Extracellular Lactate Dehydrogenase Research Articles

New Findings Regarding the Effects of Selected Blue Food Colorants (Genipin, Patent Blue V, and Brilliant Blue FCF) on the Hemostatic Properties of Blood Components In Vitro.

Natural and synthetic colorants present in food can modulate hemostasis, which includes the coagulation process and blood platelet activation. Some colorants have cardioprotective activity as well. However, the effect of genipin (a natural blue colorant) and synthetic blue colorants (including patent blue V and brilliant blue FCF) on hemostasis is not clear. In this study, we aimed to investigate the effects of three blue colorants-genipin, patent blue V, and brilliant blue FCF-on selected parameters of hemostasis in vitro. The anti- or pro-coagulant potential was assessed in human plasma by measuring the following coagulation times: thrombin time (TT), prothrombin time (PT), and activated partial thromboplastin time (APTT). Moreover, we used the Total Thrombus formation Analysis System (T-TAS, PL-chip) to evaluate the anti-platelet potential of the colorants in whole blood. We also measured their effect on the adhesion of washed blood platelets to fibrinogen and collagen. Lastly, the cytotoxicity of the colorants against blood platelets was assessed based on the activity of extracellular lactate dehydrogenase (LDH). We observed that genipin (at all concentrations (1-200 µM)) did not have a significant effect on the coagulation times (PT, APTT, and TT). However, genipin at the highest concentration (200 µM) and patent blue V at the concentrations of 1 and 10 µM significantly prolonged the time of occlusion measured using the T-TAS, which demonstrated their anti-platelet activity. We also observed that genipin decreased the adhesion of platelets to fibrinogen and collagen. Only patent blue V and brilliant blue FCF significantly shortened the APTT (at the concentration of 10 µM) and TT (at concentrations of 1 and 10 µM), demonstrating pro-coagulant activity. These synthetic blue colorants also modulated the process of human blood platelet adhesion, stimulating the adhesion to fibrinogen and inhibiting the adhesion to collagen. The results demonstrate that genipin is not toxic. In addition, because of its ability to reduce blood platelet activation, genipin holds promise as a novel and valuable agent that improves the health of the cardiovascular system and reduces the risk of cardiovascular diseases. However, the mechanism of its anti-platelet activity remains unclear and requires further studies. Its in vivo activity and interaction with various anti-coagulant and anti-thrombotic drugs, including aspirin and its derivatives, should be examined as well.

Pap12-6: A host defense peptide with potent immunomodulatory activity in a chicken hepatic cell culture.

In the fight against antimicrobial resistance, host defense peptides (HDPs) are increasingly referred to as promising molecules for the design of new antimicrobial agents. In terms of their future clinical use, particularly small, synthetic HDPs offer several advantages, based on which their application as feed additives has aroused great interest in the poultry sector. However, given their complex mechanism of action and the limited data about the cellular effects in production animals, their investigation is of great importance in these species. The present study aimed to examine the immunomodulatory activity of the synthetic HDP Pap12-6 (PAP) solely and in inflammatory environments evoked by lipoteichoic acid (LTA) and polyinosinic-polycytidylic acid (Poly I:C), in a primary chicken hepatocyte-non-parenchymal cell co-culture. Based on the investigation of the extracellular lactate dehydrogenase (LDH) activity, PAP seemed to exert no cytotoxicity on hepatic cells, suggesting its safe application. Moreover, PAP was able to influence the immune response, reflected by the decreased production of interleukin (IL)-6, IL-8, and "regulated on activation, normal T cell expressed and secreted"(RANTES), as well as the reduced IL-6/IL-10 ratio in Poly I:C-induced inflammation. PAP also diminished the levels of extracellular H2O2 and nuclear factor erythroid 2-related factor 2 (Nrf2) when applied together with Poly I:C and in both inflammatory conditions, respectively. Consequently, PAP appeared to display potent immunomodulatory activity, preferring to act towards the cellular anti-inflammatory and antioxidant processes. These findings confirm that PAP might be a promising alternative for designing novel antimicrobial immunomodulatory agents for chickens, thereby contributing to the reduction of the use of conventional antibiotics.

Ischemia does not provoke the full immune training repertoire in human cardiac fibroblasts.

