Expression Of Protein Kinase Research Articles

PKA and NBC expression patterns in cryopreserved bovine spermatozoa after kaempferol treatment

Kaempferol (KAE) is a member of the flavonoid family, which exhibits antioxidant properties. The richest sources of KAE are green leafy vegetables and herbs like kale, spinach or dill. The goal of our study was to evaluate a possible cryoprotective effects of KAE through to gene expression of protein kinase A (PKA) and sodium bicarbonate cotransporter (NBC). For the experiments we used semen samples obtained from 12 adult Holstein bulls. Before cryopreservation, all samples were divided equally and supplemented by different concentrations of KAE (0, 12.5, 25 and 50 µmol/L), except for native control. After that, RNA was isolated, and the purity and quality of RNA was verified nanophotometrically at A260/A280 nm. Next step was a transcription of RNA into cDNA, which was used for two-step qPCR while expression of PKA and NBC was quantified under specific cycling conditions. Data was evaluated by the delta delta Ct method and One-way ANOVA using Tukey’s range test. According to expression patterns, it was observed that 25 µmol/L of KAE significantly preserved (P<0.0001) the PKA compared to cryopreserved control without KAE treatment. In the case of NBC, there was slightly increase of expression in the group treated with 50 µmol/L of KAE but without significant changes. In conclusion, KAE treatment exhibit promising cryoprotective properties of PKA and NBC genes especially in higher concentration 25 and 50 µmol/L, which may successfully preserved proper expression after thawing of cryopreserved bovine spermatozoa.

PDE4B abrogation extenuates angiotensin II-induced endothelial dysfunction related to hypertension through up-regulation of AMPK/Sirt1/Nrf2/ARE signaling.

Endothelial dysfunction is commonly perceived as a precursor in the process of hypertension, a severe cardiovascular disorder. Phosphodiesterase 4B (PDE4B) inactivation has been proposed to exert cardioprotective effects and prevent pulmonary hypertension. However, the role of PDE4B in endothelial dysfunction in hypertension remains inexplicit, which will be investigated in the present work. In angiotensin II (Ang II)-induced human umbilical vein endothelial cells (HUVECs), RT-qPCR and Western blotting were used to analyze PDE4B expression. CCK-8 method was used to detect cell viability. Flow cytometry assay and Caspase 3 assay kit were used to detect cellular apoptotic level. Wound healing and tube formation assays were respectively used to detect cell migration and angiogenesis. Western blotting and corresponding assay kits were respectively used to analyze the expressions and contents of endothelial dysfunction markers. JC-1 assay, RT-qPCR and relevant assay kit were respectively used to detect mitochondrial membrane potential (ΔΨm), quantify mitochondrial DNA (mtDNA) copy number and mitochondrial permeability transition pore (mPTP) opening. Besides, Western blotting was used to analyze the expressions of endoplasmic reticulum stress (ERS) and AMP-activated protein kinase (AMPK)/sirtuin 1 (Sirt1)/nuclear factor-erythroid 2 related factor 2 (Nrf2)/antioxidant response element (ARE) signaling-associated proteins. PDE4B expression was increased in Ang II- induced HUVECs. PDE4B knockdown promoted the viability, migration, angiogenesis while inhibiting the apoptosis, endothelial dysfunction, ERS and mitochondrial damage in Ang II-induced HUVECs. Additionally, PDE4B silence activated AMPK/Sirt1/Nrf2/ARE pathway and AMPK inhibitor Compound C (CC) partially reversed the effects of PDE4B down-regulation on Ang II-induced HUVECs. Conclusively, PDE4B inhibition might protect against Ang II-induced endothelial dysfunction in HUVECs via up-regulating AMPK/Sirt1/Nrf2/ARE pathway, which might be mediated by the suppression of ERS and mitochondrial damage.

Arginine–NO pathway modulated energy metabolism in the hepatopancreas and muscle of female Litopenaeus vannamei

Currently, the regulational effect of arginine on the energy metabolism is unknown in broodstock shrimp nutrition. Accordingly, an eight-week feeding trial was conducted to confirm it in female Litopenaeus vannamei. Six diets were formulated to contain graded levels of arginine (2.90 %, 3.58 %, 4.08 %, 4.53 %, 5.04 % and 5.55 %). A total of 540 shrimp (with an initial weight of approximately 14 g) were allocated at random to six treatments, each of which consisted of three tanks with 30 shrimp each. The results showed that with the rise in the level of dietary arginine supplementation, growth performance showed an increasing trend, reaching the highest value in the 5.55 % arginine group. The gene expression of cellular energy sensor AMP-activated protein kinase was up-regulated in muscle and hepatopancreas. Dietary arginine supplementation decreased hemolymph glucose content and hepatopancreas crude lipid content, increased muscle crude lipid content, glycogen content in muscle and hepatopancreas. Dietary arginine increased muscle crude protein content, decreased hepatopancreas crude protein content and activated the target of rapamycin 1 pathway. Above results were further confirmed by gene expression. In addition, shrimp were injected with dsGFP and dsNOS for 48 h and successfully constructed the RNA interference model. The findings showed that the levels of nos, cgmp, ampk (a, b, c), genes involved in glucose metabolism, lipid metabolism and amino acid metabolism were inhibited. In the conclusion, arginine supplementation could promote growth performance through modulating energy metabolism. Arginine–NO pathway modulated energy metabolism in hepatopancreas and muscle to promote tissue glycogen synthesis, muscle protein and lipid deposition in female L. vannamei.

