Lactate Dehydrogenase Assay Research Articles

Cytocompatibility of titanium and poly(etheretherketone) surfaces after O2 non-thermal plasma sterilization.

The sterilization of medical devices is paramount to achieve an acceptable level of sterility assurance and to prevent hospital-acquired infections. However, some medical devices cannot be sterilized by usual processes such as autoclave (AC) and gamma-ray irradiation (GI). A new non-thermal plasma (NTP) process using sealed bag that preserves the sterile state of the devices could be used as an alternative sterilization method. The aim of the study was to assess the cytocompatibility of titanium and poly(etheretherketone) (PEEK) surfaces after O2-NTP sterilization compared to GI and AC. MG-63 osteoblast-like cells were seeded on titanium (TA6V) and PEEK disks sterilized by AC, GI and O2-NTP. The cells' viability and proliferation, determined by WST-1 and DNA quantification respectively, were enhanced whatever the material types from 3 to 10 days. When seeded on titanium, MG-63 cells showed a higher viability and proliferation after GI and O2-NTP treatment compared to AC treatment. When cultured on PEEK, MG-63 cells showed a higher viability after O2-NTP treatment. No difference of proliferation was observed whatever the sterilization processes. The cell colonization of the materials' surface was confirmed by scanning electron microscopy. Lactate dehydrogenase (LDH) assay revealed no cytotoxicity. Thus, O2-NTP led to similar cell responses to AC and GI and could be a cost-effective alternative process to the usual sterilization methods for fragile medical devices.

Stromal cell-derived factor-1 downregulation contributes to neuroprotection mediated by CXC chemokine receptor 4 interactions after intracerebral hemorrhage in rats.

Stromal cell-derived factor-1 (SDF-1) and CXC chemokine receptor 4 (CXCR4) have a substantial role in neuronal formation, differentiation, remodeling, and maturation and participate in multiple physiological and pathological events. In this study, we investigated the role of SDF-1/CXCR4 in neural functional injury and neuroprotection after intracerebral hemorrhage (ICH). Western blot, immunofluorescence and immunoprecipitation were used to detect SDF-1/CXCR4 expression and combination respectively after ICH. TUNEL staining, Lactate dehydrogenase assay, Reactive oxygen species assay, and Enzyme-linked immunosorbent assay to study neuronal damage; Brain water content to assay brain edema, Neurological scores to assess short-term neurological deficits. Pharmacological inhibition and genetic intervention of SDF-1/CXCR4 signaling were also used in this study. ICH induced upregulation of SDF-1/CXCR4 and increased their complex formation, whereas AMD3100 significantly reduced it. The levels of TNF-α and IL-1β were significantly reduced after AMD3100 treatment. Additionally, AMD3100 treatment can alleviate neurobehavioral dysfunction of ICH rats. Conversely, simultaneous SDF-1/CXCR4 overexpression induced the opposite effect. Moreover, immunoprecipitation confirmed that SDF-1/CXCR4 combined to initiate neurodamage effects. This study indicated that inhibition of SDF-1/CXCR4 complex formation can rescue the inflammatory response and alleviate neurobehavioral dysfunction after ICH. SDF-1/CXCR4 may have applications as a therapeutic target after ICH.

Triggering pyroptosis enhances the antitumor efficacy of PARP inhibitors in prostate cancer.

PARP inhibitors have revolutionized the treatment landscape for advanced prostate cancer (PCa) patients who harboring mutations in homologous recombination repair (HRR) genes. However, the molecular mechanisms underlying PARP inhibitors function beyond DNA damage repair pathways remain elusive, and identifying novel predictive targets that favorably respond to PARP inhibitors in PCa is an active area of research. The expression of GSDME in PCa cell lines and human PCa samples was determined by western blotting. Targeted bisulfite sequencing, gene enrichment analysis (GSEA), clone formation, construction of the stably transfected cell lines, lactate dehydrogenase (LDH) assay, western blotting as well as a mouse model of subcutaneous xenografts were used to investigate the role of GSDME in PCa. The combinational therapeutic effect of olaparib and decitabine was determined using both in vitro and in vivo experiments. We have found low expression of GSDME in PCa. Interestingly, we demonstrated that GSDME activity is robustly induced in olaparib-treated cells undergoing pyroptosis, and that high methylation of the GSDME promoter dampens its activity in PCa cells. Intriguingly, genetically overexpressing GSDME does not inhibit tumor cell proliferation but instead confers sensitivity to olaparib. Furthermore, pharmacological treatment with the combination of olaparib and decitabine synergistically induces GSDME expression and cleavage through caspase-3 activation, thus promoting pyroptosis and enhancing anti-tumor response, ultimately resulting in tumor remission. Our findings highlight a novel therapeutic strategy for enhancing the long-term response to olaparib beyond HRR-deficient tumors in PCa, underscoring the critical role of GSDME in regulating tumorigenesis.

