Phosphorylation Levels Of MAPK Research Articles

Activation patterns of intracellular signaling pathways in cardiac hypertrophy induced by different exercise modes in rats

Abstract Introduction Cardiac remodeling and hypertrophy cause as adaptive responses to physiological and pathological stimuli. Physiological cardiac hypertrophy is characterized by normal or enhanced cardiac function and myocardial metabolism, and induces concentric and eccentric hypertrophy by different exercise modes, without cardiac dysfunction and fibrosis. Each formative mechanism of cardiac hypertrophy is regulated by distinct and/or similar cellular signaling pathways. Activation of cardiac intracellular signaling pathway of phosphoinositide 3- kinase (PI3K) is involved in an underlying mechanism of developed eccentric cardiac hypertrophy by aerobic exercise training (AT). However, the activation patterns of intracellular signaling pathways of physiological cardiac hypertrophy by different exercise modes remain unclear. Purpose The purpose of this study was to investigate the activation patterns of intracellular signaling pathways in cardiac hypertrophy induced by different exercise modes, including AT, resistance training (RT), and high intensity interval training (HIIT). Methods Forty male 10-week-old Sprague-Dawley (SD) rats were divided into four groups; sedentary control (Con, n=10), AT (treadmill running, 60 min at 30 m/min, 5 days/wk for 8 wk, n=10), RT (ladder climbing, 8-10 sets/day, 3 d/wk for 8 wk, n=10), HIIT (14 repeats of 20-s swimming session with 10-s pause between sessions, 4 d/wk for 6 wk from 12-wk-old, n=10) groups. As a cardiac hypertrophic intracellular signaling pathways, PI3K protein expression level and mTOR and MAPK (ERK1/2, JNK1/2 and p38) phosphorylation levels in rat hearts were measured by Western blotting analysis. Results The hearts of rats in each training group developed cardiac hypertrophy. Moreover, the enzyme activity of oxidative phosphorylation in the skeletal muscle was promoted in the AT group, the oxidative phosphorylation and glycolytic enzyme activities were promoted in the HIIT group, and muscle hypertrophy occurred in the RT group. Levels of PI3K protein expression and mTOR phosphorylation in the heart were significantly higher in the AT, RT, and HIIT groups as compared to the Con group (each p<0.05). Additionally, p38 phosphorylation level in the heart was significantly higher in the RT group as compared to the Con, AT, and HIIT groups (each p<0.05). Furthermore, p44 ERK phosphorylation level in the heart of the RT group was significantly higher than that in the AT and HIIT groups (each p<0.05), but p42 ERK phosphorylation level did not differ among four groups. JNK1/2 (p46/p54) phosphorylation level in the heart was significantly higher in the RT group as compared to the Con group (p<0.05). Conclusion These findings suggest that the activation of PI3K-mTOR signaling pathway in the heart may be involved in the development of AT, RT and HIIT-induced cardiac hypertrophy, and the MAPK signaling activation may affect the development of cardiac hypertrophy by RT.

CRISPR/Cas9-mediated suppression of A4GALT rescues endothelial cell dysfunction in a fabry disease vasculopathy model derived from human induced pluripotent stem cells

Background and aimsThe objective of this study was to investigate the efficacy of CRISPR/Cas9-mediated A4GALT suppression in rescuing endothelial dysfunction in Fabry disease (FD) endothelial cells (FD-ECs) derived from human induced pluripotent stem cells (hiPSCs). MethodsWe differentiated hiPSCs (WT (wild-type), WTC-11), GLA-mutant hiPSCs (GLA-KO, CMC-Fb-002), and CRISPR/Cas9-mediated A4GALT-KO hiPSCs (GLA/A4GALT-KO, Fb-002-A4GALT-KO) into ECs and compared FD phenotypes and endothelial dysfunction. We also analyzed the effect of A4GALT suppression on reactive oxygen species (ROS) formation and transcriptome profiles through RNA sequencing. ResultsGLA-mutant hiPSC-ECs (GLA-KO and CMC-Fb-002) showed downregulated expression of EC markers and significantly reduced α-GalA expression with increased Gb-3 deposition and intra-lysosomal inclusion bodies. However, CRISPR/Cas9-mediated A4GALT suppression in GLA/A4GALT-KO and Fb-002-A4GALT-KO hiPSC-ECs increased expression levels of EC markers and rescued these FD phenotypes. GLA-mutant hiPSC-ECs failed to form tube-like structure in tube formation assays, showing significantly decreased migration of cells into the scratched wound area. In contrast, A4GALT suppression improved tube formation and cell migration capacity. Western blot analysis revealed that MAPK and AKT phosphorylation levels were downregulated while SOD and catalase were upregulated in GLA-KO hiPSC-ECs. However, suppression of A4GALT restored these protein alterations. RNA sequencing analysis demonstrated significant transcriptome changes in GLA-mutant EC, especially in angiogenesis, cell death, and cellular response to oxidative stress. However, these were effectively restored in GLA/A4GALT-KO hiPSC-ECs. ConclusionsCRISPR/Cas9-mediated A4GALT suppression rescued FD phenotype and endothelial dysfunction in GLA-mutant hiPSC-ECs, presenting a potential therapeutic approach for FD-vasculopathy.

