Protein Kinase Activation Research Articles

Promoting AMPK/SR-A1-mediated clearance of HMGB1 attenuates chemotherapy-induced peripheral neuropathy

BackgroundChemotherapy-induced peripheral neuropathy (CIPN) is a serious side effect of chemotherapy with poorly understood mechanisms and few treatments. High-mobility group box 1 (HMGB1)-induced neuroinflammation is the main cause of CIPN. Here, we aimed to illustrate the role of the macrophage scavenger receptor A1 (SR-A1) in HMGB1 clearance and CIPN resolution.MethodsOxaliplatin (L-OHP) was used to establish a CIPN model. Recombinant HMGB1 (rHMGB1) (his tag) was used to evaluate the phagocytosis of HMGB1 by macrophages.ResultsIn the clinic, HMGB1 expression and MMP-9 activity were increased in the plasma of patients with CIPN. Plasma HMGB1 expression was positively correlated with the cumulative dose of L-OHP and the visual analog scale. In vitro, engulfment and degradation of rHMGB1 increased and inflammatory factor expression decreased after AMP-activated protein kinase (AMPK) activation. Neutralizing antibodies, inhibitors, or knockout of SR-A1 abolished the effects of AMPK activation on rHMGB1 engulfment. In vivo, AMPK activation increased SR-A1 expression in the dorsal root ganglion, decreased plasma HMGB1 expression and MMP-9 activity, and attenuated CIPN, which was abolished by AMPK inhibition or SR-A1 knockout in the CIPN mice model.ConclusionActivation of the AMPK/SR-A1 axis alleviated CIPN by increasing macrophage-mediated HMGB1 engulfment and degradation. Therefore, promoting HMGB1 clearance may be a potential treatment strategy for CIPN.1JEzHJvY7WaTcecv7_-Z6vVideo abstract

Concomitant western diet and chronic-binge alcohol dysregulate hepatic metabolism.

There is significant overlap between non-alcoholic fatty liver disease (NAFLD) and alcohol-associated liver disease (ALD) with regards to risk factors and disease progression. However, the mechanism by which fatty liver disease arises from concomitant obesity and overconsumption of alcohol (syndrome of metabolic and alcohol-associated fatty liver disease; SMAFLD), is not fully understood. Male C57BL6/J mice were fed chow diet (Chow) or high-fructose, high-fat, high-cholesterol diet (FFC) for 4 weeks, then administered either saline or ethanol (EtOH, 5% in drinking water) for another 12 weeks. The EtOH treatment also consisted of a weekly 2.5 g EtOH/kg body weight gavage. Markers for lipid regulation, oxidative stress, inflammation, and fibrosis were measured by RT-qPCR, RNA-seq, Western blot, and metabolomics. Combined FFC-EtOH induced more body weight gain, glucose intolerance, steatosis, and hepatomegaly compared to Chow, EtOH, or FFC. Glucose intolerance by FFC-EtOH was associated with decreased hepatic protein kinase B (AKT) protein expression and increased gluconeogenic gene expression. FFC-EtOH increased hepatic triglyceride and ceramide levels, plasma leptin levels, hepatic Perilipin 2 protein expression, and decreased lipolytic gene expression. FFC and FFC-EtOH also increased AMP-activated protein kinase (AMPK) activation. Finally, FFC-EtOH enriched the hepatic transcriptome for genes involved in immune response and lipid metabolism. In our model of early SMAFLD, we observed that the combination of an obesogenic diet and alcohol caused more weight gain, promoted glucose intolerance, and contributed to steatosis by dysregulating leptin/AMPK signaling. Our model demonstrates that the combination of an obesogenic diet with a chronic-binge pattern alcohol intake is worse than either insult alone.

Investigating potential modifier genes of chronic PKA activation in heart failure

