Lactate Dehydrogenase Enzyme Activity Research Articles

Ag2 O-TiO2 -NTs enhance osteogenic activity invitro by modulating TNF-α/β-catenin signaling in bone marrow-derived mesenchymal stem cells.

The toxic effects of nanoparticles-silver oxide (Ag2 O) limited its use. However, loading Ag2 O nanoparticles into titanium dioxide (TiO2 ) nanotubes (Ag2 O-TiO2 -NTs) has more efficient biological activity and safety. The aim of this study was to observe the effect of Ag2 O-TiO2 -NTs on osteogenic differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) and its mechanism. The enzyme activity of lactate dehydrogenase (LDH) and the expression of RUNX family transcription factor 2 (Runx2), OPN, OCN in BMSCs were detected by quantitative real time polymerase chain reaction. At 14 days of induction, the mineralization ability and alkaline phosphatase (ALP) activity of cells in each group were observed by Alizarin Red S staining and ALP staining. In addition, the protein levels of tumor necrosis factor-α (TNF-α) and β-catenin in BMSCs of each group were observed by western blot. After 14 days of the induction, the mineralization ability and ALP activity of BMSCs in the Ag2 O-TiO2 -NTs group were significantly enhanced compared with those in the Ag2 O and TiO2 groups. Western blot analysis showed that the BMSCs in the Ag2 O-TiO2 -NTs group exhibited much lower protein level of TNF-α and higher protein level of β-catenin than those in the Ag2 O and TiO2 groups.Ag2 O-TiO2 -NTs enhance the osteogenic activity of BMSCs by modulating TNF-α/β-catenin signaling.

Combinations of arginine and pullulan reveal the selective effect of stabilization mechanisms on different lyophilized proteins

The stability of lactate dehydrogenase (LDH) and β-galactosidase (β-gal), incorporated in arginine/pullulan (A/P) mixtures at various weight ratios by lyophilization, was determined. The physicochemical characteristics of various A/P mixtures were assessed. With decreasing A/P ratios, the glass transition temperature of the formulations increased. Furthermore, arginine crystallization due to high relative humidity (RH) exposure was prevented at an A/P weight ratio of 4/6 or less. When stored at 0 % RH / 60 °C for 4 weeks, arginine was superior to pullulan as stabilizer. During storage at 43 % RH / 30 ℃ for 4 weeks, the enzymatic activity of LDH was best retained at an A/P weight ratio of 2/8, while β-gal activity was relatively well-retained at A/P weight ratios of both 8/2 and 2/8. LDH seemed to be more prone to degradation in the rubbery state. In the glassy state, β-gal degraded faster than LDH. Solid-state nuclear magnetic resonance spectroscopy showed that (labeled) arginine experienced a different interaction in the two protein samples, reflecting a modulation of long-range correlations of the arginine side chain nitrogen atoms (Nε, Nη). In summary, LDH stabilization in the A/P matrix requires vitrification. Further stabilization difference between LDH and β-gal may be dependent on the interaction with arginine.

Antibacterial mechanism of slightly acidic electrolyzed water against Pseudomonas arsenicoxydans A1 from greater amberjack (Seriola dumerili)

This study aimed to identify the specific spoilage organism (SSO) from the metamorphosed greater amberjack (Seriola dumerili) fish. Pseudomonas arsenicoxydans A1 was identified as SSO by culture-depending method. Further, the antibacterial efficacy of slightly acidic electrolyzed water (SAEW) was explored and the mechanism of action on P. arsenicoxydans A1 was studied. SAEW with 20–60 mg/L of available chlorine concentration (ACC) showed a good antibacterial effect on P. arsenicoxydans A1. The total number of colonies, growth curve, OD value at 260 nm and 280 nm, DNA fluorescence intensity, alkaline phosphatase (AKP), lactate dehydrogenase (LDH), superoxide dismutase (SOD), and catalase (CAT) enzyme activity and SEM results showed that SAEW could destroy the permeability and integrity of P. arsenicoxydans A1 cell membrane, and affect bacterial cell activity at metabolic, enzymatic and DNA level, which caused bacterial cell death. This study provided the application of SAEW in sterilization and preservation of raw fish fillets, which showed a promising effect on the growth of P. arsenicoxydans A1 in food cold chain.

Does chronic exposure to moderate hypoxia affect growth, physiological and oxidative stress biomarkers in adult spotted wolffish (Anarhichas minor) and the hybrid A. minor × A. lupus?

