat model of pulmona-ry arterial hypertension.
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Objective To analyze the expression of glucose transport protein (Glut)-l,Glut-3 and hexokinase (HK)-Ⅱ in non-small-cell lung cancer (NSCLC) lesions and pulmonary inflammatory lesions and discuss the correlation of them with 18F-fluorodeoxyglucose (FDG) uptake.Methods Twenty-four patients with NSCLC and 22 patients with pulmonary inflammatory lesions (25 males,21 females;age range:37-81 years) who underwent PET/CT from November 2012 to May 2016 were retrospectively analyzed.All patients had surgery and were confirmed by pathology.The expression of Glut-1,Glut-3 and HK-Ⅱ in the lesions was detected by immunohistochemistry.Immunohistochemical staining scores and maximum standardized uptake value (SUVmax) were calculated.One-way analysis of variance,the least significant difference t test,two-sample t test and Spearman correlation analysis were used.Results The SUVmax of NSCLC lesions was 8.71 ± 7.62,higher than that of pulmonary inflammatory lesions (3.29 ± 2.16;t =3.220,P＜ 0.05).Immunohistochemical staining scores of Glut-1,Glut-3 and HK-Ⅱ were 3.75±0.99,4.04±1.00 and 4.00±0.78 for NSCLC lesions respectively,and were all higher than those of pulmonary inflammatory lesions (2.32±0.65,2.89±0.83,2.41±0.50;t values:5.340,5.160,8.130,all P＜0.01).The expression of Glut-1 and HK-Ⅱ was positively correlated with SUVmax in NSCLC lesions (rs values:0.414,0.457,both P＜0.05).The expression of Glut-1,Glut-3 and HK-Ⅱ was not correlated with SUVmax(rs values:0.392,0.070,-0.066,all P＞0.05),but the expression of Glut-3 was higher than that of Glut-1 and HK-Ⅱ (F=4.123,t values:0.970,0.150,all P＜0.05) in pulmonary inflammatory lesions.Conclusions The expression of Glut-1,Glut-3 and HK-Ⅱ is higher in NSCLC lesions than that in pulmonary inflammatory lesions.Glut-1 and HK-Ⅱ are the important factors for 18F-FDG uptake in NSCLC.Glut-3 may play an important role in 18F-FDG uptake in pulmonary inflammatory lesions.
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Objective To explore the clinical characteristics and gene analysis of hexokinase deficiency (HKD). Methods Clinical symptoms, hemolysis, laboratory findings and gene analysis of a boy with HKD in our department were retrospectively analyzed, and the literatures of HKD were reviewed. Results The patient was a six months old boy presented with neonatal hyperbilirubinemia, nonspherocytic hemolyticanemia, and increased proportion of reticulocytes. Genetic testing found two compound heterozygous mutations in HK1: c.995+5G > A (intron 12) inherited from father and c.2216G C (exon 20) inherited from the mother. In the literature, clinical features of the HKD patients were mainly anemic, neonatal jaundice and hepatosplenomegaly, and the gene detection mainly includes point mutation in HK1 gene exon and intron nucleotide. Conclusions In the case with neonatal anemia, jaundice, increased indirect bilirubin, HKD should be considered. Gene analysis can be used for early diagnosis.
