Reducing lactate accumulation has always been a goal of the mammalian cell biotechnology industry. When animal cells are cultured in vitro, the accumulation of lactate is mainly the combined result of two metabolic pathways. On one hand, glucose generates lactate under the function of lactate dehydrogenase A (LDHA); on the other hand, lactate can be oxidized to pyruvate by LDHB or LDHC and re-enter the TCA cycle. This study comprehensively evaluated the effects of LDH manipulation on the growth, metabolism and human adenovirus (HAdV) production of human embryonic kidney 293 (HEK-293) cells, providing a theoretical basis for engineering the lactate metabolism in mammalian cells. By knocking out ldha gene and overexpression of ldhb and ldhc genes, the metabolic efficiency of HEK-293 cells was effectively improved, and HAdV production was significantly increased. Compared with the control cell, LDH manipulation promoted cell growth, reduced the accumulation of lactate and ammonia, significantly enhanced the efficiency of substrate and energy metabolism of cells, and significantly increased the HAdV production capacity of HEK-293 cells. Among these LDH manipulation measures, ldhc gene overexpression performed the best, with the maximum cell density increased by about 38.7%. The yield of lactate to glucose and ammonia to glutamine decreased by 33.8% and 63.3%, respectively; and HAdV titer increased by at least 16 times. In addition, the ATP production rate, ATP/O₂ ratio, ATP/ADP ratio and NADH content of the modified cell lines were increased to varying degrees, and the energy metabolic efficiency was significantly improved.
Animals ; Humans ; L-Lactate Dehydrogenase/genetics* ; Lactic Acid ; Adenoviruses, Human ; Ammonia ; HEK293 Cells ; Glucose/metabolism* ; Adenosine Triphosphate/metabolism* ; Kidney/metabolism* ; Mammals/metabolism*

Objective To prepare rabbit polyclonal antibody specifically against human lactate dehydrogenase C4 (LDHC4). Methods Site-directed mutation was performed by PCR to generate the mutated LDHC gene, and the mutated gene was ligated into the pET-28a vector to form the pET-28a-LDHC recombinant expression vector. The recombinant vector was introduced into E. coli BL21 (DE3), and LDHC4 protein was obtained by induced expression. The recombinant protein was used as an antigen to immunize New Zealand rabbits, and the antiserum was obtained after three boosted immunizations. The titer of the antiserum against LDHC4 were detected by ELISA. Western blot was used to detect the specificity of the antiserum, and immunohistochemistry was used to detect the expression of LDHC4 in human triple-negative breast cancer tissue. Results A specific rabbit anti-human LDHC4 polyclonal antibody was obtained with an antibody titer of 1:51 200. The antibody can be used for Western blot and immunohistochemistry. Conclusion The specific rabbit anti-human LDHC4 polyclonal antibody is successfully prepared.
Humans ; Rabbits ; Animals ; Escherichia coli/genetics* ; Antibodies ; Enzyme-Linked Immunosorbent Assay ; L-Lactate Dehydrogenase/metabolism* ; Blotting, Western ; Antibody Specificity