High expression of fragile X mental retardation protein inhibits ferroptosis of colorectal tumor cells by activating the RAS/MAPK signaling pathway

To investigate the mechanism by which fragile X mental retardation protein (FMRP) regulates ferroptosis evasion in colorectal cancer (CRC) cells. We examined FMRP expression levels in CRC cell lines using RT-qPCR and Western blotting and analyzed the biological functions and signaling pathways involved in FMRP-mediated regulation of CRC progression using the TCGA database. A lentiviral FMRP overexpression vector (Lv-FMRP) and 3 knockdown vectors (siFMRP-1, siFMRP-2, and siFMRP-3) were constructed, and their effects on proliferation of HCT116 cells were examined using CCK8 assay and plate clone formation assay; the changes in cell ferroptosis level was determined using MDA/ROS/GSH/Fe2+ kits, mitochondrial membrane potential changes were detected using JC-1 fluorescence staining, and the expressions of proteins associated with ferroptosis and the RAS/MAPK signaling pathway were detected using Western blotting. The subcutaneous tumorigenic potential of the transfected cells was evaluated in nude mice. Compared with normal colonic mucosal epithelial NCM460 cells, the CRC cell lines had significantly higher FMRP expression level. Bioinformatics analysis suggested the involvement of FMRP in regulation of reactive oxygen, oxidative stress-induced cell death, mitochondrial respiration, and glutathione metabolism pathways. In the cell experiments, FMRP knockdown significantly inhibited proliferation of HCT116 cells, lowered cellular GSH content, increased MDA and ROS levels, Fe2+ fluorescence intensity, and mitochondrial membrane potential, and decreased SLC7A11/GPX4 protein expressions and the phosphorylation levels of ERK, MEK, MAPK, and RAS proteins; FMRP overexpression resulted in the opposite changes in the cells. In the tumor-bearing nude mice, HCT116 cells with FMRP knockdown showed attenuated tumorigenic potential with lowered xenograft growth rate and reduced SLC7A11 expression in the xenograft. The high expression of FMRP inhibits ferroptosis in CRC cells and promotes progression of CRC by activating the RAS/MAPK signaling pathway.

TRV027 REDUCES BLOOD PRESSURE IN SHR AND RESTORES VENTRICULAR HYPERTROPHY BY AFFECTING CONNEXIN 43 PATHWAY

Objective: Hypertension is one of the common causes of cardiovascular diseases. Angiotensin II (AngII) plays a crucial role in hypertension and hypertension-induced cardiac hypertrophy. Whether TRV027, an AngII type 1 receptor biased agonist, could lower blood pressure and treat hypertensive cardiac hypertrophy is unknown. Design and method: Spontaneously hypertensive rats and Wistar-Kyoto rats were selected as the study subjects. Some SHRs were giving TRV027. H9c2 cells were selected as the in vitro study subjects to explore the mechanism of action of TRV027. Results: When compared to the WKY group, the SHR group displayed a pronounced increase in blood pressure. However, after giving subcutaneous pump of TRV027, blood pressure exhibited a marked reduction. Based on the Doppler ultrasound data, prior to the intervention, the SHR group had higher values for LVAWs, LVAWd, LVPWs, and LVPWd when contrasted with the WKY group. Moreover, the left ventricular mass in the SHR group also increased significantly (P<0.05). After the intervention with TRV027 in the SHR group, all the above-mentioned cardiac Doppler parameters were lower than those in the SHR group. HE staining of cardiac sections revealed a slight disarray in myocardial arrangement and significant myocardial hypertrophy in the SHR group. Compared with the WKY group, the level of Cx43 in the heart tissue of the SHR group was decreased, and the phosphorylation of MAPK and ERK2 was increased. After administration of TRV027, the expression levels of these proteins were restored. In the cell intervention, the expression level of Cx43 was significantly decreased, the phosphorylation levels of MAPK and ERK2 were increased, and the levels of MHC and ANP were increased in the AngII intervention group. After the intervention of TRV027 the expressions of Cx43 and MAPK/ERK pathways and MHC and ANP were restored. When Cx43 inhibitor was given to H9C2 cells,along with the decrease of Cx43 expression, the phosphorylation levels of MAPK and ERK2 were increased, while the levels of MHC and ANP were significantly increased. Conclusions: TRV027 lower blood pressure in SHR and ameliorate cardiac hypertrophy induced by hypertension. This effect is likely achieved through the Cx43 pathway.