Acupoint injection inhibiting abnormal secretion of mucin and inflammatory reaction of nasal mucosa in rats with allergic rhinitis through the p38 mitogen-activated protein kinase pathway

To observe the effects of acupoint injection on the expression of p38 mitogen-activated protein kinase (MAPK), phosphorylated (p)-p38 MAPK, mucin 5 subtype AC (MUC5AC) in the nasal mucosa, and serum inflammatory factors of rats with allergic rhinitis, so as to explore the mechanism of acupoint injection in improving inflammatory reactions in the nasal mucosa of rats with allergic rhinitis. SD rats were randomly divided into the normal group, model group, acupoint injection group, and non-acupoint group, with 7 rats in each group. The allergic rhinitis rat model was established using ovalbumin sensitization. Intervention was performed by injecting dexamethasone and 1% lidocaine mixture (0.05 mL) into the bilateral "Yingxiang"(LI20) and "Yintang"(GV24+) acupoints, with injection at non-acupoints for non-acupoint group as the control, once every 3 days for a total of 4 times. Allergic symptoms in the rat nose were evaluated using a cumulative scoring method；histopathological changes in the nasal mucosa were observed after HE staining；serum histamine (HA), interleukin (IL)-6, IL-8, and tumor necrosis factor-alpha (TNF-α) contents were detected using ELISA；real-time fluorescent quantitative PCR was used to detect the expression of MUC5AC mRNA in the nasal mucosa；Western blot was used to detect the expression of p38 MAPK, p-p38 MAPK, and MUC5AC protein in the nasal mucosa. Compared with the normal group, nasal allergic symptom score, serum HA, IL-6, IL-8, and TNF-α contents, nasal mucosal p38 MAPK, p-p38 MAPK proteins expression, MUC5AC mRNA and protein expression were significantly increased(P<0.01) in the model group. Compared with the model group and non-acupoint group, nasal allergic symptom score, serum HA, IL-6, IL-8, and TNF-α contents, nasal mucosal p38 MAPK and p-p38 MAPK proteins expression, MUC5AC mRNA and protein expression were significantly reduced (P<0.01, P<0.05) in the acupoint injection group. Acupoint injection can reduce nasal allergic symptom, and MUC5AC secretion in rats with allergic rhinitis, alleviate nasal mucosal inflammatory reactions, and its mechanism of action may be related to the inhibition of p38 MAPK phosphorylation, thereby reducing the release of inflammatory factors.

Cocaine-Induced DNA-Dependent Protein Kinase Relieves RNAP II Pausing by Promoting TRIM28 Phosphorylation and RNAP II Hyperphosphorylation to Enhance HIV Transcription

Drug abuse continues to pose a significant challenge in HIV control efforts. In our investigation, we discovered that cocaine not only upregulates the expression of the DNA-dependent protein kinase (DNA-PK) but also augments DNA-PK activation by enhancing its phosphorylation at S2056. Moreover, DNA-PK phosphorylation triggers the higher localization of the DNA-PK into the nucleus. The finding that cocaine increases the nuclear localization of the DNA-PK provides further support to our observation of enhanced DNA-PK recruitment at the HIV long terminal repeat (LTR) following cocaine exposure. By activating and facilitating the nuclear localization of the DNA-PK, cocaine effectively orchestrates multiple stages of HIV transcription, thereby promoting HIV replication. Additionally, our study demonstrates that the cocaine-induced DNA-PK promotes the hyper-phosphorylation of the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) at Ser5 and Ser2 sites, enhancing both the initiation and elongation phases, respectively, of HIV transcription. The cocaine-mediated enhancement of transcriptional initiation is supported by its activation of cyclin-dependent kinase 7 (CDK7). Additionally, the induction of transcriptional elongation is marked by higher LTR recruitment and the increased phosphorylation of CDK9, which indicates the stimulation of positive transcriptional elongation factor b (P-TEFb). We demonstrate for the first time that cocaine, through DNA-PK activation, promotes the specific phosphorylation of TRIM28 at serine 824 (p-TRIM28, S824). This modification converts TRIM28 from a transcriptional inhibitor to a transactivator for HIV transcription. Additionally, we observed that the phosphorylation of TRIM28 (p-TRIM28, S824) promotes the transition from the pausing phase to the elongation phase of HIV transcription, thereby facilitating the production of full-length HIV genomic transcripts. This finding corroborates the previously observed enhanced RNAP II CTD phosphorylation at Ser2, a marker of transcriptional elongation, following cocaine exposure. Accordingly, upon cocaine treatment, we observed the elevated recruitment of p-TRIM28-(S824) at the HIV LTR. Overall, our results unravel the intricate molecular mechanisms underlying cocaine-induced HIV transcription and gene expression. These findings hold promise for the development of highly targeted therapeutics aimed at mitigating the detrimental effects of cocaine in individuals living with HIV.