Cytotoxicity and Characterization of Ultrafine Particles from Desktop Three-Dimensional Printers with Multiple Filaments.

Previous research has indicated that ultrafine particles (UFPs, particles less than 100 nm) emitted from desktop three-dimensional (3D) printers exhibit cytotoxicity. However, only a limited number of particles from different filaments and their combinations have been tested for cytotoxicity. This study quantified the emissions of UFPs from a commercially available filament extrusion desktop 3D printer using three different filaments, including acrylonitrile butadiene Styrene (ABS), thermoplastic polyurethane (TPU), and polyethylene terephthalate glycol (PETG). In this study, controlled experiments were conducted where the particles emitted were used to expose cells grown in an air-liquid interface (ALI) system. The ALI exposures were utilized for in vitro characterization of particle mixtures, including UFPs from a 3D printer. Additionally, a lactate dehydrogenase (LDH) assay was used to evaluate the cytotoxic effects of these UFPs. A549 cells were exposed at the ALI to UFPs generated by an operational 3D printer for an average of 45 and 90 min. Twenty-four hours post-exposure, the cells were analyzed for percent cytotoxicity in a 24-well ALI insert (LDH assay). UFP exposure resulted in diminished cell viability, as evidenced by significantly increased LDH levels. The findings demonstrate that ABS has the most significant particle emission. ABS was the only filament that showed a significant difference compared to the high efficiency particulate arrestance (HEPA) following 90 min of exposure (p-value < 0.05). Both ABS and PETG exhibited a significant difference compared to the HEPA control after 45 min of exposure. A preliminary analysis of potential exposure to these products in a typical environment advises caution when operating multiple printer and filament combinations in poorly ventilated spaces or without combined gas and particle filtration systems.

Calycosin Alleviates Lupus Nephritis by Activating the Nrf2/HO-1 Signaling Pathway

Lupus nephritis is a serious condition, for which treatments are limited; hence, there is a need for new cure approaches. The aim of this study was to evaluate the therapeutic effects of calycosin against lupus nephritis induced by lipopolysaccharide (LPS) in human renal cortex proximal convoluted tubule epithelial cells (HK-2). HK-2 cells were stimulated with 1 μg/ml LPS to create a lupus nephritis cell model; the cells were pretreated with calycosin. Cell viability and apoptosis rate were determined using the cell counting kit-8 assay and flow cytometry, respectively. A caspase-3 activity detection kit was used to determine caspase-3 activity. Interleukin (IL)-6, IL-1β, and tumor necrosis factor alpha (TNF-α) levels were determined using enzyme-linked immunosorbent assay kits. Lactate dehydrogenase (LDH) level was determined using an LDH assay kit. Finally, western blotting and reverse transcription-quantitative polymerase chain reaction were performed to determine apoptosis-related protein levels and nuclear factor erythroid 2–related factor 2 (Nrf2)/heme oxygenase-1 (HO-1) signaling. Calycosin had no cytotoxic effects on HK-2 cells. Lipopolysaccharide stimulation significantly inhibited cell viability; increased the IL-6, IL-1β, and TNF-α levels; and elevated apoptosis rate, caspase3 activity, and LDH level in HK-2 cells. The protein level of cleaved caspase3 was also increased in LPS-treated HK-2 cells. In addition, the pattern of Nrf2/HO-1 signaling was disturbed by LPS. These effects were reversed by calycosin treatment. Calycosin could alleviate LPS-induced lupus nephritis and may thus be a novel agent for its treatment.Graphical

Role of mitochondrial disruption and oxidative stress in plasticizer phthalate-induced cytotoxicity to human bone osteoblasts.

