Lactate Dehydrogenase Cytotoxicity Assay Research Articles

Analysis of the chemical composition and in vitro cytotoxic activities of the essential oil of the aerial parts of Lavandula atriplicifolia Benth

Plants of the Lavandula genus have been widely used in folk and traditional medicine. The aim of this study was to evaluate the qualitative and quantitative chemical composition of the essential oil of the aerial parts of Lavandula atriplicifolia (EOAPLA) grown in Saudi Arabia and its potential cytotoxicity to cancer cell lines. The essential oil was characterized and quantified by GC–MS and GC-FID. The potential cytotoxicity of the essential oil was evaluated against colorectal (LoVo) and hepatocellular (HepG2) carcinoma cell lines by using the MTT and lactate dehydrogenase (LDH) cytotoxicity assays. A total of 36 compounds were identified that constituted 92.3% of the total oil. The chemotype of the species was dominated by C-10 massoia lactone (46.65%) as the major compound followed by oxygenated monoterpenes (28.43%). This is the first report to describe the presence of the rare compound massoia lactone in the essential oils from Lavandula species. The data revealed that EOAPLA inhibited the growth of LoVo and HepG2 cells in a dose-dependent manner. Treatment with 10 μg/mL EOAPLA for 2 days resulted in significant damage to LoVo cells in comparison with the control. In contrast to the control cells, the LoVo cells showed morphological alterations such as cytoplasmic condensation, shrinkage, and the formation of debris. Moreover, EOAPLA treatment induced apoptosis in LoVo cells since it increased the expression of caspase 3/7. The cytotoxic and anti-proliferative properties of EOAPLA make it a good candidate for treatment of various cancers.

Terminal complement complex C5b-9 reduced megalin and cubilin-mediated tubule proteins uptake in a mouse model of trichloroethylene hypersensitivity syndrome

Trichloroethylene (TCE), a commonly used industrial solvent and degreasing agent, is known to cause trichloroethylene hypersensitivity syndrome (THS) with multi-system damage, including skin, liver and kidney. Clinical evidence have shown that the kidney injury occurs in THS and our previous studies suggested that the terminal complement complex C5b-9 deposited in impaired renal tubules induced by TCE with unclear mechanisms. In the present study, we questioned whether activation of the complement system with renal deposition of C5b-9 contributes to TCE-induced kidney injury in THS. We established a BALB/c mouse model of TCE sensitization with or without pretreatment of exogenous CD59, a C5b-9 inhibitory protein. H&E staining, PAS staining, and biochemical detection of urinary proteins were performed to assess renal function. Deposition of C5b-9 and expression of CD59 were evaluated by immunohistochemistry. Sub-lytic effects of C5b-9 in tubular epithelial cells were assessed by lactate dehydrogenase (LDH) cytotoxicity assay. Expression of endocytosis receptors megalin and cubilin on proximal tubules were assessed by immunofluorescence and qRT-PCR. We found that TCE sensitization induced structural and functional changes of renal tubules in mice, associated with the deposition of sub-lytic C5b-9 on proximal tubular epithelial cells. TCE sensitization decreased proximal tubule uptake of filtered proteins and renal expression of megalin and cubilin, phenotypes that were attenuated by pretreatment with exogenous CD59. Overall, our findings reveal a novel mechanism underlying sub-lytic C5b-9 acting on megalin and cubilin, contributes to the renal tubules damage by TCE exposure.

Effect of miR‐499a‐5p on damage of cardiomyocyte induced by hypoxia‐reoxygenation via downregulating CD38 protein

The aim is to investigate the mechanism of miR-499a-5p on the damage of cardiomyocyte induced by hypoxia/reoxygenation. The activity of lactate dehydrogenase (LDH), apoptosis rate and the expression of miR-499a-5p and cluster of differentiation 38 (CD38) in hypoxia-reoxygenation model cells were detected by LDH Cytotoxicity Assay Kit, flow cytometry, real-time polymerase chain reaction, and Western blot analysis, respectively. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p or silencing of CD38 in H9c2 cells. The target relationship between miR-499a-5p and CD38 was verified by Targetscan online prediction and dual-luciferase assay. Apoptosis, the activity of LDH was detected after overexpression of miR-499a-5p and CD38. Apoptosis, the activity of LDH and the expression of CD38 were increased (P < .05) while expression of miR-499a-5p was decreased (P < .05) in hypoxia/reoxygenation model cells. Apoptosis and the activity of LDH in H9c2 cells after overexpression of miR-499a-5p or silence of CD38 were decreased (P < .05). The results of Targetscan online prediction and dual-luciferase assay indicated that CD38 was a potential target gene of miR-499a-5p. Overexpression of CD38 could reverse the inhibition of miR-499a-5p on LDH activity and apoptosis in H9c2 cells. miR-499a-5p could relief the injury of cardiomyocytes induced by hypoxia/reoxygenation via targeting CD38.

