P65 Subunit Research Articles

P66Shc Deficiency in Chronic Lymphocytic Leukemia Promotes Chemokine Receptor Expression Through the ROS-Dependent Inhibition of NF-κB.

The microenvironment of lymphoid organs is central to the pathogenesis of chronic lymphocytic leukemia (CLL). Within it, tumor cells find a favourable niche to escape immunosurveillance and acquire pro-survival signals. We have previously reported that a CLL-associated defect in the expression of the pro-apoptotic and pro-oxidant adaptor p66Shc leads to enhanced homing to and accumulation of leukemic cells in the lymphoid microenvironment. The p66Shc deficiency-related impairment in intracellular reactive oxygen species (ROS) production in CLL cells is causally associated to the enhanced expression of the chemokine receptors CCR2, CXCR3 and CCR7, that promote leukemic cell homing to both lymphoid and non-lymphoid organs, suggesting the implication of a ROS-modulated transcription factor(s). Here we show that the activity of the ROS-responsive p65 subunit of the transcription factor NF-κB was hampered in the CLL-derived cell line MEC-1 expressing a NF-κB-luciferase reporter following treatment with H2O2. Similar results were obtained when intracellular ROS were generated by expression of p66Shc, but not of a ROS-defective mutant, in MEC-1 cells. NF-κB activation was associated with increased expression of the chemokine receptors CCR2, CXCR3 and CCR7. Reconstitution of p66Shc in CLL cells normalized intracellular ROS and hampered NF-κB activation, which led to a decrease in the expression of these homing receptors. Our data provide direct evidence that the p66Shc-deficiency-related ROS depletion in CLL cells concurs to NF-κB hyperactivation and homing receptor overexpression, providing a mechanistic basis for the enhanced ability of these cells to accumulate in the pro-survival lymphoid niche.

A Study on the Anti-NF-κB, Anti-Candida, and Antioxidant Activities of Two Natural Plant Hormones: Gibberellin A4 and A7.

Introduction: Gibberellins (GA) are terpenoids that serve as important plant hormones by acting as growth and response modulators against injuries and parasitism. In this study, we investigated the in vitro anti-NF-κB, anti-Candida, and antioxidant activity of gibberellin A4 (GA4) and A7 (GA7) compounds, and further determined their toxicity in vivo. Methods: GA4 and GA7 in vitro toxicity was determined by MTT method, and nontoxic concentrations were then tested to evaluate the GA4 and GA7 anti-NF-κB activity in LPS-activated RAW-luc macrophage cell culture (luminescence assay). GA4 in silico anti-NF-κB activity was evaluated by molecular docking with the software “AutoDock Vina”, “MGLTools”, “Pymol”, and “LigPlot+”, based on data obtained from “The Uniprot database”, “Protein Data Bank”, and “PubChem database”. The GA4 and GA7 in vitro anti-Candida effects against Candida albicans (MYA 2876) were determined (MIC and MFC). GA7 was also evaluated regarding the viability of C. albicans preformed biofilm (microplate assay). In vitro antioxidant activity of GA4 and GA7 was evaluated against peroxyl radicals, superoxide anions, hypochlorous acid, and reactive nitrogen species. GA4 and GA7 in vivo toxicity was determined on the invertebrate Galleria mellonella larvae model. Results: Our data show that GA4 at 30 µM is nontoxic and capable of reducing 32% of the NF-κB activation on RAW-luc macrophages in vitro. In vitro results were confirmed via molecular docking assay (in silico), since GA4 presented binding affinity to NF-κB p65 and p50 subunits. GA7 did not present anti-NF-κB effects, but exhibited anti-Candida activity with low MIC (94 mM) and MFC (188 mM) values. GA7 also presented antibiofilm properties at 940 mM concentration. GA4 did not present anti-Candida effects. Moreover, GA4 and GA7 showed antioxidant activity against peroxyl radicals, but did not show scavenging activity against the other tested radicals. Both compounds did not affect the survival of G. mellonella larvae, even at extremely high doses (10 g/Kg). Conclusion: Our study provides preclinical evidence indicating that GA4 and GA7 have a favorable low toxicity profile. The study also points to GA4 and GA7 interference with the NF-κB via, anti-Candida activity, and a peroxyl radical scavenger, which we argue are relevant biological effects.