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Objective To study the stability of the plasma glucose in sample tubes containing different glycolysis inhibitors and anticoagulants at room temperature for up to 24 h.Methods Venous blood was collected from 20 volunteers.Each donor blood sample was divided into six tubes containing different glycolysis inhibitors and anticoagulants and kept at room temperature.Plasma glucose was measured at 0,2,4,8 and 24 h after collection.The difference of plasma glucose levels for each sample tube was com-pared with different placed time and the difference of plasma glucose levels for the same placed time was compared with different sample tubes.Results The differences of the plasma glucose results of 2 hours after sampling and of instant detecting are statisti-cally significant for each sample tube(P<0.05).The differences of the plasma glucose levels among the three sample tubes contai-ning sodium fluoride are not statistically significant at the same placed time(P>0.05).The differences of the plasma glucose results of 2,4,8,24 h after sampling for each sample tube are not statistically significant(P>0.05).The differences of the plasma glucose results for each sample tube containing sodium iodoacetate are statistically significant at different placed times(P< 0.05).The differences of plasma glucose levels of 0,2,4 hours after sampling for the three sample tubes are no statistically significant(P>0.05)and the differences of plasma glucose levels of 8,24 hours after sampling for the three sample tubes are statistically signifi-cant(P<0.05).The differences of plasma glucose levels for the two glycolytic inhibitors are statistically significant at 4,8,24 hours after sampling(P<0.01)except 2 hours after sampling(P>0.05).Conclusion Tubes containing NaF are more suitable for detec-ting venous plasma glucose within 24 h at room temperature.The type of anticoagulant has no obvious correlation with the ability of glycolytic inhibition.
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Objective To investigate the interference effects of vitamin C on three different detection methods of blood glucose.Methods According to CLSI EP7-A2 document,5% volume of vitamin C(series concentrations) solution was added to fresh mixed serum,then hexokinase method(A),glucose oxidase method(B) and glucose reductase electrode method(C) were used to detect the level of fresh mixed serum glucose.The interference level of vitamin C on three different detection methods of blood glucose was evaluated.Results In 1 250 mg/L vitamin C,there was no interference on the method A for detecting blood glucose.In 156 mg/L vitamin C,it caused unacceptable negative interference on the method B and unacceptable positive interference on the method C.Moreover the interference degree of these two methods was increased with vitamin C concentration increase,but the same concentration of vitamin C had no significant difference on detecting different blood glucose levels by the same detection method.Conclusion High concentration of vitamin C can cause significant interference on glucose detection by glucose oxidase method and glucose reductase electrode method.
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Objective:To investigate the effects of berberine on glycolysis in breast cancer cells and its effect on hexokinase Ⅱ. Methods:The inhibitory effects of berberine on the proliferation of breast cancer cells were studied by MTT with human breast cancer cell line MDA-MB-231 and MCF-7. The glucose consumption and lactic acid in breast cancer cells were detected to evaluate the effect of berberine on glycolysis in breast cancer cells. The energy supply situation of breast cancer cells was evaluated after the detection of ATP and NAD+/NADH. Finally,the hexokinase Ⅱ activity and protein content were also detected in breast cancer cells.Results:The experimental results showed that berberine had an obvious inhibitory effect on the proliferation of human breast cancer cell line MDA-MB-231 and MCF-7 cell in a concentration-dependent manner. While reducing the cell ATP content and increasing NAD+/NADH con-tent(P<0.05),berberine could clearly reduce the consumption of glucose and lactic acid content in the different breast cancer cell lines. In addition,berberine could inhibit the activity and protein content of hexokinase Ⅱ in breast cancer cells.Conclusion:Berber-ine can significantly inhibit the proliferation and the glycolysis of breast cancer cells,reduce the energy supply and obviously inhibit the expression of hexokinase Ⅱ in breast cancer cells as well.
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The energy for tumor cells mainly derives from the aerobic glycolysis,that is,the Warburg effect,which also provides a large amount of precursor substances for the growth of tumor cells.Hexokinase-Ⅱ (HK-Ⅱ),highly expressed in tumor tissue,is the rate-limiting enzyme of glycolysis and closely related to the energy metabolism of tumor.Recent studies have showed that HK-Ⅱ not only mediates Warburg effect,but also promotes tumor proliferation by inhibiting tumor cell apoptosis and regulating autophagy.It has been confirmed that blocking HK-Ⅱ gene expression and inhibiting HK-Ⅱ with small molecule inhibitor can kill tumor cells in many kinds of cancer.Agent targeting HK-Ⅱ may become a new generation of targeted drugs.
