Shank3 deficiency elicits autistic-like behaviors by activating p38α in hypothalamic AgRP neurons.

BACKGROUND: SH3 and multiple ankyrin repeat domains protein 3 (SHANK3) monogenic mutations or deficiency leads to excessive stereotypic behavior and impaired sociability, which frequently occur in autism cases. To date, the underlying mechanisms by which Shank3 mutation or deletion causes autism and the part of the brain in which Shank3 mutation leads to the autistic phenotypes are understudied. The hypothalamus is associated with stereotypic behavior and sociability. p38α, a mediator of inflammatory responses in the brain, has been postulated as a potential gene for certain cases of autism occurrence. However, it is unclear whether hypothalamus and p38α are involved in the development of autism caused by Shank3 mutations or deficiency. METHODS: Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis and immunoblotting were used to assess alternated signaling pathways in the hypothalamus of Shank3 knockout (Shank3-/-) mice. Home-Cage real-time monitoring test was performed to record stereotypic behavior and three-chamber test was used to monitor the sociability of mice. Adeno-associated viruses 9 (AAV9) were used to express p38α in the arcuate nucleus (ARC) or agouti-related peptide (AgRP) neurons. D176A and F327S mutations expressed constitutively active p38α. T180A and Y182F mutations expressed inactive p38α. RESULTS: We found that Shank3 controls stereotypic behavior and sociability by regulating p38α activity in AgRP neurons. Phosphorylated p38 level in hypothalamus is significantly enhanced in Shank3-/- mice. Consistently, overexpression of p38α in ARC or AgRP neurons elicits excessive stereotypic behavior and impairs sociability in wild-type (WT) mice. Notably, activated p38α in AgRP neurons increases stereotypic behavior and impairs sociability. Conversely, inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3-/- mice. In contrast, activated p38α in pro-opiomelanocortin (POMC) neurons does not affect stereotypic behavior and sociability in mice. LIMITATIONS: We demonstrated that SHANK3 regulates the phosphorylated p38 level in the hypothalamus and inactivated p38α in AgRP neurons significantly ameliorates autistic behaviors of Shank3-/- mice. However, we did not clarify the biochemical mechanism of SHANK3 inhibiting p38α in AgRP neurons. CONCLUSIONS: These results demonstrate that the Shank3 deficiency caused autistic-like behaviors by activating p38α signaling in AgRP neurons, suggesting that p38α signaling in AgRP neurons is a potential therapeutic target for Shank3 mutant-related autism.

Interrogating endothelial barrier regulation by temporally resolved kinase network generation.

Kinases are key players in endothelial barrier regulation, yet their temporal function and regulatory phosphosignaling networks are incompletely understood. We developed a novel methodology, Temporally REsolved KInase Network Generation (TREKING), which combines a 28-kinase inhibitor screen with machine learning and network reconstruction to build time-resolved, functional phosphosignaling networks. We demonstrated the utility of TREKING for identifying pathways mediating barrier integrity after activation by thrombin with or without TNF preconditioning in brain endothelial cells. TREKING predicted over 100 kinases involved in barrier regulation and discerned complex condition-specific pathways. For instance, the MAPK-activated protein kinase 2 (MAPKAPK2/MK2) had early barrier-weakening activity in both inflammatory conditions but late barrier-strengthening activity exclusively with thrombin alone. Using temporal Western blotting, we confirmed that MAPKAPK2/MK2 was differentially phosphorylated under the two inflammatory conditions. We further showed with lentivirus-mediated knockdown of MAPK14/p38α and drug targeting the MAPK14/p38α-MAPKAPK2/MK2 complex that a MAP3K20/ZAK-MAPK14/p38α axis controlled the late activation of MAPKAPK2/MK2 in the thrombin-alone condition. Beyond the MAPKAPK2/MK2 switch, TREKING predicts extensive interconnected networks that control endothelial barrier dynamics.

Network pharmacology analysis, molecular docking integrated experimental verification reveal ß-sitosterol as the active anti-NSCLC ingredient of Polygonatum cyrtonema Hua by suppression of PI3K/Akt/HIF-1α signaling pathway.