Cwp22, a novel peptidoglycan cross-linking enzyme, plays pleiotropic roles in Clostridioides difficile.

Clostridioides difficile is a Gram-positive, spore-forming, toxin-producing anaerobe pathogen, and can induce nosocomial antibiotic-associated intestinal disease. While production of toxin A (TcdA) and toxin B (TcdB) contribute to the main pathogenesis of C. difficile, adhesion and colonization of C. difficile in the host gut are prerequisites for disease onset. Previous cell wall proteins (CWPs) were identified that were implicated in C. difficile adhesion and colonization. In this study, we predicted and characterized Cwp22 (CDR20291_2601) from C. difficile R20291 to be involved in bacterial adhesion based on the Vaxign reverse vaccinology tool. The ClosTron-generated cwp22 mutant showed decreased TcdA and TcdB production during early growth, and increased cell permeability and autolysis. Importantly, the cwp22 mutation impaired cellular adherence in vitro and decreased cytotoxicity and fitness over the parent strain in a mouse infection model. Furthermore, lactate dehydrogenase cytotoxicity assay, live-dead cell staining and transmission electron microscopy confirmed the decreased cell viability of the cwp22 mutant. Thus, Cwp22 is involved in cell wall integrity and cell viability, which could affect most phenotypes of R20291. Our data suggest that Cwp22 is an attractive target for C. difficile infection therapeutics and prophylactics.

Development and mechanistic study of a microemulsion containing vitamin ETPGS for the enhancement of oral absorption ofcelecoxib.

Purpose: The purpose of this study was to develop a microemulsion containing D-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS) as a biodegradable surfactant to increase the oral absorption of celecoxib.Methods: This study investigated the intestinal absorption enhancement mechanism of this microemulsion by measuring transepithelial electrical resistance (TEER) values. This study also evaluated microemulsion particle–intestine interactions in terms of release and attachment processes using confocal laser scanning microscopy (CLSM).Results: The prepared microemulsion particles had a size of <300 nm with a neutral surface charge. The celecoxib-loaded microemulsion release kinetic was classified as the zero-order model. This vitamin E TPGS-based microemulsion significantly increased the in vitro intestinal absorption of celecoxib compared to celecoxib solution. The CLSM study suggested that microemulsion particles with entrapped drugs might attach to the intestinal epithelium before releasing the entrapped drug into tissues. The TEER value of the intestinal tissues treated with the celecoxib-loaded microemulsion was significantly decreased compared to the value before treatment, indicating an increase in drug transport via the paracellular pathway. The evaluation of intestinal tissue cytotoxicity using lactate dehydrogenase cytotoxicity assay suggested that the prepared celecoxib-loaded microemulsion was safe for oral route administration.Conclusions: The prepared celecoxib loaded microemulsion could increase the intestinal absorption of celecoxib compared to celecoxib solution. The intestinal absorption enhancement mechanism of this microemulsion resulted from the increase of the drug transport via the paracellular pathway.

Neurotoxicity of BPA, BPS, and BPB for the hippocampal cell line (HT-22): An implication for the replacement of BPA in plastics

Bisphenol A (BPA), a plastic additive, is ubiquitous in the environment and has endocrine disrupting effects. As many countries have prohibited the manufacture and sale of plastic products with BPA, BPA analogs have been used to replace BPA during production, including bisphenol S (BPS) and bisphenol B (BPB). To investigate the toxicities of BPA and its analogs on neurons, reactive oxygen species (ROS) assay, Annexin V-FITC (fluorescein) apoptosis detection assay, lactate dehydrogenase (LDH) cytotoxicity assay, and Cell Counting Kit-8 assay were conducted to comprehensively assess the influence of different concentrations of BPA, BPB, and BPS on ROS, apoptosis, damage, and proliferation for hippocampal HT-22 cells, respectively. Results showed that 6 h of exposure to bisphenols (BPs) could increase the ROS levels, 24 h and 48 h of exposure could induce higher apoptosis and LDH leakage rates for HT-22 cells, and 7 d of exposure could inhibit the cell proliferations. In addition, non-monotonic dose-response relationships were observed between the concentrations of bisphenols and the toxic effects mentioned above. The neurotoxic effects of BPA, BPB and BPS on HT-22 cells were in the increasing order of BPS, BPA, and BPB. In conclusion, these results showed that exposure to BPA and its analogs may result in adverse effects on hippocampal neuronal cell lines. BPS is a surrogate with lower neurotoxicity to replace BPA in production of plastic utensils.

