Mitogen-activated Protein Kinase Research Articles

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.

Involvement of sphingosine-1-phosphate receptor 1 in pain insensitivity in a BTBR mouse model of autism spectrum disorder

BackgroundAbnormal sensory perception, particularly pain insensitivity (PAI), is a typical symptom of autism spectrum disorder (ASD). Despite the role of myelin metabolism in the regulation of pain perception, the mechanisms underlying ASD-related PAI remain unclear.MethodsThe pain-associated gene sphingosine-1-phosphate receptor 1 (S1PR1) was identified in ASD samples through bioinformatics analysis. Its expression in the dorsal root ganglion (DRG) tissues of BTBR ASD model mice was validated using RNA-seq, western blot, RT-qPCR, and immunofluorescence. Pain thresholds were assessed using the von Frey and Hargreaves tests. Patch-clamp techniques measured KCNQ/M channel activity and neuronal action potentials. The expression of S1PR1, KCNQ/M, mitogen-activated protein kinase (MAPK), and cyclic AMP/protein kinase A (cAMP/PKA) signaling proteins was analyzed before and after inhibiting the S1P-S1PR1-KCNQ/M pathway via western blot and RT-qPCR.ResultsThrough integrated transcriptomic analysis of ASD samples, we identified the upregulated gene S1PR1, which is associated with sphingolipid metabolism and linked to pain perception, and confirmed its role in the BTBR mouse model of ASD. This mechanism involves the regulation of KCNQ/M channels in DRG neurons. The enhanced activity of KCNQ/M channels and the decreased action potentials in small and medium DRG neurons were correlated with PAI in a BTBR mouse model of ASD. Inhibition of the S1P/S1PR1 pathway rescued baseline insensitivity to pain by suppressing KCNQ/M channels in DRG neurons, mediated through the MAPK and cAMP/PKA pathways. Investigating the modulation and underlying mechanisms of the non-opioid pathway involving S1PR1 will provide new insights into clinical targeted interventions for PAI in ASD.ConclusionsS1PR1 may contribute to PAI in the PNS in ASD. The mechanism involves KCNQ/M channels and the MAPK and cAMP/PKA signaling pathways. Targeting S1PR1 in the PNS could offer novel therapeutic strategies for the intervention of pain dysesthesias in individuals with ASD.

Probiotic-Derived P8 Protein: Promoting Proliferation and Migration in Stem Cells and Keratinocytes.

Probiotics exert various effects on the body and provide different health benefits. Previous reports have demonstrated that the P8 protein (P8), isolated from Lactobacillus rhamnosus, has anticancer properties. However, its efficacy in stem cells and normal cells has not been reported. In this study, the effect of P8 on cell proliferation and wound healing was evaluated, investigating its underlying mechanism. Based on scratch assay results, we demonstrated that P8 treatment significantly increases wound healing by activating the cell cycle and promoting stem cell stemness. Cellular mechanisms were further investigated by culturing stem cells in a medium containing Lactobacillus-derived P8 protein, revealing its promotion of cell proliferation and migration. Also, it is found that P8 enhances the expression of stemness markers, such as OCT4 and SOX2, along with activation of the mitogen-activated protein kinase (MAPK) signaling and Hippo pathways. These results indicate that P8 can promote cell growth by increasing stem cell proliferation, migration, and stemness in a manner associated with MAPK and Hippo signaling, which could contribute to the increased wound healing after P8 treatment. Furthermore, P8 could promote wound healing in keratinocytes by activating the MAPK signaling pathways. These results suggest that P8 might be a promising candidate to enhance stem cell culture efficiency by activating cell proliferation, and enhance therapeutic effects in skin diseases.