ETHNOPHARMACOLOGICAL RELEVANCE: Polygonatum cyrtonema Hua (Huangjing) is a Chinese herb that is considered by ancient Chinese healers to have the effect of nourishing yin and moisturizing the lungs. It is clinically used to treat diseases of the pulmonary system, including non-small cell lung cancer. However, the precise active components and underlying mechanisms of Huangjing in the context of treating NSCLC remain uncertain. AIM OF THE STUDY: This study aimed to explore the active components and mechanisms of Huangjing for the treatment of NSCLC by means of data mining, network pharmacology, and in vitro and vivo experiments. MATERIALS AND METHODS: First, the main active compounds and key targets of Huangjing were predicted by network pharmacology. The potential key targets of Huangjing were molecularly docked with the main active compounds using Pymol. In vivo, we verified whether Huangjing and its main active compound have anti-lung cancer effects. Key targets were verified by PCR and immunohistochemistry. In vitro, we verified the effects of Huangjing's main active compound on the proliferation, apoptosis, and migration of A549 cells by CCK-8, colony formation, wound healing assay, and flow cytometry. Key targets and signaling pathway were validated by PCR and Western blot. RESULTS: The network pharmacology results suggested that ß-sitosterol was the main active substance. TP53, JUN, AKT1, MAPK14, ESR1, RELA, HIF1A, and RXRA were potential targets of Huangjing. Molecular docking results suggested that MAPK14, HIF-1α, and RXRA docked well with ß-sitosterol. In vivo tests also confirmed that Huangjing could significantly inhibit the growth of lung cancer tumors, while PCR and immunohistochemistry results suggested that the expression of HIF-1α was significantly decreased. Critically, KEGG analysis indicated that the PI3K/Akt/HIF-1α signaling pathway was recommended as one of the main pathways related to the anti-NSCLC effect of Huangjing. We conducted in vitro experiments to confirm the significant impact of ß-sitosterol on the proliferation, apoptosis, migration, and colony formation of A549 cells. Furthermore, our findings indicate that a high dosage of ß-sitosterol may effectively decrease the expression of HIF-1α, AKT1, JUN and RELA in A549 cells. Similarly, in vitro experiments also revealed that high doses of ß-sitosterol could inhibit the PI3K/Akt/HIF-1α signaling pathway. CONCLUSIONS: We discovered Huangjing and its main active ingredient, ß-sitosterol, can reduce HIF-1α, AKT1, JUN and RELA expression and decrease non-small cell lung cancer growth through the PI3K/Akt/HIF-1α signaling pathway.

Role of Qufeng Tongqiao Prescription in the protection of cerebral ischemia and associated molecular network mechanism.

To explore the of Qufeng Tongqiao Prescription in the treatment of cerebral ischemia-reperfusion (CIR) and associated molecular network mechanism. Venny diagram, gene ontology (GO) and kyoto encyclopedia of genes and genomes (KEGG) analysis, protein-protein interaction (PPI), hub genes mining, molecular docking, combined with animal experiments and Nissl stain were performed to determine the molecular network mechanism of Qufeng Tongqiao Prescription for CIR treatment. Fifty three intersecting genes between Qufeng Tongqiao Prescription and cerebral ischemia reperfusion were acquired from Venny analysis. GO analysis showed that the main biological process (BP) was response to lipopolysaccharide, and the main cell localization (CC) process was membrane raft, while the most important molecular function (MF) process is Cytokine receptor binding. Moreover, AGE-RAGE signaling pathway in diabetic complications is the most important signaling pathway in KEGG pathway. Through molecular docking, it was found that Astragalus membranaceus was docked with MAPK14, IL4, FOS, IL6, and JUN; pueraria membranaceus was directly docked with JUN and IL4; Acorus acorus was linked to JUN and MAPK14; Ganoderma ganoderma and human were involved in JUN docking, and Ligusticum chuanqi and pueraria could not be docked with MAPK14, respectively. The results of animal experiments showed that Qufeng Tongqiao Prescription significantly improved behavioral performance and reduced the number of neuronal deaths in rats subjected to CIR, and molecular mechanisms are associated with FOS, IL-6, IL4, JUN, and MAPK14, of there, IL-6, as a vital candidator, which has been confirmed by immunostaining detection. Together, Qufeng Tongqiao Prescription has positive therapeutic effect on CIR, and the underlying mechanism is involved MAPK14, FOS, IL4, and JUN network, while IL-6 may be as a vital target.

Distinct binding modes of a benzothiazole derivative confer structural bases for increasing ERK2 or p38α MAPK selectivity.