Trained immunity of monocytes, endothelial, and smooth muscle cells augments the cytokine response to secondary stimuli. Immune training is characterized by stabilization of hypoxia-inducible factor (HIF)-1α, mTOR activation, and aerobic glycolysis. Cardiac fibroblast (CF)-myofibroblast transition upon myocardial ischemia/reperfusion (I/R) features epigenetic and metabolic adaptations reminiscent of trained immunity. We assessed the impact of I/R on characteristics of immune training in human CF and mouse myocardium. I/R was simulated in vitro with transient metabolic inhibition. CF primed with simulated I/R or control buffer were 5days later re-stimulated with Pam3CSK for 24h. Mice underwent transient left anterior descending artery occlusion or sham operation with reperfusion for up to 5days. HIF-regulated metabolic targets and cytokines were assessed by qPCR, immunoblot, and ELISA and glucose consumption, lactate release, and lactate dehydrogenase (LDH) by chromogenic assay. Simulated I/R increased HIF-1α stabilization, mTOR phosphorylation, glucose consumption, lactate production, and transcription of PFKB3 and F2RL3, a HIF-regulated target gene, in human CF. PGK1 and LDH mRNAs were suppressed. Intracellular LDH transiently increased after simulated I/R, and extracellular LDH showed sustained elevation. I/R priming increased abundance of pro-caspase-1, auto-cleaved active caspase-1, and the expression and secretion of interleukin (IL)-1β, but did not augment Pam3CSK-stimulated cytokine transcription or secretion. Myocardial I/R in vivo increased abundance of HIF-1 and the precursor and cleaved forms of caspase-1, caspase-11, and caspase-8, but not of LDH-A or phospho-mTOR. I/R partially reproduces features of immune training in human CF, specifically HIF-1α stabilization, aerobic glycolysis, mTOR phosphorylation, and PFKB3 transcription. I/R does not augment PGK1 or LDH expression or the cytokine response to Pam3CSK. Regulation of PAR4 and inflammasome caspases likely occurs independently of an immune training repertoire.

Argininyl-fructosyl-glucose (AFG) antagonizes D-galactose-induced cellular senescence of neuro-2a via inhibiting endoplasmic reticulum stress and apoptosis

Argininyl-fructosyl-glucose (AFG), a distinctive non-saponin compound isolated from red ginseng (Panax ginseng. C.A. Meyer), demonstrates neuroprotective effects. However, the molecular mechanism underlying AFG's anti-aging properties remains unclear. This study established an in vitro D-galactose-induced N2a cells subacute aging model to investigate AFG's anti-aging activity and potential mechanisms. Results indicated that AFG treatment (8 μM) significantly reduces the overexpression of aging-related proteins (p53/p21/p16) induced by D-galactose, highlighting its anti-aging efficacy. AFG concurrently diminishes ROS accumulation, lowering extracellular lactate dehydrogenase and alleviating intracellular oxidative stress. Notably, AFG suppresses the PERK/CHOP/caspase 12 pathway, reducing intracellular Ca2+ concentration and mitigating endoplasmic reticulum (ER) stress. Additionally, AFG decreases Bax and caspase 3 expression, elevates Bcl-2 and mitochondrial membrane potential, mitigating apoptosis. NAC with 4-PBA validated the results. Overall, AFG protects N2a cells from aging by reducing ER stress and apoptosis, suggesting its potential as a key substance for treating aging and neurodegenerative diseases.

Cecropin A: investigation of a host defense peptide with multifaceted immunomodulatory activity in a chicken hepatic cell culture.