Activated charcoal supplementation in cottonseed meal-based feed improved growth performance and antioxidant capacity through enhancing intestinal barrier function in grass carp juveniles (Ctenopharyngodon idellus)

To evaluate the efficiency of activated charcoal (AC) in cottonseed meal-based feed (CSM; 38.46 % CSM, 1275 mg/kg free gossypol), experimental diets supplemented with 0 % AC, 1.5 % AC and 3.0 % AC were prepared and used to feed grass carp juveniles (Ctenopharyngodon idellus, initial weight of 5.0±0.5 g) for 8 weeks. The results showed that the 1.5 % AC group significantly increased final body weight (FBW), weight growth rate (WGR), specific growth rate (SGR), and activated the expressions of intestinal barrier-associated genes zonula occludens-1 (zo-1), zo-2 and claudin-c compared with 0 % AC group (P < 0.05). Additionally, the 1.5 % AC and the 3.0 % AC groups significantly promoted the expression of claudin-12 (P < 0.05). Compared with the 0 % AC group, the 1.5 % AC group had significantly higher total protein (TP) concentration and significantly lower glucose (GLU) concentration and aspartate aminotransferase (AST) activity (P < 0.05). Liver antioxidant results showed that supplementation with 1.5 % AC and 3.0 % AC groups significantly reduced malondialdehyde (MDA) content and increased glutathione reductase (GR) activity (P < 0.05). Total-superoxide dismutase (T-SOD) and glutathione-S-transferase (GST) activities were significantly higher in the 1.5 % AC group than those in the other groups (P < 0.05). The antioxidant gene results showed that gr and GST omega 1 (gsto1) levels were significantly higher in the 3.0 % AC group than in the 0 % AC group (P < 0.05). The 1.5 % AC group significantly promoted nuclear factor erythroid 2-related factor 2 (nrf2) expression, and the 1.5 % AC and 3.0 % AC groups significantly inhibited the expressions of kelch-like ECH-associated protein 1 (keap1a) and keap1b (P < 0.05). Liver apoptotic and inflammatory genes showed that the expressions of caspase 9 (casp9), BCL2 associated X a (baxa), nuclear factor-kappa B (nfκb) and inhibitor of kappa B (iκb) were significantly lower in the 1.5 % AC group than those in the other groups (P < 0.05). The expression of casp8 in the 1.5 % AC group was significantly lower than in the 0 % AC group (P < 0.05). Additionally, the expressions of p38 mitogen-activated protein kinase (p38MAPK) and iκb kinase-beta (ikkβ) were significantly suppressed in the 1.5 % AC group and 3.0 % AC group (P < 0.05). In summary, 1.5 % AC supplementation in CSM-based feed can enhance intestinal barrier function, alleviate CSM-based feed-induced apoptosis, improve liver antioxidant, and thus promote grass carp weight growth rate.

Endoplasmic Reticular Stress and Pathogenesis of Experimental Colitis: Mechanism of Action of 5-Amino Salicylic Acid.

Inflammatory bowel diseases which are characterized by endoplasmic reticulum (ER) stress and activation of the unfolded protein response (UPR) signaling pathway, are commonly treated with 5-amino salicylic acid (5-ASA). The objective of this study was to investigate the role of 5-amino salicylic acid in the UPR-signaling pathway in experimental colitis. Colitis was induced in male Sprague-Dawley rats by intrarectal instillation of trinitrobenzene sulfonic acid. Animals received 5-amino salicylic acid (100 mg/kg body weight) two hours before the induction of colitis and repeated daily until day 7. The animals were sacrificed on day 7 and tissues were collected for analysis. The expression of protein kinase R (PKR)-like endoplasmic reticulum (ER) kinase (PERK), a mediator of UPR-signaling increased significantly (p < 0.05), while inositol requiring enzyme type-1 (IRE1) and the CCAAT/enhancer-binding homologous protein (CHOP) remained unaltered in the inflamed colon. The expression of glucose regulated protein-78, activator of transcription factor-4 and phosphorylated eukaryotic initiation factor-2α (eIF2αP) increased (p < 0.05) in the inflamed colon. However, the levels of eIF2α protein and mRNA expression remained unchanged. Myeloperoxidase activity, colon weight and infiltration of inflammatory cells increased significantly (p < 0.05) in the submucosa whereas the body weight decreased. These changes were significantly inhibited by 5-amino salicylate treatment. These findings suggest that the anti-inflammatory properties of 5-amino salicylic acid are mediated through the inhibition of the PERK signaling pathway.