Metabolomics combined with network pharmacology reveals anti-asthmatic effects of Nepeta bracteata on allergic asthma rats

ObjectiveTo investigate the mechanisms that underlie the anti-asthmatic effects of Nepeta bracteata (DBJJ, Dabao Jingjie in Chinese) in rats by integrating metabolomics and network pharmacology. MethodsIn this study, the rat model of asthma was induced by using ovalbumin (OVA), and subsequently, the rats were treated with a decoction of N. bracteata. Pathological changes in lung tissue were observed, and the quantification of eosinophils (EOS) and white blood cells (WBC) in bronchoalveolar lavage fluid (BALF) was performed. Furthermore, the serum levels of asthma-related factors induced by OVA were assessed. The proton nuclear magnetic resonance (1H NMR) spectroscopy serum metabolomics method was utilized to identify differential metabolites and their associated metabolic pathways. Moreover, UPLC-QE-MS/MS combined with network pharmacology was employed to predict the core targets and pathways of DBJJ in its action against asthma. Furthermore, the anti-asthmatic properties of DBJJ were investigated using an integrated approach of metabolomics and network pharmacology. Subsequently, the findings were validated through molecular docking and Western blotting analysis of the key targets. ResultsThe findings indicated that the administration of DBJJ effectively alleviated OVA-induced lung histopathological changes and decreased the number of EOS and WBC in BALF. Additionally, DBJJ inhibited the OVA-induced elevation of TNF-α, IL-18, Ig-E, EOS, IL-1β, MDA, VEGF-A, and TGF-β1. A total of 21 biomarkers and 10 pathways were found by metabolomics analysis. The 29 compounds were identified by UPLC-QE-MS/MS and 13 active components were screened by oral availability and Caco-2 cell permeability, the 120 targets and 173 KEGG pathways were predicted. The integration of metabolomics and network pharmacological analysis revealed that DBJJ's primary constituents, including ferulic acid and ursolic acid, exerted their effects on four targets, namely DAO and NOS2, as well as their associated metabolites and pathways. The active constituents of DBJJ demonstrated a high binding affinity towards DAO and NOS2. Furthermore, DBJJ was observed to decrease the protein expression and phosphorylation levels of NOS2, MAPK, and STAT3. ConclusionThe administration of DBJJ demonstrates notable anti-asthma properties in rats with allergic asthma. This effect can be attributed to the modulation of various targets, including NOS2, MAPK, and STAT3, by primary constituents such as ferulic acid and ursolic acid.

Simvastatin Attenuates Cardiac Fibrosis under Pathophysiological Conditions of Heart Failure with Preserved Left Ventricular Ejection Fraction by Inhibiting TGF-β Signaling

Introduction: There is still no effective treatment for heart failure with preserved left ventricular ejection fraction (HFpEF), and therapies to improve prognosis are urgently needed. Clinical studies in patients with HFpEF have shown that statins and HMG-CoA reductase inhibitors may reduce their mortality rate. However, the mechanisms underlying the effects of statins on HFpEF remain unknown. In the present study, we examined whether simvastatin administration inhibits the development of cardiac fibrosis in HFpEF model mice. We further examined the contribution of the Smad and mitogen-activated protein (MAP) kinase pathways to the transforming growth factor-β (TGF-β) signaling pathway in the development of HFpEF. Methods: HFpEF animals were prepared by feeding C57BL/6 N mice a high-fat diet and providing water containing N[w]-nitro-l-arginine methyl ester hydrochloride (l-NAME) for 15 weeks. Simvastatin (30 mg/kg/day) or vehicle was administered orally daily during the experimental period. Cardiac function was measured by echocardiography, and cardiac fibrosis was evaluated by Masson’s trichrome staining. Changes in the TGF-β signaling proteins in myocardial tissue were examined by Western blotting. Results: A high-fat diet and l-NAME solution load induced cardiac diastolic dysfunction with cardiac fibrosis. Simvastatin treatment markedly attenuated cardiac fibrosis and reduced cardiac diastolic dysfunction. In addition, simvastatin prevented the increase in phosphorylation levels of Smad (Smad2 and Smad3) and MAPK (c-Raf, Erk1/2) pathway proteins downstream of the TGF-β receptor in cardiac tissue. Conclusions: Our present study demonstrated that simvastatin attenuated diastolic dysfunction by reducing cardiac fibrosis in HFpEF hearts. Furthermore, our findings suggest that the mechanisms by which simvastatin attenuates HFpEF development involve, at least in part, inhibition of the TGF-β signaling pathway, which is activated in the HFpEF heart.

