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Animal Science Journal = Nihon Chikusan... 2024The safety of Jatropha curcas L. cake (JCC) in animal feed remains under scrutiny, despite the advent of low phorbol ester (PE) variants. This study investigates the...
The safety of Jatropha curcas L. cake (JCC) in animal feed remains under scrutiny, despite the advent of low phorbol ester (PE) variants. This study investigates the impact of low PE JCC on swine health when used as a protein feed. Pigs were fed a 5% JCC diet with a PE concentration of 0.98 mg/kg, which surprisingly still induced toxicity. Symptoms included depression, decreased food intake, increased diarrhea, along with hypothalamus and colon lesions. The toxicity was associated with a decrease in antioxidant enzymes, an increase in inflammatory cytokines in the hypothalamus, plasma, and colon, and a rise in pro-inflammatory colon microbes and metabolites. Disturbances in neurotransmitters further suggest that this toxicity is related to disruption of the microbiota-gut-brain axis, indicating that JCC's toxic elements are not solely due to PE. The sensitivity of pigs to JCC underscores the need for thorough detoxification prior to its use as feed. These findings significantly contribute to the discourse on the safety of low PE JCC in animal feed, highlighting implications for both the feed industry and public health.
Topics: Animals; Jatropha; Animal Feed; Swine; Gastrointestinal Microbiome; Phorbol Esters; Brain-Gut Axis; Diet; Eating; Cytokines; Colon; Hypothalamus; Depression; Neurotransmitter Agents
PubMed: 38783533
DOI: 10.1111/asj.13953 -
Journal of Visualized Experiments : JoVE May 2024Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced...
Cell death is a fundamental process in all living organisms. The protocol establishes a lipopolysaccharide (LPS) and adenosine triphosphate (ATP)-induced phorbol-12-myristate-13-acetate (PMA)-differentiated lipid deposition in human monocyte (THP-1) macrophage model to observe cell death. LPS combined with ATP is a classic inflammatory induction method, often used to study pyroptosis, but apoptosis and necroptosis also respond to stimulation by LPS/ATP. Under normal circumstances, phosphatidylserine is only localized in the inner leaflet of the plasma membrane. However, in the early stages of pyroptosis, apoptosis, and necroptosis, the cell membrane remains intact and exposed to phosphatidylserine, and in the later stages, the cell membrane loses its integrity. Here, flow cytometry was used to analyze Annexin V and 7-Aminoactinomycin D (AAD) double staining to detect the cell death from the whole cells. The results show that substantial cells died after stimulation with LPS/ATP. Using scanning electron microscopy, we observe the possible forms of cell death in individual cells. The results indicate that cells may undergo pyroptosis, apoptosis, or necroptosis after stimulation with LPS/ATP. This protocol focuses on observing the death of macrophages after stimulation with LPS/ATP. The results showed that cell death after LPS and ATP stimulation is not limited to pyroptosis and that apoptosis and necrotic apoptosis can also occur, helping researchers better understand cell death after LPS and ATP stimulation and choose a better experimental method.
Topics: Humans; Macrophages; Adenosine Triphosphate; Lipopolysaccharides; THP-1 Cells; Tetradecanoylphorbol Acetate; Cell Death; Pyroptosis; Flow Cytometry; Cell Differentiation
PubMed: 38767387
DOI: 10.3791/66831 -
Histochemistry and Cell Biology May 2024As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo...
As the development of chronic wound therapeutics continues to expand, the demand for advanced assay systems mimicking the inflammatory wound microenvironment in vivo increases. Currently, this is performed in animal models or in in vitro cell-based models such as cell culture scratch assays that more closely resemble acute wounds. Here, we describe for the first time a delayed scratch closure model that mimics some features of a chronic wound in vitro. Chronic wounds such as those suffered by later stage diabetic patients are characterised by degrees of slowness to heal caused by a combination of continued localised physical trauma and pro-inflammatory signalling at the wound. To recreate this in a cell-based assay, a defined physical scratch was created and stimulated by combinations of pro-inflammatory factors, namely interferon, the phorbol ester PMA, and lipopolysaccharide, to delay scratch closure. The concentrations of these factors were characterised for commonly used human keratinocyte (HaCaT) and dermal fibroblast (HDF) cell lines. These models were then tested for scratch closure responsiveness to a proprietary healing secretome derived from human Wharton's jelly mesenchymal stem cells (MSCs) previously validated and shown to be highly effective on closure of acute wound models both in vitro and in vivo. The chronically open scratches from HaCaT cells showed closure after exposure to the MSC secretome product. We propose this delayed scratch closure model for academic and industrial researchers studying chronic wounds looking for responsiveness to drugs or biological treatments prior to testing on explanted patient material or in vivo.