The cAMP-dependent protein kinase (PKA) is a major downstream effector of β-adrenergic signaling responsible for the positive inotropic effect of catecholamines. The PKA holoenzyme is a heterotetramer composed of two catalytic (C) and two regulatory (R) subunits. Three C subunits (Cα, Cβ and Cγ) are coded by three genes and four R subunits (RIα, RIβ, RIIα and RIIβ) are encoded by four genes but the heart expresses predominantly Cα, RIα and RIIα. We have shown that conditional knock-out of RIα in cardiomyocytes from adult mice (RIα-icKO) have constitutive PKA activation and spontaneously develop heart failure with reduced ejection fraction (HFrEF) upon aging, resulting in 50% mortality after 1 year (Bedioune et al. submitted). In this work, we aimed to better characterize the HF phenotype of RIα-icKO mice. We performed echocardiography, RT-qPCR, western blot and bulk RNA sequencing in RIα-icKO mice and control littermates at 1 year of age. We show that the HF phenotype of the surviving RIα-icKO mice at 1 year is heterogeneous: among 51 mice analyzed by echocardiography and gravimetry, 23 (45%) showed clear signs of HF (EF < 50%, cardiac hypertrophy and dilatation, lung congestion, named HFKO), whereas 18 (35%) had none (named NFKO) and 10 had an intermediary phenotype (20%). RIα residual expression and PKA activity appeared similar in HFKO (n = 6–9) and NFKO (n = 6–10) Transcriptomic analysis revealed dysregulation of typical HF marker genes in NFKO that was generally exacerbated in HFKO. Gene ontology analysis also showed TOP 10 up-regulated and down-regulated pathways classically associated with HF that were similar in HFKO and NFKO. A total of 542 genes were differentially expressed (fc 1.5, P < 0.05) between HFKO and NFKO. Cross-referencing with a list of 821 genes containing genes associated with HF from NCBI and components of β-AR signaling yielded 35 differentially expressed genes, among which 9 were selected as potential modifiers of the phenotype based on the literature. Our results suggest that NFKO represent a compensated stage of HF development. Comparison with HFKO identified new genes that could modulate the impact of chronic PKA activation on HF development. Further studies are required to identify other potential candidates and validate them in neonatal cardiomyocytes with chronic PKA activation.

Creatine kinase B suppresses ferroptosis by phosphorylating GPX4 through a moonlighting function.

Activation of receptor protein kinases is prevalent in various cancers with unknown impact on ferroptosis. Here we demonstrated that AKT activated by insulin-like growth factor 1 receptor signalling phosphorylates creatine kinase B (CKB) T133, reduces metabolic activity of CKB and increases CKB binding to glutathione peroxidase 4 (GPX4). Importantly, CKB acts as a protein kinase and phosphorylates GPX4 S104. This phosphorylation prevents HSC70 binding to GPX4, thereby abrogating the GPX4 degradation regulated by chaperone-mediated autophagy, alleviating ferroptosis and promoting tumour growth in mice. In addition, the levels of GPX4 are positively correlated with the phosphorylation levels of CKB T133 and GPX4 S104 in human hepatocellular carcinoma specimens and associated with poor prognosis of patients with hepatocellular carcinoma. These findings reveal a critical mechanism by which tumour cells counteract ferroptosis by non-metabolic function of CKB-enhanced GPX4 stability and underscore the potential to target the protein kinase activity of CKB for cancer treatment.

Cell-free hemoglobin-mediated oxidation of low-density lipoprotein (oxLDL) contributes to lung microvascular endothelial dysfunction during sepsis

Introduction: Disruption of the microvascular endothelial barrier is a critical pathological feature of sepsis-induced acute lung injury. Plasma cell-free hemoglobin (CFH) is elevated in approximately 80% of patients with sepsis and is independently associated with development of acute respiratory distress syndrome (ARDS) and mortality. Oxidized CFH (ferric, 3+) can oxidize low-density lipoprotein (oxLDL), which signals through its major endothelial receptor, the lectin-like oxidized LDL receptor 1 (LOX-1), to cause endothelial dysfunction through several pro-inflammatory pathways including activation of mitogen-activated protein kinases (MAPKs). However, little is known regarding whether LOX-1 receptor signaling leads to microvascular endothelial hyperpermeability or acute lung injury, especially in the context of sepsis. We hypothesized that oxidation of LDL by CFH contributes to lung microvascular endothelial barrier dysfunction and worse outcomes during sepsis through LOX-1 and downstream p38 MAPK. Methods: To test whether generation of oxLDL by CFH is associated with endothelial injury in clinical sepsis, circulating levels of CFH, oxLDL, and vascular injury marker sVE-cadherin were measured in 24 sepsis patients via ELISA and tested for association with development of ARDS. LDL was oxidized by combining LDL with CFH in a test tube overnight at 37°C and oxLDL was quantified by TBARS assay. In primary human lung microvascular endothelial cells (HLMVEC) transendothelial electrical resistance (TER), a measure of barrier dysfunction, was assessed by Electric Cell-substrate Impedance Sensing (ECIS). LOX-1 receptor was blocked using BI-0115 (Boehringer Ingelheim, 20 μM) and p38 MAPK was inhibited using NiPp (100 μM). Results: In sepsis patients, plasma oxLDL levels correlated with CFH (r=0.686, p=0.016) and sVE-cadherin (r=0.603, p=0.012), and tended to be higher in those who developed ARDS (38 U/L [IQR 27, 45] vs. 27 U/L [IQR 19, 35], p=0.1). Oxidation of LDL by CFH exacerbated HLMVEC barrier dysfunction compared to control, LDL, or CFH. Barrier dysfunction induced by CFH or oxLDL was attenuated by blocking the LOX-1 receptor or p38 MAPK. Conclusions: Increased plasma CFH and oxLDL are associated with vascular injury during clinical sepsis; one mechanism by which CFH may cause vascular hyperpermeability and sepsis-mediated lung injury is through oxidation of LDL which can drive signaling through the endothelial LOX-1 receptor and activation of p38 MAPK. NIH R35HL150783, R21GM144915, R01HL158906, R01HL164937, T32HL094296; Parker B. Francis Fellowship This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Adaptive responses in uteroplacental metabolism and fetoplacental nutrient shuttling and sensing during placental insufficiency.