Pim1 inactivating induces RUNX3 upregulation that improves/alleviates airway inflammation and mucus hypersecretion in vitro and in vivo

PurposeOur research aimed to evaluate whether proto-oncogene serine/threonine-protein kinase Pim-1 (Pim1) inactivation could attenuate asthma by promoting runt-related transcription factor 3 (Runx3) expression and explore the underlying molecular mechanism.MethodPhorbol 12-myristate...

PKMζ, a Brain-specific PKCζ Isoform, is Required for Glycolysis and Myofibroblastic Activation of Hepatic Stellate Cells

BACKGROUND & AIMSTGFβ1 induces plasma membrane (PM) accumulation of glucose transporter 1 (Glut1) required for glycolysis of hepatic stellate cells (HSCs) and HSC activation. This study aimed to understand how Glut1 is anchored/docked onto the PM of HSCs. METHODSHSC expression of protein kinase M zeta isoform (PKMζ) was detected by RT-PCR, Western blotting, and immunofluorescence. PKMζ level was manipulated by shRNA or overexpression; HSC activation was assessed by cell expression of activation markers; PM Glut1, glucose uptake, and glycolysis of HSCs were analyzed by biotinylation, 2-NBDG-based assay, and Seahorse Glycolysis Stress Test. Phospho-mutants of vasodilator-stimulated phosphorylated protein (VASP) were created by site-directed mutagenesis. TGFβ transcriptome was obtained by RNA sequencing. Single-cell RNA sequencing datasets and immunofluorescence were leveraged to analyze PKMζ expression in cancer-associated fibroblasts (CAFs) of colorectal liver metastases. Function of HSC PKMζ was determined by tumor/HSC co-implantation study. RESULTSPrimary human and murine HSCs express PKMζ, but not full-length PKCζ. PKMζ knockdown suppresses whereas PKMζ overexpression potentiates PM accumulation of Glut1, glycolysis, and HSC activation induced by TGFβ1. Mechanistically, PKMζ binds to and induces VASP phosphorylation at serines 157 and 239 facilitating anchoring/docking of Glut1 onto the PM of HSCs. PKMζ expression is increased in the CAFs of murine and patient colorectal liver metastases compared to quiescent HSC. Targeting PKMζ suppresses transcriptome, CAF activation of HSCs, and colorectal tumor growth in mice. CONCLUSIONSSince HSCs are also a major contributor of liver fibrosis, our data highlight PKMζ and VASP as targets to inhibit metabolic reprogramming, HSC activation, liver fibrosis, and the pro-metastatic microenvironment of the liver.

Protein Kinase C Isozyme Immaturity/Deficiency in Cord Blood Monocytes and Neutrophils.

Reduced/deficient expression of Protein Kinase C (PKC)ζ in Cord blood (CB) T cells is associated with allergy development in children and a propensity to maintain an immature T-helper (Th)2 cytokine profile. In addition, other PKC isozymes are also low in CBTCs. Since previous studies have reported that cord blood/neonatal monocyte and neutrophil functions are significantly lower than cells from adults, it was of interest to see if the CBTC PKC levels were reflected in CB monocytes and neutrophils. Compared to adult blood, CB expresses low levels of PKCα, β2, ε, θ, μ, ζ and λ/ι in monocytes and PKCα, β2, η, θ, μ, ζ and λ/ι in neutrophils. The T-cell PKCζ levels were positively correlated with levels in CB monocytes but not in neutrophils. However, neither the monocytes nor the neutrophil PKCζ were associated with T-cell development towards a Th1 or Th2 cytokine propensity, based on the production of interferon-gamma and interleukin-4 in response to phytohemagglutinin and phorbol myristate acetate. The results demonstrate that some newborn babies display a deficiency in PKC isozymes in monocytes and neutrophils, as reported for T cells. However, unlike T cells, the PKCζ levels of the phagocytes did not correlate with regulation of development towards a Th1 or Th2 cytokine phenotype.

Blumea balsamifera and Sargassum aquifolium extracts reduce fatty liver damage through lipid metabolism signalling pathways

Non-alcoholic fatty liver disease (NAFLD) is a condition marked by excessive fat accumulation in the liver and poses a significant health challenge. The leaves of Blumea balsamifera and Sargassum aquifolium have been reported to have anti-atherogenic effects. This study aims to determine the effectiveness of B. balsamifera extract (BBLE) and S. aquifolium extract (SAE) in preventing and treating liver fat accumulation in Wistar rats induced by a high-cholesterol diet through the expression of the AMP-activated protein kinase (AMPK)/ Sirtuin 1 (SIRT1)/peroxisome proliferator-activated receptor γ (PPARγ) pathway, and the leptin receptor. The experimental design of this study is laboratory-based, involving, 20 Wistar rats were fed a high-cholesterol diet over a period of 21 days. The rats were divided into four groups for the evaluation of BBLE and SAE effect: negative control (P0): induced with a high-cholesterol diet + distilled water, positive control (P1): induced with a high-cholesterol diet + simvastatin, P2: induced with a high-cholesterol diet + 4 mg/kg/bw BBLE, and P3: induced with a high-cholesterol diet + 4 mg/kg/bw BBLE and 4 mg/kg/bw SAE. The treatment duration extended over three months. Immunohistochemical analyses were performed on liver tissues to measure AMPK, SIRT1, PPARγ, and leptin receptor expression. The results indicated that leptin expression was lower in the BBLE+SAE group compared to the simvastatin group, and differences were significant between the BBLE and BBLE+SAE groups. No significant differences were noted in AMPK, SIRT1, and PPARγ expression between the simvastatin and BBLE+SAE groups (p≥0.05). In conclusion, BBLE and SAE effectively reduce liver lipid accumulation and enhance fat metabolism in hypercholesterolemic rats.