Isosteroidal Alkaloids from Fritillaria verticillata Willd. and Their NMR Spectroscopic Characteristics

Comprehensive SummaryChemical investigation of Fritillaria verticillata Willd. led to the isolation of eight novel isosteroidal alkaloids (1—4, 6, 9—11) and four known alkaloids (5, 7—8, 12), including three unprecedented cis‐fused D/E (13α, 17α) cevanine alkaloids (1—3), one rarely cis‐fused E/F (22α) cevanine alkaloid (6), and one uncommon 5β‐jervine‐type isosteroidal alkaloid featuring a cis‐fused A/B ring moiety (11). In order to establish the structures of 1, the calculated NMR with DP4+ evaluation was applied from the plausible structure candidates. The characteristic proton signals for distinguishing D/E conjunction of cevanine alkaloids were summarized. In addition, some proton and carbon signals α to nitrogen in 6 and 7 are unobservable due to the 14N nuclear quadrupolar relaxation, based on the NMR experiments in different solvents, calculated NMR method and X‐ray technology, their structures were determined. The NMR characteristics of cevanine alkaloids to distinguish orientation of long‐pair on nitrogen atom with β‐hydroxyl at C‐20 were also concluded. The anti‐inflammatory effects of compounds 1 and 4 were evaluated in LPS‐activated RAW 264.7 macrophages. Compound 4 decreased LPS‐induced releases of IL‐1β and IL‐17α in RAW264.7 cells in a dose‐dependent manner. Further mechanistic study revealed that 4 suppressed the phosphorylation of IκBα and p65 subunit to regulate the NF‐κB signaling pathway.

Abstract 106: Diclofenac hybrids with oleanolic acid oximes modulate key signaling pathways in pancreatic cancer cells

Abstract Pancreatic cancer is characterized by constitutive activation of mitogen-activated protein kinases (MAPKs) like JNK and p38 determining its malignant phenotypes. Overexpression of NF-κB and COX-2 involved in inflammation contributes to increased cell growth and invasiveness. The latter might be related to the activated AKT pathway. Nonsteroidal anti-inflammatory drugs were suggested for pancreas cancers prophylaxis. Naturally occurring oleanolic acid (OA) was shown to possess anti-inflammatory and anti-tumorigenic activities. Coupling its analogs with NSAIDs may enhance this effect. Our study aimed to evaluate the effect of two oleanolic acid oximes derivatives conjugated with diclofenac (DCL), 4c (3-diclofenacoxyiminoolean-12-en-28-oic acid benzyl ester) and 4d (3-diclofenacoxyiminoolean-12-en-28-oic acid morpholide) on inflammation-related pathways, MAPK and NF-κB, cell cycle arrest and ROS formation in PSN-1 pancreatic cancer cells. PSN-1 cells were incubated with DCL, 4c or 4d at the concentration of 30µM. Activation of NF-κB was assessed by measuring binding of its p50 and p65 subunits to DNA using ELISA method. Their nuclear concentrations were determined by Western blot. RT-PCR was used to evaluate NF-κB subunits and COX-2 mRNA levels. Bead-based immunoassay determined the cytosolic levels of p38, JNK, CREB, AKT, P70S6K and STAT3 proteins. Cell cycle distribution and ROS formation were assessed using flow cytometry. Conjugate 4d significantly reduced nuclear level of p65 subunit and its mRNA expression, although compound 4c had more significant impact on COX-2 expression. Both derivatives significantly diminished p65 DNA binding, but only 4d had significant effect on p50. Both compounds reduced ROS production, but neither of them affected the cell cycle arrest. We observed decreased cytosolic AKT level along with increased levels of unphosphorylated JNK and p38 kinases which may have an antagonistic effect on proliferation by reducing activity of inflammatory mediators acting as tumor promoters. AKT decrease after treatment with 4d derivative might be the cause of increased unphosphorylated CREB level. Accumulation of p38 and JNK in cytosol could be a result of decreased ROS production and/or cause increased CREB protein level. Additionally, increased STAT3 cytosolic level suggests inhibition of mTORC1 pathway that explains reduced p70S6 kinase. Overall, these results suggest that OAO-DCL morpholide conjugate may affect both NF-κB and MAPK signaling pathways in pancreatic cancer cells. Further studies are required to explain detailed mechanism of these modifications. Funding: Studies were supported by Polish National Science Centre grant 2016/21/B/NZ7/01758. Acknowledgments: Authors thank Dr. Gary Gorbsky, Chair of Cell Cycle & Cancer Biology Program in Oklahoma Medical Research Foundation for supporting financing Maria Narozna conference participation. Citation Format: Maria Narozna, Violetta Krajka-Kuzniak, Robert Kleszcz, Wanda Baer-Dubowska. Diclofenac hybrids with oleanolic acid oximes modulate key signaling pathways in pancreatic cancer cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 106.

Synthesis, In Vitro and In Silico Analysis of New Oleanolic Acid and Lupeol Derivatives against Leukemia Cell Lines: Involvement of the NF-κB Pathway.