Glucose, lactate, and amino acids are major fetal nutrients. During placental insufficiency-induced intrauterine growth restriction (PI-IUGR), uteroplacental weight-specific oxygen consumption rates are maintained, yet fetal glucose and amino acid supply is decreased and fetal lactate concentrations are increased. We hypothesized that uteroplacental metabolism adapts to PI-IUGR by altering nutrient allocation to maintain oxidative metabolism. Here, we measured nutrient flux rates, with a focus on nutrients shuttled between the placenta and fetus (lactate-pyruvate, glutamine-glutamate, and glycine-serine) in a sheep model of PI-IUGR. PI-IUGR fetuses weighed 40% less and had decreased oxygen, glucose, and amino acid concentrations and increased lactate and pyruvate versus control (CON) fetuses. Uteroplacental weight-specific rates of oxygen, glucose, lactate, and pyruvate uptake were similar. In PI-IUGR, fetal glucose uptake was decreased and pyruvate output was increased. In PI-IUGR placental tissue, pyruvate dehydrogenase (PDH) phosphorylation was decreased and PDH activity was increased. Uteroplacental glutamine output to the fetus and expression of genes regulating glutamine-glutamate metabolism were lower in PI-IUGR. Fetal glycine uptake was lower in PI-IUGR, with no differences in uteroplacental glycine or serine flux. These results suggest increased placental utilization of pyruvate from the fetus, without higher maternal glucose utilization, and lower fetoplacental amino acid shuttling during PI-IUGR. Mechanistically, AMP-activated protein kinase (AMPK) activation was higher and associated with thiobarbituric acid-reactive substances (TBARS) content, a marker of oxidative stress, and PDH activity in the PI-IUGR placenta, supporting a potential link between oxidative stress, AMPK, and pyruvate utilization. These differences in fetoplacental nutrient sensing and shuttling may represent adaptive strategies enabling the placenta to maintain oxidative metabolism.NEW & NOTEWORTHY These results suggest increased placental utilization of pyruvate from the fetus, without higher maternal glucose uptake, and lower amino acid shuttling in the placental insufficiency-induced intrauterine growth restriction (PI-IUGR) placenta. AMPK activation was associated with oxidative stress and PDH activity, supporting a putative link between oxidative stress, AMPK, and pyruvate utilization. These differences in fetoplacental nutrient sensing and shuttling may represent adaptive strategies enabling the placenta to maintain oxidative metabolism at the expense of fetal growth.

Beneficial Effects of a Curcumin Derivative and Transforming Growth Factor-β Receptor I Inhibitor Combination on Nonalcoholic Steatohepatitis.

Curcumin 2005-8 (Cur5-8), a derivative of curcumin, improves fatty liver disease via AMP-activated protein kinase activation and autophagy regulation. EW-7197 (vactosertib) is a small molecule inhibitor of transforming growth factor β (TGF-β) receptor I and may scavenge reactive oxygen species and ameliorate fibrosis through the SMAD2/3 canonical pathway. This study aimed to determine whether co-administering these two drugs having different mechanisms is beneficial. Hepatocellular fibrosis was induced in mouse hepatocytes (alpha mouse liver 12 [AML12]) and human hepatic stellate cells (LX-2) using TGF-β (2 ng/mL). The cells were then treated with Cur5-8 (1 μM), EW-7197 (0.5 μM), or both. In animal experiments were also conducted during which, methionine-choline deficient diet, Cur5-8 (100 mg/kg), and EW-7197 (20 mg/kg) were administered orally to 8-week-old C57BL/6J mice for 6 weeks. TGF-β-induced cell morphological changes were improved by EW-7197, and lipid accumulation was restored on the administration of EW-7197 in combination with Cur5-8. In a nonalcoholic steatohepatitis (NASH)-induced mouse model, 6 weeks of EW-7197 and Cur5-8 co-administration alleviated liver fibrosis and improved the nonalcoholic fatty liver disease (NAFLD) activity score. Co-administering Cur5-8 and EW-7197 to NASH-induced mice and fibrotic hepatocytes reduced liver fibrosis and steatohepatitis while maintaining the advantages of both drugs. This is the first study to show the effect of the drug combination against NASH and NAFLD. Similar effects in other animal models will confirm its potential as a new therapeutic agent.