Host defense peptides (HDPs) are increasingly referred to as promising candidates for the reduction of the use of conventional antibiotics, thereby combating antibiotic resistance. As HDPs have been described to exert various immunomodulatory effects, cecropin A (CecA) appears to be a potent agent to influence the host inflammatory response. In the present study, a chicken primary hepatocyte-non-parenchymal cell co-culture was used to investigate the putative immunomodulatory effects of CecA alone and in inflammatory conditions evoked by polyinosinic-polycytidylic acid (Poly I:C). To examine the viability of the cells, the extracellular lactate dehydrogenase (LDH) activity was determined by colorimetric assay. Inflammatory markers interleukin (IL)-8 and transforming growth factor-ß1 (TGF-ß1) were investigated using the ELISA method, whereas concentrations of IL-6, IL-10, and interferon-γ (IFN-γ) were assayed by Luminex xMAP technology. Extracellular H2O2 and malondialdehyde levels were measured by fluorometric and colorimetric methods, respectively. Results of the lower concentrations suggested the safe application of CecA; however, it might contribute to hepatic cell membrane damage at its higher concentrations. We also found that the peptide alleviated the inflammatory response, reflected by the decreased production of the pro-inflammatory IL-6, IL-8, and IFN-γ. In addition, CecA diminished the levels of anti-inflammatory IL-10 and TGF-ß1. The oxidative markers measured remained unchanged in most cases of CecA exposure. CecA displayed a multifaceted immunomodulatory but not purely anti-inflammatory activity on the hepatic cells, and might be suggested to maintain the hepatic inflammatory homeostasis in Poly I:C-triggered immune response. To conclude, our study suggests that CecA might be a promising molecule for the development of new immunomodulatory antibiotic-substitutive agents in poultry medicine; however, there is still a lot to clarify regarding its cellular effects.

Structures, physical properties and antibacterial activity of silver nanoparticles of Lactiplantibacillus plantarum exopolysaccharide

Lactic acid bacteria (LAB) exopolysaccharide (EPS) has good water absorption, high viscosity, good stability, so it was widely used in probiotics fields. In this study, EPS-producing LAB strain Lactiplantibacillus plantarum HDL-03 was isolated and identified. Moreover, the HDL-03 EPS was used as a stabilizer and mixed with AgNO3 to synthesize a novel nanoparticle AgNPs whose structure and properties were explored. The monosaccharide composition and molecular weight indicated that HDL-03 EPS was a heteropolysaccharide composed of mannose and glucose. Fourier transform infrared spectroscopy (FT-IR), nuclear magnetic resonance (NMR) spectroscopy analysis and methylation results jointly proved it was a heteropolysaccharide containing 1,3-Manp and 1,6-Glcp. The X-Ray diffraction (XRD) results showed that this EPS has an amorphous structure, while the synthesized AgNPs have crystalline properties. The scanning electron microscopy (SEM) and transmission electron microscopy (TEM) results indicated EPS had a smooth and dense sheet structure, while the surface of AgNPs became rougher and large holes appeared after synthesis. Zeta particle size analysis suggested that the particle size of AgNPs increased by 36.63 nm compared to HDL-03 EPS. FT-IR analysis exhibited that the position of the characteristic peaks of AgNPs changed. The OH moving from a wavelength of 3388.49 cm−1 to a wavelength of 3316.79 cm−1 and telescopic vibration peak changed from 1356.07 cm−1 to 1344.22 cm−1. A plate inhibition test revealed the effect of different concentrations of EPS and AgNO3 synthesized AgNPs on the diameter of inhibition circle produced by the indicator bacteria Escherichia coli and Staphylococcus aureus. Furthermore, AgNPs were applied to the indicator bacteria, which the minimum inhibitory concentration (MIC), time-inhibitory curve, and changes in extracellular conductivity, nucleic acids, proteins, ATP, and lactate dehydrogenase (LDH) levels were determined. The AgNPs inhibited the growth of E. coli and S. aureus and exhibited outstanding antimicrobial properties. With the increase of treatment time, the degree of cell membrane damage increased, the permeability enhanced, and the intracellular substances leaked. These results indicate that HDL-03 EPS has good potential for applications in the production of food packaging, antimicrobials, catheters, textiles and coatings.

Investigation of the effects of T-2 toxin in chicken-derived three-dimensional hepatic cell cultures

Despite being one of the most common contaminants of poultry feed, the molecular effects of T-2 toxin on the liver of the exposed animals are still not fully elucidated. To gain more accurate understanding, the effects of T-2 toxin were investigated in the present study in chicken-derived three-dimensional (3D) primary hepatic cell cultures. 3D spheroids were treated with three concentrations (100, 500, 1000 nM) of T-2 toxin for 24 h. Cellular metabolic activity declined in all treated groups as reflected by the Cell Counting Kit-8 assay, while extracellular lactate dehydrogenase activity was increased after 500 nM T-2 toxin exposure. The levels of oxidative stress markers malondialdehyde and protein carbonyl were reduced by the toxin, suggesting effective antioxidant compensatory mechanisms of the liver. Concerning the pro-inflammatory cytokines, IL-6 concentration was decreased, while IL-8 concentration was increased by 100 nM T-2 toxin exposure, indicating the multifaceted immunomodulatory action of the toxin. Further, the metabolic profile of hepatic spheroids was also modulated, confirming the altered lipid and amino acid metabolism of toxin-exposed liver cells. Based on these results, T-2 toxin affected cell viability, hepatocellular metabolism and inflammatory response, likely carried out its toxic effects by affecting the oxidative homeostasis of the cells.