Nucleotide substitutions at the p.Gly117 and p.Thr180 mutational hot-spots of SKI alter molecular dynamics and may affect cell cycle.

Heterozygous deleterious variants in SKI cause Shprintzen-Goldberg Syndrome, which is mainly characterized by craniofacial features, neurodevelopmental disorder and thoracic aorta dilatations/aneurysms. The encoded protein is a member of the transforming growth factor beta signaling. Paucity of reported studies exploring the SGS molecular pathogenesis hampers disease recognition and clinical interpretation of private variants. Here, the unpublished c.349G>A, p.[Gly117Ser] and the recurrent c.539C>T, p.[Thr180Met] SKI variants were studied combining in silico and in vitro approach. 3D comparative modeling and calculation of the interaction energy predicted that both variants alter the SKI tertiary protein structure and its interactions. Computational data were functionally corroborated by the demonstration of an increase of MAPK phosphorylation levels and alteration of cell cycle in cells expressing the mutant SKI. Our findings confirmed the effects of SKI variants on MAPK and opened the path to study the role of perturbations of the cell cycle in SGS.

Anti-Inflammatory and Gut Microbiota Modulation Potentials of Flavonoids Extracted from Passiflora foetida Fruits.

This study aimed to explore the anti-inflammatory and gut microbiota modulation potentials of flavonoid-rich fraction (PFF) extracted from Passiflora foetida fruits. The results showed that PFF markedly reduced the production of nitric oxide (NO), tumor necrosis factor α (TNF-α), and interleukin 6 (IL-6) in LPS-stimulated RAW 264.7 cells. Meanwhile, PFF treatment also effectively decreased the phosphorylation levels of MAPK, PI3K/Akt, and NF-κB signaling-pathway-related proteins (ERK, JNK, p38, Akt, and p65). Moreover, PFF had an impact on microbial composition and metabolites in a four-stage dynamic simulator of human gut microbiota (BFBL gut model). Specifically, PFF exhibited the growth-promoting ability of several beneficial bacteria, including Bifidobacterium, Enterococcus, Lactobacillus, and Roseburia, and short-chain fatty acid (SCFA) generation ability in gut microbiota. In addition, spectroscopic data revealed that PFF mainly contained five flavonoid compounds, which may be bioactive compounds with anti-inflammatory and gut microbiota modulation potentials. Therefore, PFF could be utilized as a natural anti-inflammatory agent or supplement to health products.

Atg1, a key regulator of autophagy, functions to promote MAPK activation and cell death upon calcium overload in fission yeast.

Autophagy promotes or inhibits cell death depending on the environment and cell type. Our previous findings suggested that Atg1 is genetically involved in the regulation of Pmk1 MAPK in fission yeast. Here, we showed that Δatg1 displays lower levels of Pmk1 MAPK phosphorylation than did the wild-type (WT) cells upon treatment with a 1,3-β-D-glucan synthase inhibitor micafungin or CaCl2, both of which activate Pmk1. Moreover, the overproduction of Atg1, but not that of the kinase inactivating Atg1D193A activates Pmk1 without any extracellular stimuli, suggesting that Atg1 may promote Pmk1 MAPK signaling activation. Notably, the overproduction of Atg1 induces a toxic effect on the growth of WT cells and the deletion of Pmk1 failed to suppress the cell death induced by Atg1, indicating that the Atg1-mediated cell death requires additional mechanism(s) other than Pmk1 activation. Moreover, atg1 gene deletion induces tolerance to micafungin and CaCl2, whereas pmk1 deletion induces severe sensitivities to these compounds. The Δatg1Δpmk1 double mutants display intermediate sensitivities to these compounds, showing that atg1 deletion partly suppressed growth inhibition induced by Δpmk1. Thus, Atg1 may act to promote cell death upon micafungin and CaCl2 stimuli regardless of Pmk1 MAPK activity. Since micafungin and CaCl2 are intracellular calcium inducers, our data reveal a novel role of the autophagy regulator Atg1 to induce cell death upon calcium overload independent of its role in Pmk1 MAPK activation.

Transcriptomics reveals apigenin alleviates airway inflammation and epithelial cell apoptosis in allergic asthma via MAPK pathway.