OBJECTIVE: To explore the protective effect of Huoxin Pill (HXP) on acute myocardial ischemia-reperfusion (MIRI) injury in rats. METHODS: Seventy-five adult SD rats were divided into the sham-operated group, model group, positive drug group (diltiazem hydrochloride, DH), high dose group (24 mg/kg, HXP-H) and low dose group (12 mg/kg, HXP-L) of Huoxin Pill (n=15 for every group) according to the complete randomization method. After 1 week of intragastric administration, the left anterior descending coronary artery of the rat's heart was ligated for 45 min and reperfused for 3 h. Serum was separated and the levels of creatine kinase (CK), creatine kinase isoenzyme (CK-MB) and lactate dehydrogenase (LDH), superoxide dismutase (SOD), and malondialdehyde (MDA), hypersensitive C-reactive protein (hs-CRP) and interleukin-1β (IL-1β) were measured. Myocardial ischemia rate, myocardial infarction rate and myocardial no-reflow rate were determined by staining with Evans blue and 2,3,5-triphenyltetrazolium chloride (TTC). Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform (TCMSP) and Bioinformatics Analysis Tool for Molecular mechANism of Traditional Chinese Medicine (BATMAN) databases were used to screen for possible active compounds of HXP and their potential therapeutic targets; the results of anti-inflammatory genes associated with MIRI were obtained from GeneCards, Drugbank, Online Mendelian Inheritance in Man (OMIM), and Therapeutic Target Datebase (TTD) databases was performed; Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were used to analyze the intersected targets; molecular docking was performed using AutoDock Tools. Western blot was used to detect the protein expression of Toll-like receptor 4 (TLR4)/nuclear factor kappa-B (NFκB)/NOD-like receptor protein 3 (NLRP3). RESULTS: Compared with the model group, all doses of HXP significantly reduced the levels of LDH, CK and CK-MB (P<0.05, P<0.01); HXP significantly increased serum activity of SOD (P<0.05, P<0.01); all doses of HXP significantly reduced the levels of hs-CRP and IL-1β (P<0.05, P<0.01) and the myocardial infarction rate and myocardial no-reflow rate (P<0.01). GO enrichment analysis mainly involved positive regulation of gene expression, extracellular space and identical protein binding, KEGG pathway enrichment mainly involved PI3K-Akt signaling pathway and lipid and atherosclerosis. Molecular docking results showed that kaempferol and luteolin had a better affinity with TLR4, NFκB and NLRP3 molecules. The protein expressions of TLR4, NFκB and NLRP3 were reduced in the HXP group (P<0.01). CONCLUSIONS HXP has a significant protective effect on myocardial ischemia-reperfusion injury in rats, and its effect may be related to the inhibition of redox response and reduction of the inflammatory response by inhibiting the TLR4NFκB/NLRP3 signaling pathway.
Humans ; Rats ; Animals ; NF-kappa B/metabolism* ; Myocardial Reperfusion Injury/drug therapy* ; NLR Family, Pyrin Domain-Containing 3 Protein ; Rats, Sprague-Dawley ; C-Reactive Protein ; Toll-Like Receptor 4 ; Phosphatidylinositol 3-Kinases/metabolism* ; Molecular Docking Simulation ; Signal Transduction ; Myocardial Infarction/drug therapy* ; Creatine Kinase ; L-Lactate Dehydrogenase/metabolism* ; Superoxide Dismutase/metabolism*

To improve the productivity of L-phenyllactic acid (L-PLA), L-LcLDH1(Q88A/I229A), a Lactobacillus casei L-lactate dehydrogenase mutant, was successfully expressed in Pichia pastoris GS115. An NADH regeneration system in vitro was then constructed by coupling the recombinant (re) LcLDH1(Q88A/I229A) with a glucose 1-dehydrogenase for the asymmetric reduction of phenylpyruvate (PPA) to L-PLA. SDS-PAGE analysis showed that the apparent molecular weight of reLcLDH1(Q88A/I229A) was 36.8 kDa. And its specific activity was 270.5 U/mg, 42.9-fold higher than that of LcLDH1 (6.3 U/mg). The asymmetric reduction of PPA (100 mmol/L) was performed at 40 °C and pH 5.0 in an optimal biocatalytic system, containing 10 U/mL reLcLDH1(Q88A/I229A), 1 U/mL SyGDH, 2 mmol/L NAD⁺ and 120 mmol/L D-glucose, producing L-PLA with 99.8% yield and over 99% enantiomeric excess (ee). In addition, the space-time yield (STY) and average turnover frequency (aTOF) were as high as 9.5 g/(L·h) and 257.0 g/(g·h), respectively. The high productivity of reLcLDH1(Q88A/I229A) in the asymmetric reduction of PPA makes it a promising biocatalyst in the preparation of L-PLA.
L-Lactate Dehydrogenase ; genetics ; Lactobacillus casei ; enzymology ; genetics ; Phenylpyruvic Acids ; metabolism ; Pichia ; genetics ; Recombinant Proteins ; genetics ; metabolism