Using quantitative single molecule localization microscopy to optimize multivalent HER2-targeting ligands.

The progression-free survival of patients with HER2-positive metastatic breast cancer is significantly extended by a combination of two monoclonal antibodies, trastuzumab and pertuzumab, which target independent epitopes of the extracellular domain of HER2. The improved efficacy of the combination over individual antibody therapies targeting HER2 is still being investigated, and several molecular mechanisms may be in play: the combination downregulates HER2, improves antibody-dependent cell mediated cytotoxicity, and/or affects the organization of surface-expressed antigens, which may attenuate downstream signaling. By combining protein engineering and quantitative single molecule localization microscopy (qSMLM), here we both assessed and optimized clustering of HER2 in cultured breast cancer cells. We detected marked changes to the cellular membrane organization of HER2 when cells were treated with therapeutic antibodies. When we compared untreated samples to four treatment scenarios, we observed the following HER2 membrane features: (1) the monovalent Fab domain of trastuzumab did not significantly affect HER2 clustering; (2) individual therapy with either trastuzumab or (3) pertuzumab produced significantly higher levels of HER2 clustering; (4) a combination of trastuzumab plus pertuzumab produced the highest level of HER2 clustering. To further enhance this last effect, we created multivalent ligands using meditope technology. Treatment with a tetravalent meditope ligand combined with meditope-enabled trastuzumab resulted in pronounced HER2 clustering. Moreover, compared to pertuzumab plus trastuzumab, at early time points this meditope-based combination was more effective at inhibiting epidermal growth factor (EGF) dependent activation of several downstream protein kinases. Collectively, mAbs and multivalent ligands can efficiently alter the organization and activation of the HER2 receptors. We expect this approach could be used in the future to develop new therapeutics.

Cyanidin-3-glucoside inhibits ferroptosis in renal tubular cells after ischemia/reperfusion injury via the AMPK pathway

BackgroundFerroptosis, which is characterized by lipid peroxidation and iron accumulation, is closely associated with the pathogenesis of acute renal injury (AKI). Cyanidin-3-glucoside (C3G), a typical flavonoid that has anti-inflammatory and antioxidant effects on ischemia‒reperfusion (I/R) injury, can induce AMP-activated protein kinase (AMPK) activation. This study aimed to show that C3G exerts nephroprotective effects against I/R-AKI related ferroptosis by regulating the AMPK pathway.MethodsHypoxia/reoxygenation (H/R)-induced HK-2 cells and I/R-AKI mice were treated with C3G with or without inhibiting AMPK. The level of intracellular free iron, the expression of the ferroptosis-related proteins acyl-CoA synthetase long chain family member 4 (ACSL4) and glutathione peroxidase 4 (GPX4), and the levels of the lipid peroxidation markers 4-hydroxynonenal (4-HNE), lipid reactive oxygen species (ROS) and malondialdehyde (MDA) were examined.ResultsWe observed the inhibitory effect of C3G on ferroptosis in vitro and in vivo, which was characterized by the reversion of excessive intracellular free iron accumulation, a decrease in 4-HNE, lipid ROS, MDA levels and ACSL4 expression, and an increase in GPX4 expression and glutathione (GSH) levels. Notably, the inhibition of AMPK by CC significantly abrogated the nephroprotective effect of C3G on I/R-AKI models in vivo and in vitro.ConclusionOur results provide new insight into the nephroprotective effect of C3G on acute I/R-AKI by inhibiting ferroptosis by activating the AMPK pathway.

Neuroprotective Effects of a Novel Tetrapeptide SGGY from Walnut against H2O2-Stimulated Oxidative Stress in SH-SY5Y Cells: Possible Involved JNK, p38 and Nrf2 Signaling Pathways.

SGGY, an antioxidant tetrapeptide identified from walnut protein hydrolysate in our previous study, has been suggested to possess the potential to alleviate oxidative stress in cells. In this paper, the neuroprotective effects of SGGY on H2O2-stimulated oxidative stress in SH-SY5Y cells and the underlying mechanisms were investigated. Results showed that SGGY alleviated H2O2-induced oxidative stress by decreasing the intracellular reactive oxygen species (ROS) level and altering the mitochondrial membrane potential (MMP), thereby inhibiting apoptosis and increasing cell viability. SGGY significantly restored antioxidant enzyme activities and reduced malondialdehyde (MDA) content accordingly. Moreover, SGGY promoted the nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) and suppressed the H2O2-induced activation of JNK and p38 mitogen-activated protein kinases (MAPKs). Taken together, these results suggested that SGGY protected SH-SY5Y cells from H2O2-provoked oxidative stress by enhancing the ability of cellular antioxidant defense, and the possible mechanism involved MAPKs and Nrf2 signaling pathways.