Persistent chronic inflammation of the lungs and airway remodeling are important pathological features that cannot be ignored in patients with chronic asthma. Apigenin (API) is a natural small molecule compound with good anti-inflammatory and antioxidant activity that has been widely reported in recent years, but its role in chronic asthma is not well defined. Our study began with oral gavage intervention using API (10, 20 mg/kg) or dexamethasone (DEX, 2 mg/kg) in a BALB/c mouse model of ovalbumin (OVA) sensitization. Different doses of API intervention effectively reduced airway resistance in the administered group. Additionally, inflammation was downregulated, mucus secretion was reduced, and airway remodeling was inhibited in the API intervention group compared with the model group. Asthma-related inflammatory cytokines, such as IgE, IL-4, IL-5, IL-13, and IL-17, were downregulated in alveolar lavage fluid. Moreover, the apoptosis level of the administered group was found to be lower than that of the model group in the Tunel staining experiment. By analyzing transcriptome sequencing results, we found that API may exert anti-inflammatory and anti-apoptotic effects by inhibiting the MAPK pathway. Our subsequent results supported this conclusion, showing that the phosphorylation levels of ERKs, JNKs, and p38 MAPKs were inhibited in the administered group relative to the model group. Downstream expression of the apoptosis-related protein B-cell lymphoma-2 (Bcl-2) was upregulated, and the expression of Bcl-2-associated × protein (Bax) and cleaved caspase-3 was downregulated. To further investigate the specific mechanism by which API acted, we established an in vitro model with house dust mite (HDM) stimulation, using API (10, 20 μM) for administration intervention. The results showed that API was able to improve cell viability, inhibit ROS production, and reverse HDM-induced decreases in mitochondrial membrane potential (MMP) and apoptosis in airway epithelial cells via the MAPK pathway.

Pine pollen extract alleviates ethanol-induced oxidative stress and apoptosis in HepG2 cells via MAPK signaling.

Pine pollen extract (PPE) has various effects such as antioxidant, hypoglycemic and anti-obesity. However, the anti-ALD mechanism of PPE has rarely been studied. In this study, PPE was prepared and chemical components such as total protein, total sugars, total phenols, total flavonoids and polyphenol composition were determined. The effect of PPE on HepG2 cells after ethanol damage was also measured. HepG2 cells were pre-treated with 200μg/mL and 400μg/mL of PPE for 24h followed by treatment with ethanol for 24h. The results suggested that PPE significantly decreased the production of LDH, ROS and MDA. Meanwhile, the levels of GSH and SOD were also increased. The levels of Nrf2, HO-1 and Keap1 were upregulated after PPE treatment. Additionally, Bax were downregulated and Caspase3 activation was inhibited after PPE treatment. What's more, PPE also attenuated the ethanol-induced phosphorylation levels of MAPK in HepG2 cells. Three major phenols were identified as p-coumaric acid, catechins and caffeic acid. These results suggest that PPE can be effective against ALD by alleviating MAPK pathway-mediated oxidative stress and apoptosis. In conclusion, PPE is a natural product with hepatoprotective potential.

Libanoridin Isolated from Corydalis heterocarpa Inhibits Adipogenic Differentiation of Bone Marrow-Derived Mesenchymal Stromal Cells.

Bone marrow adiposity is a complication in osteoporotic patients. It is a result of the imbalance between adipogenic and osteogenic differentiation of bone marrow cells. Phytochemicals can alleviate osteoporotic complications by hindering bone loss and decreasing bone marrow adiposity. Corydalis heterocarpa is a biennial halophyte with reported bioactivities, and it is a source of different coumarin derivatives. Libanoridin is a coumarin isolated from C. heterocarpa, and the effect of libanoridin on adipogenic differentiation of human bone marrow-derived mesenchymal stromal cells (hBM-MSCs) was evaluated in the present study. Cells were induced to undergo adipogenesis, and their intracellular lipid accumulation and expression of adipogenic markers were observed under libanoridin treatment. Results showed that 10 μM libanoridin-treated adipocytes accumulated 44.94% less lipid compared to untreated adipocytes. In addition, mRNA levels of PPARγ, C/EBPα, and SREBP1c were dose-dependently suppressed with libanoridin treatment, whereas only protein levels of PPARγ were decreased in the presence of libanoridin. Fluorescence staining of adipocytes also revealed that cells treated with 10 μM libanoridin expressed less PPARγ compared to untreated adipocytes. Protein levels of perilipin and leptin, markers of mature adipocytes, were also suppressed in adipocytes treated with 10 μM libanoridin. Analysis of MAPK phosphorylation levels showed that treatment with libanoridin inhibited the activation of p38 and JNK MAPKs observed by decreased levels of phosphorylated p38 and JNK protein. It was suggested that libanoridin inhibited adipogenic differentiation of hBM-MSCs via suppressing MAPK-mediated PPARγ signaling. Future studies revealing the anti-adipogenic effects of libanoridin in vivo and elucidating its action mechanism will pave the way for libanoridin to be utilized as a nutraceutical with anti-osteoporotic properties.

