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OBJECTIVE To investigate the mechanism of catalpol affecting the differentiation of helper T cell 17 （Th17） by interfering the expressions of pyruvate kinase M2 （PKM2） and lactate dehydrogenase A （LDHA）. METHODS The naive CD4+ T cells were selected from the spleen of C57BL/6 mice， and were differentiated into Th17 cells by adding directional differentiation stimulants for 72 hours. At the same time， the cells were treated with 0 （directed control）， 20， 40 and 80 μg/mL catalpol. The flow cytometry was used to detect the proportion of Th17 cell differentiation in cells； the colorimetric method was adopted to detect the levels of pyruvate and lactate in cell culture supernatant； mRNA expressions of retinoid-related orphan nuclear receptor gamma t （RORγt）， PKM2 and LDHA were detected by qRT-PCR method； Western blot was used to detect the expression levels of PKM2， LDHA， signal transducer and activator of transcription 3 （STAT3）， and phosphorylated STAT3 （p-STAT3） proteins in cells. RESULTS Compared with the directed control group， after 72 hours of treatment with 20， 40， 80 μg/mL catalpol， the differentiation ratio of Th17 cells were decreased by 6.74%， 8.41%， 9.24%， and the levels of pyruvate and lactate in the cell culture supernatant， the mRNA expressions of PKM2， LDHA and RORγt as well as the protein expressions of PKM2 and LDHA and the phosphorylation of STAT3 were significantly reduced （P＜0.05）. CONCLUSIONS Catalpol can reduce the glycolysis level by down-regulating the expressions of PKM2 and LDHA， thereby inhibiting the differentiation of Th17 cells.
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ObjectiveTo analyze the expression of Lactate dehydrogenase A（LDHA） in both renal cell carcinoma （RCC） tissue and RCC cell lines， and to investigate the impact of LDHA expression on the progression of RCC. MethodsFrom June 2018 to June 2022， totally 52 cases of RCC tissue samples and 49 cases of para-cancerous tissue samples were collected through surgical procedures from our hospital. LDHA expression was detected using immunohistochemistry （IHC）. The expression levels of LDHA in vitro were also detected in the normal human proximal tubule epithelial cell line HK-2 and renal cell carcinoma cell lines A498， Caki-2， ACHN， and 786-O by using qRT-PCR and Western blot. A recombinant plasmid carrying LDHA-shRNA was constructed and then transfected into 786-O cells to down-regulate the expression of LDHA. Tumor proliferative capacity was monitored using CCK-8 assay， clonal formation assay and EdU assessments. Additionally， cell glycolytic activity was assessed through glucose uptake assay， lactate secretion assay， and ECAR analysis. ResultsIHC analysis revealed significantly higher expression of LDHA in RCC tissue compared to adjacent tissues（P<0.05）. Furthermore， RCC tissues with higher TNM stage exhibited greater expression of LDHA than those with lower TNM stage （P<0.05）. The results of qRT-PCR and Western blot demonstrated that the expression of LDHA in each RCC cell line was significantly higher than that in HK-2（P<0.05）. After blocking the expression of LDHA in 786-O， there was a significant down-regulation of cell proliferation and glycolysis capacity （P<0.05）. ConclusionsThe expression of LDHA in RCC tissue and RCC cell lines is significantly overexpressed compared with normal one， particularly in those with higher TNM stage. Knockdown of the expression of LDHA significantly suppresses cell proliferation and aerobic glycolysis capacity in 786-O.
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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.
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Female , Animals , Rats , Ovary , Uterus , Ovarian Follicle , Lactate Dehydrogenase 5 , GlycolysisABSTRACT
@#Oral lichen planus (OLP) is a chronic inflammatory disease of the oral mucosa. The pathogenesis of OLP is still unclear. Immune abnormalities mediated by T cells and related cytokines play a crucial role in the pathogenesis of OLP. In recent years, glycolytic metabolism-related transporters, enzymes and regulators, such as glucose transporter-1 (Glut1), glyceraldehyde 3-phosphate dehydrogenase (GAPDH), lactate dehydrogenase A (LDHA), mammalian target of rapamycin (mTOR) and hypoxia inducible factor-1α (HIF-1a), have attracted an increasing amount of attention in OLP by regulating the proliferation and differentiation of T cells and the secretion of inflammatory factors. It has been shown that 2-deoxy-D-glucose (2-DG) or rapamycin (RAPA) inhibits the glycolytic metabolism of T cells and then inhibits OLP. This article reviews the research progress of glycolytic metabolism-related transporters, enzymes and regulatory factors in OLP in recent years.
