Expression Of Protein Kinase Research Articles

LMAN2 interacts with HEATR3 to expedite HER2-positive breast cancer advancement and inflammation and Akt/ERK/NF-κB signaling.

The paper aimed to reveal the impacts and the possible mechanism of action of lectin mannose-binding 2 protein (LMAN2) in HER2-positive breast cancer (BC). The expression, prognostic potential of LMAN2, and the correlation between LMAN2 and HEAT repeat containing 3 (HEATR3) in BC were analyzed in TCGA database. Intact, Mentha, and BioGrid databases predicted LMAN2-HEATR3 interactions. Reverse transcription-quantitative PCR and Western blot examined LMAN2 expression. Cell Counting Kit-8, 5-ethynyl-2'-deoxyuridine staining, wound healing, and transwell assays, respectively, detected the aggressive cellular biological behaviors including proliferation, migration, and invasion. Western blot analyzed the expression of matrix metalloproteinases, HEATR3, and protein kinase B (Akt)/extracellular signal-regulated kinase (ERK)/nuclear factor-kappaB (NF-κB) signaling-related proteins. Co-immunoprecipitation assay was used to prove the relationship of LMAN2 with HEATR3. Enzyme-linked immunosorbent assay detected inflammatory cytokine levels. LMAN2 was overexpressed in HER2-positive BC tissues and cells and indicated unfavorable prognosis of BC patients. LMAN2 knockdown suppressed HER2-positive BC cell proliferation, migration, and invasion. LMAN2 interacted with and had a positive correlation with HEATR3. HEATR3 up-regulation reversed the repressive role of LMAN2 interference in the progression of HER2-positive BC, Akt/ERK/NF-κB signaling, and inflammatory response. Altogether, LMAN2 silencing might exert anti-tumor and anti-inflammatory properties and inactivate Akt/ERK/NF-κB signaling in HER2-positive BC via binding to HEATR3.

Threonine and tyrosine kinase (TTK) mRNA and protein expression in breast cancer; prognostic significance in the neoadjuvant setting.

Threonine and tyrosine kinase (TTK) is up-regulated in triple-negative breast cancer (TNBC), yet its expression in patients undergoing neoadjuvant chemotherapy (NACT) remains unexplored. This investigation aims to assess TTK protein expression in treatment-naïve pre-treatment cores and paired pre- and post-NACT breast cancer (BC) cohorts, as well as its correlation with microcephaly 1 (MCPH1) protein expression. Transcriptomic data were sourced from the Gene Expression Omnibus microarray database for mRNA expression analysis. TTK protein expression was evaluated using immunohistochemistry staining, employing receiver operating characteristic curve analysis to determine an optimal TTK expression cut-off point. The association between TTK expression, clinicopathological parameters and survival outcomes was examined. Additionally, MCPH1 protein expression was assessed in a pilot study. Analysis revealed a significantly elevated TTK mRNA expression in BC tissue compared to normal breast tissue, with high TTK mRNA levels predicting reduced overall survival. Notably, TTK protein expression increased significantly post-NACT in a paired cohort. Conversely, decreased TTK protein expression pre-NACT was correlated with improved overall survival. High TTK and low MCPH1 protein expression was significantly correlated, highlighting TTK's potential as a biomarker for BC and a therapeutic target for MCPH1-deficient cancer cells.

Monascus pilosus SWM-008 red mold rice and its components, monascinol and monascin, reduce obesity in a high-fat diet-induced rat model through synergistic modulation of gut microbiota and anti-lipogenesis.