Phthalates are frequently utilized in a wide range of products such as plasticizers with reported negative effects on bones. The current study evaluated the effect of butyl cyclohexyl phthalate on the human osteoblasts via different assays. MTT and lactate dehydrogenase assays were used to examine the in-vitro cytotoxic effect of butyl cyclohexyl phthalate on human bone osteoblasts in concentrations 0.1, 1, 10, and 100μM for 12 to 72h postexposures. Incubation of osteoblasts with butyl cyclohexyl phthalate significantly reduced cell viability based on its concentrations and durations of exposure. In parallel, osteoblast secretion of procollagen type 1, osteocalcin, as well as alkaline phosphatase was significantly decreased by butyl cyclohexyl phthalate in concentrations (1 or 2μM). Butyl cyclohexyl phthalate decreased ATP synthesis and mitochondrial complexes I and III activities, with increased lactate production, all of which were detrimental to cellular bioenergetics. The cellular redox defense systems were significantly depleted by increased lipid peroxidation, elevated reactive oxygen species, decreased catalase and superoxide dismutase enzymes activities, and decreased intracellular reduced glutathione (GSH). Redox stress was also induced. Interestingly, preincubating osteoblasts with reduced GSH before exposing them to butyl cyclohexyl phthalate significantly lowered the cytotoxicity of the butyl cyclohexyl phthalate, suggesting that antioxidants may play a helpful protective effect.

Use of multi-walled carbon nanotubes (MWCNTs) stabilized in Arabic gum colloidal solution to induce genotoxicity and apoptosis of human breast and lung cancer cell lines

Introduction: Increased mortality owing to breast and lung cancer is an urgent global medical issue. Nanotherapy is one of the most widely used and recent procedures for combating cancer. Therefore, this study aimed to assess the beneficial effects of multi-walled carbon nanotubes (MWCNTs) to precisely target breast (MCF-7) and lung cancer (A549) cell lines. Multi-walled carbon nanomaterials are considered as a typical biocompatible nanomaterial that can be used in drug delivery without causing side effects. On the contrary, the metallic nanoparticles, even that of noble metals, are reported to have side effects that are still unavoidable.Methods: The MWCNTs were purified and stabilized using Arabic gum in an aqueous solution under ultrasonic radiation. Scanning and transmission electron microscopy (SEM and TEM, respectively) were employed to characterize the morphology of the MWCNTs, and elemental analysis was performed using SEM to determine their chemical composition.Results: The investigated MWCNTs had an average diameter of 10–12 nm and were mainly composed of 99.4% carbon and other trace elements. The Arabic gum emulsifying agent was found a powerful stabilizing agent for the investigated MWCNTs in aqueous solutions. The cytotoxic effect of the MWCNT/ Arabic gum colloidal solution on MCF-7 and A549 was evaluated by 3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and lactate dehydrogenase assays, and the expression of apoptosis-related genes was assessed using quantitative real-time polymerase chain reaction and a human apoptosis signaling antibody array. This analysis aimed to clarify additional pathways involved, including those mediated by interleukin-1, tumor necrosis factor-α, and ceramide signaling pathways enrichments. Concerning the inhibition of cell growth, the efficacy of MWCNT/Arabic gum IC50 of MCF-7 (48.23 μg/mL) was higher than IC50 of A549 (100.1 μg/mL). MWCNT/Arabic gum enhanced the mRNA expression of apoptosis-associated genes encoding P53, Bax, and Caspases (9, 8, and 3). Upregulation of the apoptotic signaling pathway in the MCF-7 cell lysates was observed before and after exposure to the MWCNT/Arabic gum colloidal solution.Conclusion: The physicochemical properties of MWCNT/Arabic gum exhibited remarkable apoptotic activity in breast and lung cancer cell lines. However, further in vivo investigations are recommended to clarify the other molecular aspects related to the use of MWCNT/Arabic gum.

Hsa-miR-301a-3p inhibited the killing effect of natural killer cells on non-small cell lung cancer cells by regulating RUNX3.

Non-small cell lung cancer (NSCLC) is the most commonly diagnosed solid tumor. Natural killer (NK) cell-based immunotherapy is a promising anti-tumor strategy in various cancers including NSCLC. We aimed to investigate the specific mechanisms that regulate the killing effect of NK cells to NSCLC cells. Reverse transcription-quantitative PCR (RT-qPCR) assay was applied to measure the levels of hsa-microRNA (miR)-301a-3p and Runt-related transcription factor 3 (RUNX3). Enzyme-linked immunosorbent assay (ELISA) was used to measure the levels of IFN-γ and TNF-α. Lactate dehydrogenase assay was applied to detect the killing effect of NK cells. Dualluciferase reporter assay and RNA immunoprecipitation (RIP) assay were carried out to confirm the regulatory relationship between hsa-miR-301a-3p and RUNX3. A low expression of hsa-miR-301a-3p was observed in NK cells stimulated by IL-2. The levels of IFN-γ and TNF-α were increased in NK cells of the IL-2 group. Overexpression of hsa-miR-301a-3p reduced the levels of IFN-γ and TNF-α as well as the killing effect of NK cells. Furthermore, RUNX3 was identified to be a target of hsamiR-301a-3p. hsa-miR-301a-3p suppressed the cytotoxicity of NK cells to NSCLC cells by inhibiting the expression of RUNX3. We found hsa-miR-301a-3p promoted tumor growth by suppressing the killing effect of NK cells against NSCLC cells in vivo. Hsa-miR-301a-3p suppressed the killing effect of NK cells on NSCLC cells by targeting RUNX3, which may provide promising strategies for NK cell-based antitumor therapies.