The effect of micro-sized titanium dioxide on WM-266-4 metastatic melanoma cell line.

Titanium dioxide (TiO2) is widely used as an inorganic UV-filter in cosmetic products; however, it has been classified as possibly carcinogenic to humans. While numerous studies demonstrated cytotoxic and genotoxic effects of nano-sized TiO2 in different cell lines, including human skin cells, studies investigating the effects of micro-TiO2 on human keratinocytes and melanocytes, in healthy and cancer cells, are scarce. Adenosine triphosphate (ATP) binding cassette subfamily B member 5 (ABCB5) is a plasma membrane protein known for its role in the tumorigenicity, progression, and recurrence of melanoma. Here, we investigated the effect of micro-TiO2 (average particle size ≤5 µm) on the metabolic activity, cytotoxicity and ABCB5 mRNA expression in metastatic melanoma cells. Metastatic melanoma cell line WM-266-4 was treated with different concentrations of micro-TiO2 for different incubation times to obtain dose- and time-dependent responses. Untreated WM-266-4 cells, cultured under the same conditions, were used as control. The cell metabolic activity was determined by MTT assay. Cytotoxicity of micro-TiO2 was analyzed by lactate dehydrogenase (LDH) cytotoxicity assay. The ABCB5 mRNA expression in melanoma cells was analyzed using quantitative reverse transcription polymerase chain reaction (RT-qPCR). After 120 hours of exposure to micro-TiO2 the metabolic activity of melanoma cells decreased, especially at the two highest micro-TiO2 concentrations. Comparably, the cytotoxicity of micro-TiO2 on melanoma cells increased after 48 and 120 hours of exposure, in a time-dependent manner. The ABCB5 mRNA expression in micro-TiO2-treated melanoma cells also decreased significantly after 24 and 48 hours, in a time-dependent manner. Overall, our results suggest inhibitory effects of micro-TiO2 on the metabolic activity and ABCB5 mRNA expression in metastatic melanoma cells, indicating its potential use as an anticancer agent.

Metformin combined with nelfinavir induces SIRT3/mROS-dependent autophagy in human cervical cancer cells and xenograft in nude mice

The molecular mechanisms underlying the antineoplastic properties of metformin combined with nelfinavir remain elusive. To explore this question, transmission electron microscopy (TEM) was used to observe the combinatorial effect of inducing autophagosome formation in human cervical cancer cells. Western blotting respectively assayed protein expression of LC3I, LC3II, Beclin-1, Autophagy-related protein 7 (Atg7), Autophagy-related protein 3 (Atg3), NAD-dependent deacetylase sirtuin-3 (SIRT3) and major histocompatibility complex class I chain-related gene A (MICA). Lactate dehydrogenase (LDH) cytotoxicity assay evaluated natural killer (NK) cell cytotoxicity in the presence of metformin and nelfinavir in combination or each drug alone. Using tumor xenografts in a nude mouse model, antitumor efficacy of the drug combination was assessed. We found that the drug combination could induce autophagosome formation in human cervical cancer cells. The biomarker proteins of autophagy, including Beclin-1, Atg7 and Atg3, decreased, but the ratios of LC3I/II increased. We also found that this drug combination sensitizes human cervical cancer cells to NK cell-mediated lysis by increasing the protein of SIRT3 and MICA. Moreover, this drug combination markedly induced autophagy of SiHa xenografts in nude mice. Therefore, it can be concluded that metformin, in combination with nelfinavir, can induce SIRT3/mROS-dependent autophagy and sensitize NK cell-mediated lysis in human cervical cancer cells and cervical cancer cell xenografts in nude mice. Thus, our findings have revealed the detailed molecular mechanisms underlying the antitumor effects of metformin in combination with nelfinavir in cervical cancer.