Study of biocompatibility and antitumor cytotoxic activity <i>in vitro</i> of Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys strengthened by equal angular pressing

Introduction. The biological activity of potential biodegradable zinc-based alloys that are promising for oncoorthopedics was studied in this work. The alloys were processed by equal-channel angular pressing, which made it possible to increase their strength due to microstructure refinement and the ability to provide the functionality of osteosynthesis, fixed due to the metal structure developed on their basis.Aim. Investigation of effect of equal-channel angular pressing (ECAP) treatment on strength, ductility, degradation rate, biocompatibility in vitro and cytotoxicity against SKOV-3 tumor cells of the Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys.Materials and methods. The Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys in the initial state and after ECAP were used as objects of study, and blood cells of CBA mice were used as model systems. To assess the hemolytic activity, the samples were incubated with red blood cells for 4 and 24 hours at 37 °C, assessing the relative increase in the level of extracellular hemoglobin compared to the intact control. The cytotoxicity of the alloys was assessed by the change in the level of extracellular lactate dehydrogenase (LDH) activity after 24 hours of incubation with mononuclear white blood cells. The study of antitumor cytotoxic activity was carried out on human ovarian cancer cells of the SKOV-3 line in vitro, assessing their survival after 48 hours of incubation with alloy samples using the LDH test.Results. As a result of the studies, it was concluded that the studied alloys after ECAP treatment retained their biocompatibility, since there were no signs of hemolysis and cytotoxicity with respect to blood cells. However, contact with samples of all studied alloys in vitro induced a significant inhibition of the metabolic activity of the ovarian cancer cell culture in comparison with the control. Incubation with alloys samples leads to a decrease in cellular activity by an average of 49 % and 59 % for Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys, respectively. The addition of calcium to the composition of the alloy Zn – 1 %Mg contributed to the growth of antitumor cytotoxic activity.Conclusion. Thus, based on the results of assessing the hemolytic activity and cytotoxicity of the samples, we can conclude that the studied alloys are biocompatible. It was also found that Zn – 1 %Mg and Zn – 1 %Mg – 0.1 %Ca alloys had a pronounced cytotoxic effect on SKOV-3 tumor cells. The obtained data indicate the prospects for the development of a new type of medical devices based on the studied alloys, promising, in particular, for oncoorthopedics: a metal structure developed on their basis can ensure the strength of osteosynthesis, reduce the risk of local recurrence of oncological disease and does not require a second operation to remove the device.

Intervention effects of sulfate glycosaminoglycan from swim bladder against arsenic-induced damage in IEC-6 cells

In this study, a purified macromolecular sulfate glycosaminoglycan whose structural characterization is similar to chondroitin sulfate from the swim bladder of Aristichthys nobilis, named SBSG, was used to explore the intervention effects on arsenic-induced intestinal epithelial cells (IEC-6) damage. Arsenic exposure led to cell membrane rupture, mitochondrial dysfunction, oxidative damage, and down-regulation of tight junction proteins expression. Treatment with SBSG could alleviate arsenic exposure-induced cell damage by decreasing the extracellular lactate dehydrogenase activity and influencing mitochondrial membrane potential, reactive oxygen species level, malondialdehyde content, and anti-oxidative enzyme activity. On the other hand, SBSG could promote nitric oxide production to achieve potential immunoregulation. The Western blot showed that intervention of SBSG mainly could restrain the activation of the JNK signaling pathway and up-regulate the expression of ZO-1 against arsenic-induced cell damage. This study provides a new perspective for understanding the heavy metal detoxification of SBSG on the intestinal and indicates that SBSG could be used as natural antioxidant resistant to heavy metal toxicity.

Toxic Effects of Two Redox States of Thallium on Immortalised Hypothalamic GT1-7 Neuronal Cells.