Oleanolic acid (OA) and Lupeol (LU) belong to the class of natural triterpenes and are endowed with a wide range of biological activities, including cytotoxicity toward several cancer cell lines. In this context, we investigated a set of compounds obtained from the two natural precursors for the cytotoxicity against leukemia HL60 cells and the multidrug-resistant (MDR) variant HL60R. Six new semi-synthetic triterpenes have been synthetized, fully characterized, and were investigated together with other triterpenes compounds for their pharmacological mechanism of action. The interaction of the more cytotoxic compounds with the nuclear factor kappa B (NF-κB) pathway has been also evaluated with the aid of docking. The lupane-like compounds were more active than the precursor, while the oleane-like compounds showed more complex behavior. Both OA and LU derivatives possess a similar interaction pattern with the p65 subunit of NF-κB, justifying the similar trend in their ability to inhibit the binding of p65 to DNA. Further, some of the derivatives tested were able to increase IκB-α levels preventing the translocation of NF-κB to the nucleus. In conclusion, this study offers a deeper insight on the pharmacological action of triterpenes toward leukemia cells, and it improves the background useful for the development of new anti-cancer drugs.

Endothelial ACKR3 drives atherosclerosis by promoting immune cell adhesion to vascular endothelium

Atherosclerosis is the foundation of potentially fatal cardiovascular diseases and it is characterized by plaque formation in large arteries. Current treatments aimed at reducing atherosclerotic risk factors still allow room for a large residual risk; therefore, novel therapeutic candidates targeting inflammation are needed. The endothelium is the starting point of vascular inflammation underlying atherosclerosis and we could previously demonstrate that the chemokine axis CXCL12–CXCR4 plays an important role in disease development. However, the role of ACKR3, the alternative and higher affinity receptor for CXCL12 remained to be elucidated. We studied the role of arterial ACKR3 in atherosclerosis using western diet-fed Apoe−/− mice lacking Ackr3 in arterial endothelial as well as smooth muscle cells. We show for the first time that arterial endothelial deficiency of ACKR3 attenuates atherosclerosis as a result of diminished arterial adhesion as well as invasion of immune cells. ACKR3 silencing in inflamed human coronary artery endothelial cells decreased adhesion molecule expression, establishing an initial human validation of ACKR3’s role in endothelial adhesion. Concomitantly, ACKR3 silencing downregulated key mediators in the MAPK pathway, such as ERK1/2, as well as the phosphorylation of the NF-kB p65 subunit. Endothelial cells in atherosclerotic lesions also revealed decreased phospho-NF-kB p65 expression in ACKR3-deficient mice. Lack of smooth muscle cell-specific as well as hematopoietic ACKR3 did not impact atherosclerosis in mice. Collectively, our findings indicate that arterial endothelial ACKR3 fuels atherosclerosis by mediating endothelium-immune cell adhesion, most likely through inflammatory MAPK and NF-kB pathways.

Positive allosteric γ-aminobutyric acid type A receptor modulation prevents lipotoxicity-induced injury in hepatocytes in vitro.

To determine if a novel positive allosteric modulator of the γ-aminobutyric acid type A (GABAA ) receptor, the thioacrylamide-derivative HK4, which does not penetrate the blood-brain barrier, protects human hepatocytes against lipotoxicity-induced injury. Allosteric modulation of the GABAA receptor by HK4 was determined by patch clamp in HEK-293 cells, calcium influx in INS-1E cells and by using the specific GABAA channel blockers picrotoxin and tert-butylbicyclophosphorothionate (TBPS) in HepG2 cells. Apoptosis was analysed using caspase 3/7, terminal deoxynucleotidyl transferase-dUTP nick end labelling (TUNEL) and array assays in HepG2 cells and/or human primary hepatocytes. Phosphorylation of STAT3 and the NF-κB subunit p65, protein disulphide isomerase (PDI) and poly-ADP-ribose polymerase-1 (PARP-1) was detected by Western blotting. Patch clamping, calcium influx measurements and apoptosis assays with the non-competitive GABAA channel blockers picrotoxin and TBPS proved HK4 as a selective positive allosteric modulator of the GABAA receptor. In HepG2 cells, which expressed the main GABAA receptor subunits, HK4 prevented palmitate-induced apoptosis. This protective effect was mediated by downregulation of caspase 3/7 activity and was additionally verified by TUNEL assay. HK4 effectively prevented palmitate-induced apoptosis in human primary hepatocytes. HK4 reduced STAT3 and NF-κB phosphorylation, reduced cleaved PARP-1 expression and upregulated the endoplasmic reticulum (ER) chaperone PDI. HK4 reduced lipotoxic-induced apoptosis by preventing inflammation, DNA damage and ER stress. We propose that the effect of HK4 is mediated by STAT3 and NF-κB. It is suggested that thioacrylamide compounds represent an innovative pharmacological tool to treat or prevent non-alcoholic steatohepatitis as first-in-class drugs.

Extracellular ST6GAL1 regulates monocyte-macrophage development and survival.