Hyperlipidemia exacerbates acute pancreatitis via interactions between P38MAPK and oxidative stress

BackgroundThe mechanisms involved in the hyperlipidemia-associated acute pancreatitis (HLAP) is not yet fully understood. AimsTo investigate the role of P38MAPK (mitogen-activated protein kinases) and oxidative stress in the pathogenesis of HLAP. MethodsIn AP (acute pancreatitis) patients, the GEO database retrieved gene expression profiles of cytokines, MAPK14, nuclear factor kappa B subunit 1 (NF-κB 1) and superoxide dismutase 2 (SOD 2). GeneMANIA has been used for the prediction of potential interaction mechanisms. Validation was carried out using an experimental AP model and a bi-directional Mendelian randomization (MR) analysis. ResultsCompared to mild AP, patients with severe AP had higher gene expression of MAPK14, NF-κB1, SOD2, IL-1β and IL-6R. GeneMANIA revealed 77.6 % physical interactions among MAPK14, NF-κB1, SOD2, IL-1β and IL-6R. Our results indicated that HLAP group had a more severe pancreatic injury, a stronger inflammatory response with higher serum levels of TNF-α, IL-6 and IL-1β in comparison with the AP group, which were significantly mitigated in HLAP-Pi group. Furthermore, SB 203580 inhibited increasing levels of malondialdehyde (MDA) in serum and of inducible nitric oxide synthase (iNOS), P38MAPK, p-P38MAPK and NF-κB p65 in pancreatic tissue as well as decreasing serum values of SOD and GSH-PX in HLAP group. MR analysis suggested that MAPK14 levels were negatively associated with the SOD levels, by using the inverse variance weighted (IVW) method (b = −0.193: se = 0.225; P = 1.03e-17). Reverse MR analysis indicated that SOD was negatively associated with the MAPK14 levels in the IVW analysis (b = −0.163: se = 0.020; P = 1.38e-15). ConclusionInteractions between P38MAPK and oxidative stress may play an important role in the pathogenesis of HLAP.

Pim1 is Critical in T-cell-independent B-cell Response and MAPK Activation in B Cells

The Pim family consists of three members that encode a distinct class of highly conserved serine/threonine kinases. In this study, we generated and examined mice with hematopoiesis-specific deletion of Pim1 and bone marrow (BM) chimeric mice with B-cell-specific targeted deletion of Pim1. Pim1 was expressed at all stages of B-cell development and hematopoietic-specific deletion of Pim1 altered B-cell development in BM, spleen and peritoneal. However, Pim1 deficiency did not affect T-cell development. Studies of BM chimeric mice showed that Pim1 is required in a cell-intrinsic manner to maintain normal B-cell development. Pim1 deficiency led to significant changes in B cell antibody responses. Additionally, Pim1 deficiency resulted in reduced B cell receptor (BCR)-induced cell proliferation and cell cycle progression. Examination of the various BCR-activated signaling pathways in Pim1-deficient B cells reveals defective activation of mitogen-activated protein kinases (MAPKs), which are known to regulate genes involved in cell proliferation and survival. qRT-PCR analysis of BCR-engaged B cells from Pim1-deficient B cells revealed reduced expression of cyclin-dependent kinase (CDK) and cyclin genes, including CDK2, CCNB1 and CCNE1. In conclusion, Pim1 plays a crucial role in B-cell development and B cell activation.

β-aminobutyric acid-induced resistance in postharvest peach fruit involves interaction between the MAPK cascade and SNARE13 protein in salicylic acid-dependent pathway.