Thallium (Tl), is a highly toxic heavy metal that exists in monovalent (Tl(I)) and trivalent (Tl(III)) ionic states. This study aimed to compare the toxicities of Tl(I) and Tl(III) in a mouse hypothalamic GT1-7 neuronal cell line. Decreased viability and increased cytotoxicity were observed in the GT1-7 cells 16 h after Tl(I) or Tl(III) treatment. Tl(III) was more cytotoxic, than Tl(I), as indicated by extracellular lactate dehydrogenase levels. Both treatments induced caspase 3 activity, DNA fragmentation, malondialdehyde (MDA) production, and superoxide dismutase activity in the cells. MDA production was higher after Tl(III) than after Tl(I) treatment. Moreover, co-treatment with antioxidants, such as mannitol, ascorbic acid, or tocopherol, significantly attenuated the Tl-induced decrease in GT1-7 cell numbers. Therefore, both treatments induced oxidative stress-related apoptosis. Furthermore, Tl(III) reduced the cell viability more subtly than Tl(I) after 1 and 3 h of treatment. This effect was enhanced by co-treatment with maltol or citric acid, which promoted the influx of metallic elements into the cells. Thus, Tl(III) entered GT1-7 cells later than Tl(I) and had a delayed onset of toxicity. However, Tl(III) likely produces more extracellular lipid peroxides, which may explain its stronger cytotoxicity.

Luteolin: A Phytochemical to Mitigate S. Typhimurium Flagellin-Induced Inflammation in a Chicken In Vitro Hepatic Model.

The use of natural feed supplements is an alternative tool to diminish the damage caused by certain bacteria, improving animal health and productivity. The present research aimed to investigate the proinflammatory effect of flagellin released from the bacterial flagellum of Salmonella enterica serovar Typhimurium and to attenuate the induced inflammation with luteolin as a plant-derived flavonoid on a chicken primary hepatocyte-non-parenchymal cell co-culture. Cells were cultured in a medium supplemented with 250 ng/mL flagellin and 4 or 16 µg/mL luteolin for 24 h. Cellular metabolic activity, lactate dehydrogenase (LDH) activity, interleukin-6, 8, 10 (IL-6, IL-8, IL-10), interferon-α, γ (IFN-α, IFN-γ), hydrogen peroxide (H2O2) and malondialdehyde (MDA) concentrations were determined. Flagellin significantly increased the concentration of the proinflammatory cytokine IL-8 and the ratio of IFN-γ/IL-10, while it decreased the level of IL-10, indicating that the model served adequate to study inflammation in vitro. Luteolin treatment at 4 µg/mL did not prove to be cytotoxic, as reflected by metabolic activity and extracellular LDH activity, and significantly reduced the flagellin-triggered IL-8 release of the cultured cells. Further, it had a diminishing effect on the concentration of IFN-α, H2O2 and MDA and restored the level of IL-10 and the ratio of IFN-γ/IL-10 when applied in combination with flagellin. These results suggest that luteolin at lower concentrations may protect hepatic cells from an excessive inflammatory response and act as an antioxidant to attenuate oxidative damage.

Icariin protects cerebral neural cells from ischemia‑reperfusion injury in an invitro model by lowering ROS production and intracellular calcium concentration.

Ischemia is one of the major causes of stroke. The present study investigated the protection of cultured neural cells by icariin (ICA) against ischemia-reperfusion (I/R) injury and possible mechanisms underlying the protection. Neural cells were isolated from neonatal rats and cultured in vitro. The cells were subjected to oxygen-glucose deprivation and reoxygenation (OGD-R) as an I/R mimic to generate I/R injury, and were post-OGD-R treated with ICA. Following the treatments, cell viability, apoptosis, reactive oxygen species (ROS), lactate dehydrogenase (LDH), superoxide dismutase (SOD) and Ca2+ concentration were assessed using Cell Counting Kit-8 assay, flow cytometry, CyQUANT™ LDH Cytotoxicity Assay, H2DCFDA and SOD colorimetric activity kit. After OGD-R, considerable I/R injury was observed in the neural cells, as indicated by reduced cell viability, increased apoptosis and increased production of ROS and LDH (P<0.05). Cellular Ca2+ concentration was also increased, while SOD activity remained unchanged. Post-OGD-R ICA treatments increased cell viability up to 87.1% (P<0.05) and reduced apoptosis as low as 6.6% (P<0.05) in a concentration-dependent manner. The treatments also resulted in fewer ROS (P<0.05), lower extracellular LDH content (440.5 vs. 230.3 U/l; P<0.05) and reduced Ca2+ increase (P<0.05). These data suggest that ICA protects the neural cells from I/R injury in an in vitro model through antioxidation activity and maintaining cellular Ca2+ homeostasis. This function may be explored as a potential therapeutic strategy for ischemia-related diseases after further in vivo studies.