Combined Treatment of Caffeic Acid Phenethyl Ester With Docetaxel Inhibits Survival of Non-small-cell Lung Cancer Cells via Suppression of c-MYC.

Non-small-cell lung cancer (NSCLC) comprises approximately 85% of lung cancer. Treatment with docetaxel prolongs the survival of patients with NSCLC. However, the development of resistance to docetaxel has compromised its efficacy. Caffeic acid phenethyl ester (CAPE) has been reported to suppress survival and radiotherapy resistance in lung cancer cells. We determined in this study if combination treatment of docetaxel with CAPE suppresses the proliferation and the survival of NSCLC cells more effectively. Proliferation, viability, flow cytometric and comet assays were used to examine the difference in anticancer effects of combined treatment as compared to docetaxel treatment alone. Western blot and gene overexpression were used to unravel the underlying molecular mechanism. Treatment with docetaxel or CAPE alone dose-dependently suppressed the proliferation and survival of H1299 and A549 cells. Combined treatment of docetaxel with CAPE caused greater inhibition of survival of H1299 and A549 cells. Docetaxel alone and the combined treatment both dose-dependently increased apoptosis of H1299 cells; however, combined treatment induced much more apoptosis than docetaxel alone. Combined treatment suppressed the protein expression of phospho-protein kinase B (AKT, Ser 473), S-phase protein 2 (SKP2), MYC proto-oncogene bHLH transcription factor (c-MYC), epidermal growth factor receptor (EGFR), phospho-EGFR (Tyr 1045, and Tyr 992) but increased levels of cleaved caspase 3 and cytochrome c proteins in H1299 and A549 cells. The inhibition of expression of SKP2, c-MYC, phospho-EGFR (Tyr 992) proteins by combined treatment was significantly greater than that with treatment using either CAPE or docetaxel alone. Overexpression of c-MYC in rescued proliferation of H1299 cells under combination treatment. Our study revealed that the combination of CAPE with docetaxel is more effective at reducing the proliferation and survival of NSCLC cells, and this is via inhibition of c-MYC. Combined therapy of docetaxel and CAPE may benefit patients with NSCLC.

CSWT improves ventricular function by promoting angiogenesis through Rho/ROCK/ERK1/2 signal pathway in infarcted myocardium in rats

Abstract Objective This study aimed to investigate the effects of extracorporeal cardiac shock wave therapy (CSWT) on myocardial ischemia and cardiac function in rats with myocardial infarction, and to explore its impact on the expression of myocardial vascular endothelial growth factor and the extent of myocardial fibrosis, thereby elucidating the mechanism of angiogenesis. Methods A rat model of myocardial infarction was induced by ligating the left anterior descending branch of the coronary artery, confirmed by echocardiography. Animals were randomly assigned to five groups: sham operation group, myocardial infarction group, myocardial infarction + CSWT group, myocardial infarction + Y-27632 group, and myocardial infarction + CSWT + Y-27632 group. Cardiac function was evaluated by echocardiography on the 16th and 72nd day post-surgery. Myocardial morphology was assessed by HE staining, and the extent of myocardial fibrosis was determined by Masson's trichrome staining. Immunohistochemical staining for factor VIII was conducted to observe angiogenesis in each infarcted area. Western blotting was employed to detect the protein expression of extracellular regulated kinase (ERK1/2) and pro-angiogenic cytokines in the infarct border zone. Results CSWT treatment significantly reduced myocardial fibrosis and improved cardiac function compared to the myocardial infarction group. Additionally, CSWT upregulated ERK expression and angiogenesis-related factors, promoting angiogenesis. The beneficial effects of CSWT were abolished by inhibiting the Rho/ROCK/ERK1/2 pathway. Furthermore, the area of myocardial fibrosis was significantly reduced in the MI+Y-27632 group compared to the myocardial infarction group (p &amp;lt; 0.01), and left ventricular systolic function index was significantly higher in the myocardial infarction group. Conclusions CSWT may promote angiogenesis post-myocardial infarction by activating the Rho/ROCK/ERK1/2 pathway. Additionally, Y-27632 may improve cardiac function in rats with myocardial infarction, potentially through mitigating myocardial fibrosis.echo images of ratsprotein expression by WB

Sinensetin inhibits the movement ability and tumor immune microenvironment of non-small cell lung cancer through the inactivation of AKT/β-catenin axis.