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Objective: To investigate the activity of celastrol on the proliferation and the mechanism of energy metabolism to human gastric cancer cells (SGC-7901) and human umbilical vein endothelial cells (ECV304). Methods: SGC-7901 and ECV304 were determined to analyze the proliferation inhibitory rate of celastrol to two kinds of cells by MTT method and growth curve; HE staining method was used to observe the morphological changes; Using spectrophotometric method, the activities of the enzymes in glycolytic pathway (hexokinase, pyruvate kinase, and lactate dehydrogenase) were determined, the enzyme in the tricarboxylic acid cycle (succinate dehydrogenase), and the level of ATP which was the end-products in energy metabolism were determined; The Western blotting method was used to determine the expression levels of hypoxia inducible factor (HIF-1α) and single carboxyl transporter (MCF-4). Results: The proliferation inhibition of celastrol to SGC-7901 and ECV304 cells showed in a time-and dose-dependent manner, led to morphologic changes of cells, reduced the activity of HK, LDH, and SDH, lowered the level of ATP; There was no effect to PK. The protein expression levels of HIF-1α and MCF-4 were significantly reduced. The inhibition of celastrol to SGC-7901 was stronger than that of ECV304 cells. Conclusion: Celastrol influence energy metabolism of the two cells by reducing the expression levels of HIF-1α and MCF-4 is significant, and then proliferation can be inhibited. It shows a double inhibition on human gastric cancer cells and angiogenesis of tumor.
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Objective To investigate the role of hypoxia-inducible factor-1α/ hexokinase Ⅱ (HIF-1α/HKⅡ) signaling pathway in the inhibition of hypoxia/reoxygenation (H/R)-induced apoptosis in the rat cardiomyocytes by hypoxic preconditioning (HPC).Methods Primarily cultured cardiomyocytes obtained from the neonatal rats were seeded in culture dishes at the density of 5× 105cells/ml.The cardiomyocytes were attached to the wall for 72 h and then randomly divided into 6 groups (n=15 each) using a random number table:control group (group C);group H/R;group HPC;HIF-1o inhibitor YC-1 group (group YC-1);group HPC + YC-1;dimethyl sulfoxide (DMSO) group.The ceils were exposed to D-Hank solution saturated with 95％ N2 and 5％ CO2 for 4 h,and then cultured in the normal culture atmosphere for 2 h.The cells in YC-1 and DMSO groups were incubated in the culture medium containing 10 μmol/l YC-1 and 0.1％ DMSO (100 μl) for 24 b,respectively.HPC was induced by 3 cycles of 10 min hypoxia followed by 30 min reoxygenation,and H/R injury model was then established in group HPC.In group HPC+YC-1,the cells were incubated for 5 min in the M199 culture medium supplemented with 10 μmol/L YC-1 and 10％ fetal bovine serum,and the other treatments were similar to those previously described in group HPC.After the end of treatments,the apoptosis in cardiomyocytes was detected by TUNEL,the expression of HIF-1α,HKⅡ and cytochrome c was detected by Western blot,and the mitochondrial membrane potential was quantitatively measured using fluorescence.Apoptotic rate was calculated.Results Compared with group C,the apoptotic rate was significantly increased,the mitochondrial membrane potential was significantly decreased,and the expression of HIF-1α,HKⅡ and cytochrome c was significantly up-regulated in group H/R (P＜0.05).Compared with group H/R,the apoptotic rate was significantly decreased,and the mitochondrial membrane potential was significantly increased,and the expression of HIF-1αt and HKⅡ was significantly up-regulated,and the expression of cytochrome c was significantly down-regulated in group HPC (P＜0.05).Compared with group HPC,the apoptotic rate was significantly increased,the mitochondrial membrane potential was significantly decreased,the expression of HIF-1α and HKⅡ was significantly down-regulated,and the expression of cytochrome c was significantly up-regulated in group HPC+YC-1 (P＜0.05).Conclusion The mechanism by which HPC inhibits H/R-induced apoptosis in rat cardiomyocytes is associated with activation of HIF-1α/HKⅡ signaling pathway.