This study is the first to explore the effects of the novel yellow pigment monascinol (Msol) from red mold rice (RMR) on reducing body fat and to compare its effects with those of monascin (MS) and ankaflavin (AK). In a high-fat diet-induced rat model, different doses of RMR fermented rice (RL, RM, RH) and purified Msol, MS, and AK were administered over an 8-week period. The results showed that all treatment groups significantly reduced body weight and fat mass. Msol, in particular, activated acetyl-CoA carboxylase (ACC), inhibiting fatty acid synthesis and reducing triglyceride accumulation. All treatments suppressed the differentiation of preadipocytes into mature adipocytes by inhibiting CCAAT/enhancer-binding proteins β (C/EBPβ) and C/EBPα, as well as peroxisome proliferator-activated receptor γ (PPARγ). In the liver, RL, RM, RH, MS, and AK enhanced the expression of AMP-activated protein kinase (AMPK), ACC, peroxisome proliferator-activated receptor α (PPARα), and carnitine palmitoyl transferase-1α (CPT-1α), thereby promoting fatty acid metabolism. Additionally, RMR and its active components, MS and Msol, reduced body fat by modulating gut microbiota. These compounds significantly decreased the abundance of bacteria associated with fat storage, such as Oliverpabstia intestinalis, while increasing the abundance of bacteria linked to energy expenditure and lipid breakdown, such as Akkermansia muciniphila and Ruminococcus callidus. Moreover, MS and Msol upregulated proteins involved in fat degradation, such as UCP1, thereby enhancing fat burning and reducing fat accumulation. These regulatory effects suggest that Monascus and its components have potential in managing metabolic health and reducing obesity.

Association of AMP-activated protein kinase and uncoupling protein-2 gene expressions with hypertension in obese patients

Association of AMP-activated protein kinase and uncoupling protein-2 gene expressions with hypertension in obese patients

"Shugan Tiaoshen" needling improves depression after ischemic stroke by regulating AMPK-dependent autophagy

To observe the effect of "Shugan Tiaoshen"(soothing the liver and regulating the mental activities) needling on the expressions of AMP-activated protein kinase (AMPK) and autophagy-related proteins in the prefrontal cortex of rats with depression after ischemic stroke (post-stroke depression), in order to explore its mechanisms underlying improvement of post-stroke depression behaviors. A total of 60 male SD rats were randomly divided into sham operation (control), stroke, post-stroke depression, inhibitor, acupuncture, and acupuncture + inhibitor groups. The post-stroke depression model was established by occlusion of the middle cerebral artery and reperfusion (MCAO/R), combined with chronic unpredictable mild stimulation (CUMS). Rats in the inhibitor group received bilateral lateral ventricular injection of AMPK inhibitor (0.1 μmol, 10 μL). Manual acupuncture stimulation was applied to "Baihui" (GV20), "Yintang" (GV24+), "Hegu" (LI4), and "Taichong" (LR3) for the acupuncture group, once daily for 14 d. Rats in the acupuncture + inhibitor group received acupuncture treatment after 3 d of AMPK inhibitor injection. Neurologic impairment was assessed by Longa score. Behavioral assessment of depression was conducted by sucrose preference test and open field test. H.E. staining was used to observe the morphological changes of prefrontal cortex. Western blot was used to detect the protein expression levels of AMPK, p-AMPK, Beclin1, P62 and LC3 in the prefrontal cortex tissue. Transmission electron microscopy (TEM) was used to observe the ultrastructural changes of prefrontal neurons. Compared with the control group, only the total locomotor distance, the percentage of central area dwell time within 5 min and the p-AMPK/AMPK were significantly decreased (P<0.01) in the stroke group while Longa score was increased (P<0.01). Compared with the control group, the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, levels of p-AMPK/AMPK, Beclin1 and LC3Ⅱ/Ⅰ were significantly decreased (P<0.01, P<0.05), while Longa score and P62 expression was significantly up-regulated (P<0.01) in the post-stroke depression group. Compared with the post-stroke depression group, the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, and the levels of p-AMPK/AMPK, Beclin1, and LC3Ⅱ/Ⅰ were considerably increased (P<0.01), while P62 expression was notably decreased (P<0.05) in the acupuncture group. Comparison between the inhibitor group and the acupuncture+inhibitor group showed that the sucrose preference, total locomotor distance and the percentage of central area dwell time within 5 min, and the expression level of p-AMPK/AMPK were even lower in the former group than those in the latter group (P<0.05, P<0.01), and the expression level of P62 was higher in the former group than in the latter group (P<0.01). H.E. staining showed loose arrangement of prefrontal cortex neurons, reduced number, pyknotic and hyperchromatic nuclei, even with degeneration and necrosis of some neurons in the stroke, post-stroke depression and the inhibitor groups, while increase in neuronal number, with relatively complete structure and uniform stain observed in the acupuncture group. Results of TEM showed disrupted neuronal nuclear membrane, reduction in the number of mitochondria with moderate to severe swelling, and decrease or disappearance of the cristae, and transparent matrix with vacuolation in the post-stroke depression group, which was relatively milder (mild swelling, nearly normal structure and more autophagosome forming) in the acupuncture group and acupuncture+inhibitor group. "Shugan Tiaoshen" needling can improve the depressive symptoms in post-stroke depression rats, which may be related to its function in activating AMPK-dependent autophagy process.