Antibacterial activity of reduced graphene oxide prepared by microbe

In previous work, we successfully bioreduced graphene oxide (B-rGO) utilizing organisms, but the antibacterial properties of B-rGO are still unknown. In this study, Escherichia coli (E. coli) was used as a test target and the antibacterial activity of B-rGO was compared to that of commercial reduced graphene oxide (C-rGO), in order to understand its toxicity. Data from levels of extracellular lactate dehydrogenase (LDH), and intracellular reactive oxygen species (ROS) content demonstrate that cell membrane stress and oxidative stress play a role in this antibacterial process. However, when the concentration of co-existing B-rGO was 50.0 mg/L, E. coli damaged easily, the LDH release concentration was 98.75 U/L, the ROS fluorescence signal was 57.75/106 cells, and the phenomenon known as ‘wrapping’ occurred. Indicated here is that B-rGO does indeed demonstrate very good antibacterial activity as evidenced when using Scanning Electron Microscopy (SEM). Meanwhile, numerous oxygen-containing functional groups existed on the surface of B-rGO as confirmed by Fourier transform infrared spectroscopy (FTIR). The power-time curve of microcalorimetry and flow cytometry analysis confirmed that the antibacterial activity of B-rGO was inadequate when compared to that of C-rGO. Despite E. coli mortality being dosage-dependent for both B-rGO and C-rGO, this implies that B-rGO does in fact achieve good biocompatibility. The outcomes reveal that B-rGO has great potential for environmental remediation.

Effect of Bacillus Subtilis on Immune Function of Hd11 Chicken Macrophages

Poultry is frequently contaminated by Salmonella, a pathogen leading to human health concern worldwide. This study aimed to evaluate the effect of Bacillus subtilis (BS)strain 048 (BS048) on the activation, phagocytosis, sterilization, cytokine secretion, and nitrogen oxide synthesis of HD11 chicken macrophages subjected to Salmonella enteritidis challenge, using lipopolysaccharide treatment as a negative control. The results showed: (1) BS048 had no significant effect on extracellular lactate dehydrogenase activity (p>0.05), while lipopolysaccharide treatment significantly increased extracellular lactate dehydrogenase activity (p<0.05);(2)BS048 significantly upregulated the expression levels of pro-inflammatory cytokines (interleukin (IL)-1â and IL-6), anti-inflammatory cytokines(IL-10 and transforming growth factor-â1), and anti-viral cytokine, interferon-â (p<0.01); (3)BS048 significantly upregulated the mRNA expression level of the inducible nitric oxide synthase and its activity as well as extracellular nitrogen oxide level (p<0.01). In conclusion, BS048 could improve anti-inflammatory and immune functions of HD11 chicken macrophages, without cytotoxic effects on these cells.

Immunomodulatory effects of chicken cathelicidin-2 on a primary hepatic cell co-culture model.

Cathelicidin-2 is an antimicrobial peptide (AMP) produced as part of the innate immune system of chickens and might be a new candidate to combat infection and inflammation within the gut-liver axis. Studying the hepatic immune response is of high importance as the liver is primarily exposed to gut-derived pathogen-associated molecular patterns. The aim of the present study was to assess the effects of chicken cathelicidin-2 alone or combined with lipoteichoic acid (LTA) or phorbol myristate acetate (PMA) on cell viability, immune response and redox homeostasis in a primary hepatocyte—non-parenchymal cell co-culture of chicken origin. Both concentrations of cathelicidin-2 decreased the cellular metabolic activity and increased the extracellular lactate dehydrogenase (LDH) activity reflecting reduced membrane integrity. Neither LTA nor PMA affected these parameters, and when combined with LTA, cathelicidin-2 could not influence the LDH activity. Cathelicidin-2 had an increasing effect on the concentration of the proinflammatory CXCLi2 and interferon- (IFN-)γ, and on that of the anti-inflammatory IL-10. Meanwhile, macrophage colony stimulating factor (M-CSF), playing a complex role in inflammation, was diminished by the AMP. LTA elevated IFN-γ and decreased M-CSF levels, while PMA only increased the concentration of M-CSF. Both concentrations of cathelicidin-2 increased the H2O2 release of the cells, but the concentration of malondialdehyde as a lipid peroxidation marker was not affected. Our findings give evidence that cathelicidin-2 can also possess anti-inflammatory effects, reflected by the alleviation of the LTA-triggered IFN-γ elevation, and by reducing the M-CSF production induced by PMA. Based on the present results, cathelicidin-2 plays a substantial role in modulating the hepatic immune response with a multifaceted mode of action. It was found to have dose-dependent effects on metabolic activity, membrane integrity, and reactive oxygen species production, indicating that using it in excessively high concentrations can contribute to cell damage. In conclusion, cathelicidin-2 seems to be a promising candidate for future immunomodulating drug development with an attempt to reduce the application of antibiotics.