PubMed: 38713267
DOI: 10.1007/s00418-024-02292-y -
Fitoterapia Jul 2024In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The...
In Brazil, latex from Euphorbia umbellata (African milk tree) has been increasingly used in folk medicine to treat several types of cancer, including melanoma. The effect of lyophilized latex (LL), its hydroethanolic extract (E80), triterpene (F-TRI)- and diterpene (F-DIT)-enriched fractions, along with six isolated phorbol esters from LL and phorbol 12-myristate 13-acetate (PMA) on J774A.1, THP-1, SK-MEL-28, and B16-F10 cell line viability were evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) method. The compounds were identified by 2D-NMR and HRESIMS. The effect of the LL, extract and fractions on cell viability was also assessed through a resazurin reduction assay. At 100 μg/ml, LL, and its fractions moderately inhibited J774A.1 (37.5-59.5%) and THP-1 (12.6-43.6%) metabolism. LL (IC 70 μg/ml) and F-TRI (IC 68 μg/ml) were barely more effective against B16-F10 cells, and only F-TRI exerted an inhibitory effect on SK-MEL-28 cells (IC 66-75 μg/ml). The samples did not effectively inhibit THP-1 growth (IC 69-87 μg/ml, assessed by MTT). B16-F10 was susceptible to PMA (IC 53 μM) and two 12-phenylacetate esters (IC 56-60 μM), while SK-MEL-28 growth was inhibited (IC 58 μM) by one of these kinds of esters with an additional 4β-deoxy structure. Synagrantol A (IC 39 μM) was as effective as PMA (IC 47 μM) in inhibiting J774A.1 growth in a dose-dependent manner. Furthermore, an in silico study with target receptors indicated a high interaction of the compounds with the PKC proteins. These results provide useful knowledge on the effect of tigliane-type diterpenes on tumor cell from the perspective of medicinal chemistry.
Topics: Euphorbia; Latex; Phorbol Esters; Humans; Mice; Animals; Cell Line, Tumor; Molecular Structure; Plant Extracts; Brazil; Monocytes; Phytochemicals; Cell Survival; Diterpenes; Terpenes; Antineoplastic Agents, Phytogenic; Tetradecanoylphorbol Acetate; Melanoma
PubMed: 38703916
DOI: 10.1016/j.fitote.2024.105987 -
Human Cell Jul 2024Gingival wound healing plays a critical role in maintaining oral health. However, this process can be delayed by oxidative stress and excessive inflammatory responses....
Gingival wound healing plays a critical role in maintaining oral health. However, this process can be delayed by oxidative stress and excessive inflammatory responses. In this study, we established a human inflammatory gingival tissue equivalent (iGTE) to investigate the inhibitory effects of hydrogen-rich water (HW), enzyme-digested edible bird's nest (EBND) and sialic acid (SA) on PMA (an inducer of oxidative free radicals)- and LPS (an inflammatory stimulus)-impaired wound healing. The iGTE was constructed by human gingival fibroblasts (hGFs), keratinocytes and macrophages under three-dimensional conditions. Wounds in the iGTE and hGF/keratinocyte monolayers were created by mechanical injury. Tissues and cells were pretreated with HW, EBND, and SA, and then exposed to the inflammatory and oxidative environment induced by PMA (10 ng/mL) and LPS (250 ng/mL). The inflammatory cytokines IL-6 and IL-8 were quantitatively analyzed by ELISA. Histopathological image analysis was performed by HE and immunofluorescence staining. In the iGTE, PMA/LPS significantly reduced the epithelial thickness while causing a decrease in K8/18, E-cadherin, laminin and elastin expression and an increase in COX-2 expression along with ulcer-like lesions. In mechanically scratched hGFs and keratinocyte monolayers, PMA/LPS significantly impaired wound healing, and promoted the secretion of IL-6 and IL-8. Pretreatment of HW, EBND, and SA significantly suppressed PMA/LPS-induced wound healing delay and inflammatory responses in cell monolayers, as well as in the iGTE. Remarkably, the combined use of HW and EBND exhibited particularly robust results. Combined use of HW and EBND may be applied for the prevention and treatment of wound healing delay.