The inducer β-aminobutyric acid (BABA) is capable of immune response in various plants. However, the specific mitogen-activated protein kinase (MAPK) cascade involved in BABA-induced resistance (BABA-IR) has not yet been elucidated. Here, peach fruits treated with the BABA exhibited a pattern-triggered immunity (PTI) defense against Rhizopus stolonifer, accompanied by the generation of reactive oxygen species (ROS) and activation of MAPK cascade. Transcriptome sequencing suggested a total of fifteen PpMAPKKK/PpMAPKK/PpMAPK genes involved in BABA-IR in peach fruit. Further qRT-PCR analysis showed that the transcript profiles of PpMAPKKK3, PpMAPKK5 and PpMAPK1 were obviously potentiated. Subsequently, yeast two-hybrid (Y2H), luciferase complementation imaging (LCI), pull-down and in vitro phosphorylation assays were conducted to characterize the complete MAPK cascade (PpMAPKKK3-PpMAPKK5-PpMAPK1) involved in peach fruit. Moreover, the downstream events of MAPK1 include the involvement of SNARE13 and the corresponding NPR1-responsive defense. Single silencing of MAPKKK3, MAPKK5 or MAPK1 and double silencing of MAPKKK3/MAPKK5 or MAPKK5/MAPK1 resulted in enhanced susceptibility to the fungus R. stolonifer in mutants and attenuated salicylic acid (SA)-dependent defense gene expression; in contrast, the homologous or heterologous overexpression of PpSNARE13 in peach fruit or Arabidopsis led to an enhanced SA pool and elevated expression of PR genes. Reciprocally, the ppsnare13cas9 mutants are generally compromised in the priming of SA-dependent resistance. Therefore, the MAPKKK3-MAPKK5-MAPK1 cascade contributes to PTI signal transduction in BABA-elicited peach fruit, by combination with downstream events such as SNARE13, NPR1, and SA-dependent signaling.

Alternative splicing is coupled to gene expression in a subset of variably expressed genes

Numerous factors regulate alternative splicing of human genes at a co-transcriptional level. However, how alternative splicing depends on the regulation of gene expression is poorly understood. We leveraged data from the Genotype-Tissue Expression (GTEx) project to show a significant association of gene expression and splicing for 6874 (4.9%) of 141,043 exons in 1106 (13.3%) of 8314 genes with substantially variable expression in nine GTEx tissues. About half of these exons demonstrate higher inclusion with higher gene expression, and half demonstrate higher exclusion, with the observed direction of coupling being highly consistent across different tissues and in external datasets. The exons differ with respect to multiple characteristics and are enriched for hundreds of isoform-specific Gene Ontology annotations suggesting an important regulatory mechanism. Notably, splicing-expression coupling of exons with roles in JUN and MAP kinase signalling could play an important role during cell division.

The endosomal-vacuolar transport system acts as a docking platform for the Pmk1 MAP kinase signaling pathway in Magnaporthe oryzae.

In Magnaporthe oryzae, the Pmk1 MAP kinase signaling pathway regulates appressorium formation, plant penetration, effector secretion, and invasive growth. While the Mst11-Mst7-Pmk1 cascade was characterized two decades ago, knowledge of its signaling in the intracellular network remains limited. In this study, we demonstrate that the endosomal surface scaffolds Pmk1 MAPK signaling and Msb2 activates Ras2 on endosomes in M. oryzae. Protein colocalization demonstrated that Msb2, Ras2, Cap1, Mst50, Mst11, Mst7, and Pmk1 attach to late endosomal membranes. Damage to the endosome-vacuole transport system influences Pmk1 phosphorylation. When Msb2 senses a plant signal, it internalizes and activates Ras2 on endosome membrane surfaces, transmitting the signal to Pmk1 via Mst11 and Mst7. Signal-sensing and delivery proteins are ubiquitinated and sorted for degradation in late endosomes and vacuoles, terminating signaling. Plant penetration and lowered intracellular turgor are required for the transition from late endosomes to vacuoles in appressoria. Our findings uncover an effective mechanism that scaffolds and controls Pmk1 MAPK signaling through endosomal-vacuolar transport, offering new knowledge for the cytological and molecular mechanisms by which the Pmk1 MAPK pathway modulates development and pathogenicity in M. oryzae.