Overexpression of microRNA-211 in Functional Dyspepsia via Downregulation of the Glial Cell Line-Derived Neurotrophic Factor (GDNF) by Increasing Phosphorylation of p38 MAPK Pathway.

Overexpression of miRNA-211 suppresses the differentiation of bone marrow stem cells into intestinal ganglion cells via downregulation of GDNF, a regulator of intestine barrier function. The study aimed to investigate the interaction between miR-211 and GDNF on intestinal epithelial cells. The expression levels of miR-211 and GDNF in duodenal biopsy specimens from FD patients and healthy controls were compared. Enteric glia cell (EGCs) cell line transfected with miR-211 mimics and inhibitors were used to clarify the expression levels of GDNF were analyzed by qRT-PCR and ELISA. Intestine epithelial cell (IECs) cell line cultured in medium from ECGs in different transfection conditions were used in wound healing assay, cell proliferation assay, and western blotting for evaluation of p38 MAPK phosphorylation level. MiR-211 expression was significantly upregulated in the duodenal tissue of patients with FD compared to healthy subjects, whereas GDNF expression was significantly downregulated (both p < 0.05). Transfection with miR-211 mimics significantly decreased GDNF mRNA expression and protein secretion (p < 0.001). An inhibited intestinal epithelial cell wound healing (p < 0.05) and increased expression levels of phosphorylated p38 MAPK (p < 0.05) were found in IECs cultured with medium from EGCs transfected with miR-211 mimics. MiR-211 may downregulates GDNF mRNA and protein expression via activation of the pp38 MAPK signaling pathway. Targeting miR-211 or the MAPK pathway may be a potential intervention for FD.

Nicotinamide Mononucleotide Attenuates LPS-Induced Acute Lung Injury With Anti-Inflammatory, Anti-Oxidative and Anti-Apoptotic Effects

IntroductionNicotinamide mononucleotide (NMN) is a nucleotide that is commonly recognized for its role as an intermediate of nicotinamide adenine dinucleotide (NAD+) biosynthesis with multiple pharmacological effects. The purpose of this study was to evaluate the protective effect of nicotinamide mononucleotide (NMN) against lipopolysaccharide (LPS)–induced acute lung injury (ALI). MethodsWe investigated the effect of NMN on ALI-induced inflammatory response, oxidative stress, and cell apoptosis. The ALI mouse model was performed by injecting LPS intratracheally at a dose of 10 mg/kg in 50 μL saline. Flow cytometry was used to detect neutrophil infiltration in bronchoalveolar lavage fluid (BALF), and ELISA was used to detect the contents of inflammatory cytokines TNF-α, IL-1β and IL-6 in BALF. Oxidative stress was evaluated by determining the superoxide dismutase (SOD) activity and malondialdehyde (MDA) content in lung tissue. ROS formation was analyzed by immunofluorescence. Western blotting was performed to detect apoptotic levels and p38MAPK/NF-κB phosphorylation levels in lung tissue. ResultsIn the ALI mouse model, NMN showed a significant therapeutic effect compared to the LPS group. NMN attenuated the pathological damage and cell apoptosis in lung tissue, decreased the levels of TNF-α, IL-1β, and IL-6 in BALF, and reduced the number of total cells and neutrophils in BALF. In addition, NMN attenuated the LPS-induced elevation of dry-to-wet ratio, MDA content, p38 MAPK and p65 NF-κB phosphorylation levels, and the SOD activity was increased by NMN treatment. ConclusionsIn conclusion, the present study showed that NMN exerted a protective effect on LPS-induced ALI with anti-inflammatory, antioxidative, and antiapoptotic effects.