Topics: Wound Healing; Gingiva; Humans; Keratinocytes; Lipopolysaccharides; Fibroblasts; Hydrogen; Animals; Water; Cells, Cultured; Tetradecanoylphorbol Acetate; Macrophages; Oxidative Stress; Birds; Inflammation; Interleukin-6
PubMed: 38679666
DOI: 10.1007/s13577-024-01065-y -
Pharmaceuticals (Basel, Switzerland) Mar 2024Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca concentration ([Ca]) in pulmonary artery...
Exposure to hypoxia results in the development of pulmonary arterial hypertension (PAH). An increase in the intracellular Ca concentration ([Ca]) in pulmonary artery smooth muscle cells (PASMCs) is a major trigger for pulmonary vasoconstriction and proliferation. This study investigated the mechanism by which KMUP-1, a xanthine derivative with phosphodiesterase inhibitory activity, inhibits hypoxia-induced canonical transient receptor potential channel 1 (TRPC1) protein overexpression and regulates [Ca] through store-operated calcium channels (SOCs). Ex vivo PASMCs were cultured from Sprague-Dawley rats in a modular incubator chamber under 1% O/5% CO for 24 h to elucidate TRPC1 overexpression and observe the Ca release and entry. KMUP-1 (1 μM) inhibited hypoxia-induced TRPC family protein encoded for SOC overexpression, particularly TRPC1. KMUP-1 inhibition of TRPC1 protein was restored by the protein kinase G (PKG) inhibitor KT5823 (1 μM) and the protein kinase A (PKA) inhibitor KT5720 (1 μM). KMUP-1 attenuated protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 1 μM)-upregulated TRPC1. We suggest that the effects of KMUP-1 on TRPC1 might involve activating the cyclic guanosine monophosphate (cGMP)/PKG and cyclic adenosine monophosphate (cAMP)/PKA pathways and inhibiting the PKC pathway. We also used Fura 2-acetoxymethyl ester (Fura 2-AM, 5 μM) to measure the stored calcium release from the sarcoplasmic reticulum (SR) and calcium entry through SOCs in hypoxic PASMCs under treatment with thapsigargin (1 μM) and nifedipine (5 μM). In hypoxic conditions, store-operated calcium entry (SOCE) activity was enhanced in PASMCs, and KMUP-1 diminished this activity. In conclusion, KMUP-1 inhibited the expression of TRPC1 protein and the activity of SOC-mediated Ca entry upon SR Ca depletion in hypoxic PASMCs.
PubMed: 38675401
DOI: 10.3390/ph17040440 -
International Journal of Molecular... Apr 2024Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under...
Mertk, a type I receptor tyrosine kinase and member of the TAM family of receptors, has important functions in promoting efferocytosis and resolving inflammation under physiological conditions. In recent years, Mertk has also been linked to pathophysiological roles in cancer, whereby, in several cancer types, including solid cancers and leukemia/lymphomas. Mertk contributes to oncogenic features of proliferation and cell survival as an oncogenic tyrosine kinase. In addition, Mertk expressed on macrophages, including tumor-associated macrophages, promotes immune evasion in cancer and is suggested to act akin to a myeloid checkpoint inhibitor that skews macrophages towards inhibitory phenotypes that suppress host T-cell anti-tumor immunity. In the present study, to better understand the post-translational regulation mechanisms controlling Mertk expression in monocytes/macrophages, we used a PMA-differentiated THP-1 cell model to interrogate the regulation of Mertk expression and developed a novel Mertk reporter cell line to study the intracellular trafficking of Mertk. We show that PMA treatment potently up-regulates Mertk as well as components of the ectodomain proteolytic processing platform ADAM17, whereas PMA differentially regulates the canonical Mertk ligands Gas6 and Pros1 (Gas6 is down-regulated and Pros1 is up-regulated). Under non-stimulated homeostatic conditions, Mertk in PMA-differentiated THP1 cells shows active constitutive proteolytic cleavage by the sequential activities of ADAM17 and the Presenilin/γ-secretase complex, indicating that Mertk is cleaved homeostatically by the combined sequential action of ADAM17 and γ-secretase, after which the cleaved intracellular fragment of Mertk is degraded in a proteasome-dependent mechanism. Using chimeric Flag-Mertk-EGFP-Myc reporter receptors, we confirm that inhibitors of γ-secretase and MG132, which inhibits the 26S proteasome, stabilize the intracellular fragment of Mertk without evidence of nuclear translocation. Finally, the treatment of cells with active γ-carboxylated Gas6, but not inactive Warfarin-treated non-γ-carboxylated Gas6, regulates a distinct proteolytic itinerary-involved receptor clearance and lysosomal proteolysis. Together, these results indicate that pleotropic and complex proteolytic activities regulate Mertk ectodomain cleavage as a homeostatic negative regulatory event to safeguard against the overactivation of Mertk.