Temporal variation in p38-mediated regulation of DUX4 in facioscapulohumeral muscular dystrophy

Facioscapulohumeral muscular dystrophy (FSHD) is a degenerative muscle disease caused by loss of epigenetic silencing and ectopic reactivation of the embryonic double homeobox protein 4 gene (DUX4) in skeletal muscle. The p38 MAP kinase inhibitor losmapimod is currently being tested in FSHD clinical trials due to the finding that p38 inhibition suppresses DUX4 expression in preclinical models. However, the role of p38 in regulating DUX4 at different myogenic stages has not been investigated. We used genetic and pharmacologic tools in FSHD patient-derived myoblasts/myocytes to explore the temporal role of p38 in differentiation-induced DUX4 expression. Deletion of MAPK14/11 or inhibition of p38α/β caused a significant reduction in early differentiation-dependent increases in DUX4 and DUX4 target gene expression. However, in MAPK14/11 knockout cells, there remains a differentiation-associated increase in DUX4 and DUX4 target gene expression later in differentiation. Furthermore, pharmacologic inhibition of p38α/β only partially decreased DUX4 and DUX4 target gene expression in late differentiating myotubes. In xenograft studies, p38α/β inhibition by losmapimod failed to suppress DUX4 target gene expression in late FSHD xenografts. Our results show that while p38 is critical for DUX4 expression during early myogenesis, later in myogenesis a significant level of DUX4 expression is independent of p38α/β activity.

Kehuang capsule inhibits MAPK and AKT signaling pathways to mitigate CCl4-induced acute liver injury

Background and aimsKehuang (KH) capsule is an herbal medical product approved for the treatment of liver diseases, including liver injury, in China. However, the mechanism is still unclear. This study aimed to elucidate the protective effects of KH capsule against carbon tetrachloride (CCl4)-induced acute liver injury (ALI) in a murine model. MethodsMice were randomly divided into control, model (CCl4), CCl4+KH_Low and CCl4+KH_High group. Liver enzyme levels and histological changes were assessed to evaluate liver injury. Oxidative stress markers and inflammatory cell infiltration in liver tissues were measured. Additionally, network pharmacology was employed to explore the potential mechanisms of KH capsule. ResultsKH capsule significantly reduced serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels, as well as the necrotic area in liver tissue. KH capsule also decreased the infiltration of macrophages and neutrophils, thereby inhibiting the expression of interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and interleukin-1 beta (IL-1β). Furthermore, KH capsule decreased liver malondialdehyde (MDA) levels and increased superoxide dismutase (SOD) activity. The number of terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end labeling (TUNEL)-positive cells in liver tissue was also reduced. The expression of nuclear factor erythroid 2 related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) proteins was significantly elevated, while the protein expression of cytochrome P450 2E1 (CYP2E1) was significantly reduced. Mass spectrometry identified genistein, galangin, wogonin, skullcapflavone II, and hispidulin as potential active ingredients of KH capsule. Network pharmacology analysis revealed enrichment in the mitogen-activated protein kinase (MAPK) and phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT) signaling pathways. Western blot analysis confirmed that KH capsule suppressed AKT, extracellular signal-regulated kinase (ERK), and p38 signaling. ConclusionsThese findings suggest that KH capsule exerts protective effects against CCl4-induced ALI, with the inhibition of MAPK and PI3K-AKT signaling pathways playing a crucial role in its mechanism of action.

Network pharmacology and experimental verification unraveled the mechanism of Bailing Capsule against asthma.

Asthma is a serious public health challenge around the world. Recent studies into traditional Chinese medicine preparations for asthma have yielded promising findings regarding Bailing Capsule's potential in bronchial asthma prevention and treatment. This study aims to initially clarify the potential mechanism of Bailing Capsule in the treatment of asthma using network pharmacology and in vitro experimental approaches. Network pharmacology was adopted to detect the active ingredients of Bailing Capsule via Traditional Chinese Medicine Systems Pharmacology Database, and the key targets and signaling pathways in the treatment of asthma were predicted. Docking and molecular dynamics simulations were conducted to verify the most important interactions formed by these probes within different regions of the binding site. The predicted targets were validated in lipopolysaccharide-induced 16HBE cell experiment. Seven active ingredients were screened from Bailing Capsule, 294 overlapping targets matched with asthma were considered potential therapeutic targets, such as SRC, TP53, STAT3, and E1A binding protein P300. The main functional pathways involving these key targets include phosphatidylinositol 3-kinase/protein kinase B, mitogen-activated protein kinase, renin-angiotensin system and other signaling pathways, which were mainly involved in the inflammatory response, apoptosis, and xenobiotic stimulus. Moreover, molecular docking showed that Cerevisterol have higher affinity for SRC, TP53, STAT3, and E1A binding protein P300 than other main active components, which is close to the docking results of the co-crystallized ligands to proteins. Consequently, Cerevisterol was selected for molecular dynamics simulation and the results show that Cerevisterol can bind most tightly to SRC, TP53, and STAT3. Bailing Capsule can promote the growth of 16HBE cell, reduce the production of IL-4, TNF-α and IL-6, and down-regulate the levels of SRC and STAT3 mRNA. This study preliminarily reveals the potential mechanism of Bailing Capsule against asthma with the aid of network pharmacology and in vitro cell experiment, which provided reference and guidance for in-depth research and clinical application.