Although current treatment strategies have improved clinical outcomes of non-small cell lung cancer (NSCLC) patients, side effect and prognosis remain a hindrance. Thus, safer and more effective therapeutical drugs are needed for NSCLC. Sinensetin (Sin) is a flavonoid from citrus fruits, which exhibits antitumor effect on diverse cancers. However, the effect and mechanism of Sin on NSCLC remain unknown. In this study, NSCLC cell lines, and tumor-bearing mice were treated with Sin. The effect and mechanism of Sin were addressed using cell counting kit-8, transwell, enzyme-linked immunosorbent assay, hematoxylin and eosin, immunohistochemistry, and western blot analysis assays in both cell and animal models. Sin reduced the cell viability of A549 and H1299, with the IC50 of 81.46 µM and 93.15 µM, respectively. Sin decreased invaded cell numbers, the expression of N-cadherin and vascular endothelial growth factor A (VEGFA), while increased the E-cadherin level, the cytotoxicity of CD8+ T cells, and the concentration of interferon-γ (IFN-γ), interleukin-2 (IL-2), and tumor necrosis factor-α (TNF-α) in NSCLC cells. Mechanistically, Sin declined the expression of protein kinase B (AKT)/β-catenin pathway, which was restored with the application of SC79, an activator of AKT. The inhibitory role of Sin in NSCLC cell proliferation, invasion, epithelial-mesenchymal transition (EMT) and immune escape was reversed by the management of SC79. In vivo, Sin reduced tumor size and weight, and the expression of N-cadherin, VEGFA, and AKT/β-catenin pathway, but enhanced the level of E-cadherin and IFN-γ. Taken together, Sin suppressed cell growth, invasion, EMT and immune escape via AKT/β-catenin pathway in NSCLC.

The Effects of Warm Acupuncture on the Expression of AMPK in High-Fat Diet-Induced MAFLD Rats.

This study investigated the effects of acupuncture and warm acupuncture on the expression and mechanism of the AMP-activated protein kinase (AMPK) signalling pathway associated with lipid accumulation in the liver tissue of rats with metabolic dysfunction-associated fatty liver disease (MAFLD) induced by a high-fat diet. Sprague-Dawley rats were categorised into four groups: control (CON), untreated MAFLD (MAFLD), and two MAFLD groups treated with acupuncture (ACU) and warm acupuncture (WA). The treatment groups underwent 16 application sessions over 8 weeks at the SP9 and BL18 acupoints. We measured the expression levels of AMPK, sterol regulatory element-binding protein1 (SREBP1), acetyl-coenzyme A carboxylase (ACC), peroxisome proliferator-activated receptorα (PPARα), carnitine palmitoyltransferase1 (CPT1), and CPT2. AMPK was activated in both ACU and WA groups. WA downregulated both SREBP1 and ACC expression at the protein level, whereas the acupuncture treatment downregulated SREBP1 expression. Additionally, WA selectively induced the activation of signalling pathways related to AMPK, PPARα, CPT1, and CPT2 at the mRNA level. Histological observations confirmed that fat accumulation was reduced in both the ACU and the WA groups compared to the MAFLD group. The WA treatment-promoted amelioration of HFD-induced MAFLD may be related to the activation of the AMPK/SREBP1/ACC pathway in the liver.

Spinal lumbar Urocortin 3-expressing neurons are associated with both scratching and Compound 48/80-induced sensations.

Urocortin 3 is a neuropeptide that belongs to the corticotropin-releasing hormone family and is involved in mechanosensation and stress regulation. In this study, we show that Urocortin 3 marks a population of excitatory neurons in the mouse spinal cord, divided into 2 nonoverlapping subpopulations expressing protein kinase C gamma or calretinin/calbindin 2, populations previously associated with mechanosensation. Electrophysiological experiments demonstrated that lumbar spinal Urocortin 3 neurons receive both glycinergic and GABAergic local tonic inhibition, and monosynaptic inputs from both Aβ and C fibers, which could be confirmed by retrograde trans-synaptic rabies tracing. Furthermore, fos analyses showed that subpopulations of lumbar Urocortin 3 neurons are activated by artificial scratching or Compound 48/80-induced sensations. Chemogenetic activation of lumbar Urocortin 3-Cre neurons evoked a targeted biting/licking behavior towards the corresponding dermatome and chemogenetic inhibition decreased Compound 48/80-induced behavior. Hence, spinal lumbar Urocortin 3 neurons represent a mechanically associated population with roles in both scratching and Compound 48/80-induced sensations.

Dietary niacin supplementation improves meat quality, muscle fiber type, and mitochondrial function in heat-stressed Taihe black-bone silky fowls.