Topics: Humans; c-Mer Tyrosine Kinase; ADAM17 Protein; Amyloid Precursor Protein Secretases; Proteolysis; Intercellular Signaling Peptides and Proteins; THP-1 Cells; Macrophages; Protein S; Monocytes; Tetradecanoylphorbol Acetate
PubMed: 38673989
DOI: 10.3390/ijms25084404 -
Chinese Journal of Natural Medicines Apr 2024Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is...
Phorbol esters are recognized for their dual role as anti-HIV-1 agents and as activators of protein kinase C (PKC). The efficacy of phorbol esters in binding with PKC is attributed to the presence of oxygen groups at positions C20, C3/C4, and C9 of phorbol. Concurrently, the lipids located at positions C12/C13 are essential for both the anti-HIV-1 activity and the formation of the PKC-ligand complex. The influence of the cyclopropane ring at positions C13 and C14 in phorbol derivatives on their anti-HIV-1 activity requires further exploration. This research entailed the hydrolysis of phorbol, producing seco-cyclic phorbol derivatives. The anti-HIV-1 efficacy of these derivatives was assessed, and the affinity constant (K) for PKC-δ protein of selected seco-cyclic phorbol derivatives was determined through isothermal titration calorimetry. The findings suggest that the chemical modification of cyclopropanols could affect both the anti-HIV-1 activity and the PKC binding affinity. Remarkably, compound S11, with an EC50 of 0.27 μmol·L and a CC of 153.92 μmol·L, demonstrated a potent inhibitory effect on the intermediate products of HIV-1 reverse transcription (ssDNA and 2LTR), likely acting at the viral entry stage, yet showed no affinity for the PKC-δ protein. These results position compound S11 as a potential candidate for further preclinical investigation and for studies aimed at elucidating the pharmacological mechanism underlying its anti-HIV-1 activity.
Topics: HIV-1; Humans; Anti-HIV Agents; Phorbol Esters; Molecular Structure; Protein Kinase C; Structure-Activity Relationship
PubMed: 38658099
DOI: 10.1016/S1875-5364(24)60630-8 -
Journal of Interferon & Cytokine... Apr 2024This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from...
Activated and Naïve Allogenic Human Placental Mesenchymal Stromal Cells Exert an Immunomodulatory Effect on Hidradenitis Suppurativa Patient Peripheral Blood Mononuclear Cells.
This pilot study aimed to evaluate the immunomodulatory effect of placental mesenchymal stem/stromal cells (MSCs) on peripheral blood mononuclear cells (PBMCs) from patients with hidradenitis suppurativa (HS). Blood samples were collected from 3 healthy and 3 patients with HS. Isolated PBMCs were stained with carboxyfluorescein succinimidyl ester (CFSE) and stimulated with phorbol 12-myristate 13-acetate (PMA)/Ionomycin solution. The PBMCs of patients with HS were co-cultured with naïve MSCs (n-MSCs), activated with tumor necrosis factor (TNF)-α (10 ng/mL) and interferon (IFN)-γ (10 ng/mL) MSCs (a-MSCs), or adalimumab (30 μg/mL). The division index (proliferation inhibition) of PBMCs was analyzed by flow cytometry using the Proliferation Modeling tool after 5 days of coculture. The relative inflammatory gene expression dynamics and cytokine secretion were quantified in triplicate using real-time polymerase chain reaction (PCR) and Luminex assays. PBMCs from the HS control group showed statistically significant increases in interleukin (IL)-6 and IFN-γ cytokine concentrations and gene expression when compared with healthy subjects. Statistically significant reduction of the division index was found in the a-MSCs group ( = 0.04). Also, the Luminex assay revealed significantly reduced proinflammatory cytokine concentrations of IL-9 ( = 0.022) and IL-17A ( = 0.022) in the a-MSCs group with the same trend of numerical lowering in n-MSCs group when compared to HS control. The results of real-time PCR revealed a numerical increase in the expression of the , and genes in both the a-MSCs and n-MSCs groups compared with the HS control. In conclusion, our findings suggest that MSCs can effectively curb PBMCs proliferation and suppress the production of inflammatory cytokines. Moreover, the preactivation of MSCs with IFN-γ and TNF-α before use can enhance their therapeutic effectiveness. Nevertheless, a larger sample size is imperative to validate these results.