This study was to investigate the mechanism of safflower yellow injection for regulating inflammatory response against myocardial ischemia-reperfusion injury( MIRI) in rats. Male Wistar rats were randomly divided into sham operation group,model group,Hebeishuang group,safflower yellow injection high,medium and low dose groups. MIRI model was established by ligating left anterior descending coronary artery. Myocardial histopathological changes were observed by HE staining; myocardial infarct size was detected by TTC staining; content and changes of tumor necrosis factor-α( TNF-α) and interleukin-6( IL-6),serum creatine kinase( CK),aspartate aminotransferase( AST),and lactate dehydrogenase( LDH) were detected by biochemical method or enzyme-linked immunosorbent assay( ELISA). Western blot assay was used to detect the protein expression of Toll-like receptor 4( TLR4) and nuclear factor-κB( NF-κB p65) in myocardial tissues. The results showed that as compared with the sham operation group,the myocardial arrangement of the model group was disordered,with severe edemain the interstitial,significantly increased area of myocardial infarction,increased activities of AST,CK and LDH in serum,and significantly increased contents of TNF-α and IL-6; the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were also increased. As compared with the model group,the myocardial tissues were arranged neatlyin the Hebeishuang group and safflower yellow injection high,medium and low dose groups; the edema was significantly reduced; the myocardial infarct size was significantly reduced; the serum AST,CK,LDH activity and TNF-α,IL-6 levels were significantly decreased,and the expression levels of TLR4 and NF-κB( p65) protein in myocardial tissues were decreased. As compared with the Hebeishuang group,the myocardial infarct size was larger in the safflower yellow injection high,medium and low dose groups; the activities of AST,CK and LDH in serum and the contents of TNF-α and IL-6 in serum were higher,but there was no statistically significant difference in the expression levels of TLR4 and NF-κB( p65) protein in tissues. It is suggested that safflower yellow injection has a significant anti-MIRI effect,and its mechanism may be related to the regulation of TLR-NF-κB pathway to inhibit inflammatory response.
Animals ; Anti-Inflammatory Agents ; pharmacology ; Aspartate Aminotransferases ; blood ; Chalcone ; analogs & derivatives ; pharmacology ; Creatine Kinase ; blood ; Interleukin-6 ; metabolism ; L-Lactate Dehydrogenase ; blood ; Male ; Myocardial Reperfusion Injury ; drug therapy ; Rats ; Rats, Wistar ; Toll-Like Receptor 4 ; metabolism ; Transcription Factor RelA ; metabolism ; Tumor Necrosis Factor-alpha ; metabolism

metabolism and development. The lncRNA small nucleolar RNA host gene 7 (SNHG7) was reported to be upregulated in colorectal cancer and contribute to its progression. In the current study, we investigated the role of lncRNA-SNHG7 in breast cancer and explored the underlying mechanism.METHODS: We monitored the expression of lncRNA-SNHG7 in breast cancer tissues and breast cancer cell lines. We evaluated the effects of lncRNA-SNHG7 on cell proliferation and glycolysis in breast cancer cells by knocking down or overexpressing lncRNA-SNHG7. We searched for the potential microRNA (miRNA) target of lncRNA-SNHG7 and evaluated the effects of the target miRNA on glycolysis. We evaluated the potential regulation of lncRNA-SNHG7 by c-Myc.RESULTS: LncRNA-SNHG7 was up-regulated in both breast cancer tissues and breast cancer cell lines. Knocking down lncRNA-SNHG7 inhibited breast cancer cell proliferation while overexpressing lncRNA-SNHG7 enhanced cell proliferation. Knocking down lncRNA-SNHG7 resulted in decreased expression of lactate dehydrogenase A (LDHA) and decreased glycolysis. LncRNA-SNHG7 targeted miR-34a-5p to regulate LDHA expression and glycolysis. c-Myc bound to promoter of lncRNA-SNHG7 and positively regulated lncRNA-SNHG7 expression.CONCLUSION: We demonstrated that c-Myc regulated glycolysis through the lncRNA-SNHG7/miR-34a-5p/LDHA axis in breast cancer cells.]]>
Breast Neoplasms ; Breast ; Cell Line ; Cell Proliferation ; Colorectal Neoplasms ; Glycolysis ; L-Lactate Dehydrogenase ; Metabolism ; MicroRNAs ; Proto-Oncogene Proteins c-myc ; RNA, Long Noncoding ; RNA, Small Nucleolar