Mitogen-activated protein kinases (MAPKs), including extracellular signal-regulated kinase 2 (ERK2) and p38α MAP kinase (p38α MAPK), regulate various cellular responses. ERK2 is a drug target for treating many diseases, such as cancer, whereas p38α has attracted much attention as a promising drug target for treating inflammatory disorders. ERK2 is a critical off-target for p38α MAPK and vice versa. In this study, an allosteric ERK2 inhibitor with a benzothiazole moiety (compound 1) displayed comparable inhibitory activity against p38α MAPK. Crystal structures of these MAPKs showed that compound 1 bound to the allosteric site of ERK2 and p38α MAPK in distinct manners. Compound 1 formed a covalent bond with Cys162 of p38α MAPK, whereas this covalent bond was absent in the ERK2 complex even though the corresponding cysteine is conserved in ERK2. Structural dissection combined with computational simulations indicated that an amino acid difference in the allosteric site is responsible for the distinct binding modes of compound 1 with ERK2 and p38α MAPK. These structural insights underline the feasibility of developing highly selective and potent ERK2 and p38α MAPK inhibitors.

Design, Synthesis, and Molecular Docking of Novel Miscellaneous Chalcones as p38α Mitogen-Activated Protein Kinase Inhibitors.

New chalcones were synthesized and evaluated to serve as p38-α type of mitogen-activated protein kinase (MAPK) inhibitors. According to the National Cancer Institute, the findings indicated that at a 10 µM dosage, compounds 3a and 6 were the most active among all the compounds examined, with mean growth inhibition% of 94.83 and 58.49, respectively. In 5-dose testing, they showed anticancer activity in the micro-molar range with GI50 in the range of 1.41-46.1 and 2.07-31.3 µM, respectively. Besides, powerful activity, especially against the leukaemia cell lines and good selectivity to cancer cells compared to normal PCS-800-017 with a selectivity index=12.41 and 23.77, respectively. Compounds 3a and 6 inhibited p38α MAPK with IC50 values of 0.1462±0.0063 and 0.4356±0.0189 µM, correspondingly. 3a showed good inhibition for HL-60(TB) cells and induced cell cycle arrest in HL-60(TB) cells at the G2/M phase. Besides, it elevated the total apoptosis by 14.68-fold and increased the caspase-3 level by 3.52-fold compared with doxorubicin, which raised it by 4.30-fold, inducing apoptosis by acting as caspase-dependent inducers. These results suggest that 3a is a promising antiproliferative and p38α MAPK inhibitor, confirmed by molecular docking with high compatibility 3a with the p38α MAPK binding site.

Identification of new 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids as p38α MAPK inhibitors: Design, synthesis, antitumor evaluation, molecular docking and in silico studies.

In pursuit of discovering novel scaffolds that demonstrate potential inhibitory activity against p38α MAPK and possess strong antitumor effects, we herein report the design and synthesis of new series of 17 final target 5-(2,6-dichlorophenyl)-3-oxo-2,3-dihydro-5H-thiazolo[3,2-a]pyrimidine-7-carboxylic acids (4-20). Chemical characterization of the compounds was performed using FT-IR, NMR, elemental analyses and mass spectra of some representative examples. With many compounds showing potential inhibitory activity against p38α MAPK, two derivatives, 8 and 9, demonstrated the highest activity (>70 % inhibition) among the series. Derivative 9 displayed IC50 value nearly 2.5 folds more potent than 8. As anticipated, they both showed explicit interactions inside the kinase active site with the key binding amino acid residues. Screening both compounds for cytotoxic effects, they exhibited strong antitumor activities against lung (A549), breast (MCF-7 and MDA MB-231), colon (HCT-116) and liver (Hep-G2) cancers more potent than reference 5-FU. Their noticeable strong antitumor activity pointed out to the possibility of an augmented DNA binding mechanism of antitumor action besides their kinase inhibition. Both 8 and 9 exhibited strong ctDNA damaging effects in nanomolar range. Further mechanistic antitumor studies revealed ability of compounds 8 and 9 to arrest cell cycle in MCF-7 cells at S phase, while in HCT-116 treated cells at G0-G1 and G2/M phases. They also displayed apoptotic induction effects in both MCF-7 and HCT-116 with total cell deaths more than control untreated cells in reference to 5-FU. Finally, the compounds were tested for their anti-migratory potential utilizing wound healing assay. They induced a significant decrease in wound closure percentage after 24 h treatment in the examined cancer cells when compared to untreated control MCF-7 and HCT-116 cells better than 5-FU. In silico computation of physicochemical parameters revealed the drug-like properties of 8 and 9 with no violation to Lipinski's rule of five as well as their tolerable ADMET parameters, thus suggesting their utilization as potential future drug leads amenable for further optimization and development.