A recent study has shown that niacin supplementation induces the conversion of type II to type I muscle fibres, thereby promoting a phenotypic shift in oxidative metabolism in porcine skeletal muscle. These effects may be mediated by modulation of the AMPK1/SIRT1 pathway, which activates peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α), a key regulator of fibre conversion, thereby promoting skeletal muscle mitochondrial biogenesis and myofibre conversion. In this study, we explored how niacin (NA) supplementation impacts the quality of meat and the characteristics of muscle fibers in Taihe Black-bone Silky Fowls (TBsf) exposed to heat conditions. Chickens were rationally assigned to five different treatment groups with five replicates of six chickens each: thermophilic (TN), heat stress (HS) and HS + NA (HN) groups, with the HN group being supplemented with 200, 400 and 800 mg/kg (HS + NA0.02, HS + NA0.04 and HS + NA0.08) NA in the premix, respectively. The results of the experiment showed that addition of 800 mg/kg NA to the diet significantly improved TBsf muscle tenderness compared to HS. Dietary enrichment with 200-800 mg/kg NA significantly increased total antioxidant capacity, superoxide dismutase, and glutathione peroxidase activities, while significantly decreasing malondialdehyde compared to HS. Incorporation of 200-800 mg/kg NA into the diet significantly reduced lactate dehydrogenase activity and myosin heavy chain (MyHC-IIB) gene expression. Furthermore, adding 800 mg/kg NA can significantly enhance the mRNA expression of mitochondrial transcription factors (TFAM and TFB1M) in TBsf skeletal muscle. Adding 400 and 800 mg/kg of NA significantly increased the mRNA expression of AMP-activated protein kinase 1 (AMPK1), PGC-1α, cytochrome c oxidase (Cytc), and nuclear respiratory factor (NRF-1) in the skeletal muscle of TBsf. Supplementing NA at 200-400 mg/kg significantly increased the expression of Sirtuin 1 (SIRT1) mRNA in TBsf skeletal muscle. The experimental results showed that the addition of NA to the diet reduced the shear force of TBsf muscle under heat exposure conditions. It increased the proportion of type I muscle fibres by increasing the antioxidant capacity of the muscle and by promoting mitochondr fibreial biogenesis. Considering the results of this study, it is recommended that TBsf be supplemented with 400-800 mg/kg of NA in the diet to reduce the adverse effects of heat stress on meat quality.

(E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone, a Major Homoisoflavonoid, Attenuates Free Fatty Acid-Induced Hepatic Steatosis by Activating AMPK and PPARα Pathways in HepG2 Cells.

(E)-5-hydroxy-7-methoxy-3-(2-hydroxybenzyl)-4-chromanone (HMC), a homoisoflavonoid isolated from Portulaca oleracea, has significant anti-adipogenesis potential; it regulates adipogenic transcription factors. However, whether HMC improves hepatic steatosis in hepatocytes remains vague. This study investigated whether HMC ameliorates hepatic steatosis in free fatty acid-treated human hepatocellular carcinoma (HepG2) cells, and if so, its mechanism of action was analyzed. Hepatic steatosis was induced by a free fatty acid mixture in HepG2 cells. Thereafter, different HMC concentrations (10, 30, and 50 µM) or fenofibrate (10 µM, a PPARα agonist, positive control) was treated in HepG2 cells. HMC markedly decreased lipid accumulation and triglyceride content in free fatty acid-treated HepG2 cell; it (10 and 50 μM) markedly upregulated protein expressions of pAMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase. HMC (10 and 50 μM) markedly inhibited the expression of sterol regulatory element-binding protein-1c, fatty acid synthase, and stearoyl-coA desaturase 1, which are the enzymes involved in lipid synthesis. Furthermore, HMC (10 and 50 μM) markedly upregulated the protein expression of peroxisome proliferator-activated receptor alpha (PPARα) and enhanced the protein expressions of carnitine palmitoyl transferase 1 and acyl-CoA oxidase 1. HMC inhibits lipid accumulation and promotes fatty acid oxidation by AMPK and PPARα pathways in free fatty acid-treated HepG2 cells, thereby attenuating hepatic steatosis.

Hesperidin facilitating gastrointestinal motility by “Gut-brain axis” and “SCF/C-Kit signaling pathways”