Novel anti-Acanthamoebic properties of raloxifene sulfonate/sulfamate derivatives

Acanthamoeba are known to cause a vision threatening eye infection typically due to contact lens wear, and an infection of the central nervous system. The ability of these amoebae to switch phenotypes, from an active trophozoite to a resistant cyst form is not well understood; the cyst stage is often resistant to chemotherapy, which is of concern given the rise of contact lens use and the ineffective disinfectants available, versus the cyst stage. Herein, for the first time, a range of raloxifene sulfonate/sulfamate derivatives which target nucleotide pyrophosphatase/phosphodiesterase enzymes, were assessed using amoebicidal and excystation tests versus the trophozoite and cyst stage of Acanthamoeba. Moreover, the potential for cytopathogenicity inhibition in amoebae was assessed. Each of the derivatives showed considerable anti-amoebic activity as well as the ability to suppress phenotypic switching (except for compound 1a). Selected raloxifene derivatives reduced Acanthamoeba-mediated host cell damage using lactate dehydrogenase assay. These findings suggest that pyrophosphatase/phosphodiesterase enzymes may be valuable targets against Acanthamoeba infections.

Toxicity assessment of CeO₂ and CuO nanoparticles at the air-liquid interface using bioinspired condensational particle growth

CeO2 and CuO nanoparticles (NPs) are used as additives in petrodiesel to enhance engine performance leading to reduced diesel combustion emissions. Despite their benefits, the additive application poses human health concerns by releasing inhalable NPs into the ambient air. In this study, a bioinspired lung cell exposure system, Dosimetric Aerosol in Vitro Inhalation Device (DAVID), was employed for evaluating the toxicity of aerosolized CeO2 and CuO NPs with a short duration of exposure (≤10 min vs. hours in other systems) and without exerting toxicity from non-NP factors. Human epithelial A549 lung cells were cultured and maintained within DAVID at the air-liquid interface (ALI), onto which aerosolized NPs were deposited, and experiments in submerged cells were used for comparison. Exposure of the cells to the CeO2 NPs did not result in detectable IL-8 release, nor did it produce a significant reduction in cell viability based on lactate dehydrogenase (LDH) assay, with a marginal decrease (10%) at the dose of 388 μg/cm2 (273 cm2/cm2). In contrast, exposure to CuO NPs resulted in a concentration dependent reduction in LDH release based on LDH leakage, with 38% reduction in viability at the highest dose of 52 μg/cm2 (28.3 cm2/cm2). Cells exposed to CuO NPs resulted in a dose dependent cellular membrane toxicity and expressed IL-8 secretion at a global dose five times lower than cells exposed under submerged conditions. However, when comparing the ALI results at the local cellular dose of CuO NPs to the submerged results, the IL-8 secretion was similar. In this study, we demonstrated DAVID as a new exposure tool that helps evaluate aerosol toxicity in simulated lung environment. Our results also highlight the necessity in choosing the right assay endpoints for the given exposure scenario, e.g., LDH for ALI and Deep Blue for submerged conditions for cell viability.

Sesamin attenuates UVA-induced keratinocyte injury via inhibiting ASK-1-JNK/p38 MAPK pathways.