Crosstalk between mitochondrial homeostasis and AMPK pathway mediate the receptor-mediated cardioprotective effects of estradiol in ovariectomized female rats.

Estrogen (E2) deficiency is a risk factor for cardiovascular disease (CVD), however, the exact mechanism for the E2 protective effect on CVD remains unclear. This study aimed to investigate the estrogen receptor (ER) and non-receptor mediated effects of E2 treatment on the cardiac expression of adenosine monophosphate-dependent protein kinase (AMPK), autophagic, mitophagy and mitochondrial homeostasis-regulating genes in ovariectomized (OVX) rats. Female rats were divided into two main groups; sham and bilaterally OVX rats, then each group was subdivided into four subgroups according to treatment; untreated, subcutaneously treated with E2 (30 μg/kg), or Fulvestrant (F) (5 mg/Kg), or a combination of both drugs for 28 days. The OVX rats or F-treated sham rats showed dyslipidemia, and marked disturbances in parameters of AMPK signaling, autophagy, mitophagy, mitochondrial fission, fusion and biogenesis. E2 administration to OVX or F-treated sham rats has corrected the disturbed lipid and cardiac profiles, increased AMPK, and restored the balance of cardiac autophagy, mitophagy, and mitochondrial dynamics and homeostasis. Most of these effects in OVX rats were blocked by the ER antagonist (F). Estrogen treatment has cardioprotective effects in OVX females through modulating cardiac mitochondrial homeostasis, mitophagy and autophagy and restoring the AMPK signaling pathway. As witnessed by Fulvestrant, these effects suggest the main role of ER-mediated signaling in regulating mitophagy and plasma and cardiac lipids along with the existence of a post-translational control mechanism or the involvement of estrogenic non-receptor pathway controlling the postmenopausal cardiac mitochondrial energy production machinery that needs further investigation.

RIPK4 Downregulation Reduces ABCG2 Expression, Increasing BRAF-Mutated Melanoma Cell Susceptibility to Cisplatin- and Doxorubicin-Induced Apoptosis.

Melanoma cells remain resistant to chemotherapy with cisplatin (CisPt) and doxorubicin (DOX). The abnormal expression of Receptor-Interacting Protein Kinase 4 (RIPK4) in certain melanomas contributes to tumour growth through the NFκB and Wnt/β-catenin signalling pathways, which are known to regulate chemoresistance and recurrence. Despite this, the role of RIPK4 in response to chemotherapeutics in melanoma has not been reported. In this study, we examined how the downregulation and overexpression of RIPK4 affect the sensitivity of BRAF-mutated melanoma cells (A375 and WM266.4) to CisPt and DOX along with determining the underlying mechanism. Using two RIPK4 silencing methods (siRNA and CRISPR/Cas9) and overexpression (dCas9-VPR), we assessed CisPt and DOX-induced apoptosis using caspase 3/7 activity, annexin V/7AAD staining, and FASC analysis. In addition, qRT-PCR and Western blotting were used to detect apoptosis-related genes and proteins such as cleaved PARP, p53, and cyclin D1. We demonstrated that the overexpression of RIPK4 inhibits, while its downregulation enhances, CisPt- or DOX-induced apoptosis in melanoma cells. The effects of downregulation are similar to those observed with pre-incubation with cyclosporin A, an ABCG2 inhibitor. Additionally, our findings provide preliminary evidence of crosstalk between RIPK4, BIRC3, and ABCG2. The results of these studies suggest the involvement of RIPK4 in the observed resistance to CisPt or DOX.