Crosstalk with renal proximal tubule cells drives acidosis-induced inflammatory response and dedifferentiation of fibroblasts via p38-singaling.

BACKGROUND: Tubulointerstitial kidney disease associated microenvironmental dysregulation, like acidification, inflammation and fibrosis, affects tubule cells and fibroblasts. Micromilieu homeostasis influences intracellular signaling and intercellular crosstalk. Cell-cell communication in turn modulates the interstitial microenvironment. We assessed the impact of acidosis on inflammatory and fibrotic responses in proximal tubule cells and fibroblasts as a function of cellular crosstalk. Furthermore, cellular signaling pathways involved were identified. METHODS: HK-2 (human proximal tubule) and CCD-1092Sk (human fibroblasts), in mono and coculture, were exposed to acidic or control media for 3 or 48 h. Protein expression of inflammation markers (TNF, TGF-ß and COX-2), dedifferentiation markers (N-cadherin, vinculin, ß-catenin and vimentin), fibrosis markers (collagen III and fibronectin) and phospho- as well as total MAPK levels were determined by western blot. Secreted collagen III and fibronectin were measured by ELISA. The impact of MAPK activation was assessed by pharmacological intervention. In addition, necrosis, apoptosis and epithelial permeability were determined. RESULTS: Independent of culture conditions, acidosis caused a decrease of COX-2, vimentin and fibronectin expression in proximal tubule cells. Only in monoculture, ß-Catenin expression decreased and collagen III expression increased in tubule cells during acidosis. By contrast, in coculture collagen III protein expression of tubule cells was reduced. In fibroblasts acidosis led to an increase of TNF, COX-2, vimentin, vinculin, N-cadherin protein expression and a decrease of TGF-ß expression exclusively in coculture. In monoculture, expression of COX-2 and fibronectin was reduced. Collagen III expression of fibroblasts was reduced by acidosis independent of culture conditions. In coculture, acidosis enhanced phosphorylation of ERK1/2, JNK1/2 and p38 transiently in proximal tubule cells. In fibroblasts, acidosis enhanced phosphorylation of p38 in a sustained and very strong manner. ERK1/2 and JNK1/2 were not affected in fibroblasts. Inhibition of JNK1/2 and p38 under coculture conditions reduced acidosis-induced changes in fibroblasts significantly. CONCLUSIONS: Our data show that the crosstalk between proximal tubule cells and fibroblasts is crucial for acidosis-induced dedifferentiation of fibroblasts into an inflammatory phenotype. This dedifferentiation is at least in part mediated by p38 and JNK1/2. Thus, cell-cell communication is essential for the pathophysiological impact of tubulointerstitial acidosis.

E. hellem Ser/Thr protein phosphatase PP1 targets the DC MAPK pathway and impairs immune functions.

Microsporidia are difficult to be completely eliminated once infected, and the persistence disrupts host cell functions. Here in this study, we aimed to elucidate the impairing effects and consequences of microsporidia on host DCs. Enterocytozoon hellem, one of the most commonly diagnosed zoonotic microsporidia species, was applied. In vivo models demonstrated that E. hellem-infected mice were more susceptible to further pathogenic challenges, and DCs were identified as the most affected groups of cells. In vitro assays revealed that E. hellem infection impaired DCs' immune functions, reflected by down-regulated cytokine expressions, lower extent of maturation, phagocytosis ability, and antigen presentations. E. hellem infection also detained DCs' potencies to prime and stimulate T cells; therefore, host immunities were disrupted. We found that E. hellem Ser/Thr protein phosphatase PP1 directly interacts with host p38α (MAPK14) to manipulate the p38α(MAPK14)/NFAT5 axis of the MAPK pathway. Our study is the first to elucidate the molecular mechanisms of the impairing effects of microsporidia on host DCs' immune functions. The emergence of microsporidiosis may be of great threat to public health.

Regulatory mechanisms underlying interleukin-6 expression in murine brown adipocytes.