AbstractBoth in their natural habitat (estuaries, marine coastal environment, ocean depths and freshwater lakes) or in captivity conditions (land‐based, open‐sea aquaculture and aquariums), exposure to hypoxia can have direct detrimental effects on fish growth, reproduction, behaviour and health. The objective of this study was to investigate the effects of chronic exposure of spotted wolffish (Anarhichas minor) (∼1.4 kg) and the interspecific hybrid (A. minor × A. lupus) (∼600 g) to moderate hypoxia levels (dissolved oxygen [DO]: 40%, 50% and 60% saturation) in comparison to a normoxia group (control 100%) over a period of 14 weeks at ∼7.5°C. The trials were conducted as a common‐garden experiment (both fish groups reared together) in quadruplicate (4 tanks per DO level = 16 tanks). Fish performance (specific growth rate, feed intake [FI]) and physiological status (condition factor [K] and hepatosomatic index, haematocrit) were monitored at different intervals. The metabolic enzyme activity of citrate synthase (CS), lactate dehydrogenase (LDH) and pyruvate kinase (PK) was measured as well as antioxidant enzymes glutathione reductase and catalase. Oxidative stress indices were assessed by the quantification of thiobarbituric acid reactive substances (TBARS) and the measurement of the enzymatic activity level of aconitase. Significant reduction in growth rate occurred at DO 40%. DO was identified as a major driver of FI. Our results indicate a very strong adaptation to hypoxia as (1) exposures to DO 40% were not severe enough to affect aerobic or anaerobic metabolism capacity or to significantly induce oxidative stress and (2) metabolic organization of the muscle tissue, as expressed by LDH, CS and PK is not affected by DO levels down to 40%. However, at the lowest DO levels (40% and 50%), hybrid fish displayed significantly better growth than the spotted wolffish.

Probing human heart TCA cycle metabolism and response to glucose load using hyperpolarized [2-13 C]pyruvate MRS.

The healthy heart has remarkable metabolic flexibility that permits rapid switching between mitochondrial glucose oxidation and fatty acid oxidation to generate ATP. Loss of metabolic flexibility has been implicated in the genesis of contractile dysfunction seen in cardiomyopathy. Metabolic flexibility has been imaged in experimental models, using hyperpolarized (HP) [2-13 C]pyruvate MRI, which enables interrogation of metabolites that reflect tricarboxylic acid (TCA) cycle flux in cardiac myocytes. This study aimed to develop methods, demonstrate feasibility for [2-13 C]pyruvate MRI in the human heart for the first time, and assess cardiac metabolic flexibility. Good manufacturing practice [2-13 C]pyruvic acid was polarized in a 5 T polarizer for 2.5-3 h. Following dissolution, quality control parameters of HP pyruvate met all safety and sterility criteria for pharmacy release, prior to administration to study subjects. Three healthy subjects each received two HP injections and MR scans, first under fasting conditions, followed by oral glucose load. A 5 cm axial slab-selective spectroscopy approach was prescribed over the left ventricle and acquired at 3 s intervals on a 3 T clinical MRI scanner. The study protocol, which included HP substrate injection, MR scanning, and oral glucose load, was performed safely without adverse events. Key downstream metabolites of [2-13 C]pyruvate metabolism in cardiac myocytes include the glycolytic derivative [2-13 C]lactate, TCA-associated metabolite [5-13 C]glutamate, and [1-13 C]acetylcarnitine, catalyzed by carnitine acetyltransferase (CAT). After glucose load, 13 C-labeling of lactate, glutamate, and acetylcarnitine from 13 C-pyruvate increased by an average of 39.3%, 29.5%, and 114% respectively in the three subjects, which could result from increases in lactate dehydrogenase, pyruvate dehydrogenase, and CAT enzyme activity as well as TCA cycle flux (glucose oxidation). HP [2-13 C]pyruvate imaging is safe and permits noninvasive assessment of TCA cycle intermediates and the acetyl buffer, acetylcarnitine, which is not possible using HP [1-13 C]pyruvate. Cardiac metabolite measurement in the fasting/fed states provides information on cardiac metabolic flexibility and the acetylcarnitine pool.