Hydrogen helps to ameliorate Staphylococcus aureus-induced mastitis in mice

Many studies have shown that hydrogen has anti-inflammatory and anti-oxidant effects. Because of its ability to quickly pass through cell membranes, hydrogen has become a hot spot in the research of inflammatory diseases. Vitamin E glycerin (VEG) and hydrogen-rich Vitamin E glycerin (HR-VEG) were prepared, aiming to explore their anti-inflammatory activities in mice mastitis induced by Staphylococcus aureus (S. aureus). In the early part of this study, the prepared vitamin E medium (VEM) and hydrogen-rich vitamin E medium (HR-VEM) were added to mammary epithelial cells infected with S. aureus. HR-VEM was found to be more effective in reducing the phosphorylation of p65 and p38 and in reducing the production of interleukin-1 beta (IL-1β) than VEM. Whereafter, the mice model of mastitis was established by injecting S. aureus from the mammary duct. Then VEG and HR-VEG were applied to the mammary gland for seven consecutive days. After that, the clinical symptoms, histopathology, bacterial load, inflammatory factors, as well as the related pathway were analyzed. The results showed that HR-VEG can more significantly alleviate the damage of mammary tissue than VEG, and reduce the production of tumor necrosis factor-alpha (TNF-α), IL-1β and interleukin 6 (IL-6). In addition, HR-VEG inhibited the TLR2 and Nod2 signaling pathways and reduced the phosphorylation level of MAPK and NF-κB signaling pathways in S. aureus-induced murine mastitis. This study indicates that hydrogen helps to ameliorate S. aureus-induced mastitis in mice through attenuating TLR2 and Nod2 mediated NF-κB and MAPK activation.

PD-L1 Inhibits T Cell-Induced Cytokines and Hyaluronan Expression via the CD40-CD40L Pathway in Orbital Fibroblasts From Patients With Thyroid Associated Ophthalmopathy

Thyroid associated ophthalmopathy (TAO), characterized by T cell infiltration and orbital fibroblast activation, is an organ-specific autoimmune disease which is still short of effective and safety therapeutic drugs. The PD-1/PD-L1 pathway has been reported hindering the progression of Graves’ disease to some extent by inhibiting T cell activity, and tumor therapy with a PD-1 inhibitor caused some adverse effects similar to the symptoms of TAO. These findings suggest that the PD-1/PD-L1 pathway may be associated with the pathogenesis of TAO. However, it remains unknown whether the PD-1/PD-L1 pathway is involved in orbital fibroblast activation. Here, we show that orbital fibroblasts from patients with TAO do not express PD-L1. Based on in vitro OF-T cell co-culture system, exogenous PD-L1 weakens T cell-induced orbital fibroblast activation by inhibiting T cell activity, resulting in reduced production of sICAM-1, IL-6, IL-8, and hyaluronan. Additionally, exogenous PD-L1 treatment also inhibits the expression of CD40 and the phosphorylation levels of MAPK and NF-κB pathways in orbital fibroblasts of the OF-T cell co-culture system. Knocking down CD40 with CD40 siRNA or down-regulating the phosphorylation levels of MAPK and NF-κB pathways with SB203580, PD98059, SP600125, and PDTC can both reduce the expression of these cytokines and hyaluronan. Our study demonstrates that the orbital immune tolerance deficiency caused by the lack of PD-L1 in orbital fibroblasts may be one of the causes for the active orbital inflammation in TAO patients, and the utilization of exogenous PD-L1 to reconstruct the orbital immune tolerance microenvironment may be a potential treatment strategy for TAO.

The Inhibition of LPS-Induced Oxidative Stress and Inflammatory Responses Is Associated with the Protective Effect of (-)-Epigallocatechin-3-Gallate on Bovine Hepatocytes and Murine Liver.

This study aimed to evaluate whether (-)-epigallocatechin-3-gallate (EGCG) alleviates hepatic responses to lipopolysaccharide (LPS)-induced inflammation and oxidation. Isolated bovine hepatocytes and BALB/c mice were used for LPS challenge and EGCG pretreatment experiments in vitro and in vivo. LPS-challenged (6 μg/mL) hepatocytes exhibited increased levels of NF-κB (p65 and IκBα) and MAPK (p38, ERK, JNK) phosphorylation as well as increased binding activity of p65 to target pro-inflammatory gene promoters, and these effects were suppressed by pretreatment with 50 μM EGCG. Moreover, the reduction in Nrf2 signaling and antioxidant enzyme activities induced by LPS stimulation were reversed upon EGCG treatment. In vivo experiments demonstrated the protective role of EGCG in response to GalN/LPS-induced mortality and oxidative damage. Together, our results suggest that EGCG is hepatoprotective via inhibition of MAPK/NF-κB signaling and activation of the Nrf2 cascade. This information might help design strategies for counteracting hepatitis in ruminants and monogastric animals.

MET Exon 14 Splice-Site Mutations Preferentially Activate KRAS Signaling to Drive Tumourigenesis.