Interaction of immune cells with the systemic environment is necessary for the coordinated development and execution of immune responses. Monocyte-macrophage lineage cells reside at the junction of innate and adaptive immunity. Previously we reported that the sialyltransferase ST6GAL1 in the extracellular milieu modulates B cell development and IgG production, granulocyte production, and attenuates acute airway inflammation to bacterial challenge in mouse models. Here, we report that extracellular ST6GAL1 also elicits profound responses in monocyte-macrophage lineage cells. We show that recombinant ST6GAL1 adheres to subsets of thioglycolate-elicited inflammatory cells in the mouse peritoneum and to cultured human monocyte THP-1 cells. Exposure of the inflammatory cells to recombinant ST6GAL1 elicited wholesale changes in the gene expression profile of primary mouse myeloid cells; most notable was the striking up-regulation of monocyte-macrophage and monocyte-derived dendritic cell development pathway signature genes and transcription factors PU.1, NFκB and their target genes, driving increased monocyte-macrophage population and survival ex vivo. In the cultured human monocyte cells, the essential cell surface receptor of the monocyte-macrophage lineage, the M-CSF receptor (M-CSF-R, Csfr1) was a target of extracellular ST6GAL1 catalytic activity. Extracellular ST6GAL1 activated the M-CSF-R and initiated intracellular signaling events, namely, the nuclear translocation of NFκB subunit p65, and phosphorylation of ERK 1/2 and AKT. The findings implicate extracellular ST6GAL1 in monocyte development by a mechanism initiated at the cell surface and support an emerging paradigm of an extracellular glycan-modifying enzyme as a central regulator coordinating immune hematopoietic cell development and function.

Increased TNF-α production in response to IL-6 in patients with systemic inflammation without infection.

Acute systemic inflammation can lead to life-threatening organ dysfunction. In patients with sepsis, systemic inflammation is triggered in response to infection, but in other patients, a systemic inflammatory response syndrome (SIRS) is triggered by non-infectious events. IL-6 is a major mediator of inflammation, including systemic inflammatory responses. In homeostatic conditions, when IL-6 engages its membrane-bound receptor on myeloid cells, it promotes pro-inflammatory cytokine production, phagocytosis, and cell migration. However, under non-physiologic conditions, such as SIRS and sepsis, leucocyte dysfunction could modify the response of these cells to IL-6. So, our aim was to evaluate the response to IL-6 of monocytes from patients diagnosed with SIRS or sepsis. We observed that monocytes from patients with SIRS, but not from patients with sepsis, produced significantly more TNF-α than monocytes from healthy volunteers, after stimulation with IL-6. Monocytes from SIRS patients had a significantly increased baseline phosphorylation of the p65 subunit of NF-κB, with no differences in STAT3 phosphorylation or SOCS3 levels, compared with monocytes from septic patients, and this increased phosphorylation was maintained during the IL-6 activation. We found no significant differences in the expression levels of the membrane-bound IL-6 receptor, or the serum levels of IL-6, soluble IL-6 receptor, or soluble gp130, between patients with SIRS and patients with sepsis. Our results suggest that, during systemic inflammation in the absence of infection, IL-6 promotes TNF-α production by activating NF-κB, and not the canonical STAT3 pathway.

Chuanzhi Tongluo Capsules antagonizes cerebral ischemia-reperfusion injury in mice by inhibiting neuroinflammation and oxidative stress

Chuanzhi Tongluo Capsules(CZTL) is effective in activating blood, resolving stasis, replenishing Qi, and dredging collaterals, which has been widely used in clinical treatment of stroke(cerebral infarction) differentiated into the syndrome of wind striking meridian and collateral in the recovery stage characterized by blood stasis and Qi deficiency. However, its modern pharmacological mechanisms of action remain unclear. This study duplicated the middle cerebral artery occlusion and reperfusion(MCAO/R) model in mice using the suture-occluded method to explore the protective effect and mechanism of CZTL on ischemic stroke. The mice were divided into the sham-operation group, model group, and CZTL group. The ones in the CZTL group were gavaged with 0.3 g·kg~(-1)·d~(-1) CZTL for three successive days. One hour after the last intragastric administration, those in the model and CZTL groups were subjected to MCAO/R. After 24 h reperfusion, the effects of CZTL on neurological deficit score, cerebral infarction area, brain edema, and brain histopathology were evaluated. The levels of reactive oxygen species(ROS), malondialdehyde(MDA), interleukin-6(IL-6), interleukin-1β(IL-1β), and tumor necrosis factor-α(TNF-α) and the activity of superoxide dismutase(SOD) in brain tissue homogenate were detected using corresponding assay kits. The expression of B-cell lymphoma-2(Bcl-2), Bcl-2-associated X protein(Bax), Toll like receptor 4(TLR4), and phosphorylated nuclear factor-κB P65 subunit(p-NF-κB P65) were assayed by Western blot. The results indicated that CZTL significantly reduced the neurological deficit score, brain edema, and infarct volume, improved the brain histopathology, inhibited the expression of ROS, MDA, IL-6, IL-1β, and TNF-α in the brain tissue, and up-regulated the activity of SOD, down-regulated the expression of pro-apoptotic protein Bax, promoted the expression of anti-apoptotic protein Bcl-2, and suppressed the expression of TLR4 and p-NF-κB P65 phosphorylation of MCAO/R mice. All these have demonstrated that CZTL has a significant protective effect against MCAO/R injury in mice, which may be related to its resistance to neuroinflammation and oxidative stress.