Oxidative Stress Accompanies HIF1-Dependent Impairment of Glucose Metabolism in the Hippocampus of Adult Rats That Survived Prenatal Severe Hypoxia

Introduction: Many socially significant diseases are associated with prenatal developmental disorders. Previously, we showed the pathological role of hypoxia-inducible factor-1 (HIF1) in post-hypoxic reoxygenation. This study aimed to investigate the effect of prenatal severe hypoxia (PSH) on HIF1α protein expression as well as on HIF1-dependent activity of the pentose phosphate pathway (PPP) and anaerobic glycolysis in the hippocampus (HPC) of offspring that reached adulthood. Methods: PSH was induced during the critical period of fetal hippocampal formation on gestation days 14–16 in a hypobaric chamber (180 Torr, 5% oxygen, 3 h). Subsequent studies were conducted on both the HPC of adult control and PSH rats under normal conditions, as well as in response to severe hypoxia (SH) or psycho-emotional stress (“learned helplessness” [LH] model). We evaluated HIF1α protein levels using both immunohistochemistry and Western blotting techniques. The amount of glucose-6-phosphate dehydrogenase (G6PD) was also determined by Western blotting. Colorimetric enzymatic assays were employed to analyze enzymatic activity of lactate dehydrogenase (LDH), the concentration of lactate, NADPH, reduced glutathione (GSHred), and malonic dialdehyde (MDA). Results: We showed that PSH caused a stable increase in the content of HIF1α protein in the HPC, which was accompanied by an increase in the efficiency of anaerobic glycolysis. This was confirmed by increased LDH activity and lactate concentration. At the same time, the amounts of G6PD, NADPH, and GSHred decreased in the HPC of PSH rats, whereas the concentration of MDA, an oxidative stress marker, exceeded the control values. In a series of experiments using the LH or SH stress, it was shown that in the HPC of control rats, there was an increase in the amount of HIF1α in response to stress, which was also accompanied by more efficient anaerobic glycolysis and decrease of PPP-dependent NADPH production, similar to the intact PSH rats. In PSH rats, emotional stress resulted in higher HIF1α levels without affecting glycolysis or PPP. Conclusion: Therefore, the increased content and activity of the transcription factor HIF1α in the HPC of adult rats exposed to prenatal hypoxia leads to an imbalance between glycolysis and PPP, which is accompanied by oxidative stress.

Taurine promotes muscle fiber type transformation through CaN/NFATc1 signaling in porcine myoblasts.

This study investigated the effect of taurine (TAU) on the muscle fiber type transformation in porcine myoblasts and its molecular mechanisms. The findings revealed that TAU augmented the protein expression of slow MyHC and the enzyme activities of oxidative metabolism markers like malate dehydrogenase and succinic dehydrogenase. Conversely, it curtailed the expression of fast MyHC and glycolytic metabolism enzyme activity of lactate dehydrogenase. Moreover, TAU elevated the expression of genes associated with oxidative fiber while diminishing the expression of those linked to glycolytic fibers, suggesting that TAU promoted the muscle fiber type transformation from glycolytic fiber to oxidative fiber. Additionally, TAU notably enhanced the expression of key molecules of calcineurin (CaN)/nuclear factor of activated T cells c1 (NFATc1) signaling and the CaN activity in porcine myoblasts. However, CaN inhibitor cyclosporine A abolished these effects induced by TAU. Our results indicated that TAU regulated the muscle fiber type transformation from glycolytic to oxidative fiber by activation of CaN/NFATc1 signaling.

Histone Deacetylase Inhibitor (SAHA) Reduces Mortality in an Endotoxemia Mouse Model by Suppressing Glycolysis.

Sepsis is a life-threatening medical emergency triggered by excessive inflammation in response to an infection. High mortality rates and limited therapeutic options pose significant challenges in sepsis treatment. Histone deacetylase inhibitors (HDACi), such as suberoylanilide hydroxamic acid (SAHA), have been proposed as potent anti-inflammatory agents for treating inflammatory diseases. However, the underlying mechanisms of sepsis treatment remain poorly understood. In this study, we investigated the effects of SAHA treatment in the lipopolysaccharide (LPS)-induced endotoxemia mouse model as it closely mimics the early stages of the systemic inflammation of sepsis. Our results demonstrate a reduced inflammatory mediator secretion and improved survival rates in mice. Using quantitative acetylomics, we found that SAHA administration increases the acetylation of lactate dehydrogenase (LDHA), and consequently inhibits LDHA activity. Notably, the reduced enzyme activity of LDHA results in a reduced rate of glycolysis. Furthermore, our experiments with bone marrow-derived macrophages (BMDMs) show that SAHA administration reduced oxidative stress and extracellular ATP concentrations, ultimately blunting inflammasome activation. Overall, our study provides insights into the mechanism underlying SAHA's therapeutic effects in sepsis treatment and highlights LDHA as a potential target for developing novel sepsis treatment.