Phenylalanine Regulates Milk Protein Synthesis via LAT1-mTOR Signaling Pathways in Bovine Mammary Epithelial Cells.

Phenylalanine (Phe) is a potentially limiting amino acid for lactating cows. The mechanism by which Phe regulates milk protein synthesis remains unclear. The present study elucidates the mechanisms by which phenylalanine affects milk protein synthesis, amino acid utilization, and related signaling pathways in bovine mammary epithelial cells (BMECs). The BMECs were treated with five concentrations (0, 0.22, 0.44, 0.88, 1.76 mM, and serum free). Rapamycin inhibitors and RNA interference (RNAi) were used to inhibit the phosphorylation of the mammalian target of rapamycin (mTOR) signaling pathway and the expression of relevant amino acid transporters, respectively. The results showed that 4×Phe (0.88 mM) significantly increased (p < 0.05) both the mRNA and protein expression of α-casein (CSN1S1), β-casein (CSN2), and κ-casein (CSN3), as well as L-type amino acid transporter-1 (LAT1) mRNA expression. Protein expression and modification assays of mTOR-related proteins showed that 4×Phe could increase (p < 0.05) the expression of α-casein and eukaryotic initiation factor 4E-binding protein-1 (4EBP1) and tended to increase the expression of ribosomal protein S6 protein kinase (S6K1, p = 0.054). The general control nonderepressible 2 (GCN2) signaling pathway factor, eukaryotic initiation factor 2 (eIF2α), was downregulated by 4×Phe treatment (p < 0.05). The rapamycin inhibition test showed that Phe regulated casein synthesis via the mTOR signaling pathway. RNAi experiments showed that LAT1 mediated the entry of Phe into cells. Moreover, 4×Phe treatment tended to decrease (0.05 < p < 0.10) the consumption of valine, leucine, histidine, tyrosine, cysteine, alanine, asparagine, and serine in the medium. Collectively, phenylalanine enhanced α-casein synthesis by regulating the phosphorylation of 4EBP1 and eIF2α and promoting the formation of the mTOR-centered casein translation initiation complex.

Evaluation of betanin-loaded liposomal nanocarriers in AlCl3-induced Alzheimer's disease in rats: Impact on cognitive function, neurodegeneration, and TREM2/ADAM10 pathways.

Betanin (BET) has been studied for its therapeutic benefits in various diseases, but its low bioavailability and uncertain brain penetration limit its efficacy. Accordingly, this study aimed to explore BET-loaded liposomal nanocarriers (LPN) as a novel treatment for Alzheimer's disease (AD), focusing on the triggering receptor expressed on myeloid cells 2 (TREM2)/DNAX-activating protein of 12 kDa (DAP12) and extracellular signal-regulated kinase 1/2 (ERK1/2) pathways implicated in AD. In an AlCl3-induced AD rat model, 48 male Wistar rats were divided into four groups: control, AlCl3 (50 mg/kg, intraperitoneal), AlCl3+BET (100 mg/kg, per os), and AlCl3+BET LPN (25 mg/kg, intranasally), with treatments administered for 28 days. Morris water maze test and histopathological examination showed that BET LPN-treated rats had improved spatial and learning memory and less hippocampal and cortical degeneration compared with the AlCl3 and oral BET groups. Mechanistically, BET LPN treatment corrected AD biomarkers, increased miR-132 and ADAM10 expression, and reduced oxidative stress, inflammation, and apoptosis. Additionally, BET LPN treatment suppressed the expression of TREM2, DAP12, ERK1/2, and mitogen-activated protein kinase 1/2 (MEK1/2), showing greater improvement than oral BET. These findings suggest that BET LPN enhances cognitive function and neuroprotection in AD by modulating miR-132 and ADAM10 and inhibiting ERK1/2 and TREM2/DAP12 pathways, providing a more effective treatment compared with traditional oral BET administration.