Fucoxanthin Alleviates Lipopolysaccharide-Induced Inflammation and Immunosuppression in RAW264.7 Macrophages.

Fucoxanthin (FX) has been reported to reduce mortality in mouse models of sepsis, but its exact cause remains to be determined. In the present study, we evaluated the immunomodulatory properties of FX in lipopolysaccharide (LPS)-stimulated RAW 264.7 cells. Our results showed that FX could not only suppress the immune activation responses caused by LPS primary stimulation, but also antagonize LPS restimulation-induced immunosuppression in macrophages. The immunomodulatory capabilities of FX was mainly demonstrated by regulating the production of the inflammatory mediator under different LPS stimuli. Furthermore, we found that adenosine monophosphate-activated protein kinase (AMPK) activation was required for FX's anti-inflammatory and anti-immunosuppressive activities. Our results complement existing data supporting the clinical potential for FX in treating sepsis.

PD03-02 NOVEL STRUCTURAL VARIANTS IN CTNNB1 AND MOLECULAR CLASSIFICATION IN CORTISOL-PRODUCING ADENOMA

You have accessJournal of UrologyCME1 Apr 2023PD03-02 NOVEL STRUCTURAL VARIANTS IN CTNNB1 AND MOLECULAR CLASSIFICATION IN CORTISOL-PRODUCING ADENOMA Yoichi Fujii, Seiichiro Higuchi, Yusuke Sato, Yuichi Shiraishi, Kenichi Yoshida, Satoru Miyano, Haruki Kume, Tomohiko Ichikawa, Atsushi Iwama, Tomoaki Tanaka, and Seishi Ogawa Yoichi FujiiYoichi Fujii More articles by this author , Seiichiro HiguchiSeiichiro Higuchi More articles by this author , Yusuke SatoYusuke Sato More articles by this author , Yuichi ShiraishiYuichi Shiraishi More articles by this author , Kenichi YoshidaKenichi Yoshida More articles by this author , Satoru MiyanoSatoru Miyano More articles by this author , Haruki KumeHaruki Kume More articles by this author , Tomohiko IchikawaTomohiko Ichikawa More articles by this author , Atsushi IwamaAtsushi Iwama More articles by this author , Tomoaki TanakaTomoaki Tanaka More articles by this author , and Seishi OgawaSeishi Ogawa More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000003220.02AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Cortisol-producing adenoma (CPA) is the most common hormone-producing adrenal tumor. Although several hotspot mutations of driver genes (PRKACA, GNAS, and CTNNB1) have been identified in the previous studies, genotypes have not been confirmed in approximately 30% of CPAs. In addition, the association between genotypes and clinical characteristics of CPAs are poorly understood. METHODS: Genomic DNA and RNA was extracted from 125 surgical specimens of adrenal CPAs, including 60 cases with Cushing’s syndrome (CS) and 65 with subclinical Cushing’s syndrome (SCS), and subjected to targeted-capture sequencing and RNA sequencing. RESULTS: Novel structural variants (SVs) in CTNNB1, including long deletion and inversion, were identified in 8% (10/125) of CPA cases. All these SVs resulted in the lack of exon 3, which encodes the phosphorylation site of beta-catenin and plays an important role in its degradation, leading to the constitutive activation of the Wnt pathway by disrupting the degradation of beta-catenin. In addition, several mutations in PRKACA, which encodes the catalytic subunit of cyclic adenosine monophosphate (cAMP)–dependent protein kinase (PKA), were identified around the known hotspot p.L206. Subsequent functional assay showed that these PRAKACA mutants abolished its binding ability to PRKAR1A, the regulatory subunit of PKA, leading to the constitutive, cAMP-independent PKA activation. In total, 82.4% (103/125) of CPAs had alterations in PRKACA, GNAS, and wnt pathway genes (CTNNB1, APC, and MED12) in almost mutually exclusive manner, based on which CPAs are classified into three distinct subgroups. These mutational subtypes showed distinct genetic and clinical characteristics. RNA sequencing revealed that the expression level of steroidogenic enzymes such as CYP17A1 and CYP21A2 were more upregulated in PRKACA-mutated subtype than wnt-mutated subtype. Consistent with this observation, PRKACA-mutated subtype showed significantly higher cortisol-producing autonomous secretion level, more frequent CS than wnt-mutated subtype. In contrast, wnt-mutated subtype more tended to be SCS, showed lower cortisol-producing ability and largar tumor volume compared to the other two subtypes. CONCLUSIONS: Novel CTNNB1 structural variants were identified. CPAs showed distinct genetic landscape, associated with various clinical features. Source of Funding: No © 2023 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 209Issue Supplement 4April 2023Page: e74 Advertisement Copyright & Permissions© 2023 by American Urological Association Education and Research, Inc.MetricsAuthor Information Yoichi Fujii More articles by this author Seiichiro Higuchi More articles by this author Yusuke Sato More articles by this author Yuichi Shiraishi More articles by this author Kenichi Yoshida More articles by this author Satoru Miyano More articles by this author Haruki Kume More articles by this author Tomohiko Ichikawa More articles by this author Atsushi Iwama More articles by this author Tomoaki Tanaka More articles by this author Seishi Ogawa More articles by this author Expand All Advertisement PDF downloadLoading ...