Time-dependent changes in the glycolytic pathway in activated T cells are independent of tumor burden or anti-cancer chemotherapy

Although activated adoptive T cells therapy (ATC) is an effective approach for cancer treatment, it is not clear how modulation of T cell activation impacts their biochemical signature which significantly impacts the cell function. This study is aimed to investigate the impact of polyclonal activation on the metabolic signature of T cells from tumor-bearing mice under different settings of treatment with chemotherapy. Thirty female Swiss albino mice were divided into 5 groups (n = 6/each), Gp1(PBS), groups Gp2 were inoculated intraperitoneal (i.p) with 1 × 106 cells/mouse Ehrlich ascites carcinoma (EAC), Gp3-Gp5 were treated with cisplatin (20 mg/mice) which were represented as EAC/CIS/1wk Or EAC/CIS/2wk 3 times every other day. Splenocytes were cultured in or presence of concanavalin-A (Con-A) and IL-2 for 24 h or 72 h, then cells were harvested, and processed to determine the enzyme activities of hexokinase (HK), phosphofructokinase (PFK), lactate dehydrogenase (LDH) and glucose 6 phosphate dehydrogenase(G6PD) enzymes. The results showed that before culture, T cells harvested from EAC/PBS/1wk of mice or inoculated with EAC/CIS/1wk showed higher activity in HK, PFK, LDH, and G6PH as compared to naive T cells. After 24, and 72 h of culture and activation, the enzyme activities in T cells harvested from EAC/CIS/2wk mice or EAC/CIS/3wk mice decreased compared with their control. The late stage of the tumor without chemotherapy gives a low glycolic rate. In late activation, naive and early stages of the tumor with chemotherapy can give high glycolic metabolism. These results show great significance as an application of adoptive T-cell therapy.

The effect of electrical stimulation of skeletal muscle on cardioprotection and on muscle-derived myokine levels in rats: A pilot study.

Electrical muscle stimulation (EMS) is a widely used method in sports and rehabilitation therapies to simulate physical exercise. EMS treatment via skeletal muscle activity improves the cardiovascular functions and the overall physical condition of the patients. However, the cardioprotective effect of EMS has not been proven so far, therefore, the aim of this study was to investigate the potential cardiac conditioning effect of EMS in an animal model. Low-frequency 35-min EMS was applied to the gastrocnemius muscle of male Wistar rats for three consecutive days. Their isolated hearts were then subjected to 30 min global ischemia and 120 min reperfusion. At the end of reperfusion cardiac specific creatine kinase (CK-MB) and lactate dehydrogenase (LDH) enzyme release and myocardial infarct size were determined. Additionally, skeletal muscle-driven myokine expression and release were also assessed. Phosphorylation of cardioprotective signaling pathway members AKT, ERK1/2, and STAT3 proteins were also measured. EMS significantly attenuated cardiac LDH and CK-MB enzyme activities in the coronary effluents at the end of the ex vivo reperfusion. EMS treatment considerably altered the myokine content of the stimulated gastrocnemius muscle without altering circulating myokine levels in the serum. Additionally, phosphorylation of cardiac AKT, ERK1/2, and STAT3 was not significantly different in the two groups. Despite the lack of significant infarct size reduction, the EMS treatment seems to influence the course of cellular damage due to ischemia/reperfusion and favorably modifies skeletal muscle myokine expressions. Our results suggest that EMS may have a protective effect on the myocardium, however, further optimization is required.

Investigations on some indicators in the blood of cattle with orosthenic activity tongue rolling.