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&lt;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.

Empagliflozin promotes skin flap survival by activating AMPK signaling pathway.

Flaps are widely used in surgical wound repair, yet distal necrosis poses a significant postoperative challenge, stemming from potential factors such as inadequate blood perfusion, inflammation, ischemia/reperfusion (I/R) injury, mitochondrial impairment, and subsequent ferroptosis. Empagliflozin (EMPA), a sodium-glucose cotransporter 2 inhibitor, has pharmacological activities that promote angiogenesis, mitophagy, and inhibit inflammation, I/R injury, and ferroptosis. However, it is unclear whether EMPA can enhance flap survival. Here, we established a modified McFarlane flap model and applied EMPA to demonstrate its mechanism of action. 24 rats were evenly divided into four groups: the control, low-dose EMPA (10 mg/kg), high-dose EMPA (30 mg/kg), and inhibitor groups. Molecular biology experiments demonstrated that EMPA promoted the expression of angiogenesis-related factors vascular endothelial growth factor (VEGF) and CD34. Additionally, it also increased superoxide dismutase (SOD) activity and reduced malondialdehyde (MDA) levels, thus suppressing oxidative stress. EMPA further alleviated inflammation by downregulating the expression of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6). In vitro experiments showed that EMPA promoted the proliferation of human umbilical vein endothelial cells (HUVECs) and reduce their reactive oxygen species (ROS) production. Further investigation demonstrated that EMPA improves flap prognosis by inducing the expression of the adenosine monophosphate-activated protein kinase (AMPK) signaling pathway, further promoting mitophagy and inhibiting ferroptosis. These effects collectively contributed to the survival of the skin flap. Overall, our research elucidates the protective effects of EMPA on flap survival and its specific mechanisms, offering new insights into solving post-transplant flap necrosis.

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.

Muscarinic acetylcholine type 1 receptor antagonism activates TRPM3 to augment mitochondrial function and drive axonal repair in adult sensory neurons.

Antagonism of the muscarinic acetylcholine type 1 receptor (M1R) promotes sensory axon repair and is protective in peripheral neuropathy, however, the mechanism remains elusive. We investigated the role of the heat-sensing transient receptor potential melastatin-3 (TRPM3) cation channel in M1R antagonism-mediated nerve regeneration and explored the potential of TRPM3 activation to facilitate axonal plasticity. Dorsal root ganglion (DRG) neurons from adult control or diabetic rats were cultured and treated with TRPM3 agonists (CIM0216, pregnenolone sulfate) and M1R antagonists pirenzepine (PZ) or muscarinic toxin 7 (MT7). Ca2+ transients, mitochondrial respiration, AMP-activated protein kinase (AMPK) expression, and mitochondrial inner membrane potential were analyzed. The effect of M1R activation or blockade on TRPM3 activity mediated by phosphatidylinositol 4,5-bisphosphate (PIP2) was studied. Metabolic profiling of DRG neurons and human neuroblastoma SY-SY5Y cells was conducted. M1R antagonism induced by PZ or MT7 increased Ca2+ influx in DRG neurons and was inhibited by TRPM3 antagonists or in the absence of extracellular Ca2+. TRPM3 agonists elevated Ca2+ levels, augmented mitochondrial respiration, AMPK activation and neurite outgrowth. M1R antagonism stimulated TRPM3 channel activity through inhibition of PIP2 hydrolysis to activate Ca2+/calmodulin-dependent protein kinase kinase β (CaMKKβ)/AMPK, leading to augmented mitochondrial function and neuronal metabolism. DRG neurons with AAV-mediated shRNA knockdown of TRPM3 exhibited suppressed antimuscarinic drug-induced neurite outgrowth. TRPM3 agonists increased glycolysis and TCA cycle metabolites, indicating enhanced metabolism in DRG neurons and SH-SY5Y cells. Activation of the TRPM3/CaMKKβ/AMPK pathway promoted collateral sprouting of sensory axons, positioning TRPM3 as a promising therapeutic target for peripheral neuropathy.

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.