Traditional Chinese Herbal Medicine: Therapeutic Potential in Chronic Obstructive Pulmonary Disease

Chronic obstructive pulmonary disease (COPD) is characterized by persistent airflow obstruction and manifested by symptoms such as chronic cough, sputum, and dyspnea. Traditional Chinese medicine (TCM) has long been used to treat COPD. This article summarizes potential Chinese herbal medicines (CHM) for COPD treatment through a literature review and collation. The findings highlight 14 kinds of effective CHM for COPD, including Scutellaria radix, Salvia miltiorrhiza radix et rhizoma, Hippophae fructus, Ginseng radix et rhizoma, and Astragali radix. Flavonoids, terpenoids, alkaloids, and volatile oil constituents are the main bioactive components for the treatment of COPD. They mainly inhibit the tumor necrosis factor alpha/nuclear factor kappa B (TNF-α/NF-κB) signaling pathway by reducing the production and release of inflammatory factors such as interleukin 1 beta (IL-1β), interleukin 6 (IL-6), interleukin 8 (IL-8), TNF-α, and NF-κB, thereby reducing inflammation in lungs of rats with COPD and protect the lung tissues. Some active ingredients can inhibit toll-like receptor 4 (TLR4), mitogen-activated protein kinase (MAPK), phosphoinositide 3-kinase-protein kinase B (PI3K-Akt), Janus kinase-signal transducer and activator of transcription (JAK-STAT), and transforming growth factor beta (TGF-β) signaling pathways to express anti-inflammatory activity. This work establishes a foundation for using CHM in COPD treatment and offers new insights and approaches for developing novel anti-COPD drugs.

Lysosomal damage triggers a p38 MAPK-dependent phosphorylation cascade to promote lysophagy via the small heat shock protein HSP27.

Maintenance of lysosomal integrity is essential for cell viability. Upon injury, lysosomes may be targeted for degradation via a selective form of autophagy known as lysophagy. The engulfment of a damaged lysosome by an autophagosome is mediated by the recruitment of adaptor proteins, including SQSTM1/p62. p62 promotes lysophagy via the formation of phase-separated condensates in a mechanism that is regulated by the heat shock protein HSP27. Here, we demonstrate a direct interaction between HSP27 and p62. We used structural modeling to predict the binding interface between HSP27 and p62 and identify several disease-associated mutations that map to this interface. We used proteomics to identify post-translational modifications of HSP27 that regulate HSP27 recruitment to stressed lysosomes, finding robust phosphorylation at several serine residues. Next, we characterized the upstream signaling mechanism leading to HSP27 phosphorylation and found that p38 mitogen-activated protein kinase (MAPK) and its effector kinase MAP kinase-activated protein kinase 2 (MK2) are activated upon lysosomal damage by the kinase mTOR and the production of intracellular reactive oxygen species (ROS). Increased ROS activates p38 MAPK, which in turn allows MK2-dependent phosphorylation of HSP27. Depletion of HSP27 or the inhibition of HSP27 phosphorylation alters the dynamics of p62 condensates on stressed lysosomes, significantly inhibiting p62-dependent lysophagy. Thus, we define a novel lysosomal quality control mechanism in which lysosomal injury triggers a p38 MAPK/MK2 signaling cascade promoting p62-dependent lysophagy. Further, this signaling cascade is activated by many cellular stressors, including oxidative and heat stress, suggesting that other forms of selective autophagy may be regulated by p38 MAPK/MK2/HSP27.