Hematological studies were carried out in cattle with tongue-rolling hyperactivity in order to establish the etiopathogenetic mechanisms of this behavioral disorder. Cattle were divided into two groups: Group 1 (n = 12), control animals that did not demonstrate the tongue-rolling orosthenic activity, and Group 2 (n = 12), cattle that manifested this stereotyped behavior. Blood samples were collected from all cattle twice at 10-day intervals from Vena jugularis for analysis of red blood cell counts, hemoglobin (HGB) content, mean corpuscular volume (MCV), mean corpuscular HGB (MCH), mean corpuscular HGB concentration (MCHC), hematocrit (HCT), white blood cell counts (WBC), and differential white cell counts [lymphocytes (LYM), monocytes (MON), and granulocytes (GRAs)]. Some blood biochemical constituents were assayed: calcium, inorganic phosphorus, magnesium, plasma total protein, blood glucose, total bilirubin, urea, creatinine, chlorides, cholesterol, triglycerides, and albumin, as well as the enzyme activities of aspartate amino-transferase (AST), alanine amino-transferase (ALT), alkaline phosphatase (ALP), gamma-glutamyltransferase (γ-GT), lactate dehydrogenase (LDH), and creatine kinase (CK). Blood concentrations of the hormones adrenaline, noradrenaline, cortisol, adrenocorticotropic hormone (ACTH), dopamine, serotonin, free thyroxine, thyroid-stimulating hormone, and trace elements manganese, copper, and iron were determined. It was found out that the cattle with tongue-rolling hyperactivity did not demonstrate changes in red blood picture (HGB, HCT, MCV, MCH, MCHC), white blood picture (WBC, LYM, MON, GRA), blood biochemistry (Ca, P, Mg, TP, Glu, TB, urea, creatine, Cl, Chole, TG, and Alb), AST, ALT, ALP, γ-GT, LDH, and CK activities, Cu, and Fe levels. In the study, increased concentrations of adrenaline, noradrenaline, cortisol, ACTH, and serotonin were established. The data demonstrating increased levels of adrenaline, noradrenaline, cortisol, and ACTH confirmed the etiological role of environmental stressors in the emergence of oral hyperactivity. Increased serotonin concentrations in the blood suggest that cattle with the stereotype are probably "happy" with tongue rolling. The lack of changes in blood trace elements manganese, copper, and iron allowed excluding their deficiency as a probable cause in the etiology of the disorder.

Resveratrol reduces platelet storage lesion by preventing free mitochondrial DNA (mtDNA) accumulation in platelet concentrates during storage.

Platelet activation and mitochondrial damage are among the crucial events leading to the quality reduction of platelet concentrates (PCs) during preparation and storage, called platelet storage lesion. Platelet activation results in the clearance of transfused platelets. Oxidative stress and platelet activation trigger mitochondrial DNA (mtDNA) release into the extracellular milieu which is associated with adverse transfusion reactions. Therefore, we aimed to investigate the effects of resveratrol, an antioxidant polyphenol, on platelet activation markers and mtDNA release. Ten PCs were divided equally into two bags each, one of them was allocated to the control group (n = 10) and another to the case group (resveratrol-treated, n = 10). Free mtDNA level and CD62P (P-selectin) expression level were measured by absolute quantification Real-Time PCR, and flow cytometry on days 0 (the receiving day), 3, 5, and 7 of storage respectively. Moreover, Lactate dehydrogenase (LDH) enzyme activity, pH, platelet count, mean platelet volume (MPV), and platelet distribution width (PDW) were assessed as well. Treatment of PCs with resveratrol can significantly decrease mtDNA release during storage compared to the control. In addition, platelet activation was significantly mitigated. We also observed significantly lower MPV, PDW, and LDH activity in resveratrol-treated PCs compared to the control group on days 3, 5, and 7. Furthermore, resveratrol maintained the pH of PCs on day 7.Resveratrol diminished free mtDNA and maintained biochemical parameters in PCs, possibly by reducing platelet activation. Therefore, resveratrol might be a possible additive solution for improving the quality of stored PCs.

Biochemical responses of the giant clam (Tridacna squamosa Lamarck, 1819) and the burrowing sea urchin (Echinometra mathaei (de Blainville, 1825)) to spatial and seasonal variations on the Red Sea coast of Egypt

This study aims to investigate the spatial and seasonal impacts on the biochemical responses of two bioindicators: Tridacna squamosa and Echinometra mathaei which inhabit the Red Sea coast of Egypt. Samples were collected from three sites during the summer of 2019 and the winter of 2020. Total protein, MDA, and enzyme activity of pyruvate kinase (PK) and lactate dehydrogenase (LDH) were measured in the gonads of the collected samples. The results confirmed that environmental variables vary at both spatial and seasonal levels, where the two species exhibited different patterns of biochemical response to internal and external stresses. In addition, both studied species did not resort to anaerobic respiration as a defense mechanism. Therefore, this study confirms that the use of biomarkers in the field shall be regarded as a general warning of internal and external stress.