Simple SummaryMET exon 14 splice-site mutations occur in ~3–4% of lung adenocarcinoma cases, defining a cohort of patients which might benefit from anti-MET targeted therapy. Such therapies have yielded mixed results, however, pointing to the need for better treatment design. Our study sought to aid this by characterizing key changes in mutant MET signaling behaviour. We first compared the transcriptional profiles of lung tumours with either METΔex14 or wild-type MET-amplification. METΔex14-mutant tumours exhibited increased activation of the Ras-MAPK pathway, consistent with our observations in an isogenic model system. Furthermore, sustained activity of this pathway is necessary for proliferation and maintenance of METΔex14 tumours, while forced reactivation of this pathway is sufficient to restore growth in the absence of MET activity. Our findings suggest that the MAPK pathway represents a main effector of METΔex14-driven cancer, lending credence to the possibility of combined MET-MAPK inhibition to improve therapeutic outcomes.Targeted therapies for MET exon 14-skipping (METΔex14)-driven lung cancers have generated some promising results but response rates remain below that seen for other kinase-driven cancers. One strategy for improving treatment outcomes is to employ rational combination therapies to enhance the suppression of tumour growth and delay or prevent the emergence of resistance. To this end, we profiled the transcriptomes of MET-addicted lung tumours and cell lines and identified the RAS-mitogen-activated protein kinase (MAPK) pathway as a critical effector required for METΔex14-dependent growth. Ectopic expression of MET in an isogenic cell line model showed that overexpression of the mutant MET receptor led to higher levels of MAPK phosphorylation and nuclear import, resulting in increased expression and phosphorylation of nuclear MAPK targets. In comparison, other known MET effectors were unaffected. Inhibition of this pathway by KRAS knockdown in MET-addicted cells in vitro led to decreased viability in only the METΔex14-mutant cells. Conversely, decoupling RAS-MAPK axis, but not other effector pathways, from MET activity via the introduction of constitutively active mutants conferred resistance to MET inhibitors in vitro. Our results suggest that aberrant hyperactivity of the MET receptor caused by the exon 14-skipping mutation does not uniformly upregulate all known downstream effectors, rather gaining a predilection for aberrantly activating and subsequently relying on the RAS-MAPK pathway. These findings provide a rationale for the co-targeting of the RAS-MAPK pathway alongside MET to prolong therapeutic response and circumvent resistance to improve patient survival.

Antifibrogenic and apoptotic effects of Ocoxin in cultured rat hepatic stellate cells.

Ocoxin is a nutritional supplement that has been shown to exert antioxidant and immunomodulatory responses in patients with chronic hepatitis C. The present work aimed to determine the effects of Ocoxin on activated hepatic stellate cells (HSC), the cell type mainly responsible for collagen deposition in the fibrotic liver. Ocoxin was found to reduce the survival of a cell line of immortalized non-tumoral rat HSC in a dose-response fashion and to diminish collagen type I levels. This latter effect was observed even at doses not affecting cell survival, pointing to an antifibrogenic action for the supplement. The decrease in viability exerted by Ocoxin on HSC correlated with an increase in histone-associated fragments in the cytoplasm and with increased activity of caspase-3, indicating the induction of apoptosis. To determine the molecular mechanisms mediating Ocoxin-induced apoptosis, the activation of members of the MAPK family was analyzed. Incubation of HSC with Ocoxin caused a transient and dramatic enhancement on ERK, JNK, and p38 MAPK phosphorylation levels. Using specific inhibitors for these enzymes, p38 MAPK was identified as a key mediator of the apoptotic effect of Ocoxin on HSC.

SJMHE1 protects against excessive iodine-induced pyroptosis in human thyroid follicular epithelial cells through a toll-like receptor 2-dependent pathway.

To elucidate the effect of Schistosoma japonicum peptide (SJMHE1) on pyroptosis in thyroid follicular epithelial cells (TFCs) induced by excessive iodine and the potential mechanism, the effects of SJMHE1 were investigated in NaI-treated Nthy-ori 3-1 cells; and the involvement of the ROS/MAPK/NF-κB signaling pathways in these effects was evaluated by employing CCK-8 assays, flow cytometry, ELISA, and Western blotting experiments. We found that SJMHE1 significantly reduced NLRP3, N-terminus of gasdermin D (GSDMD-N) and cleaved caspase-1 (C-caspase-1) expression, and decreased IL-1β secretion in TFCs. SJMHE1 also markedly reduced reactive oxygen species (ROS) production, and decreased the phosphorylation levels of MAPK and NF-κB pathway members. Moreover, blocking of the Toll-like receptor 2 significantly impaired SJMHE1-mediated protection from excessive iodine-induced pyroptosis in TFCs. Therefore, our results suggested a protective role of SJMHE1 in excessive iodine-induced pyroptosis in TFCs, which might be attributed to its suppression for ROS/MAPK/NF-κB signaling pathway by binding of SJMHE1 with TLR2.