Three types of adipocytes, white, brown, and beige, regulate the systemic energy balance through the storage and expenditure of chemical energy. In addition, adipocytes produce various bioactive molecules known as adipokines. In contrast to white adipocyte-derived molecules, less information is available on the adipokines produced by brown adipocytes (batokine). This study explored the regulatory expression of interleukin (IL)-6 in cell culture studies. Norepinephrine or a nonselective ß-adrenergic receptor agonist increased the expression of IL-6 in primary brown adipocytes and HB2 brown adipocytes. Treatment with forskolin (Fsk), an activator of the cAMP-dependent protein kinase (PKA) pathway (downstream signaling of the ß-adrenergic receptor), efficiently stimulated IL-6 expression in brown adipocytes and myotubes. Phosphorylated CREB and phosphorylated p38 MAP kinase levels were increased in Fsk-treated brown adipocytes within 5 min. In contrast, a long-term (∼60 min and ∼4 h) treatment with Fsk was required for increase in STAT3 phosphorylation and C/EBPß expression, respectively. The PKA, p38 MAP kinase, STAT3, and C/EBPß pathways are required for the maximal IL-6 expression induced by Fsk, which were verified by use of various inhibitors of these signal pathways. Vitamin C enhanced Fsk-induced IL-6 expression through the extracellular signal-regulated kinase activity. The present study provides basic information on the regulatory expression of IL-6 in activated brown adipocytes.

Connective tissue growth factor enhances TGF-ß1-induced osteogenic differentiation via activation of p38 MAPK in mesenchymal stem cells.

OBJECTIVES: Cellular differentiation is based on the effects of various growth factors. Transforming growth factor (TGF)-ß1 plays a pivotal role in inducing osteogenic differentiation of mesenchymal stem cells (MSCs). In this study, we investigated the influence of connective tissue growth factor (CTGF), known to function synergistically with TGF-ß1, on osteogenic differentiation in MSCs. METHODS: UE7T-13 cells were treated with TGF-ß1 and/or CTGF. Subsequently, protein levels of intracellular signaling pathway molecules were determined through western blot analysis. The mRNA expression levels of osteogenic differentiation markers were investigated using reverse transcription-quantitative polymerase chain reaction. Bone matrix mineralization was evaluated through alizarin red staining. RESULTS: Co-treatment with TGF-ß1 and CTGF resulted in the suppression of TGF-ß1-induced phosphorylation of extracellular signal-regulated kinase 1/2, an intracellular signaling pathway molecule in MSCs, while significantly enhancing the phosphorylation of p38 mitogen-activated protein kinase (MAPK). In MSCs, co-treatment with CTGF and TGF-ß1 led to increased expression levels of alkaline phosphatase and type I collagen, markers of osteogenic differentiation induced by TGF-ß1. Osteopontin expression was observed only after TGF-ß1 and CTGF co-treatment. Notably, bone sialoprotein and osteocalcin were significantly upregulated by treatment with CTGF alone. Furthermore, CTGF enhanced the TGF-ß1-induced mineralization in MSCs, with complete suppression observed after treatment with a p38 MAPK inhibitor. CONCLUSIONS: CTGF enhances TGF-ß1-induced osteogenic differentiation and subsequent mineralization in MSCs by predominantly activating the p38 MAPK-dependent pathway.

The P38MAPK/ATF2 signaling pathway is involved in PND in mice.

Accumulating evidence indicates that microglia-mediated neuroinflammation in the hippocampus contributes to the development of perioperative neurocognitive disorder (PND). P38MAPK, a point of convergence for different signaling processes involved in inflammation, can be activated by various stresses. This study aims to investigate the role of the P38MAPK/ATF2 signaling pathway in the development of PND in mice. Aged C57BL/6 mice were subjected to tibial fracture surgery under isoflurane anesthesia to establish a PND animal model. The open field test was used to evaluate the locomotor activity of the mice. Neurocognitive function was assessed with the Morris water maze (MWM) and fear conditioning test (FCT) on postoperative days 1, 3 and 7. The mice exhibited cognitive impairment accompanied by increased expression of proinflammatory factors (IL-1ß, TNF-α), proapoptotic molecules (caspase-3, bax) and microglial activation in the hippocampus 1, 3 and 7 days after surgery. Treatment with SB239063 (a P38MAPK inhibitor) decreased the expression of proinflammatory factors, proapoptotic molecules and Iba-1 in the CA1 region of the hippocampus. The number of surviving neurons was significantly increased. Inhibition of the P38MAPK/ATF2 signaling pathway attenuates hippocampal neuroinflammation and neuronal apoptosis in aged mice with PND, thus improving the perioperative cognitive function of the mice.