Sugarcane mosaic virus orchestrates the lactate fermentation pathway to support its successful infection.

Viruses often establish their own infection by altering host metabolism. How viruses co-opt plant metabolism to support their successful infection remains an open question. Here, we used untargeted metabolomics to reveal that lactate accumulates immediately before and after robust sugarcane mosaic virus (SCMV) infection. Induction of lactate-involved anaerobic glycolysis is beneficial to SCMV infection. The enzyme activity and transcriptional levels of lactate dehydrogenase (LDH) were up-regulated by SCMV infection, and LDH is essential for robust SCMV infection. Moreover, LDH relocates in viral replicase complexes (VRCs) by interacting with SCMV-encoded 6K2 protein, a key protein responsible for inducing VRCs. Additionally, lactate could promote SCMV infection by suppressing plant defense responses. Taken together, we have revealed a viral strategy to manipulate host metabolism to support replication compartment but also depress the defense response during the process of infection.

Hazelnut Skin in Ewes' Diet: Effects on Colostrum Immunoglobulin G and Passive Transfer of Immunity to the Lambs.

Passive immunity transfer has a pivotal role in newborn lambs, where the colostrum represents the primary source of immunoglobulins. This study hypothesized that the high content in polyphenolic compounds, mono- and poly-unsaturated fatty acids, and vitamin E of hazelnut skin affects blood and colostrum immunoglobulin G (IgG) concentration and related gamma-glutamyl-transferase (GGT) and lactate dehydrogenase (LDH) levels in sheep and their lambs. In the last 45 days of pregnancy, ewes were divided into a control (CTR) and a hazelnut skin supplemented group (HZN). Blood and colostrum were collected from ewes and lambs before the first suckling, at 24 and 48 h after birth, then IgG concentration, GGT and LDH activity levels were measured. IgG concentration in the colostrum and in lamb's serum were significantly greater in HZN than CTR. No significant difference was detected for ewe's blood. A significant positive correlation was found between IgG and GGT in lambs' serum and colostrum, between IgG and LDH, as well as between GGT and LDH in lambs' serum and colostrum. Our results suggest that hazelnut skin supplementation influences IgG colostrum concentration, with improved immune passive transfer to the suckling lambs. The transfer of maternal derived immune factors is confirmed by the GGT and LDH enzyme activity levels.

L-carnitine modulates free mitochondrial DNA DAMPs and platelet storage lesions during storage of platelet concentrates.

Platelet storage lesions may occur in Platelet concentrates (PCs) storage time, reducing PCs' quality. Mitochondrial damage causes mitochondrial DNA (mtDNA) to be released into the extracellular space. In this study, we evaluated the effect of L-carnitine (LC) as an antioxidant on free mtDNA DAMPs release in PCs during storage. Ten PCs prepared by the PRP method were studied. The copy numbers of free mtDNA, total reactive oxygen species (ROS), lactate dehydrogenase (LDH) enzyme activity, pH, and platelet counts were measured on days 0, 3, 5, and 7 of PCs storage in LC-treated and untreated platelets. LDH activity was significantly lower than the control group during 7 days of PCs storage (p = 0.041). Also, ROS production decreased in LC-treated PCs compared to the control group during storage (p = 0.026), and the difference mean of ROS between the two groups was significant on day 3, 5, and 7 (Pday3 = 0.02, Pday5 = 0.0001, Pday7 = 0.031). Moreover, LC decreased the copy numbers of free mtDNA during 7 days of storage (p = 0.021), and the difference mean of the copy numbers of free mtDNA in LC-treated PCs compared to the control group was significant on day 5 and 7 (Pday5 = 0.041، Pday7 = 0.022). It seems that LC can maintain the metabolism and antioxidant capacity of PCs and thus can reduce mitochondrial damage and mtDNA release; consequently, it can decrease DAMPs in PCs. Therefore, it may be possible to use this substance as a platelet additive solution in the future.