Low-intensity pulsed ultrasound enhances immunomodulation and facilitates osteogenesis of human periodontal ligament stem cells by inhibiting the NF-κB pathway.

Human periodontal ligament stem cells (hPDLSCs) are vital in cellular regeneration and tissue repair due to their multilineage differentiation potential. Low intensity pulsed ultrasound (LIPUS) has been applied for treating bone and cartilage defects. This study explored the role of LIPUS in the immunomodulation and osteogenesis of hPDLSCs. hPDLSCs were cultured in vitro, and the effect of different intensities of LIPUS (30, 60, and 90 mW/cm2) on hPDLSC viability was measured. hPDLSCs irradiated by LIPUS and stimulated by lipopolysaccharide (LPS) and LIPUS (90 mW/cm2) were co-cultured with peripheral blood mononuclear cells (PBMCs). Levels of immunomodulatory factors in hPDLSCs and inflammatory factors in PBMCs were estimated, along with determination of osteogenesis-related gene expression in LIPUS-irradiated hPDLSCs. The mineralized nodules and alkaline phosphatase (ALP) activity of hPDLSCs and levels of IκBα, p-IκBα, and p65 subunits of NF-κB were determined. hPDLSC viability was increased as LIPUS intensity increased. Immunomodulatory factors were elevated in LIPUS-irradiated hPDLSCs, and inflammatory factors were reduced in PBMCs. Osteogenesis-related genes, mineralized nodules, and ALP activity were promoted in LIPUS-irradiated hPDLSCs. The cytoplasm of hPDLSCs showed increased IκBα and p65 and decreased p-IκBα at increased LIPUS intensity. After LPS and LIPUS treatment, the inhibitory effect of LIPUS irradiation on the NF-κB pathway was partially reversed, and the immunoregulation and osteogenic differentiation of hPDLSCs were decreased. LIPUS irradiation enhanced immunomodulation and osteogenic differentiation abilities of hPDLSCs by inhibiting the NF-κB pathway, and the effect is dose-dependent. This study may offer novel insights relevant to periodontal tissue engineering.Graphical

Identification of lesional attributes of dysmenorrhea severity and the serum antimüllerian hormone levels in women with ovarian endometriomas

To investigate whether lesional immunostaining of putative biomarkers of recurrence and the extent of lesional and cortical fibroses are correlated with the severity of dysmenorrhea and serum antimüllerian hormone (AMH) levels in women with ovarian endometriomas (OEs). Retrospective cohort study. Academic hospital. A total of 313 women with histologically confirmed OEs were recruited. Their demographic and clinical information and data on their preoperative AMH levels were collected. Additionally, samples of their lesional tissues and ovarian cortex tissues adjacent to the OE lesions were procured for histologic and immunohistochemistry analyses. None. All OE tissue samples were stained for phosphorylated nuclear factor κB p65 subunit, progesterone receptor isoform B, Slit2, and α-smooth muscle actin. In addition, the extent of lesional and cortical fibroses was quantitated by Masson trichrome staining. We evaluated the relationship between the lesion size; laterality; extent of lesional and cortical fibroses, along with the putative markers of recurrence; and severity of dysmenorrhea and preoperative serum AMH levels in patients with OE. We found that the extent of lesional fibrosis was positively correlated with the severity of dysmenorrhea but had no impact on the AMH levels. On the other hand, the extent of cortical fibrosis, along with age, was negatively correlated with the AMH levels. The correlation between lesional fibrosis and the severity of dysmenorrhea and between cortical fibrosis and the AMH levels would call an early intervention once OE is diagnosed or suspected to prevent further pain and diminished ovarian reserve.

Rack1 regulates pro-inflammatory cytokines by NF-κB in diabetic nephropathy.

Diabetic nephropathy (DN) is one of the chronic microvascular diseases of diabetes. Studies revealed that inflammation is involved in the development of DN. However, its mechanisms are not fully clear. Here, we screened DN-related mRNAs by RNA sequencing in the renal tissues of db/db DN mice and normal control mice. The Swiss-Model, ZDOCK 3.0.2 and PyMOL 2.3.2 were applied for bioinformatics analysis. In total, we obtained 6,820 mRNAs that were dysexpressed in DN. Among them, Receptor for Activated C Kinase 1 (Rack1) was focused on for its high fold changes and high values of fragments per kilobase million (FPKM) in both two groups (FPKM >100). Moreover, Rack1 was highly expressed in DN in vivo and in vitro. Results displayed that the expressions of pro-inflammatory cytokines Mcp-1 and Tnf-α were increased when Rack1 was overexpressed in cells cultured with low glucose while the expressions of Mcp-1 and Tnf-α were decreased when Rack1 was silenced in cells cultured with high glucose. Furthermore, results showed that the established DN inflammatory factor nuclear factor NF-kappa-B (NF-κB) was regulated by Rack1 via the direct interaction between Rack1 and NF-κB subunits P50 and P65. In summary, this identified Rack1 could play an important role in the inflammation of DN via NF-κB, which can provide new insight for DN research.