Ultraviolet (UV) exposure-stimulated reactive oxygen species (ROS) formation in keratinocytes is a crucial factor in skin aging. Phytochemicals have become widely popular for protecting the skin from UV-induced cell injury. Sesamin (SSM) has been shown to play a role in extensive pharmacological activity and exhibit photoprotective effects. To assess the protective effect of SSM on UVA-irradiated keratinocytes and determine its potential antiphotoaging effect. HaCaT keratinocytes pretreated with SSM were exposed to UVA radiation at 8 J/cm2 for 10 min. Cell viability and oxidative stress indicators were evaluated using a cell counting kit-8 and lactate dehydrogenase (LDH), malondialdehyde (MDA), glutathione (GSH), and superoxide dismutase (SOD) assay kits. Apoptosis and intracellular ROS levels were analyzed using annexin V-fluorescein isothiocyanate/propyridine iodide and dichlorodihydrofluorescein diacetate staining, respectively. Protein levels of matrix metalloprotein-1 (MMP-1), MMP-9, Bax/Bcl-2, and mitogen-activated protein kinase (MAPK) pathway proteins, phospho-apoptosis signal-regulating kinase-1 (p-ASK-1)/ASK-1, phospho-c-Jun N-terminal protein kinase (p-JNK)/JNK, and p-p38/p38 were determined using western blotting. Sesamin showed no cytotoxicity until 160 μmol/L on human keratinocytes. Sesamin pretreatment (20 and 40 μM) reversed the suppressed cell viability, increased LDH release and MDA content, decreased cellular antioxidants GSH and SOD, and elevated intracellular ROS levels, which were induced by UVA irradiation. Additionally, SSM inhibited the expression of Bax, MMP-1, and MMP-9 and stimulated Bcl-2 expression. In terms of the regulatory mechanisms, we demonstrated that SSM inhibits the phosphorylation of ASK-1, JNK, and p38. The results suggest that SSM attenuates UVA-induced keratinocyte injury by inhibiting the ASK-1-JNK/p38 MAPK pathways.

Xanthatin suppresses pancreatic cancer cell growth via the ROS/RBL1 signaling pathway: In vitro and in vivo insights

BackgroundAs a malignant digestive system tumor, pancreatic cancer has a high mortality rate. Xanthatin is a sesquiterpene lactone monomer compound purified from the traditional Chinese herb Xanthium strumarium L. It has been reported that Xanthatin exhibits inhibitory effects on various cancer cells in retinoblastoma, glioma, hepatoma, colon cancer, lung cancer, as well as breast cancer. However, in pancreatic cancer cells, only one report exists on the suppression of Prostaglandin E2 synthesis and the induction of caspase 3/7 activation in Xanthatin-treated MIA PaCa-2 cells, while systematic in vitro and in vivo investigations and related mechanisms have yet to be explored. PurposeThis research aims to explore the in vitro and in vivo effects of Xanthatin on pancreatic cancer and its molecular mechanisms. MethodsThe anticancer effects and mechanisms of Xanthatin on pancreatic cancer cells were assessed through employing cell counting kit-8 (CCK-8) assay, lactate dehydrogenase (LDH) assay, carboxyfluorescein diacetate succinimidyl ester (CFDA SE) cell proliferation assay, colony formation assay, wound healing assay, transwell assay, Annexin V-FITC/propidium iodide (PI) dual staining, Hoechst nuclear staining, Western blot analysis, phosphoproteomics, and reactive oxygen species (ROS) measurement. The in vivo anticancer effects of Xanthatin on pancreatic cancer cells were studied using a nude mouse model. ResultsThe present study showed that Xanthatin can prevent the proliferation and metastasis of pancreatic cancer cells and trigger the exposure of phosphatidylserine (PS), chromatin condensation, and caspase activation, thereby inducing apoptosis. Phosphoproteomic analysis indicated that Xanthatin inhibits the phosphorylation of the proliferation-associated protein RBL1, and oxidative stress can lead to RBL1 dephosphorylation. Further investigation revealed that Xanthatin significantly upregulates ROS levels in pancreatic cancer cells, and the antioxidant N-acetylcysteine (NAC) can reverse Xanthatin-induced cell proliferation inhibition and apoptosis. In addition, Xanthatin can suppress pancreatic cancer cell growth in a xenograft nude mouse model with low toxicity to the mice. ConclusionXanthatin may inhibit the proliferation of pancreatic cancer cells and trigger apoptosis through the ROS/RBL1 signaling pathway.

Antimalarial Potential of Fraction 5 from Ethanolic Leaves Extract of Artocarpus Altilis