Atractylenolide III (ATL-III), a sesquiterpene compound isolated from Rhizoma Atractylodis Macrocephalae, has revealed a number of pharmacological properties including anti-inflammatory, anti-cancer activity, and neuroprotective effect. This study aimed to evaluate the cytoprotective efficiency and potential mechanisms of ATL-III on corticosterone injured rat phaeochromocytoma (PC12) cells. Our results demonstrate that ATL-III increases cell viability and reduces the release of lactate dehydrogenase (LDH). The results suggest that ATL-III protects PC12 cells from corticosterone-induced injury by inhibiting the intracellular Ca overloading, inhibiting the mitochondrial apoptotic pathway and modulating the MAPK/NF-ΚB inflammatory pathways. These findings provide a novel insight into the molecular mechanism by which ATL-III protected the PC12 cells against corticosterone-induced injury for the first time. Our results provide the evidence that ATL-III may serve as a therapeutic agent in the treatment of depression.
Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Survival ; drug effects ; Corticosterone ; toxicity ; Inflammation Mediators ; metabolism ; L-Lactate Dehydrogenase ; metabolism ; Lactones ; pharmacology ; Mitochondria ; drug effects ; metabolism ; Mitogen-Activated Protein Kinases ; metabolism ; NF-kappa B ; metabolism ; Neuroprotective Agents ; pharmacology ; PC12 Cells ; Phosphorylation ; drug effects ; Rats ; Sesquiterpenes ; pharmacology ; Signal Transduction ; drug effects

Human seminal plasma is rich in potential biological markers for male infertility and male reproductive system diseases, which have an application value in the diagnosis and treatment of male infertility. The methods for the detection of semen biochemical markers have been developed from the manual, semi-automatic to the present automatic means. The automatic detection of semen biochemical markers is known for its advantages of simple reagent composition and small amount of reagents for each test, simple setting of parameters, whole automatic procedure with few errors, short detection time contributive to batch detection and reduction of manpower cost, simple calibration and quality control procedure to ensure accurate and reliable results, output of results in the order of the samples in favor of clinical diagnosis and treatment, and open reagents applicable to various automatic biochemistry analyzers. At present, the automatic method is applied in the detection of such semen biochemical markers as seminal plasma total and neutral alpha-glucosidase, acid phosphatase, fructose, γ-glutamyl transpeptidase, zinc, citric acid, uric acid, superoxide dismutase and carnitine, sperm acrosin and lactate dehydrogenase C4, and semen free elastase, which can be used to evaluate the secretory functions of the epididymis, seminal vesicle and prostate, sperm acrosome and energy metabolism function, seminal plasma antioxidative function, and infection or silent infection in the male genital tract.
Acid Phosphatase ; analysis ; Biomarkers ; analysis ; Carnitine ; analysis ; Citric Acid ; analysis ; Epididymis ; metabolism ; Fructose ; analysis ; Humans ; Infertility, Male ; diagnosis ; Isoenzymes ; L-Lactate Dehydrogenase ; Male ; Prostate ; metabolism ; Semen ; chemistry ; Seminal Vesicles ; Spermatozoa ; chemistry ; alpha-Glucosidases ; analysis ; gamma-Glutamyltransferase ; analysis