Sinulariolide Suppresses Inflammation of Fibroblast-Like Synoviocytes in Rheumatoid Arthritis and Mitigates Collagen-Induced Arthritis Symptoms in Mice.

Rheumatoid arthritis (RA) is a systemic inflammatory disease characterized by active polyarthritis, which leads to functional loss and joint deformities. Natural compounds derived from marine organisms are considered valuable immune-modulating agents. This study aimed to assess the anti-inflammatory effect of sinulariolide, a soft coral-derived compound, on RA fibroblast-like synoviocytes and its therapeutic efficacy against collagen-induced arthritis (CIA). To determine the effects of sinulariolide on tumor necrosis factor-alpha (TNF-α)-induced inflammation, MH7A cells pre-treated with 10 ng/mL TNF-α for 24h were treated with sinulariolide. The effect of sinulariolide on proinflammatory cytokine expressions at both the mRNA and protein levels in the MH7A cells was assessed using real-time-polymerase chain reaction and enzyme-linked immunosorbent assay (ELISA). Further, we analyzed the effect of sinulariolide on the activation of mitogen-activated protein kinase (MAPK) and nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) pathways using Western blotting and the TransAM NF-κB p65 kit. To comprehensively evaluate the potential application of sinulariolide in the treatment of inflammatory diseases, we used a well-established collagen-induced arthritis (CIA) mouse model. We examined the tissue sections of the ankle joints of the mice, assessed synovial hyperplasia, inflammatory cell infiltration, and cartilage damage, and used ELISA to analyze changes in cytokine expression in the hind paw tissues. MH7A cells treated with sinulariolide showed a notable reduction in the expression of proinflammatory cytokines, which could be due to decreased activation of the MAPK and NF-kB pathways. Additionally, sinulariolide-treated mice showed significantly reduced joint swelling and lower clinical arthritis scores than those in the normal and control groups. Significant reductions in synovial hyperplasia, inflammatory cell infiltration, and cartilage damage were observed in the tissue sections of the ankle joints of the mice treated with sinulariolide. Furthermore, the expression of inflammatory cytokines in the hind paw tissue of the mice treated with sinulariolide was significantly decreased. Sinulariolide inhibited the progression of inflammation in MH7A cells. Sinulariolide treatment significantly reduced clinical arthritis symptoms and histological inflammatory responses in mice with CIA. Sinulariolide may serve as a potential therapeutic agent for RA.

Upregulation of TLR2 in keratinocytes activates the MAPK pathway and plays a role in the pathogenesis of FHS

BackgroundMutations in gamma-secretase complex (GSC) genes are associated with hidradenitis suppurativa (HS), and toll-like receptor (TLR) 2 is elevated in HS lesions. However, it remains unclear whether TLR2 is upregulated in the skin lesions of patients with HS with GSC gene variants, and the role of its upregulation in the pathogenesis of this disease are unknown. ObjectiveTo investigate the role of TLR2 upregulation in NCSTN and PSENEN knockdown keratinocytes. MethodsHuman immortalized keratinocyte line (HaCaTs) was treated with potent short-hairpin RNA targeting NCSTN or PSENEN. RNA sequencing was used to assess the effects of PAM2CSK4 treatment on gene expression in HaCaT cells. Altered signaling pathways were confirmed in both HaCaT cells, as well as in skin lesions from patients with HS and in Ncstn keratinocyte-specific knockout (NcstnΔKC) mice. ResultsTLR2 agonist stimulation exacerbated the increased phosphorylation levels of extracellular signal-regulated kinase 1/2 and p38 mitogen-activated protein kinase (MAPK) in NCSTN- and PSENEN- knockdown keratinocytes. Similar findings were observed in skin lesions from patients with HS and NcstnΔKC mice. ConclusionNovel variations were identified within the GSC gene in Chinese patients with HS. Moreover, our study indicates that TLR2/MAPK signaling pathways play a key role in the pathogenesis of HS associated with GSC gene mutations and represent a crucial therapeutic target.