Melatonin shapes bacterial clearance function of porcine macrophages during enterotoxigenic Escherichia coli infection.

Due to the immature gastrointestinal immune system, weaning piglets are highly susceptible to pathogens, e.g., enterotoxigenic Escherichia coli (ETEC). Generally, pathogens activate the immune cells (e.g., macrophages) and shape intracellular metabolism (including amino acid metabolism); nevertheless, the metabolic cues of tryptophan (especially melatonin pathway) in directing porcine macrophage function during ETEC infection remain unclear. Therefore, this study aimed to investigate the changes in the serotonin pathway of porcine macrophages during ETEC infection and the effect of melatonin on porcine macrophage functions. Porcine macrophages (3D4/21 cells) were infected with ETEC, and the change of serotonin pathway was analysed by reverse transcription PCR and metabolomic analysis. The effect of melatonin on porcine macrophage function was also studied with proteomic analysis. In order to investigate the effect of melatonin on bacterial clearance function of porcine macrophages during ETEC infection, methods such as bacterial counting, reverse transcription PCR and western blotting were used to detect the corresponding indicators. The results showed that ETEC infection blocked melatonin production in porcine macrophages (P < 0.05) which is largely associated with the heat-stable enterotoxin b (STb) of ETEC (P < 0.05). Interestingly, melatonin altered porcine macrophage functions, including bacteriostatic and bactericidal activities based on proteomic analysis. In addition, melatonin pre-treatment significantly reduced extracellular lactate dehydrogenase (LDH) activity (P < 0.05), indicating that melatonin also attenuated ETEC-triggered macrophage death. Moreover, melatonin pre-treatment resulted in the decrease of viable ETEC in 3D4/21 cells (P < 0.05), suggesting that melatonin enhances bacterial clearance of porcine macrophages. These results suggest that melatonin is particularly important in shaping porcine macrophage function during ETEC infection.

Bexarotene-induced cell death in ovarian cancer cells through Caspase-4-gasdermin E mediated pyroptosis

Bexarotene selectively activates retinoid X receptor, which is a commonly used anticancer agent for cutaneous T-cell lymphoma. In this study, we aimed to investigate the anticancer effect of bexarotene and its underlying mechanism in ovarian cancer in vitro. The ES2 and NIH:OVACAR3 ovarian cancer cell lines were treated with 0, 5, 10, or 20 µM of bexarotene. After 24 h, cell number measurement and lactate dehydrogenase (LDH) cytotoxicity assay were performed. The effect of bexarotene on CDKN1A expression, cell cycle-related protein, cell cycle, pyroptosis, and apoptosis was evaluated. Bexarotene reduced cell proliferation in all concentrations in both the cells. At concentrations of > 10 µM, extracellular LDH activity increased with cell rupture. Treatment using 10 µM of bexarotene increased CDKN1A mRNA levels, decreased cell cycle-related protein expression, and increased the sub-G1 cell population in both cells. In ES2 cells, caspase-4 and GSDME were activated, whereas caspase-3 was not, indicating that bexarotene-induced cell death might be pyroptosis. A clinical setting concentration of bexarotene induced cell death through caspase-4–mediated pyroptosis in ovarian cancer cell lines. Thus, bexarotene may serve as a novel therapeutic agent for ovarian cancer.