Background: Artocarpus altilis leaf extract (AAL.E) was separated by VLC, and six fractions were obtained. Fraction 5 (AAL.E.5) showed antimalarial activity with an IC50 value of 3.71 µg/mL. Objective: This study aimed to determine the antimalarial activity of AAL.E.5 subfractions against P. falciparum, the mechanism of action against Plasmodium Falciparum Malate quinone oxidoreductase (PfMQO), and the active substances. Methods: The AAL.E.5 was separated by open-column chromatography and eluted with chloroform-methanol gradient elution in order of increasing polarity. The antimalarial activity of all subfractions was assessed using a lactate dehydrogenase (LDH) assay against P. falciparum and the mechanism of action of the PfMQO enzyme. The profiles of the most active subfractions were analyzed using High-Performance Liquid Chromatography (HPLC). Results: The separation of fraction 5 (AAL.E.5) yielded 11 subfractions (AAL.E.5.1–AAL.E.5.11). Screening antimalarial activity at 10 μg/mL in this subfraction showed that only five subfractions (AAL.E.5.6-AAL. E.5.10) inhibited P. falciparum and two subfractions (AAL.E.5.6 and AAL.E.5.10) inhibited the PfMQO enzyme. Only subfraction 6 (AAL.E.5.6) inhibited both, with IC50 values of 6.609 µg/mL and 20.34 µg/mL. The thin layer chromatography profile of AAL.E.5.6 revealed reddish-orange spots, indicating the presence of flavonoid compounds, and was also presumed from the UV-visible to HPLC chromatogram for band I in the 300 – 400 nm range and band II in the 240–285 nm range. Conclusion: Subfraction 6 has antimalarial activity against P. falciparum and is thought to have a mechanism of action in PfMQO. Based on the TLC, HPLC, and UV-Vis spectra, subfraction 6 was assumed to be a flavonoid.

Mechanism of bilobalide promoting neuroprotection of macrophages

This study aims to explore the neuroprotective effect of bilobalide(BB) and the mechanisms such as inhibiting inflammatory response in macrophage/microglia, promoting neurotrophic factor secretion, and interfering with the activation and differentiation of peripheral CD4~+ T cells. BB of different concentration(12.5, 25, 50, 100 μg·mL~(-1)) was used to treat the RAW264.7 and BV2 cells for 24 h. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide(MTT) assay and cell counting kit-8(CCK-8) were employed to detect the cytotoxicity of BB and appropriate concentration was selected for further experiment. Lipopolysaccharide(LPS) was applied to elicit inflammation in RAW264.7 and BV2 cells, mouse bone marrow-derived macrophages(BMDMs), and primary microglia, respectively. The effect of BB on cell proliferation and secretion of inflammatory cytokines and neurotrophic factors was detected by enzyme-linked immunosorbent assay(ELISA). Spleen monocytes of C57BL/6 female mice(7-8 weeks old) were isolated, and CD4~+ T cells were separated by magnetic beads under sterile conditions. Th17 cells were induced by CD3/CD28 and the conditioned medium for eliciting the inflammation in BMDMs. The content of IL-17 cytokines in the supernatant was detected by ELISA to determine the effect on the activation and differentiation of CD4~+ T cells. In addition, PC12 cells were incubated with the conditioned medium for eliciting inflammation in BMDMs and primary microglia and the count and morphology of cells were observed. The cytoto-xicity was determined by lactate dehydrogenase(LDH) assay. The result showed that BB with the concentration of 12.5-100 μg·mL~(-1) had no toxicity to RAW264.7 and BV2 cells, and had no significant effect on the activity of cell model with low inflammation. The 50 μg·mL~(-1) BB was selected for further experiment, and the results indicated that BB inhibited LPS-induced secretion of inflammatory cytokines. The experiment on CD4~+ T cells showed that the conditioned medium for LPS-induced inflammation in BMDMs promoted the activation and differentiation of CD4~+ T cells, while the conditioned medium of the experimental group with BB intervention reduced the activation and differentiation of CD4~+ T cells. In addition, BB also enhanced the release of neurotrophic factors from BMDMs and primary microglia. The conditioned medium after BB intervention can significantly reduce the death of PC12 neurons, inhibit neuronal damage, and protect neurons. To sum up, BB plays a neuroprotective role by inhibiting macrophage and microglia-mediated inflammatory response and promoting neurotrophic factors.

Some Wild Mushrooms with High Antioxidant Capacity Exhibit Potent Anticancer Activity on Cancer Cells using the Apoptotic and Antimigration Cell Death Mechanisms.