Hesperidin shows promising results as a potential feed additive for enhancing gastrointestinal motility in animals. Gastrointestinal function plays a pivotal role in animal growth and the digestibility of dietary nutrients, with gastrointestinal motor function serving as a crucial component. However, limited research has been conducted on the application of hesperidin as a feed additive to promote gastrointestinal motility. The present study aims to assess the efficacy of Hesperidin as a feed additive in promoting gastrointestinal motility and elucidating its underlying mechanism. A total of 200 newly hatched (1-day-old) broilers with similar body weight were randomly allocated into 4 groups as follows: the control group receiving only the basal diet, and the other 3 groups supplemented with 50, 100, and 150 mg of hesperidin per kg of the basal diet, respectively. Each group consisted of 5 replicates with ten broilers per replicate, and the feeding trial lasted for a duration of 21 d. At 21 d of age, a 5% w/v Evans Blue solution in distilled water was utilized to measure intestinal transit rates (ITR). Gastric emptying (GE) was evaluated by administering a phenol red solution at a concentration of 0.05% w/v (1 mL/broiler). Fifteen broilers from each group were euthanized and immediately dissected to obtain gizzard, hypothalamus, duodenum, and jugular blood samples. Jugular blood samples were collected for brain-gut peptide content analysis, while gizzard, hypothalamus, and duodenum samples were used for immunohistochemical analysis. Real-time qPCR was performed on gizzard samples. The results demonstrated a significant improvement in the GE and ITR of broilers in all treatment groups compared to the control group (P < 0.05), particularly in the 100mg/Kg and 150mg/Kg hesperidin group. Incorporation of hesperidin into the broilers' diet significantly enhances serum levels of ghrelin, encompassing serotonin (5-HT), motilin (MTL), cholecystokinin (CCK), and Stem Cell Factor (SCF) as well as substance P (SP) in the gizzard and duodenal tissues while reducing vasoactive intestinal peptide (VIP) levels (P < 0.05). The group administered a dosage of 150mg/Kg exhibited the most pronounced effect.Immunohistochemistry analysis revealed that hesperidin supplementation up-regulated SP protein content and down-regulated VIP protein content in the hypothalamus, gizzard, and duodenum of broilers (P < 0.05), with the most pronounced effect illustrated in the 150mg/Kg hesperidin group. Furthermore, addition of hesperidin to broiler feed resulted in a significant up-regulation of protein expression and gene expression related to SCF and The protein expression of Receptor tyrosine kinase (C-Kit) was significantly upregulated in the 150mg/Kg group, while the gene expression of C-Kit was significantly upregulated in the 50 mg/Kg group (P < 0.05). In conclusion, hesperidin exhibits promising potential as a feed additive for broilers, as its dietary supplementation of hesperidin improves gastrointestinal motility through modulation of both "gut-brain axis" signaling pathways and "SCF/C-Kit signaling pathways" within broiler chicken's digestive system. Notably, basal diet supplemented with 150mg/Kg hesperidin demonstrates superior efficacy.

Qiwei Jinggan Ling regulates oxidative stress and lipid metabolism in alcoholic liver disease by activating AMPK

BackgroundAlcoholic liver disease (ALD) is a severe public health concern worldwide and there is still a lack of effective treatments. Qiwei Jinggan Ling (QJL) has protective effects against various liver injuries, but its pharmacological action on ALD has received little attention. PurposeTo investigate the effect and mechanism of QJL on ALD in vivo and in vitro. MethodsIn vivo, an ALD mouse model was established by alcohol combined with a high-fat diet (HFD) and treated with QJL. Biochemical indicators, HE staining, and Oil Red O staining were employed to assess hepatic oxidative stress, steatosis, and alcohol metabolism. RNA sequencing analysis was performed, and the results were verified by qRT-PCR and Western blot to elucidate the hepatoprotective mechanism of QJL. In vitro, HepG2 cells were co-stimulated with NaOA (sodium oleate) and EtOH (ethanol), followed by intervention with Compound C (CC, AMPK inhibitor) and QJL-containing serum. Oil Red O, BODIPY (boron-dipyrromethene), and ROS (reactive oxygen species) staining were applied to validate the efficacy and mechanism of QJL-containing serum. The expression of AMP-activated protein kinase (AMPK) pathway-related factors was analyzed through qRT-PCR and Western blot for additional corroboration. Moreover, the key pharmacodynamic components of QJL were identified by UPLC-MS/MS and molecular docking. ResultsIn vivo, QJL ameliorated liver structural disorders, steatosis, oxidative stress, and impaired alcohol metabolism, as indicated by biochemical indicators and histopathological assays. RNA sequencing analysis revealed that QJL reversed the expression of genes related to alcohol metabolism, fatty acid metabolism, and cholesterol metabolism. The results of qRT-PCR and Western blot were in line with those of RNA sequencing. Furthermore, it was discovered that QJL significantly upregulated the expression of p-AMPK and downregulated the expression of sterol regulatory element binding transcription factor 1 (SREBP-1c). In vitro, biochemical indicators and staining assays demonstrated that QJL-containing serum inhibited lipid accumulation and oxidative stress. The qRT-PCR and Western blot analysis revealed that QJL-containing serum markedly enhanced the expression of p-AMPK and carnitine palmitoyltransferase 1a (Cpt1a), while suppressing the expression of SREBP-1c, fatty acid synthase (Fasn), and acetyl-coenzyme A carboxylase 1 (ACC-1). However, CC inhibited the above pharmacological activities of QJL-containing serum. Additionally, (2S)-Liquiritigenin, Glycyrrhetinate, Isovitexin, Taxifolin, and Yohimbine were proved to be the key active components of QJL. ConclusionQJL had the potential to be a therapeutic drug for ALD by activating the AMPK pathway, thereby regulating lipid metabolism and inhibiting oxidative stress.