Clerodendrum trichotomum Extract Attenuates UV-B-Induced Skin Impairment in Hairless Mice by Inhibiting MAPK Signaling.

Exposure to solar ultraviolet-B (UV-B) radiation significantly accelerates skin aging by inducing the expression of matrix metalloproteinases (MMPs) such as MMP-1, leading to alterations in the extracellular matrix and consequent photoaging. Some plant components, renowned for their UV-absorbing and antioxidative properties, show potential for mitigating photoaging by reducing UV-B-induced MMP levels. In this context, we explored the inhibitory effects of Clerodendrum trichotomum extract (CTE) on UV-B-induced skin damage. The mechanism of CTE was predicted using network pharmacology approach. Also, antiaging efficacy was evaluated by mouse model and cellular system using human epidermal keratinocytes (HEKa), including its modulation of mitogen-activated protein kinase (MAPK) signaling pathways. CTE effectively counters UV-B-induced skin damage, as evidenced by the suppression of MMP-9 and MMP-1 expression in mice. We found that each fraction and chemical constituents of CTE suppressed UV-B-induced MMP-1 secretion in HEKa cells. CTE inhibits UV-B-induced skin aging by partially suppressing MMP-1 and MMP-9 secretion via the modulation of MAPK signaling pathways.

Integrative pathway analysis across humans and 3D cellular models identifies the p38 MAPK-MK2 axis as a therapeutic target for Alzheimer's disease.

Alzheimer's disease (AD) presents a complex pathological landscape, posing challenges to current therapeutic strategies that primarily target amyloid-β (Aβ). Using a novel integrative pathway activity analysis (IPAA), we identified 83 dysregulated pathways common between both post-mortem AD brains and three-dimensional AD cellular models showing robust Aβ42 accumulation. p38 mitogen-activated protein kinase (MAPK) was the most upregulated common pathway. Active p38 MAPK levels increased in the cellular models, human brains, and 5XFAD mice and selectively localized to presynaptic dystrophic neurites. Unbiased phosphoproteomics confirmed increased phosphorylation of p38 MAPK substrates. Downstream activation of MAPK-activated protein kinase 2 (MK2) plays a crucial role in Aβ42-p38 MAPK-mediated tau pathology. Therapeutic targeting of the p38 MAPK-MK2 axis with selective inhibitors significantly reduced Aβ42-driven tau pathology and neuronal loss. IPAA prioritizes the best models to derisk target-drug discovery by integrating human tissue gene expression with functional readouts from cellular models, enabling the identification and validation of high-confidence AD therapeutic targets.

RIPK2 Is Crucial for the Microglial Inflammatory Response to Bacterial Muramyl Dipeptide but Not to Lipopolysaccharide.

Receptor-interacting serine/threonine protein kinase 2 (RIPK2) is a kinase that is essential in modulating innate and adaptive immune responses. As a downstream signaling molecule for nucleotide-binding oligomerization domain 1 (NOD1), NOD2, and Toll-like receptors (TLRs), it is implicated in the signaling triggered by recognition of microbe-associated molecular patterns by NOD1/2 and TLRs. Upon activation of these innate immune receptors, RIPK2 mediates the release of pro-inflammatory factors by activating mitogen-activated protein kinases (MAPKs) and nuclear factor-kappa B (NF-κB). However, whether RIPK2 is essential for downstream inflammatory signaling following the activation of NOD1/2, TLRs, or both remains controversial. In this study, we examined the role of RIPK2 in NOD2- and TLR4-dependent signaling cascades following stimulation of microglial cells with bacterial muramyl dipeptide (MDP), a NOD2 agonist, or lipopolysaccharide (LPS), a TLR4 agonist. We utilized a highly specific proteolysis targeting chimera (PROTAC) molecule, GSK3728857A, and found dramatic degradation of RIPK2 in a concentration- and time-dependent manner. Importantly, the PROTAC completely abolished MDP-induced increases in iNOS and COX-2 protein levels and pro-inflammatory gene transcription of Nos2, Ptgs2, Il-1β, Tnfα, Il6, Ccl2, and Mmp9. However, increases in iNOS and COX-2 proteins and pro-inflammatory gene transcription induced by the TLR4 agonist, LPS, were only slightly attenuated with the GSK3728857A pretreatment. Further findings revealed that the RIPK2 PROTAC completely blocked the phosphorylation and activation of p65 NF-κB and p38 MAPK induced by MDP, but it had no effects on the phosphorylation of these two mediators triggered by LPS. Collectively, our findings strongly suggest that RIPK2 plays an essential role in the inflammatory responses of microglia to bacterial MDP but not to LPS.