Design, synthesis, biological evaluation, and in silico studies of quinoxaline derivatives as potent p38α MAPK inhibitors.

A new series of quinoxaline derivatives possessing the hydrazone moiety were designed, synthesized, and screened for in-vitro anti-inflammatory activity by the bovine serum albumin (BSA) denaturation technique, and for antioxidant activity, by the (2,2'-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay. The synthesized compounds were also tested for p38α mitogen-activated protein (MAP) kinase inhibition. The in-vivo anti-inflammatory activity was assessed by the carrageenan-induced rat paw edema inhibition method. All the compounds (4a-n) exhibited moderate to high in-vitro anti-inflammatory activity. Compound 4a displayed the highest inhibitory activity in the BSA assay (83.42%) in comparison to the standard drug diclofenac sodium (82.90%), while 4d exhibited comparable activity (81.87%). The DPPH assay revealed that compounds 4a and 4d have free radical scavenging potential (74.70% and 74.34%, respectively) comparable to the standard butylated hydroxyanisole (74.09%). Furthermore, the p38α MAP kinase inhibition assay demonstrated that compound 4a is highly selective against p38α MAP kinase (IC50 = 0.042) in comparison to the standard SB203580 (IC50 = 0.044). The five most active compounds (4a-4d and 4f) with good in-vitro profiles were selected for in-vivo anti-inflammatory studies. Compounds 4a and 4d were found to display the highest activity (83.61% and 82.92% inhibition, respectively) in comparison to the standard drug diclofenac sodium (82.65% inhibition). These compounds (4a and 4d) also exhibited better ulcerogenic and lipid peroxidation profiles than diclofenac sodium. The molecular docking and molecular dynamics simulation studies were also performed and found to be in agreement with the p38α MAP kinase inhibitory activity.

LC-QToF chemical profiling of Euphorbia grantii Oliv. and its potential to inhibit LPS-induced lung inflammation in rats via the NF-κB, CY450P2E1, and P38 MAPK14 pathways.

Acute lung injury (ALI) is a life-threatening syndrome that causes high morbidity and mortality worldwide. The aerial parts of Euphorbia grantii Oliv. were extracted with methanol to give a total methanolic extract (TME), which was further fractionated into dichloromethane (DCMF) and the remaining mother liquor (MLF) fractions. Biological guided anti-inflammatory assays in vitro revealed that the DCMF showed the highest activity (IC50 6.9 ± 0.2 µg/mL and 0.29 ± 0.01 µg/mL) compared to. celecoxib (IC50 of 88.0 ± 1 µg/mL and 0.30 ± 0.01 µg/mL) on COX-1 and COX-2, respectively. Additionally, anti-LOX activity was IC50 = 24.0 ± 2.5 µg/mL vs. zileuton with IC50 of 40.0 ± 0.5 µg/mL. LC-DAD-QToF analysis of TME and the active DCMF resulted in the tentative identification and characterization of 56 phytochemical compounds, where the diterpenes were the dominated metabolites. An LPS-induced inflammatory model of ALI (10 mg/kg i.p) was used to assess the anti-inflammatory potential of DCMF in vivo at dose of 200 mg/kg and 300 mg/kg compared to dexamethasone (5 mg/kg i.p). Our treatments significantly reduced the pro-inflammatory cytokines (TNF-α, IL-1, IL-6, and MPO), increased the activity of antioxidant enzymes (SOD, CAT, and GSH), decreased the activity of oxidative stress enzyme (MDA), and reduced the expression of inflammatory genes (p38.MAPK14 and CY450P2E1). The western blotting of NF-κB p65 in lung tissues was inhibited after orally administration of the DCMF. Histopathological study of the lung tissues, scoring, and immunohistochemistry of transforming growth factor-beta 1 (TGF-ß1) were also assessed. In both dose regimens, DCMF of E. grantii prevented further lung damage and reduced the side effects of LPS on acute lung tissue injury.

Involvement of CD44 and MAPK14-mediated ferroptosis in hemorrhagic shock.