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Objective:To investigate the effect of tumor-associated macrophage(TAM) on proliferation of renal carcinoma cells and its related mechanism.Methods:The model of TAM was established by stimulating human monocytic leukemia cell line THP-1 with phorbol myristate acetate (PMA), bacterial endotoxin (LPS) and interferon-γ (IFN- γ). Then the TAM model was co-cultured with carcinoma cell lines ACHN and 786-O in vitro .The cytokines IL-6, TNF-α and IL-1β in TAM supernatant were detected by enzyme-linked immunosorbent assay (ELISA). MTT method was used to detect the proliferation of ACHN and 786-O cells treated with supernatant of TAM or TAM/Tocilizumab. Western blot was used to detect lactate dehydrogenase A (LDHA) expression of both renal cancer cells co-cultured with TAM or TAM/Tocilizumab. The ACHN and 786-O cells with LDHA-overexpression and LDHA-knockdown were cultured in TAM supernatant in vitro. The cell proliferation was detected by MTT and the relative proliferation rate was calculated.Results:THP-1 cells was differentiated into TAM through the treatment of 80 ng/ml PMA combined with 20 ng/ml LPS and 20 ng/ml IFN- γ.The expression rate of CD68, a cell surface marker on TAM, was (36.2 ±4.5)%. When TAM was co-cultured with ACHN cells, the results of ELISA showed that the secretion of IL-6 in the supernatant was significantly elevated compared with that in the supernatant when ACHN cells cultured alone [(138.0 ±12.4) pg/ml and (19.7±4.9) pg/ml], and the secretion of TNF- α [(122.5 ±14.2) pg/ml and (12.6 ±2.3) pg/ml] and IL-1 β [(89.2 ±6.4) pg/ml and (69.2 ±3.5) pg/ml] were also significantly increased. The secretion of IL-6 [(119.2 ±14.8) pg/ml and (17.1 ±3.3) pg/ml], TNF- α [(122.6 ±14.4) pg/ml and (45.7 ±7.2) pg/ml] and IL-1 β [(95.1 ±11.8) pg/ml and (88.2 ±12.7) pg/ml] in the supernatant were also significantly elevated when 786-O cells co-cultured with TAM compared with 786-O cells cultured alone. After treated with the supernatant of TAM for 72 hours, the relative proliferation rates of ACHN and 786-O cells [(128.6 ±21.4)% and (124.2 ±19.7)%] were significantly higher than that of the control group (100.0%). At the same time, the expression of LDHA in ACHN and 786-O cells increased significantly. After 72 hours of treatment with the supernatant of TAM combined with tocilizumab, the relative proliferation rates of ACHN and 786-O cells [(76.5±13.7)% and (74.8±12.5)%] were significantly lower than that of the control group(100.0%), and the expression of LDHA was also significantly decreased at the same time. The relative proliferation rates of ACHN and 786-O cells in LDHA overexpression group [(121.5 ±17.2)% and (122.7±21.6)%]were significantly higher than that in blank-vector-transfection group[(93.3±10.7)% and (89.8±11.2)%], while the relative proliferation rates in LDHA-knockdown group [(61.4±11.2)% and (58.0 ±10.6)% ]were significantly lower than that in blank-vector-transfection group.Conclusions:By secreting IL-6, TAM can up-regulate the expression of LDHA and promote the proliferation of renal cancer cells.
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Objective To construct the eukaryotic expression vector of LDHA with Flag label and detect its effects on the growth of human choroidal melanoma (CM) MUM-2B cells.Methods CM cells line MUM-2B subcultured by the Military Academy of Sciences were divided into two groups:experimental group and control group.The experimental group was transiently transfected with Flag-LDHA plasmid,and the control group was transiently transfected with Flag plasmid.Using the Flag-LDHA with GST label as a template,the LDHA gene was amplified by polymerase chain reaction (PCR),which then was inserted into eukaryotic expression vector of Flag,and the recombinant plasmid Flag-LDHA was identified by bacterial liquid PCR,double enzyme digestion and sequencing,both which were transiently transfected into human CM MUM-2B cells after successful identification,and finally,its expression was determined by Western blot.The biology behaviors of melanoma cell line MUM-2B transfected with Flag-LDHA and Flag plasmid were analyzed by counting Kit-8 (CCK8) assays.Results The coding region sequence of LDHA gene of approximately 1000 bp was harvested from PCR amplification,which was successfully cloned into the Flag vector.Compared with the control group,the PCR result of the bacterial liquid in the experimental group was positive.The double enzyme digestion results showed that eukaryotic expression vector of Flag with a length of about 4000 bp Flag vector and a 1000 bp LDHA gene band.And the sequencing results indicated that the inserted sequence was completely in consonance with the coding sequence of the LDHA gene.Western blot results showed the successful expression of recombinant plasmid Flag-LDHA in MUM-2B melanoma cells.CCK8 assays demonstrated that Flag-LDHA recombinant plasmid could promote the growth of melanoma cell line MUM-2B.Conclusion The eukaryotic expression vector of Flag-LDHA was successfully constructed,which can promote the growth of melanoma cell line MUM-2B.This will lay the foundation for studying the function of LDHA in the initiation and progression of human CM.