OBJECTIVE: To establish an animal model for loaded swimming, so as to investigate the energy metabolism effects of soybean isoflavones (SI) on swimming mice. METHODS: Thirty male Kunming mice were randomly divided into three groups:normal control, swimming group, and swimming+SI group. The normal control group mice were fed a basic AIN-93M diet, the SI groups were supplied with soybean isoflavones(4 g/kg).Two weeks later, the mice were forced to swim for an hour,and then all the mice were killed, the samples of blood, liver and muscles of hind were collected.The serum contents of lactic acid(Lac), the activities of lactic dehydrogenase (LDH), succinate dehydrogenase (SDH), creatine kinase (CK) and ATPase were measured. RESULTS: Compared with normal control,the serum content of Lac was significantly improved in the group of the swimming control and SI(<0.05),the activity of LDH in the serum was obviously improved in the group of the swimming control and SI, and the activity of CK and SDH were both significantly improved in the group of the swimming control and SI except the activity of SDH in the liver of the group SI; compared with the swimming control,the serum contents of Lac,the activities of LDH, ATPase, SDH, CK were obviously improved(<0.05). CONCLUSIONS Soybean isoflavones can improve the energy metabolism,antioxidant capacity of the swimming mice.
Adenosine Triphosphatases ; blood ; Animals ; Creatine Kinase ; blood ; Energy Metabolism ; Isoflavones ; pharmacology ; L-Lactate Dehydrogenase ; blood ; Lactic Acid ; blood ; Male ; Mice ; Random Allocation ; Soybeans ; chemistry ; Succinate Dehydrogenase ; blood ; Swimming

OBJECTIVE: To investigate the effects of excessive endoplasmic reticulum stress on lung ischemia/reperfusion (I/R) induced myocardial injury in mice. METHODS: Forty healthy SPF male C57BL/6J mice were divided into 4 groups randomly (=10):sham operation group (Sham group), lung I/R group (I/R group), endoplasmic reticulum stress (ERS) pathway agonist Tunicamycin group (TM) and ERS inhibitor 4-phenyl butyric acid group (4-PBA). The model of lung I/R injury was established by clamping the left hilum of lung for 30 min followed by 180 min of reperfusion. In sham group, only sternotomy was performed, the hilum of lung was not clamped, and the mice were mechanically ventilated for 210 min. In TM and 4-PBA groups, TM 1mg/kg and 4-PBA 400 mg/kg were injected intraperitoneally, respectively, at 30 min before establishment of the model. At 180 min of reperfusion, blood samples were collected from the orbit for determination of myocardial enzyme. The animals were then sacrificed, and hearts were removed for determination of light microscope, TUNEL, Caspase 3 enzymatic activity, real-time polymerase chain reaction and Western blot. RESULTS: Compared with sham group, the cardiomyocytes had obvious damage under light microscope, and the serum creatine kinase-MB (CK-MB) and lactic dehydrogenase (LDH) activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-Jun N-terminalkinase(p-JNK), Caspase 12, CCAAT/enhancer-binding protein homologous protein (CHOP) and glucose regulated protein 78(GRP78) protein and mRNA were up-regulated in I/R, TM and 4-PBA groups (<0.01). Compared with I/R group, the cardiomyocytes damage was obvious under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were increased significantly, the expressions of p-JNK, Caspase 12, CHOP and GRP78 protein and mRNA were up-regulated in group TM; while all above changes were relieved in group 4-PBA (<0.01). Compared with TM group, the cardiomyocytes damage was relieved under light microscope, and the serum CK-MB and LDH activities, apoptosis index and Caspase 3 enzymatic activity were decreased significantly, the expressions of p-JNK, Caspase 12,CHOP and GRP78 protein and mRNA were down-regulated in group 4-PBA. CONCLUSIONS The excessive endoplasmic reticulum stress participates in myocardial injury induced by lung ischemia/reperfusion (I/R) and inhibit excessive endoplasmic reticulum stress response can relieved myocardial injury.
Animals ; Apoptosis ; Caspase 12 ; Caspase 3 ; metabolism ; Creatine Kinase, MB Form ; blood ; Endoplasmic Reticulum Stress ; Heart Injuries ; physiopathology ; Heat-Shock Proteins ; metabolism ; L-Lactate Dehydrogenase ; blood ; Lung ; pathology ; MAP Kinase Kinase 4 ; metabolism ; Male ; Mice ; Mice, Inbred C57BL ; Myocardium ; pathology ; Random Allocation ; Reperfusion Injury ; Transcription Factor CHOP ; metabolism