Alantolactone induces concurrent apoptosis and GSDME-dependent pyroptosis of anaplastic thyroid cancer through ROS mitochondria-dependent caspase pathway

BackgroundAnaplastic thyroid cancer (ATC) is one of the fatal cancers and has not effective treatments. Alantolactone (ATL), a terpenoid extracted from traditional Chinese medicinal herb Inula helenium L., confers significant anti-inflammatory, antibacterial and antitumor activity. However, the activity and mechanisms of ATL in ATC remain unclear. PurposeTo investigate the potential anti-ATC effects in vitro and in vivo and the mechanisms involved. MethodsThe anti-proliferative activity of Alantolactone (ATL) against ATC cells was analyzed through CCK-8 and colony formation assays. Flow cytometry assay was performed to assess the cell cycle, cell apoptosis, ROS, and mitochondrial membrane potential (ΔΨm), whereas the cellular localization of cytochrome c and calreticulin were determined using cellular immunofluorescence assays. The lactate dehydrogenase (LDH) enzyme activity in the cell culture medium was measured using a commercial LDH kit, whereas ELISA was conducted to assess the secretory function of IL-1β. Western blot assays were conducted to determine the expression or regulation of proteins associated with apoptosis and pyroptosis. Subcutaneous tumor model of nude mice was established to evaluate the anticancer activity of ATL in vivo. The expression of Ki67, cyclin B1, cleaved-PARP, cleaved-caspase 3, and IL-1β in the animal tumor tissues was profiled using immunohistochemistry analyses. ResultsOur data showed that ATL significantly inhibited the proliferation and colony formation activity of ATC cells. ATL induced ATC cell cycle arrest at G2/M phase, and downregulated the expression of cyclin B1 and CDC2. Furthermore, ATL induced concurrent apoptosis and pyroptosis in the ATC cells, and the cleavage of PARP and GSDME. It also significantly increased the release of LDH and IL-1β. Mechanically, ATL-mediated increase in ROS suppressed the Bcl-2/Bax ratio, downregulated the mitochondrial membrane potential and increased the release of cytochrome c, leading to caspase 9 and caspase 3 cleavage. We also found that ATL induced the translocation of an immunogenic cell death marker (calreticulin) to the cell membrane. In addition, it inhibited the growth of the ATC subcutaneous xenograft model, and activated proteins associated with apoptosis and pyroptosis, with a high safety profile. ConclusionTaken together, these results firstly demonstrated that ATL exerted an anti-ATC activity by inducing concurrent apoptosis and GSDME-dependent pyroptosis through ROS-mediated mitochondria-dependent caspase activation. Meanwhile, these cell deaths exhibited obvious characteristics of immunogenic cell death, which may synergistically increase the potential of cancer immunotherapy in ATC. Further studies are needed to explore deeper mechanisms for the anti- ATC activity of ATL.

TGF-β3 induces lactate production in Sertoli cell through inhibiting the Notch pathway.

In the testis, spermatocytes and spermatids rely on lactate produced by Sertoli cells (SCs) as energy source. Transforming growth factor-beta 3 (TGF-β3) is one of the generally accepted paracrine regulatory factors of SC-created blood-testis barrier (BTB), yet its role in SC glycolysis and lactate production still remains unclear. To investigate the effect of TGF-β3 on glycolysis and lactate production in SCs and determine the role of lethal giant larvae 2 (Lgl2) and Notch signaling activity during this process. Primary cultured rat SCs and TM4 cells were treated with different concentrations of TGF-β3. In some experiments, cells were transfected with siRNA specifically targeting Lgl2 and then treated with TGF-β3 or N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester. Lactate concentration, glucose and glutamine (Gln) consumption in the culture medium, activity of phosphofructokinase (PFK), lactate dehydrogenase (LDH), and glutaminase (Gls), ATP level, oxygen consumption, extracellular acidification, and mitochondrial respiration complex activity were detected using commercial kits. The protein level of Lgl2, LDH, monocarboxylate transporter 4 (MCT4), and activity of Akt, ERK, p38 MAPK, and Notch pathway were detected by Western blot. The stage-specific expression of Jagged1 was examined by immunohistochemistry (IHC) and qPCR after laser capture microdissection. Spermatogenesis in rat testis injected with recombinant Jagged1 (re-Jagged1) was observed by HE staining, and lactate concentration in testis lysate was measured at a different day point after re-Jagged1 treatment. Significant enhancement of lactate concentration was detected in a culture medium of both primary SCs and TM4 cells treated with TGF-β3 at 3 or 5ng/ml. Besides, other parameters of glycolysis, that is, glucose and Gln consumption, enzyme activity of PFK, LDH, and Gls displayed different levels of increment in primary SCs and TM4 cells after TGF-β3 treatment. Mitochondria respiration of SCs was shown to decrease in response to TGF-β3. Lgl2, MCT4, activity of ERK, and p38 MAPK were up-regulated, whereas Akt and Notch pathway activity were inhibited by TGF-β3. Silencing of Lgl2 in SCs affected lactate production and attenuated the previous effects of TGF-β3 on SC glycolysis except for Gln consumption, Gls activity, and activity of Akt, ERK, and p38. N-[N-(3,5-difluorophenacetyl)-l-alanyl]-S-phenylglycine t-butyl ester (DAPT) treatment in SCs antagonized glycolysis suppression caused by Lgl2-silencing. In vivo analysis revealed a stage-specific expression of Jagged1 in contrary with TGF-β3. Activating Notch signaling by re-Jagged1 resulted in restorable hypospermatogenesis and lowered lactate level in rat testis. TGF-β3 induces lactate production in SC through up-regulating Lgl2, which weakened the Notch signaling activity and intensified glycolysis in SCs. Thus, besides the known function of TGF-β3 as the BTB regulator, TGF-β3-Lgl2-Notch may be considered an important pathway controlling SC glycolysis and spermatogenesis.