A review of the protective effects of chlorogenic acid against different chemicals.

Chlorogenic acid (CGA) is a naturally occurring non-flavonoid polyphenol found in green coffee beans, teas, certain fruits, and vegetables, that exerts antiviral, antitumor, antibacterial, and antioxidant effects. Several in vivo and in vitro studies have demonstrated that CGA can protect against toxicities induced by chemicals of different classes such as fungal/bacterial toxins, pharmaceuticals, metals, pesticides, etc., by preservation of cell survival via reducing overproduction of nitric oxide and reactive oxygen species and suppressed pro-apoptotic signaling. CGA antioxidant effects mediated through the Nrf2-heme oxygenase-1 signaling pathway were shown to enhance the levels of antioxidant enzymes such as superoxide dismutase, catalase, glutathione-S-transferases, glutathione peroxidase, and glutathione reductase as well as glutathione content. Also, CGA could suppress inflammation via inhibition of toll-like receptor 4 and MyD88, and the phosphorylation of inhibitor of kappa B and p65 subunit of NF-κB, resulting in diminished levels of downstream inflammatory factors including interleukin (IL)-1 β, IL-6, tumor necrosis factor-α, macrophage inflammatory protein 2, cyclooxygenase-2, and prostaglandin E2. Moreover, CGA inhibited apoptosis by reducing Bax, cytochrome C, and caspase 3 and 9 expression while increasing Bcl-2 levels. The present review discusses several mechanisms through which CGA may exert its protective role against such agents. Chemical and natural toxic agents affect human health. Phenolic antioxidant compounds can suppress free radical production and combat these toxins. Chlorogenic acid is a plant polyphenol present in the human diet and exerts strong antioxidant properties that can effectively help in the treatment of various toxicities.

The licorice flavonoid isoliquiritigenin attenuates Mycobacterium tuberculosis-induced inflammation through Notch1/NF-κB and MAPK signaling pathways

Ethnopharmacological relevanceThe genus Glycyrrhiza is a small perennial herb that has been traditionally used to treat many diseases across the world. Licorice (Gancao in Chinese) is the dried root and rhizome of G. glabra, G. uralensis or G. inflata. Licorice plays an important role in traditional Chinese medicine (TCM), and is the most frequently used in Chinese herbal formulas. Isoliquiritigenin (ISL) is a flavonoid extracted from licorice, and has been evaluated for its various biological activities, including anti-inflammatory, anti-tumor and anti-oxidant activities. Excessive and persistent inflammation in the Mycobacterium tuberculosis (Mtb) infection is not conducive to the elimination of Mtb, but contributes to serious pulmonary dysfunction. Aim of the studyThis study aimed to examine the anti-inflammatory effects of ISL in the Mtb infection. MethodsIn vitro models of Mtb-infected macrophages were established. Murine macrophage Raw 264.7 cells and primary peritoneal macrophages were used in this study. Cell viability was determined by the cell counting kit-8 (CCK-8) assay. The effects of ISL on the secretion levels of interleukin -1β (IL-1β), tumor necrosis factor -α (TNF-α), and interleukin −6 (IL-6) were detected by the enzyme-linked immunosorbent assay (ELISA). The expression levels of inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX2) were measured by the real time quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. Western blot was used to assess the effects of ISL on the activation of NLRP3 inflammasome and Notch1/NF-κB and MAPK signaling pathways. Immunofluorescence assays was used to detected the translocation of phosphorylation of p65 subunit of NF-κB. ResultsIt was revealed that ISL inhibited the secretion of IL-1β and the activation of pore-forming protein (gasdermin D, GSDMD) by suppressing the activation of NLPR3 inflammasome induced by Mtb infection. ISL was also shown to have promising inhibitory effects on inflammatory factors, such as TNF-α, IL-6, iNOS and COX2. Regarding the anti-inflammatory mechanism of ISL, it was found that ISL exerted its anti-inflammatory effects by inhibiting the activation of Notch1/NF-κB and MAPK signaling pathways. ConclusionISL reduced Mtb-induced inflammation through the Notch1/NF-κB and MAPK signaling pathways. ISL might be used as a potential adjuvant drug to treat tuberculosis by adjusting host immune responses.