Shengmai injection alleviates H2O2‑induced oxidative stress through activation of AKT and inhibition of ERK pathways in neonatal rat cardiomyocytes

Ethnopharmacological relevanceShengmai injection (SMI) is a classical traditional Chinese medicine (TCM) officially recorded in Pharmacopoeia of the People's Republic of China (version 2015) and has long been used to treat heart failure in China. However scientific evidence for the anti-oxidative stress potential of SMI used in traditional medicine is lacking. Aim of studyThe present study aimed to evaluate the efficacy of SMI in alleviating H2O2‑induced Oxidative Stress the underlying mechanisms Materials and methodsH2O2-induced oxidative stress model of cardiomyocytes was established with primary cultured neonatal rat cardiomyocytes. CCK8 cell viability assay and lacatate dehydrogenase cytotoxicity assay were performed to ensure the safety dose and lowest effective dose for the mode employing CCK-8 cell viability assay kit and lactate dehydrogenase cytotoxicity assay kit. ROS levels were determined using CM-H2DCFDA fluorescent probe in cardiomyocytes with H2O2-induced oxidative stress. The change of NAD(P)H level in cardiomyocytes was evaluated during the process of oxidative stress. The content of myocardial cytosolic Ca2+ and Ca2+ was determined using Fura-2/AM and Rhod 2-AM fluorescent probe in mitochondrial in the process of oxidative stress. Annexin V-FITC/PI double staining was applied to examine the apoptotic cells in cardiomyocytes with oxidative stress. To identify the apoptosis after oxidative stress myocardial cells with the application of Annexin V-FITC/PI double staining apoptosis detection kit. Quantitative polymerase chain reaction (RT-PCR) was applied to measure the expression of antioxidant enzymes: catalase (CAT), superoxide dismutase (SOD), glutathione reductase (GSR). Western blot was performed to observe the phosphorylation of AKT and ERK1/2. ResultsSMI was shown to significantly attenuate oxidative stress-induced cell proliferation arrest and apoptosis in neonatal rat cardiomyocytes. In addition, SMI treatment could decrease the production of reactive oxygen species (ROS), nicotinamide adenine dinucleotide (NADH) and malondialdehyde (MDA), and reduce the overloads of cytoplasmic Ca2+ and mitochondrial Ca2+ induced by H2O2. SMI could also restore the mRNA expression and activities of SOD, GSR, and CAT suppressed by H2O2. Mechanistically, SMI upregulated intracellular AKT phosphorylation and downregulate ERK1/2 phosphorylation in H2O2-treated cardiomyocytes. Pretreatment with LY294002, an AKT phosphorylation inhibitor, suppressed the protective role of SMI in cardiomyocytes, while pretreatment with PD98059, an ERK1/2 phosphorylation inhibitor, enhanced the effect of SMI. ConclusionsIn conclusion, SMI may attenuate oxidative stress-induced damage in cardiomyocytes potentially through the AKT and ERK1/2 pathway and can function as a promising injectable traditional Chinese medicine to treat oxidative stress-induced injury.

MiR-218-5p Suppresses the Killing Effect of Natural Killer Cell to Lung Adenocarcinoma by Targeting SHMT1.

PurposeLung adenocarcinoma (LA) is one of the major types of lung cancer. MicroRNAs (miRNAs) play an essential role in regulating responses of natural killer (NK) cells to cancer malignancy. However, the mechanism of miR-218-5p involved in the killing effect of NK cells to LA cells remains poorly understood.Materials and MethodsThe expression of miR-218-5p was examined by quantitative real-time polymerase chain reaction (qRT-PCR). Serine hydroxymethyl transferase 1 (SHMT1) level was detected by qRT-PCR or western blots. Cytokines production of interferon-γ (IFN-γ) and tumor necrosis factor-α (TNF-α) were detected by ELISA. The killing effect of NK cells to LA cells was investigated using lactate dehydrogenase cytotoxicity assay kit. The interaction of miR-218-5p and SHMT1 was probed by luciferase activity assay. Xenograft model was established to investigate the killing effect of NK cells in vivo.ResultsmiR-218-5p was enhanced and SHMT1 was inhibited in NK cells of LA patients, whereas stimulation of interleukin-2 (IL-2) reversed their abundances. Addition of miR-218-5p reduced IL-2-induced cytokines expression and cytotoxicity in NK-92 against LA cells. Moreover, SHMT1 was negatively regulated by miR-218-5p and attenuated miR-218-5p-mediated effect on cytotoxicity, IFN-γ and TNF-α secretion in IL-2-activated NK cells. In addition, miR-218-5p exhaustion inhibited tumor growth by promoting killing effect of NK cells.ConclusionmiR-218-5p suppresses the killing effect of NK cells to LA cells by targeting SHMT1, providing a potential target for LA treatment by ameliorating NK cells function.