A p38 MAPK-ROS axis fuels proliferation stress and DNA damage during CRISPR-Cas9 gene editing in hematopoietic stem and progenitor cells

Exvivo activation is a prerequisite to reaching adequate levels of gene editing by homology-directed repair (HDR) for hematopoietic stem and progenitor cell (HSPC)-based clinical applications. Here, we show that shortening culture time mitigates the p53-mediated DNA damage response to CRISPR-Cas9-induced DNA double-strand breaks, enhancing the reconstitution capacity of edited HSPCs. However, this results in lower HDR efficiency, rendering exvivo culture necessary yet detrimental. Mechanistically, exvivo activation triggers a multi-step process initiated by p38 mitogen-activated protein kinase (MAPK) phosphorylation, which generates mitogenic reactive oxygen species (ROS), promoting fast cell-cycle progression and subsequent proliferation-induced DNA damage. Thus, p38 inhibition before gene editing delays G1/S transition and expands transcriptionally defined HSCs, ultimately endowing edited cells with superior multi-lineage differentiation, persistence throughout serial transplantation, enhanced polyclonal repertoire, and better-preserved genome integrity. Our data identify proliferative stress as a driver of HSPC dysfunction with fundamental implications for designing more effective and safer gene correction strategies for clinical applications.

Determining the mechanism of Shuxuening injection against liver cirrhosis through network pharmacology and animal experiments

ObjectiveTo screen and identify the key active molecules, signaling pathways, and therapeutic targets of Shuxuening (SXN) injection for treating liver cirrhosis (LC) and to evaluate its therapeutic potential using a mouse model. MethodsTarget genes of SXN and LC were retrieved from public databases, and enrichment analysis was performed. A protein–protein interaction (PPI) network was constructed using the Search Tool for the Retrieval of Interacting Genes/Proteins (STRING), and hub genes were identified using Molecular Complex Detection (MCODE). LC was induced in rats and mice via intraperitoneal injections of diethylnitrosamine and carbon tetrachloride (CCl4) for 12 weeks. Starting at week 7, SXN was administered intraperitoneally to the mice in the treatment group. Serum and liver tissues of the mice were collected for the detection of indicators, pathological staining, and expression analysis of hub targets using quantitative real-time polymerase chain reaction (qRT-PCR). ResultsWe identified 368 overlapping genes (OLGs) between SXN and LC targets. These OLGs were subsequently used to build a PPI network and to screen for hub genes. Enrichment analysis showed that these genes were associated with cancer-related pathways, including phosphoinositide-3-kinase/Akt and mitogen-activated protein kinase signaling and various cellular processes, such as responses to chemicals and metabolic regulation. In vivo experiments demonstrated that SXN treatment significantly improved liver function and pathology in CCl4-induced LC mice by reducing inflammation and collagen deposition. Furthermore, qRT-PCR demonstrated that SXN regulated the expression of MAPK8, AR and CASP3 in the livers of LC mice. ConclusionThis study highlighted the therapeutic effects of SXN in alleviating LC using both bioinformatics and experimental methods. The observed effect was associated with modulation of hub gene expression, particularly MAPK8, and CASP3.