Artemisia dracunculus L. Ethanolic Extract and an Isolated Component, DMC2, Ameliorate Inflammatory Signaling in Pancreatic β-Cells via Inhibition of p38 MAPK

Non-resolving pancreatic islet inflammation is widely viewed as a contributor to decreases in β-cell mass and function that occur in both Type 1 and Type 2 diabetes. Therefore, strategies aimed at reducing or eliminating pathological inflammation would be useful to protect islet β-cells. Herein, we described the use of 2′,4′-dihydroxy-4-methoxydihydrochalcone (DMC2), a bioactive molecule isolated from an ethanolic extract of Artemisia dracunculus L., as a novel anti-inflammatory agent. The ethanolic extract, termed PMI 5011, reduced IL-1β-mediated NF-κB activity. DMC2 retained this ability, indicating this compound as the likely source of anti-inflammatory activity within the overall PMI 5011 extract. We further examined NF-κB activity using promoter-luciferase reporter constructs, Western blots, mRNA abundance, and protein secretion. Specifically, we found that PMI 5011 and DMC2 each reduced the ability of IL-1β to promote increases in the expression of the Ccl2 and Ccl20 genes. These genes encode proteins that promote immune cell recruitment and are secreted by β-cells in response to IL-1β. Phosphorylation of IκBα and the p65 subunit of NF-κB were not reduced by either PMI 5011 or DMC2; however, phosphorylation of p38 MAPK was blunted in the presence of DMC2. Finally, we observed that while PMI 5011 impaired glucose-stimulated insulin secretion, insulin output was preserved in the presence of DMC2. In conclusion, PMI 5011 and DMC2 reduced inflammation, but only DMC2 did so with the preservation of glucose-stimulated insulin secretion.

Nitrated fatty acid, 10-nitrooleate protects against hyperoxia-induced acute lung injury in mice

The antioxidant and anti-inflammatory effects of electrophilic nitrated fatty acid (NFA); 10-nitrooleate, have been reported. The present study investigated whether 10-nitrooleate has a protective role against hyperoxic-induced acute lung injury (HALI). Using a C57BL/6 mice model of HALI, we investigated the protective effect of 10-nitrooleate. C57BL/6 mice were administered with NFA intratracheally, exposed to hyperoxia for 48 h to induce HALI, and kept at room air for 24 h. Bronchoalveolar lavage (BAL) fluid and lung samples were collected after 24 h of post hyperoxia to analyze markers associated with HALI. Intratracheal (IT) and intraperitoneal (IP) administration of NFA notably attenuated hyperoxia-induced infiltration of inflammatory cells, alveolar-capillary leakage, upregulation of proinflammatory cytokine levels (IL-6 and TNFα) into the BAL fluid, and resolution of inflammation in the lung. Western blot analyses showed that 10-nitrooleate reduced the expression of the inflammatory transcription factor NFκB p65 subunit and increased antioxidant proteins HO-1 and NQO1 expression in the lung tissues compared to vehicle-treated animals. Moreover, 10-nitrooleate reversed the hyperoxia-induced expression of mitophagy-associated markers (PINK1 and p62/SQSTM1), thereby protecting the HALI/ acute respiratory distress syndrome (ARDS). IT and IP delivery of 10-nitrooleate reduces hyperoxia-induced ALI/ARDS by regulating the antioxidant pathways and restoring the mitochondrial homeostasis by regulating mitophagy. It is suggested that NFAs can be further evaluated as supplementary therapy for critically ill patients like COVID-19/ARDS.

Protective Roles of Sodium Butyrate in Lipopolysaccharide-Induced Bovine Ruminal Epithelial Cells by Activating G Protein-Coupled Receptors 41

This study aimed to evaluate whether sodium butyrate (SB) attenuates the ruminal response to LPS-stimulated inflammation by activating GPR41 in bovine rumen epithelial cells (BRECs). We examined the SB regulation of GPR41 and its impact on LPS-induced inflammation using GPR41 knockdown BRECs. The LPS-induced BRECs showed increases in the expression of genes related to pro-inflammation and decreases in the expression of genes related to tight junction proteins; these were attenuated by pretreatment with SB. Compared with that in LPS-stimulated BRECs, the ratio of phosphorylated NF-κB (p65 subunit) to NF-κB (p65 subunit) and the ratio of phosphorylated IκBα to IκBα were suppressed with SB pretreatment. The LSB group abated LPS-induced apoptosis and decreased the expression of Bax, Caspase 3, and Caspase 9 mRNA relative to the LPS group. In addition, the LSB group had a lower proportion of cells in the G0–G1 phase and a higher proportion of cells in the S phase than the LPS group. The mRNA expression of ACAT1 and BDH1 genes related to volatile fatty acid (VFA) metabolism were upregulated in the LSB group compared to those in LPS-induced BRECs. In addition, pretreatment with SB promoted the gene expression of GPR41 in the LPS-induced BRECs. Interestingly, SB pretreatment protected BRECs but not GPR41KD BRECs. Our results suggest that SB pretreatment protects against the changes in BRECs LPS-induced inflammatory response by activating GPR41.