Targeting the crosstalk between cytokine-induced killer cells and myeloid-derived suppressor cells in hepatocellular carcinoma

Cytokine-induced killer (CIK) cell-based immunotherapy is effective as an adjuvant therapy in early stage hepatocellular carcinoma (HCC) but lacks efficacy in advanced HCC. We aimed to investigate immune suppressor mechanisms in HCC, focusing on the role of myeloid-derived suppressor cells (MDSCs) in response to CIK therapy. MDSCs were quantified by flow cytometry and quantitative real-time PCR. Cytokines were detected by cytokine array. A lactate dehydrogenase cytotoxicity assay was performed in the presence or absence of MDSCs to study CIK function against HCC cells in vitro. An FDA-approved PDE5 inhibitor, tadalafil, was used to target MDSCs in vitro and in vivo. Two different murine HCC cell lines were tested in subcutaneous and orthotopic tumor models in C57BL/6 and BALB/c mice. The antitumor effects of human CIKs and MDSCs were also tested in vitro. Adoptive cell transfer of CIKs into tumor-bearing mice induced inflammatory mediators (e.g., CX3CL1, IL-13) in the tumor microenvironment and an increase of tumor-infiltrating MDSCs, leading to impaired antitumor activity in 2 different HCC models. MDSCs efficiently suppressed the cytotoxic activity of CIKs in vitro. In contrast, treatment with a PDE5 inhibitor reversed the MDSC suppressor function via ARG1 and iNOS blockade and systemic treatment with a PDE5 inhibitor prevented MDSC accumulation in the tumor microenvironment upon CIK cell therapy and increased its antitumor efficacy. Similar results were observed when human CIKs were tested in vitro in the presence of CD14+HLA-DR-/low MDSCs. Treatment of MDSCs with a PDE5 inhibitor suppressed MDSC suppressor function and enhanced CIK activity against human HCC cell lines in vitro. Our results suggest that targeting MDSCs is an efficient strategy to enhance the antitumor efficacy of CIKs for the treatment of patients with HCC. Cytokine-induced killer cells are a mixture of immune cells given to eliminate cancer cells. However, not all patients respond to this treatment. Herein, we show in 2 different liver cancer models that myeloid-derived suppressor cells are increased in response to cytokine-induced killer cell therapy. Targeting these myeloid-derived suppressor cells may provide an additional therapeutic benefit alongside cytokine-induced killer cell therapy.

A defucosylated bispecific multivalent molecule exhibits broad HIV-1-neutralizing activity and enhanced antibody-dependent cellular cytotoxicity against reactivated HIV-1 latently infected cells.

Current treatments cannot completely eradicate HIV-1 owing to the presence of latently infected cells, which harbor transcriptionally silent HIV-1. However, defucosylated antibodies can readily kill latently infected cells after their activation to express envelope glycoprotein (Env) through antibody-dependent cellular cytotoxicity (ADCC). We herein aimed to test a defucosylated bispecific multivalent molecule consisting of domain-antibody and single-domain CD4, LSEVh-LS-F, for its HIV-1 neutralizing activity and ADCC against the reactivated latently infected cells, compared with the nondefucosylated molecule LSEVh-LS. LSEVh-LS-F's neutralizing activity against a panel of newly characterized Chinese HIV-1 clinical isolates was assessed by using TZM-bl-based and PBMC-based assays. LSEVh-LS-F-mediated ADCC in the presence of natural killer cells against cell lines that stably express Env proteins, HIV-1-infected cells and LRA-reactivated HIV-1 latent cells, was measured using a lactate dehydrogenase (LDH) cytotoxicity assay or flow cytometry. LSEVh-LS-F and LSEVh-LS were equally effective in neutralized infection of all HIV-1 isolates tested with IC50 and IC90 values 3∼4-fold lower than those of VRC01. LSEVh-LS-F was more effective in natural killer-mediated killing of HIV-1 Env-expressing cell lines, HIV-1-infected cells, latency reactivation agents-reactivated ACH2 cells and reactivated latently infected resting CD4+ T cell line as well as resting CD4+ T lymphocytes isolated from patients receiving HAART. LSEVh-LS-F exhibits broad HIV-1 neutralizing activity and enhanced ADCC against HIV-1-infected cells, reactivated latently infected cell lines and primary CD4+ T cells, thus being a promising candidate therapeutic for eradicating the HIV-1 reservoir.