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Objective To investigate the clinicopathological significances of LDHA/mutant p53 co-expres-sion in gliomas. Methods According to the 2016 WHO CNS,archived 68 gliomas were collected and analyzed retrospectively. The co-expression of LDHA/mutant p53 was detected by immunohistochemical staining. Results High expression of LDHA alone was always found in high grade gliomas(48.5%). Mutant p53 high expression was usually observed in glioblastomas (26.5%). There was a close relationship between co-expression of LDHA/mutant p53 in glioblastoma(27.9%,P = 0.005),or gliomas with high histological grading(27.9%,P = 0.002). Conclusions Co-expression of LDHA/mutant p53 in tumor cells might be a specific immunohistochemical pheno-type of gliomas,and may help for distinguishing glioblastoma and other high grade gliomas from low grade gliomas.
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OBJECTIVE To observe the regulation effect of chidamide on energy metabolism in HCT-8 and HT-29 cells. METHODS HCT-8 and HT-29 cells were treated with chidamide 5,10 and 20 μmol · L-1. Morphological changes of these cells were observed under an ordinary optical microscope. Cell proliferation was detected by MTT. ATP production was determined by CellTiter-Glo? assay kit. Metabolic changes were tested by glycolytic stress kit. The mRNA level of lactate dehydrogenase A (LDH-A)was analyzed by real-time quantitative PCR,whereas the protein level of LDH-A was analyzed by Western blotting. RESULTS Compared with control group,cell morphology of HCT-8 and HT-29 cells in chidamide treated group was irregular,accompanied by deformation,shrinkage and cell debris, and the inhibitory rate of proliferation increased(P<0.05). There was no significant difference in ATP total content between chidamide 5 and 10 μmol · L-1 16 h treatment groups,but in chidamide 20 μmol · L-1 treatment group it was decreased(P<0.05). Chidamide 20μmol · L-1 had no effect on oxygen consumption rate, but glycolysis ATP generation rate was reduced by 30.7% and 37.9%(P<0.05),respectively. Chidamide 20μmol · L-1 had no effect on LDH-A mRNA level,but it decreased the protein level of LDH-A(P<0.01). CONCLUSION Chidamide can abate the respiratory metabolic ability of HCT-8 and HT-29 cells. The mechanism may be related to the down-regulation of LDH-A.
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The structure and properties about encoding protein of lactate dehydrogenase A from Taenia solium(Ts LDH-A)were analyzed and predicted by bioinformatics in this study.The immunological characteristics of this novel gene were also analyzed by cloning and expressing.The full-length cDNA encoding Ts LDH-A was identified from the cDNA plasmid library by blastx and rpsblast programs provided by NCBI.The physico-chemical properties and structures of Ts LDH-A were analyzed by tools provided by ExPASy.And the B cell epitopes of Ts LDH-A were predicted by the B Cell Epitope Prediction Tools provided by IEDB Analysis Resource.The PCR amplified coding region of Ts LDH-A was cloned into the prokaryotic expression vector pET-28a (+) and expressed in E.coli BL21 with IPTG induction.The immunogenicity of the purified recombinant protein was analyzed by Western Blotting.It was demonstrated that the amino acid sequence of Ts LDH-A had identity with that of LDH-A from other specie and there was a conserved LDH domain in the deduced amino acid sequence.The full-length cDNA sequence encoding Ts LDH-A included a complete open reading frame(ORF)of 1332 bp and coded to a putative protein with 331 amino acids.The molecular weight of Ts LDH-A was predicted to be 35461.1 Da and the coding protein was demonstrated to contain 3 trans-membrane regions and 4 main B cell epitopes.The active site of L-lactate dehydrogenase located at the epitope aa190-199.The 3 key residues in the catalytic site of enzyme were conserved in different species and located near to each other in spatial position.PCR,double enzyme restriction and DNA sequencing were used to identify pET28a (+)-Ts LDH-A.The recombinant protein could react with the rat's sera as well as the sera from the patients and the swine infected Taenia solium.It is clear that the full-length cDNA sequence encoding Ts LDH-A can be screened from the cDNA library of adult Taenia solium by bioinformatics analysis and can be used to investigate the structure and properties about gene and encoding protein of Ts LDH-A as well as the immunological activities of gene expression in the prokaryotic system.