This study aims to provide in vitro experimental evidence that wild mushrooms have the potential to be used as a pharmaceutical that could be effective against various types of cancer. Throughout human history, besides food, traditional medicine and natural poisons obtained from mushrooms have been used for the treatment of many diseases. Clearly, edible and medicinal mushroom preparations have beneficial health effects without the known severe adverse side effects. This study was designed to reveal the cell growth inhibitory potential of five different edible mushrooms and the biological activity of Lactarius zonarius was shown here for the first time. The mushrooms fruiting bodies were dried and powdered then extracted with hexane, ethyl acetate, and methanol. The mushroom extracts were screened for possible antioxidant activities by the free radical scavenging activity (DPPH) method. Antiproliferative activity and cytotoxicity of the extracts were investigated in vitro on A549 (human lung carcinoma), HeLa (human cervix carcinoma), HT29 (human colon carcinoma), Hep3B (human hepatoma), MCF7 (human breast cancer), FL (human amnion cells), and Beas2B (normal human cells) cells lines by using MTT cell proliferation assay, lactate dehydrogenase (LDH) assay, DNA degradation, TUNEL, and cell migration assay. Using proliferation, cytotoxicity, DNA degradation, TUNEL, and migration assay, we displayed that hexane, ethyl acetate, and methanol extracts of the Lactarius zonarius, Laetiporus sulphureus, Pholiota adiposa, Polyporus squamosus, and Ramaria flava were effective on the cells even so at low doses (< 45.0 - 99.6 µg/mL) by acting in a way that represses migration, as a negative inducer of apoptosis. It was also demonstrated that mushroom extracts with high antioxidant effect have within the acceptable cytotoxic activity of 20%-30% on the cell membrane at concentrations higher than 60 µg/mL. Overall, all of the mushroom extracts with high antioxidant effects had strong antiproliferative activity and low toxicity for cells. These findings, at least, highlight that these mushroom extracts can be used for the treatment of cancer disease, especially as a supportive therapy against colon, liver, and lung cancer.

Amygdalin Reverses Macrophage PANoptosis Induced by Drug-Resistant Escherichia coli.

Infectious diseases caused by drug-resistant Escherichia coli (E. coli) pose a critical concern for medical institutions as they can lead to high morbidity and mortality rates. In this study, amygdalin exhibited anti-inflammatory and antioxidant activities, as well as other potentials. However, whether it could influence the drug-resistant E. coli-infected cells remained unanswered. Amygdalin was therefore tested in a cellular model in which human macrophages were exposed to resistant E. coli. Apoptosis was measured by flow cytometry and the lactate dehydrogenase (LDH) assay. Western immunoblotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to quantify interleukin-18 (IL-18), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The production of reactive oxygen species (ROS) in macrophages was detected by ROS kit. The expression of panapoptotic proteins in macrophages was measured by qRT-PCR and Western immunoblotting. Drug-Resistant E. coli inhibited cell viability and enhanced apoptosis in the cellular model. In cells treated with amygdalin, this compound can inhibit cell apoptosis and reduce the expression of pro - inflammatory cytokines such as IL-1β, IL-18 and IL-6. Additionally, it decreases the production of PANoptosis proteins, Furthermore, amygdalin lowered the levels of reactive oxygen species induced by drug-resistant E. coli, in cells, demonstrating its antioxidant effects. Amygdalin, a drug with a protective role, alleviated cell damage caused by drug-resistant E. coli in human macrophages by inhibiting the PANoptosis signaling pathway.

Amygdalin Reverses Macrophage PANoptosis Induced by Drug-Resistant Escherichia coli.

Infectious diseases caused by drug-resistant Escherichia coli (E. coli) pose a critical concern for medical institutions as they can lead to high morbidity and mortality rates. In this study, amygdalin exhibited anti-inflammatory and antioxidant activities, as well as other potentials. However, whether it could influence the drug-resistant E. coli-infected cells remained unanswered. Amygdalin was therefore tested in a cellular model in which human macrophages were exposed to resistant E. coli. Apoptosis was measured by flow cytometry and the lactate dehydrogenase (LDH) assay. Western immunoblotting and quantitative reverse-transcription polymerase chain reaction (qRT-PCR) were used to quantify interleukin-18 (IL-18), interleukin-1β (IL-1β), and interleukin-6 (IL-6). The production of reactive oxygen species (ROS) in macrophages was detected by ROS kit. The expression of panapoptotic proteins in macrophages was measured by qRT-PCR and western immunoblotting. Drug-resistant E. coli inhibited cell viability and enhanced apoptosis in the cellular model. In amygdalin-treated cells, amygdalin can inhibit cell apoptosis and pro-inflammatory cytokine expression of IL-1β, IL-18, and IL-6, and it reduces the production of pan-apoptotic proteins. In addition, amygdalin reduced the levels of E. coli-induced ROS in cells, indicating that it has antioxidant effects. Amygdalin, a compound with a protective role, alleviated cell damage caused by drug-resistant E. coli in human macrophages by inhibiting the PANoptosis signaling pathway.