Exposure to nanoplastic induces cell damage and nitrogen inhibition of activated sludge: Evidence from bacterial individuals and groups

Wastewater treatment plants (WWTPs) are almost the only place where plastic fragments are artificially removed, resulting in mass accumulation of nanoplastics (NPs). In this research, four different concentrations (0 mg/L, 0.1 mg/L, 1 mg/L, 10 mg/L) of polystyrene nanoplastics (PS-NPs) were used to investigate the cell damage and nitrogen inhibition of activated sludge, exposed in a self-assembled SBR reactor for 30 days. Intracellular reactive oxides (ROS) and extracellular lactate dehydrogenase (LDH) increased with the rise of exposure concentration, and morphological analysis disclosed the creases, collapse, and even rupture of cell membranes. However, exposure damage (PS-NPs ≤ 1 mg/L) appeared to be reversible, attributed to that extracellular polymeric substances (EPS) secretion can thicken the three protective layers outside the membrane. PS-NPs did not disrupt the EPS chemical structure, but increased humic acid content. Prolonged exposure time (from 15 to 30 days) was directly related to the nitrogen inhibition. Due to the habitat changes under PS-NPs exposure, abundance and diversity of microorganisms in the original activated sludge decreased significantly, and the dominant phylum was occupied by Patescibacteria (PS-NPs = 10 mg/L). Changes in enzyme activities of AMO, NR, NIR, and NOR with exposure concentration may explain the conversion of nitrogen in SBR. This research broadens our horizons to understand the response mechanism of activated sludge bacteria to PS-NPs exposure individually and collectively.

Protective effects of curcumin/cyclodextrin polymer inclusion complex against hydrogen peroxide-induced LO2 cells damage.

The objective of the present study was to explore the protective effects of the curcumin/cyclodextrin polymer (CUR/CDP) inclusion complex on hydrogen peroxide (H2O2)‐induced LO2 cells damage. In this study, a H2O2‐induced cells oxidative injury model was established to test the protective effects of the CUR/CDP inclusion complex. The cell viability of cells was detected by the thiazolyl blue tetrazolium bromide (MTT) assay. The extracellular lactate dehydrogenase (LDH) activity, catalase (CAT) activity, and malondialdehyde (MDA) level were detected by assay kits. The cellular reactive oxygen species (ROS) level was detected using the dichlorodihydrofluorescein (DCF) fluorescence assay. Western blotting analysis was conducted to assess the changes of phosphorylated‐p53 and caspase‐3. The results showed that 700 μM H2O2‐treated LO2 cells for 3 h resulted in a significant decrease of cell viability to 53.00 ± 1.68%, which established the cell oxidative injury model. Cells treated with H2O2 led to a significant increase of extracellular LDH activity, MDA content, and ROS level, and decreased CAT activity. Treatment with CUR/CDP significantly reversed the changes of the above indicators. Moreover, CUR/CDP treatment at 20 and 40 μg/ml inhibited H2O2‐induced increase in phosphorylated‐p53 and caspase‐3 expression, indicating that CUR/CDP suppressed cell apoptosis to alleviate liver injury. The results of those studies demonstrated that CUR/CDP had a protective effect on the oxidative damage of LO2 cells, and it could be developed as a new type of natural liver protection product to apply in the prevention of liver injury.

Inhibition Activity of Plantaricin Q7 Produced by Lactobacillus plantarum Q7 against Listeria monocytogenes and Its Biofilm

Plantaricin Q7 is a broad-spectrum antimicrobial peptide produced by Lactobacillus plantarum Q7. The effects of plantaricin Q7 on Listeria monocytogenes and its biofilm were investigated. The results showed that plantaricin Q7 changed the cell membrane permeability and integrity of Listeria monocytogenes significantly. The extracellular lactate dehydrogenase activity increased from 156.74 U/L to 497.62 U/L, and the K+ concentration was increased rapidly from 0.02 g/L to 0.09 g/L. Furthermore, the flagellum motility of Listeria monocytogenes reduced and the relative adhesion rate decreased about 30% after treatment with plantaricin Q7. Meanwhile, the morphology and structure of Listeria monocytogenes cell and biofilm were damaged. These findings suggested that plantaricin Q7 exhibited significant inhibitory effects on not only Listeria monocytogenes cell but also its biofilm, which might be used as a natural and effective biological preservative for food storage.