Effect of PD-1 inhibitor Nivolumab on the proliferation and cytotoxicity of anti-CD19 chimeric antigen receptor T cells

目的评估程序性细胞死亡受体1（PD-1）抑制剂Nivolumab对CD19-CAR-T细胞体外增殖和杀伤活性的影响。方法收集5例外周血PD-1高表达恶性淋巴瘤患者的外周血T细胞制备CD19-CAR-T细胞，在培养第8天加入终浓度分别为72、36、18 µg/ml的Nivolumab，同时设患者T细胞联合72 µg/ml Nivolumab及正常人CD19-CAR-T细胞为对照，采用CCK-8法、LDH细胞毒性检测、ELISA法比较各组的增殖活性、杀伤活性及炎症因子水平。结果①PD-1高表达患者CD19-CAR-T细胞转染率与正常人接近[（32.80±7.22）%对（35.10±5.84）%，t=−0.554，P=0.593]。②72 µg/ml Nivolumab联合CD19-CAR-T细胞不影响其增殖，但联合应用24、48 h对Pfeiffer细胞的杀伤率均优于单用患者CD19-CAR-T细胞及患者来源T细胞+72 µg/ml的Nivolumab（P值均<0.001），其中48 h时各组的杀伤率分别为（71.61±9.50）%、（6.77±1.26）%、（15.33±4.11）%。72、36 µg/ml Nivolumab联合来源CD19-CAR-T细胞对Pfeiffer细胞的杀伤率差异无统计学意义（P值分别为0.281、0.267），二者均高于患者来源CD19-CAR-T细胞+18 µg/ml Nivolumab组（P值均<0.001）。③不同剂量Nivolumab联合患者来源CD19-CAR-T细胞，不影响炎症因子IFN-γ、TNF-α水平（P值均>0.05）。结论终浓度为36 µg/ml的Nivolumab与CD19-CAR-T细胞联合应用，可在减轻药物毒副作用同时增强CD19-CAR-T细胞的杀伤活性。

Enterococcus faecalis induces apoptosis and pyroptosis of human osteoblastic MG63 cells via the NLRP3 inflammasome.

To investigate the ability of E.faecalis to cause apoptosis and pyroptosis of human osteoblastic MG63 cells and whether the inflammasome is involved in this process. MG63 cells were infected with E.faecalis at a multiplicity of infection (MOI) of 10, 100, 500 and 1000 for 6 and 12h. The cell proliferation was determined with the Cell Counting Kit-8. Apoptosis and pyroptosis after E.faecalis infection was evaluated by flow cytometry and a lactate dehydrogenase (LDH) cytotoxicity assay kit. Then, MG63 cells transfected with specific small interfering RNAs (siRNAs) targeting the NLR family pyrin domain-containing 3 (NLRP3) gene were infected with E.faecalis and subjected to the LDH release and apoptotic cell assays. One-way anova test and Student-Newman-Keuls test were used to evaluate the differences amongst groups in each experiment. An independent sample t-test was performed to analyse the differences between the 12- and 6-h time-points. P<0.05 was regarded as significant. E.faecalis induced apoptosis and inflammatory cell death (pyroptosis) of MG63 cells in a MOI dose-dependent manner. The NLRP3 inflammasome and caspase-1 were activated in E.faecalis-infected MG63 cells. However, the percentages of induced apoptotic and pyroptic cell death decreased significantly when the NLRP3 inflammasome was downregulated using siRNAs (P<0.05). E.faecalis promoted apoptosis and pyroptosis of MG63 cells via the NLRP3 inflammasome. This indicates that E.faecalis infection may result in the delayed reconstruction of periapical lesions, and NLRP3 is the potential target of new intracanal therapeutic agents.

Bovine macrophage-derived extracellular traps act as early effectors against the abortive parasite Neospora caninum

Macrophages are multipurpose phagocytes and are considered to be irreplaceable during the early host innate immune response against microbial and parasitic pathogens. However, no report has investigated the novel anti-parasitic mechanism of macrophage-derived extracellular traps (ETs) against the abortive apicomplexan parasite Neospora caninum (N. caninum) in cattle. Scanning electron microscopy (SEM) was used to visualize and characterize N. caninum tachyzoite-induced macrophage-triggered ETs in exposed bovine macrophages. Fluorescence confocal microscopy was used to confirm the classical backbone structure of DNA embedded with histone 3 (H3) and myeloperoxidase (MPO) in N. caninum tachyzoite-induced macrophage-derived ETs. Furthermore, the lactate dehydrogenase (LDH) levels in the supernatants of parasite-exposed macrophages were detected by a LDH Cytotoxicity Assay® kit. The results clearly demonstrated that N. caninum tachyzoites triggered bovine macrophage-derived ET-like structures. Inhibiting assays revealed that N. caninum tachyzoite-induced macrophage-mediated ET formation may be an ERK 1/2- and p38 MAPK-dependent cell death process. In conclusion, the present study is the first report on the formation of ETs in bovine macrophages against N. caninum tachyzoites and adds new data on the possible role of macrophages in vivo infection by capturing invasive stages and exposing them to other leukocytes.