3,4,5‑Trihydroxycinnamic acid suppresses phorbol‑12‑myristate‑13‑acetate and A23187‑induced mast cell activation in RBL‑2H3 cells.

Previously, anti-inflammatory properties of 3,4,5-Trihydroxycinnamic acid (THC) has been reported in lipopolysaccharide (LPS)-induced RAW264.7 murine macrophage cells and in an LPS-induced sepsis BALB/c mice animal model. However, the effect of THC on the anti-allergic effect in mast cells has not been elucidated. The current study aimed to demonstrate the anti-allergic properties of THC and its underlying mechanism. Rat basophilic leukemia (RBL-2H3) cells were treated with phorbol-12-myristate-13-acetate (PMA) and A23187, a calcium ionophore, to be activated. The anti-allergic effect of THC was determined by measuring cytokine and histamine release. Western blotting was conducted to determine mitogen-activated protein kinases (MAPKs) activation and nuclear factor-κB (NF-κB) translocation. THC significantly suppressed PMA/A23187-induced tumor necrosis factor α secretion and THC also significantly attenuated degranulation, releasing β-hexosaminidase and histamine in concentration-dependent manners. Furthermore, THC significantly attenuated PMA/A23187-induced cyclooxygenase 2 expression and nuclear translocation of NF-κB. THC significantly suppressed PMA/A23187-induced increased phosphorylation of p38 mitogen-activated protein kinase, phosphorylated (p-)extracellular signal-regulated kinase 1/2 and p-c-Jun N-terminal kinase in RBL-2H3 cells. Overall, the results demonstrated that THC exhibited anti-allergic action by significantly attenuating degranulation of mast cells through the inhibition of MAPKs/NF-κB signaling pathway in RBL-2H3 cells.

Compound 13 Promotes Epidermal Healing in Mouse Fetuses via Activation of AMPK

Unlike adults, early developing fetuses can completely regenerate tissue, and replicating this could lead to the development of treatments to reduce scarring. Mice epidermal structures, including wound healing patterns, are regenerated until embryonic day (E) 13, leaving visible scars thereafter. These patterns require actin cable formation at the epithelial wound margin through AMP-activated protein kinase (AMPK) activation. We aimed to investigate whether the administration of compound 13 (C13), a recently discovered AMPK activator, to the wound could reproduce this actin remodeling and skin regeneration pattern through its AMPK activating effect. The C13 administration resulted in partial formations of actin cables, which would normally result in scarring, and scar reduction during the healing of full-layer skin defects that occurred in E14 and E15 fetuses. Furthermore, C13 was found to cause AMPK activation in these embryonic mouse epidermal cells. Along with AMPK activation, Rac1 signaling, which is involved in leaflet pseudopodia formation and cell migration, was suppressed in C13-treated wounds, indicating that C13 inhibits epidermal cell migration. This suggests that actin may be mobilized by C13 for cable formation. Administration of C13 to wounds may achieve wound healing similar to regenerative wound healing patterns and may be a potential candidate for new treatments to heal scars.

Effect of Ketogenic Diet on the Progression of 7, 12-Dimethylbenz(Α) Anthracene –Induced Mammary Cancer in Female Wistar Rats

In the present research, the level of cancer antigen 125 (CA125) significantly increased in DMBA experimental groups (group II) compared to normal control group. However the level of CA125 decreased in dose dependent manner with 10% carbohydrate ketogenic diet having the most significant effect. Also, the 7,12-Dimethylbenz(α) anthracene (DMBA) caused significant increase in liver enzymes (Alanine amino transferase, Aspartate amino transferase and Bilirubin) of 41±1.06, 40±1.16, 20.80 ±1.47 and 25±1.34, 27±143, 12.17±1.08 compared to normal control at P&lt;0.05 respectively. Furthermore, with the group treatment 10%, 20% and 30% carbohydrate ketogenic diets also elevated the liver enzymes. More so, a significant decrease was observed in total protein, albumin, RBC, Hb, PCV, MCH superoxide dismutase, glutathione reductase and glutathione peroxidase with a corresponding increase in white blood cells. Interestingly, ketogenic diets significantly increase total protein, albumin, superoxide dismutase, glutathione reductase, and glutathione peroxidase with a decrease in white blood cells, total cholesterol, triglyceride, low density lipoprotein bilirubin concentration. It can therefore be concluded that, the administration of ketogenic diets slows the progression of breast cancer through inhibiting the downstream of insulin receptors of both mammalian target of rapamycin signal cascade and motogen activator protein kinase pathways.