Fluted pumpkin seeds protect the spermatogenesis score index and testicular histology of caffeine treated rats.

The present study was designed to evaluate the protective effect of fluted pumpkin seeds (FPS) against caffeine (CAFF) induced testicular toxicity in rats. Thirty young healthy male Wistar rats (196 ± 12 g) were randomly organized into five sets of six animals per each group: control, caffeine (CAFF; 50 mg kg-1 bw) and FPS co-treatment groups (CAFF + 50 mg FPS, CAFF + 100 mg FPS and CAFF + 200 mg FPS kg-1 bw). CAFF and FPS were administered daily and twice per week respectively by oral gavage for 40 days. CAFF treatment decreased testicular lactate dehydrogenase enzyme activity level, which was attenuated on co-administration with FPS at 50 and 100 mg kg-1 bw. Furthermore, CAFF decreased seminiferous epithelia thickness and spermatogenesis score index and increased the number of tubules with abnormal histological features, which were attenuated on co-administration with FPS at 50 mg kg-1 bw much more than at the higher doses (p < 0.05). CAFF did not affect malondialdehyde and glutathione concentrations and glutathione peroxidase (GSH-Px) activity in the testes whereas FPS co-treatment at the higher doses elevated glutathione level and GSH-Px activity and did not affect spermatogenesis score index at the highest dose (200 mg kg-1 bw). Testicular malondialdehyde concentrations remained unaffected in all FPS co-treatment groups. Overall, FPS is able to minimize the CAFF-induced testicular injury at lower than at the higher tested doses.

Increasing of LDH Specific Activity and PEPCK Level Play a Role on Activation of Gluconeogenesis Pathway in Early Onset Pre-Eeclampsia Placenta.

Recent advancement on experiment concluded that etiology of pre-eclampsia (PE) could be explained by the "two-stage" theory. The theory of which explained that pre-eclampsia occurs due to abnormalities in spiral arteries development and release of inflammatory response. Failure of spiral arteries development, the lesion of damage could be due to ischemia-reperfusion or hypoxia-reoxygenation. Hypoxia in pre-eclamptic placenta leads to metabolic change to anaerobic in glycolysis. Lactate dehydrogenase (LDH) has important role in anaerobic glycolysis that catalyzes the conversion of lactate to pyruvate during hypoxia. On the other hand, phosphoenolpyruvate carboxy kinase (PEPCK) is merely an enzyme of gluconeogenesis. This research conduct to reveal that in early onset pre-eclampsia the placenta still hypoxic and undergoes gluconeogenesis even after delivery, through metabolic enzyme of LDH and PEPCK level. This cross-sectional study compared early onset PE (< 34 weeks) with normal term placenta. We measured LDH enzyme activity using colorimetric assay and PEPCK protein using ELISA method. Result show that placental LDH specific activity was increased significantly in PE with median 2.750 (0.030 - 5.680) U/mg compared to normal term placenta 0.255 (0.032 - 1.194) U/mg (Mann-Whitney, p< 0.001). PEPCK level was significantly increased in PE 8.998 (1.737-44.914) ng/mg compared to normal term placenta 1.552 (0.741-8.832) ng/mg (Mann-Whitney, p< 0.001). We conclude that anaerobic glycolysis and gluconeogenesis pathway are increased in early onset PE placenta as adaptation mechanism to hypoxic condition.