Characterization of the Cellular Responses of Dental Mesenchymal Stem Cells to the Immune System

IntroductionDental stem cells have gained importance recently and are being used for various purposes in regenerative medicine and dentistry. Although much research has been done to show the various properties of these dental stem cells, the immunomodulatory properties of some of these stem cells are still unknown. This is important considering these cells are being used routinely. Therefore, the aim of this study was to investigate the interactions between the activated immune cells and 3 types of dental-derived mesenchymal stem cells: dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and stem cells of the apical papilla (SCAP). MethodsSCAP, dental pulp stem cells, stem cells from human exfoliated deciduous teeth, and periodontal ligament fibroblasts were cultured, and various assays were performed including a proliferation assay, flow cytometric analysis, lactate dehydrogenase and chromium-51 cytotoxicity assays, and an enzyme-linked immunosorbent assay to evaluate the interactions of these dental stem cells when cocultured with either peripheral blood mononuclear cells or natural killer cells. ResultsSCAP were less resistant to immune cell–mediated cytotoxicity as seen from the results obtained from the LDH and chromium-51 cytotoxicity assays. The flow cytometric analysis showed a lower resilience of SCAP to cytotoxic compounds. The enzyme-linked immunosorbent assay results demonstrated that the SCAP induced high levels of proinflammatory cytokine secretion compared with the other dental stem cells. ConclusionsSCAP did not perform as well as the other dental stem cells. This could in turn affect their survival and differentiation abilities as well as their functionality. This may be an important aspect to consider when selecting dental stem cells for various regenerative procedures.

Identification of the Multifaceted Chemopreventive Activity of Curcumin Against the Carcinogenic Potential of the Food Additive, KBrO3.

Background: Potassium bromate (KBrO3), a food additive, has been used in many bakery products as an oxidizing agent. It has been shown to induce renal cancer in many in-vitro and in-vivo experimental modelsObjectives: This study evaluated the carcinogenic potential of potassium bromate (KBrO3) and the chemopreventive mechanisms of the anti-oxidant and anti-inflammatory phytochemical, curcumin against KBrO3-induced carcinogenicity.Method: Lactate dehydrogenase (LDH) cytotoxicity assay and morphological characteristics were used to assess curcumin's cytoprotective potential against KBrO3 toxicity. To assess the chemopreventive potential of curcumin against KBrO3-induced oxidative insult, intracellular H2O2 and the nuclear concen-tration of the DNA adduct 8-OHdG were measured. PCR array, qRT-PCR, and western blot analysis were used to identify dysregulated genes by KBrO3 exposure. Furthermore, immunofluorescence was used to evaluate the ciliary loss and the disturbance of cellular tight junction induced by KBrO3.Results: Oxidative stress assays showed that KBrO3 increased the levels of intracellular H2O2 and the DNA adduct 8-OHdG. Combination of curcumin with KBrO3 efficiently reduced the level of H2O2 and 8-OHdG while up-regulating the expression of catalase. PCR array, qRT-PCR, and western blot analysis revealed that KBrO3 dysregulated multiple genes involved in inflammation, proliferation, and apoptosis, namely CTGF, IL-1, and TRAF3. Moreover, qRT-PCR and immunofluorescence studies showed that KBrO3 negatively affected the tight junctional protein (ZO-1) and induced a degeneration of primary ciliary proteins. The negative impact of KBrO3 on cilia was markedly repressed by curcumin.Conclusion: Curcumin could potentially be used as a protective agent against carcinogenicity of KBrO3.

Cytotoxic Effect of Aeruginosin-865, Resveratrol and Capsaicin on Mouse Fibroblasts and Cells Derived from Fallow Deer