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After renal injury, selective damage occurs in the proximal tubules as a result of inhibition of glycolysis. The molecular mechanism of damage is not known. Poly(ADP-ribose) polymerase (PARP) activation plays a critical role of proximal tubular cell death in several renal disorders. Here, we studied the role of PARP on glycolytic flux in pig kidney proximal tubule epithelial LLC-PK1 cells using XFp extracellular flux analysis. Poly(ADP-ribosyl)ation by PARP activation was increased approximately 2-fold by incubation of the cells in 10 mM glucose for 30 minutes, but treatment with the PARP inhibitor 3-aminobenzamide (3-AB) does-dependently prevented the glucose-induced PARP activation (approximately 14.4% decrease in 0.1 mM 3-AB-treated group and 36.7% decrease in 1 mM 3-AB-treated group). Treatment with 1 mM 3-AB significantly enhanced the glucose-mediated increase in the extracellular acidification rate (61.1±4.3 mpH/min vs. 126.8±6.2 mpH/min or approximately 2-fold) compared with treatment with vehicle, indicating that PARP inhibition increases only glycolytic activity during glycolytic flux including basal glycolysis, glycolytic activity, and glycolytic capacity in kidney proximal tubule epithelial cells. Glucose increased the activities of glycolytic enzymes including hexokinase, phosphoglucose isomerase, phosphofructokinase-1, glyceraldehyde-3-phosphate dehydrogenase, enolase, and pyruvate kinase in LLC-PK1 cells. Furthermore, PARP inhibition selectively augmented the activities of hexokinase (approximately 1.4-fold over vehicle group), phosphofructokinase-1 (approximately 1.6-fold over vehicle group), and glyceraldehyde-3-phosphate dehydrogenase (approximately 2.2-fold over vehicle group). In conclusion, these data suggest that PARP activation may regulate glycolytic activity via poly(ADP-ribosyl)ation of hexokinase, phosphofructokinase-1, and glyceraldehyde-3-phosphate dehydrogenase in kidney proximal tubule epithelial cells.
Subject(s)
Animals , Cell Death , Epithelial Cells , Glucose , Glucose-6-Phosphate Isomerase , Glycolysis , Hexokinase , Kidney , LLC-PK1 Cells , Oxidoreductases , Phosphofructokinase-1 , Phosphopyruvate Hydratase , Poly Adenosine Diphosphate Ribose , Poly(ADP-ribose) Polymerases , Pyruvate Kinase , SwineABSTRACT
BACKGROUND: Low survival rate of transplanted cells compromises the efficacy of cell therapy. Hexokinase II (HKII) is known to have anti-apoptotic activity through its interaction with mitochondria. The objective was to identify miRNAs targeting HKII and investigate whether miRNA-mediated modulation of HKII could improve the survival of mesenchymal stem cells (MSCs) exposed to H2O2. The expression of HKII in MSCs exposed to H2O2 was evaluated, and HKII-targeting miRNA was screened based on miRNA-target prediction databases. The effect of H2O2 on the expression of the selected HKII-targeting miRNA was examined and the effect of modulation of the selected HKII-targeting miRNA using anti-miRNA on H2O2-induced apoptosis of MSC was evaluated. RESULTS: H2O2 (600 µM) induced cell death of MSCs and decreased mitochondrial HKII expression. We have identified miR-181a as a HKII-targeting miRNA and H2O2 increased the expression of miR-181a in MSCs. Delivery of anti-miR-181a, which neutralizes endogenous miR-181a, significantly attenuated H2O2-induced decrease of HKII expression and disruption of mitochondrial membrane potential, improving the survival of MSCs exposed to H2O2. CONCLUSIONS: These findings suggest that H2O2-induced up-regulation of miR-181a contributes to the cell death of MSCs by down-regulating HKII. Neutralizing miR-181a can be an effective way to prime MSCs for transplantation into ischemic tissues.