PROPHYLACTIC n CMT-3 ATTENUATES SEPSIS-INDUCED ACUTE KIDNEY INJURY IN ASSOCIATION WITH NLRP3 INFLAMMASOME ACTIVATION AND APOPTOSIS.

Background: The kidney is the most common extrapulmonary organ injured in sepsis. The current study examines the ability of aerosolized nanochemically modified tetracycline 3 (nCMT-3), a pleiotropic anti-inflammatory agent, to attenuate acute kidney injury (AKI) caused by intratracheal LPS. Methods: C57BL/6 mice received aerosolized intratracheal nCMT-3 (1 mg/kg) or saline, followed by intratracheal LPS (2.5 mg/kg) to induce acute lung injury-induced AKI. Tissues were harvested at 24 h. The effects of nCMT-3 and LPS on AKI were assessed by plasma/tissue levels of serum urea nitrogen, creatinine, neutrophil gelatinase-associated lipocalin, kidney injury molecule 1, and renal histology. Renal matrix metalloproteinase (MMP) level/activity, cytochrome C, Bax, Bcl-2, caspase-3, p38 mitogen-activated protein kinase activation, NLRP3, and caspase-1 were also measured. Apoptotic cells in kidney were determined by TUNEL assay. Renal levels of IL-1β and IL-6 were measured to assess inflammation. Results: Acute lung injury-induced AKI was characterized by increased plasma blood urea nitrogen, creatinine, injury biomarkers (neutrophil gelatinase-associated lipocalin, kidney injury molecule 1), and histologic evidence of renal injury. Lipopolysaccharide-treated mice demonstrated renal injury with increased levels of inflammatory cytokines (IL-1β, IL-6), active MMP-2 and MMP-9, proapoptotic proteins (cytochrome C, Bax/Bcl-2 ratio, cleaved caspase-3), apoptotic cells, inflammasome activation (NLRP3, caspase-1), and p38 signaling. Intratracheal nCMT-3 significantly attenuated all the measured markers of renal injury, inflammation, and apoptosis. Conclusions: Pretreatment with aerosolized nCMT-3 attenuates LPS-induced AKI by inhibiting renal NLRP3 inflammasome activation, renal inflammation, and apoptosis.

Radiofrequency Irradiation Attenuated UVB-Induced Skin Pigmentation by Modulating ATP Release and CD39 Expression.

Hyperpigmentation stimulated by ultraviolet (UV)-induced melanin overproduction causes various cosmetic problems. UV radiation's activation of the cyclic adenosine monophosphate (cAMP)-mediated cAMP-dependent protein kinase (PKA)/cAMP response element-binding protein (CREB)/microphthalmia-associated transcription factor (MITF) pathway is the main pathway for melanogenesis. However, the secretion of adenosine triphosphate (ATP) from keratinocytes due to UV radiation also leads to melanogenesis. Adenosine, converted from ATP by CD39 and CD73, can activate adenylate cyclase (AC) activity and increase intracellular cAMP expression. cAMP-mediated PKA activation results in dynamic mitochondrial changes that affect melanogenesis via ERK. We evaluated whether radiofrequency (RF) irradiation could decrease ATP release from keratinocytes and suppress the expression of CD39, CD73, and A2A/A2B adenosine receptors (ARs) and the activity of AC and downregulate the PKA/CREB/MITF pathway, which would eventually decrease melanogenesis in vitro in UV-irradiated cells and animal skin. Our results indicate that RF decreased ATP release from UVB-irradiated keratinocytes. When conditioned media (CM) from UVB-irradiated keratinocytes (CM-UVB) were administered to melanocytes, the expressions of CD39, CD73, A2A/A2BARs, cAMP, and PKA increased. However, the expression of these factors decreased when CM from UVB and RF-irradiated keratinocytes (CM-UVB/RF) was administered to melanocytes. The phosphorylation of DRP1 at Ser637, which inhibits mitochondrial fission, increased in UVB-irradiated animal skin and was decreased by RF irradiation. The expression of ERK1/2, which can degrade MITF, was increased using RF treatment in UVB-irradiated animal skin. Tyrosinase activity and melanin levels in melanocytes increased following CM-UVB administration, and these increases were reversed after CD39 silencing. Tyrosinase activity and melanin levels in melanocytes were decreased by CM-UVB/RF irradiation. In conclusion, RF irradiation decreased ATP release from keratinocytes and the expressions of CD39, CD73, and A2A/A2BARs, which decreased AC activity in melanocytes. RF irradiation downregulated the cAMP-mediated PKA/CREB/MITF pathway and tyrosinase activity, and these inhibitory effects can be mediated via CD39 inhibition.