Shenfu injection attenuates cardiac dysfunction and inhibits apoptosis in septic mice.

BackgroundCardiac dysfunction frequently occurs after sepsis and septic shock and contributes to multiple organ failure. Shenfu injection, a well-known Chinese medicine formulation, has recently been demonstrated to possess potential cardio-protective effects, although its effect and mechanism in sepsis-induced myocardial dysfunction remains unclear.MethodsTo investigate whether Shenfu injection could alleviate myocardial injury and improve cardiac function, a model of sepsis was induced by cecal ligation and puncture (CLP) surgery in C57BL/6 mice. Cardiac function, inflammatory factors, and apoptosis were evaluated in the myocardium.ResultsOur results showed that the survival and left ventricular function markedly declined when exposed to CLP compared with the sham procedure. In contrast, Shenfu injection lowered mortality and prevented CLP from triggering left ventricular dysfunction. Moreover, mice treated with Shenfu injection exhibited noticeably decreased myocardial inflammatory cytokines [tumor necrosis factor-α (TNF-α), interleukin (IL)-1β, IL-6, and monocytechemoattractantprotein-1 (MCP-1)] and reduced apoptotic myocardiocytes death compared with mice that had CLP. The activation of myocardial NF-кB detected in septic mice was suppressed by the presence of Shenfu injection, as evidenced by decreased nuclear translocation of the NF-кB p65 subunit. Moreover, Shenfu injection also prevented sepsis-caused decreases in myocardial phospho-Akt and phospho-GSK-3β in septic mice.ConclusionsThese data suggest that administration of Shenfu injection attenuates cardiac dysfunction, protects against inflammatory injury, and reduces apoptosis of myocardiocytes during sepsis by activating the AKT/GSK-3β pathway.

Single‐Cell RNA‐Seq Profiling Identifies L1 Cell Adhesion Molecule (L1cam) as a Mediator in Colonic Tuft Cells ‐ Innate Lymphoid Cell (ILCs) Signaling in the Hnrnp I Knockout Mice

ObjectivesPreviously we found that ablation of heterogeneous nuclear ribonucleoprotein I, (Hnrnp I) in intestinal epithelial cells (IECs) in colon significantly upregulates inflammatory cytokines and chemokines expression and increases number of tuft cells and innate lymphoid cells (ILCs, including ILC1, ILC2 and ILC3) in colon. The objective of this current study is to investigate the cell‐cell communications and to identify the mediators involved in the colon of Hnrnp I knockout (KO) mice. We hypothesize that signal transduction between colonic tuft cells and ILCs stimulates cytokine expression in the knockout mice.MethodsSingle‐cell RNAseq libraries (n = 3) were prepared from total RNA extracted from colon using Chromium Single‐Cell Gene Expression NextGem (v3.1, 10X Genomics). Analyses from raw counts were performed using the Seurat (v3.2.0) R package using default parameters. A computational cell calling algorithm was used to identify cell types using two up‐to‐date annotated mouse cell datasets. Analyses of cell‐cell communications and identification of significant pathways were performed using CellChat (v1.1.3). Immunofluorescence staining of colon tissue was performed with anti‐DCAMKL1 antibody (Abcam, ab31704). Colon cytokine levels were examined using cytokine 32‐plex array (Eve Technologies) using total protein extracted from colon tissues.ResultsImmunofluorescence staining of DCAMKL1, a tuft cell marker, confirms that the number of Tuft cells in colon (normalized to number of crypt) is significantly increased in Hnrnp I KO mice comparing to Hnrnp I wildtype (WT). Results from CellChat suggest communications between tuft cells and immune cells (ILCs, cytotoxic T cells, helper T cells, active lymphocytes and myeloid progenitors) are mainly through L1 Cell Adhesion Molecule (L1cam) – Integrins interaction, which is KO‐specific because the ablation of Hnrnp I results in the expression of L1cam in tuft cells in KO mice. Expression of integrin αv and β3 subunits (Itgav and Itgb3) are also higher in ILCs in the KO mice. NF‐κB subunits P65, and NF‐κB pathway target genes P21 and c‐Myc, also show higher expression levels in ILCs in KO mice comparing to WT. Cytokines assay results show that IL4, IL6 and IL13 all significantly increased, while IFNγ unchanged, and IL17 decreased in colon in KO mice comparing to WT.ConclusionsOur results demonstrate an Hnrnp I KO‐specific communication in colon between tuft cells and ILCs via L1cam‐integrin αv and β3 subunits interaction. The interaction further activates NF‐κB signaling in ILCs, regulating cell cycle of ILCs. The activation of ILCs further triggers immune responses in KO mice as indicated by increased proinflammatory cytokine expressions. Our results also suggest that among ILC1, ILC2 and ILC3, ILC2 is the major cell type involved in this communication because those increased cytokines – IL4, IL6 and IL13 observed in KO mice are all effectors secreted by mature ILC2.