Pharmacokinetic investigation on the mechanism of interaction of anti-breast cancer calycosin with albumin: In vitro

It has been documented that calycosin induces anticancer effects against a wide range of cancer cells, in vitro. However, pharmacokinetic characteristics of calycosin based on its interaction with albumin as a main carrier protein as well as corresponding anticancer mechanisms remain largely unknown. Herein, we inquired into the interaction of calycosin with human serum albumin (HSA) by a wide range collection of spectroscopic and theoretical data. Also, anticancer effects of calycosin on breast cancer cells, MDA-MB 231, were explored by cell viability, lactate dehydrogenase (LDH), caspase-3 activity, and real-time PCR assays. The findings demonstrated that the quenching process resulting from the interaction of calycosin and HSA was largely static in nature and that the calycosin-HSA system spontaneously forms. Cellular and molecular studies revealed that calycosin did not induce a significant cytotoxic effect on the viability of normal human breast epithelial cells, (MCF-10A), even at 200 μM concentration at which it was able to inhibit the proliferation of breast cancer MDA-MB 231 cells up to 50%. Then, it was disclosed that calycosin can disrupt the membrane integrity and induces apoptosis in MDA-MB 231 cells through overexpression of caspase-3 mRNA, down regulation of Bcl-2, and elevation of caspase-3 mRNA and activity mediated by downregulation of mTOR and Akt mRNA. Therefore, it was realized that in breast cancer MDA-MB 231 cells, calycosin is able to suppress cellular proliferation via apoptosis, which is controlled by the Akt/mTOR signaling cascade.

A rapid and reliable detection procedure of Atlantic trout introgression at the diagnostic lactate dehydrogenase chain‐1 gene

AbstractThe Italian‐native Mediterranean brown trout (Salmo ghigii) is a seriously threatened freshwater fish, especially by anthropogenic hybridisation with the domestic strains of Atlantic origin that have been repeatedly released into the wild for angling. A PCR‐restriction fragment length polymorphism (RFLP) assay of the diagnostic lactate dehydrogenase chain‐1 (LDH‐C1) gene sequences has been routinely applied to distinguish exotic from native brown trout lineages and detect Atlantic introgression signals in the Mediterranean wild populations. Here, we used dermal swab DNA obtained from 28 wild trout to improve laboratory procedures to genetically characterise trout samples at the LDH‐C1gene through (1) a capillary electrophoresis analysis of the RFLP fragments and (2) the optimisation of a diagnostic single nucleotide polymorphism analysable through mini‐sequencing approaches. The developed methods were fully consistent with those obtained through the traditional approach, but their analytical process is almost entirely automated and digitalised, thus improving result readability and accuracy in the detection of alien introgressed traces in wild Mediterranean brown trout populations.

Salidroside suppresses the multiple oncogenic activates and immune escape of lung adenocarcinoma through the circ_0009624-mediated PD-L1 pathway.

Lung adenocarcinoma (LUAD) is a fatal malignancy all over the world. Salidroside (SAL) is an active component extracted from Rhodiola rosea that has been reported to exert antitumor activity against several human cancers, containing lung adenocarcinoma (LUAD). The purpose of this study was to explore the effect and underlying mechanism of SAL in LUAD. Cell viability, proliferation, migration, and invasion were measured using cell counting kit-8 (CCK-8), 5-ethynyl-2'-deoxyuridine (EdU), and transwell assays. Effects of LUAD cells on the cytotoxicity, percentage, and death of CD8+ cells were detected using lactate dehydrogenase (LDH) and flow cytometry assays. Programmed cell death ligand 1 (PD-L1) protein level was examined using western blot. Circ_0009624, enolase 1 (ENO1), and PD-L1 levels were determined using real-time quantitative polymerase chain reaction (RT-qPCR). The biological role of SAL on LUAD tumor growth was assessed using the xenograft tumor model in vivo. SAL restrained LUAD cell proliferation, migration, invasion, and immune escape in vitro via modulating PD-L1. Circ_0009624 expression was increased in LUAD. Applying SAL repressed circ_0009624 and PD-L1 expression in LUAD cells. SAL treatment hindered suppressed various oncogenic activates and immune escape of LUAD cells by regulating the circ_0009624/PD-L1 pathway. SAL blocked LUAD xenograft growth in vivo. Applying SAL might constrain malignant phenotypes and immune escape of LUAD cells partially through the circ_0009624-mediated PD-L1 pathway, providing a novel insight for LUAD treatment.