Metformin May Alter the Metabolic Reprogramming in Cancer Cells by Disrupting the L-Arginine Metabolism: A Preliminary Computational Study.

Metabolic reprogramming in cancer is considered to be one of the most important hallmarks to drive proliferation, angiogenesis, and invasion. AMP-activated protein kinase activation is one of the established mechanisms for metformin's anti-cancer actions. However, it has been suggested that metformin may exert antitumoral effects by the modulation of other master regulators of cellular energy. Here, based on structural and physicochemical criteria, we tested the hypothesis that metformin may act as an antagonist of L-arginine metabolism and other related metabolic pathways. First, we created a database containing different L-arginine-related metabolites and biguanides. After that, comparisons of structural and physicochemical properties were performed employing different cheminformatic tools. Finally, we performed molecular docking simulations using AutoDock 4.2 to compare the affinities and binding modes of biguanides and L-arginine-related metabolites against their corresponding targets. Our results showed that biguanides, especially metformin and buformin, exhibited a moderate-to-high similarity to the metabolites belonging to the urea cycle, polyamine metabolism, and creatine biosynthesis. The predicted affinities and binding modes for biguanides displayed good concordance with those obtained for some L-arginine-related metabolites, including L-arginine and creatine. In conclusion, metabolic reprogramming in cancer cells by metformin and biguanides may be also driven by metabolic disruption of L-arginine and structurally related compounds.

AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation.

Bone metastasis of cancer cells leads to severe pain by disrupting bone structure and inducing central sensitization. Neuroinflammation in the spinal cord plays a decisive role in the maintenance and development of pain. In the current study, male Sprague-Dawley (SD) rats are used to establish a cancer-induced bone pain (CIBP) model by intratibial injection of MRMT-1 rat breast carcinoma cells. Morphological and behavioral analyses verify the establishment of the CIBP model, which represents bone destruction, spontaneous pain and mechanical hyperalgesia in CIBP rats. Activation of astrocytes marked by upregulated glial fibrillary acidic protein (GFAP) and enhanced production of the proinflammatory cytokine interleukin-1β (IL-1β) are accompanied by increased inflammatory infiltration in the spinal cord of CIBP rats. Furthermore, activation of the NOD-like receptor pyrin domain-containing protein 3 (NLRP3) inflammasome is consistent with increased neuroinflammation. Adenosine monophosphate-activated protein kinase (AMPK) activation is involved in attenuating inflammatory pain and neuropathic pain. Intrathecal injection of the AMPK activator AICAR in the lumbar spinal cord reduces dynamin-related protein 1 (Drp1) GTPase activity and suppresses NLRP3 inflammasome activation. This effect consequently alleviates pain behaviors in CIBP rats. Cell research on C6 rat glioma cells indicates that AICAR treatment restores IL-1β-induced impairment of mitochondrial membrane potential and elevation of mitochondrial reactive oxygen species (ROS). In summary, our findings indicate that AMPK activation attenuates cancer-induced bone pain by reducing mitochondrial dysfunction-mediated neuroinflammation in the spinal cord.

IRE1α arm of unfolded protein response in muscle-specific TGF-β signaling-mediated regulation of muscle cell immunological properties

Endoplasmic reticulum stress (ERS) and the unfolded protein response (UPR) are involved in various muscle pathological states. The IRE1α arm of UPR can affect immunological properties of myofiber through restraining p38 mitogen-activated protein kinases (MAPK) activation under inflammatory milieu. However, the relevant pathway molecules regulating the initiation of the IRE1α arm in myofiber remain unclear. In this work, expression of transforming growth factor-beta (TGF-β) and TGF-β receptor II (TGF-βr2), and UPR pathway activation were examined in cardiotoxin (CTX)-damaged mouse muscle, which revealed the activation of TGF-β signaling and UPR in CTX-damaged muscle and in regenerating myofibers. Using control or transgenic mice with TGF-βr2 deleted in skeletal muscle (SM TGF-βr2−/−) and the derived primary differentiating myogenic precursor cells (MPCs) treated with/without ERS activator or inhibitor, IRE1α pathway inhibitor, or TGF-β signaling activator, this study further revealed an essential role of intrinsic TGF-β signaling in regulating muscle cell to express inflammation-related molecules including H-2Kb, H2-Eα, TLR3, and special myokines. TGF-β signaling prompted UPR IRE1α arm and restrained p38 MAPK activation in myofiber under inflammatory milieu. This study uncovers a previously unrecognized function of TGF-β signaling acting as an upstream factor controlling myofiber immune capacities in the inflamed state through the UPR–IRE1α–p38 MAPK pathway.