PubMed: 38607317
DOI: 10.1089/jir.2024.0035 -
Zhongguo Xue Xi Chong Bing Fang Zhi Za... Feb 2024To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis...
OBJECTIVE
To investigate the expression of neutrophil extracellular traps (NETs) and phagocytic function in the peripheral blood of patients with hepatic alveolar echinococcosis (HAE), and to examine their correlations with clinical inflamma tory indicators and liver functions.
METHODS
A total of 50 patients with HAE admitted to Department of Hepatobiliary and Pancreatic Surgery, The Affiliated Hospital of Qinghai University from August 2022 to June 2023 were enrolled, while 50 age- and gender-matched healthy individuals from the Centre for Healthy Examinations of the hospital during the same period served as controls. The levels of NETs markers neutrophil myeloperoxidase (MPO) and neutrophil elastase (NE) were measured using enzyme-linked immunosorbent assay (ELISA). Peripheral blood neutrophils were isolated using density gradient centrifugation, stimulated using phorbol 12-myristate 13 acetate (PMA), and the levels of MPO and citrullination histone H3 (CitH3) released by neutrophils were quantified using flow cytometry. The phagocytic functions of neutrophils were examined using flow cytometry. In addition, the correlations of MPO and NE levels with clinical inflammatory indicators and liver biochemical indicators were examined using Spearman correlation analysis among HAE patients.
RESULTS
The peripheral blood plasma MPO[(417.15 ± 76.08) ng/mL vs. (255.70 ± 80.84) ng/mL; = 10.28, < 0.05], NE[(23.16 ± 6.75) ng/mL vs. (11.92 ± 3.17) ng/mL; = 10.65, < 0.05]and CitH3 levels[(33.93 ± 18.93) ng/mL vs. (19.52 ± 13.89) ng/mL; = 4.34, < 0.05]were all significantly higher among HAE patients than among healthy controls, and a lower phagocytosis rate of neutrophils was detected among HAE patients than among healthy controls[(70.85 ± 7.32)% vs. (94.04 ± 3.90)%; = 20.18, < 0.05], and the ability to produce NETs by neutrophils was higher among HAE patients than among healthy controls following PMA stimulation. Pearson correlation analysis showed that the phagocytosis rate of neutrophils correlated negatively with platelet-to-lymphocyte ratio (PLR), neutrophil-to-lymphocyte ratio (NLR), interleukin-6 (IL-6) level and C-reactive protein (CRP) level ( = -0.515 to -0.392, all values < 0.05), and the MPO and NE levels positively correlated with inflammatory markers NLR, PLR, CRP and IL-6 ( = 0.333 to 0.445, all values < 0.05) and clinical liver biochemical indicators aspartic transaminase, alanine aminotransferase, direct bilirubin and total bilirubin among HAE patients ( = 0.290 to 0.628, all values < 0.001).
CONCLUSIONS
Excessive formation of NETs is found among HAE patients, which affects the phagocytic ability of neutrophils and results in elevated levels of inflammatory indicators. NETs markers may be promising novel biomarkers for early diagnosis, monitoring, and severity assessment of liver disease.
Topics: Humans; Extracellular Traps; Interleukin-6; Echinococcosis, Hepatic; Neutrophils; Tetradecanoylphorbol Acetate; Bilirubin
PubMed: 38604682
DOI: 10.16250/j.32.1374.2023172