Natural substances offer interesting bioactivity patterns including antiproliferative, antioxidant or cytotoxic effects. However, the safety profile of many of them has not been extensively determined. In this study, the cytotoxic effect of Aeruginosin-865, resveratrol and capsaicin at different concentrations was tested on normal mouse cells (NIH/3T3) and tumour fibroblasts (WEHI-13VAR) as well as on liver- and kidney-derived cells from fallow deer. A lactate dehydrogenase cytotoxicity assay kit was used to measure cell death in response to treatment with the test substances. It was found that NIH/3T3 cells tolerated Aeruginosin-865 (10-200 μM) and resveratrol (5-100 μM) treatment without any cytotoxic effect, while capsaicin exerted a cytotoxic effect only at the highest tested concentration (200 μ M). Mouse fibrosarcoma cells were more sensitive to the cytotoxic effect of all three compounds where Aeruginosin-865 (100-200 μM) and resveratrol (50–100 μM) showed high-dose cytotoxicity and capsaicin showed low- and high-dose cytotoxicity (25 μM and 200 μ M). The three tested compounds at the highest concentrations were found to be cytotoxic to both liver- and kidney-derived cells from fallow deer. Overall, the results indicate that the cytotoxic effects of the three tested natural substances on cells derived from fallow deer and mouse tumour fibroblasts differ significantly from those exerted on normal fibroblasts. The results demonstrate the potential of these natural compounds as therapeutic agents and pave the way for future in vivo toxicological investigations.

The specific cytotoxicities of chimeric antigen receptor-engineered T cells on different lymphomas

目的探讨共刺激分子为4-1BB的二代CD19嵌合抗原受体T细胞（CAR-T细胞）对不同侵袭性淋巴瘤细胞的体内、体外杀伤活性以及CAR-T细胞在体内生存的时间。方法采用慢病毒包装并感染T细胞的方法制备CD19 CAR-T细胞，采用CCK-8、ELISA和乳酸脱氢酶细胞毒性检测法检测CD19 CAR-T细胞的增殖能力、炎症因子释放水平和对Raji（套细胞淋巴瘤细胞株）、Pfeiffer（弥漫大B细胞淋巴瘤细胞株）、EHEB（慢性淋巴细胞白血病细胞株）细胞的杀伤活性，采用流式细胞术分析CD19 CAR-T细胞治疗前后荷瘤裸鼠的肿瘤负荷和CAR-T细胞残留水平。结果①与二代CAR-T细胞比较，三代CAR-T细胞的体外（24、48 h）增殖能力（P值均<0.05）、对瘤细胞的杀伤活性（P值均<0.05）较强，差异有统计学意义；而炎症因子的释放水平差异无统计学意义（P值均>0.05）。②效靶比为4∶1且共培养48 h时，二代CD19 CAR-T细胞对Raji细胞[（72.36±2.98）%]、Pfeiffer细胞[（55.92±4.16）%]、EHEB细胞[（35.53±3.93）%]的杀伤活性从强到弱，差异有统计学意义（P=0.013）。③裸鼠体内试验结果显示二代CAR-T细胞在EHEB荷瘤小鼠体内存活时间长于Raji荷瘤小鼠（P=0.046）。结论共刺激分子为4-1BB的二代CD19 CAR-T细胞对惰性淋巴瘤细胞株的杀伤活性稍低于对侵袭性淋巴瘤细胞株，但在惰性淋巴瘤细胞株荷瘤小鼠体内存活时间更长，可能更适合惰性淋巴瘤的治疗。

Investigation of antidiabetic potential of Phyllanthus niruri L. using assays for α-glucosidase, muscle glucose transport, liver glucose production, and adipogenesis

Phyllanthus niruri is used in herbal medicine for treatment of diabetes. The objective of this study was to investigate the antidiabetic potential of P. niruri, using assays for α-glucosidase, muscle glucose transport, liver glucose production and adipogenesis. α-Glucosidase inhibitory activity was performed on aqueous and ethanolic extract of aerial parts of P. niruri. The aqueous and ethanolic extract of P. niruri showed α-glucosidase inhibitory activity with IC50 values of 3.7 ± 1.1 and 6.3 ± 4.8 μg/mL, respectively. HR-bioassay/HPLC-HRMS and NMR analysis was used for identification of compounds. Corilagin (1) and repandusinic acid A (2) were identified as α-glucosidase inhibitors in the water extract of P. niruri with IC50 values of 0.9 ± 0.1 and 1.9 ± 0.02 μM, respectively. In in vitro cell-based bioassays, cells were treated for 18 h with maximal non-toxic concentrations of the ethanolic extract of P. niruri, which were determined by the lactate dehydrogenase cytotoxicity assay. The ethanolic extract of P. niruri was not able to reduce glucose-6-phosphatase activity. However, the extract increased deoxyglucose uptake in C2C12 muscle cells and enhanced adipogenesis in 3T3-L1 fat cells which has been reported for the first time. The present study demonstrated that P. niruri may thus have potential application for treatment and/or management of type 2 diabetes.