To elucidate the induction of ferroptotic pathways and the transcriptional modulation of pivotal genes in the context of hemorrhagic shock. The R software was used to analyze the GSE64711 dataset, isolating genes relevant to ferroptosis. Enrichment analyses and protein interaction networks were assembled. Using WGCNA hub genes were identified and intersected with ferroptosis-related genes, highlighting hub genes CD44 and MAPK14. In a rat hemorrhagic shock model, cardiac ROS, Fe2+, MDA, and GSH levels were assessed. Key ferroptotic proteins (SLC7A11/GPX4) in myocardial tissues were examined via western blot. Hub genes, CD44 and MAPK14, expressions were confirmed through immunohistochemistry. Analyzing the GSE64711 dataset revealed 337 differentially expressed genes, including 12 linked to ferroptosis. Enrichment analysis highlighted pathways closely related to ferroptosis. Using Genemania, we found these genes mainly affect ROS metabolism and oxidative stress response. WGCNA identified CD44 and MAPK14 as hub genes. Rat myocardial tissue validation showed significant cardiac damage and elevated ROS and MDA levels, and decreased GSH levels in the hemorrhagic shock model. The ferroptotic pathway SLC7A11/GPX4 was activated, and immunohistochemistry showed a significant increase in the expression levels of CD44 and MAPK14 in the hemorrhagic shock rat model. We demonstrated the presence of tissue ferroptosis in hemorrhagic shock by combining bioinformatics analysis with in vivo experimentation. Specifically, we observed the activation of the SLC7A11/GPX4 ferroptotic pathway. Further, CD44 and MAPK14 were identified as hub genes in hemorrhagic shock.

Fangji Fuling Decoction Alleviates Sepsis by Blocking MAPK14/FOXO3A Signaling Pathway.

OBJECTIVE: To examine the therapeutic effect of Fangji Fuling Decoction (FFD) on sepsis through network pharmacological analysis combined with in vitro and in vivo experiments. METHODS: A sepsis mouse model was constructed through intraperitoneal injection of 20 mg/kg lipopolysaccharide (LPS). RAW264.7 cells were stimulated by 250 ng/mL LPS to establish an in vitro cell model. Network pharmacology analysis identified the key molecular pathway associated with FFD in sepsis. Through ectopic expression and depletion experiments, the effect of FFD on multiple organ damage in septic mice, as well as on cell proliferation and apoptosis in relation to the mitogen-activated protein kinase 14/Forkhead Box O 3A (MAPK14/FOXO3A) signaling pathway, was analyzed. RESULTS: FFD reduced organ damage and inflammation in LPS-induced septic mice and suppressed LPS-induced macrophage apoptosis and inflammation in vitro (P<0.05). Network pharmacology analysis showed that FFD could regulate the MAPK14/FOXO signaling pathway during sepsis. As confirmed by in vitro cell experiments, FFD inhibited the MAPK14 signaling pathway or FOXO3A expression to relieve LPS-induced macrophage apoptosis and inflammation (P<0.05). Furthermore, FFD inhibited the MAPK14/FOXO3A signaling pathway to inhibit LPS-induced macrophage apoptosis in the lung tissue of septic mice (P<0.05). CONCLUSION: FFD could ameliorate the LPS-induced inflammatory response in septic mice by inhibiting the MAPK14/FOXO3A signaling pathway.

Function and inhibition of P38 MAP kinase signaling: Targeting multiple inflammation diseases.

Inflammation is a natural host defense mechanism that protects the body from pathogenic microorganisms. A growing body of research suggests that inflammation is a key factor in triggering other diseases (lung injury, rheumatoid arthritis, etc.). However, there is no consensus on the complex mechanism of inflammatory response, which may include enzyme activation, mediator release, and tissue repair. In recent years, p38 MAPK, a member of the MAPKs family, has attracted much attention as a central target for the treatment of inflammatory diseases. However, many p38 MAPK inhibitors attempting to obtain marketing approval have failed at the clinical trial stage due to selectivity and/or toxicity issues. In this paper, we discuss the mechanism of p38 MAPK in regulating inflammatory response and its key role in major inflammatory diseases and summarize the synthetic or natural products targeting p38 MAPK to improve the inflammatory response in the last five years, which will provide ideas for the development of novel clinical anti-inflammatory drugs based on p38 MAPK inhibitors.

Structural basis of a redox-dependent conformational switch that regulates the stress kinase p38α.

Many functional aspects of the protein kinase p38α have been illustrated by more than three hundred structures determined in the presence of reducing agents. These structures correspond to free forms and complexes with activators, substrates, and inhibitors. Here we report the conformation of an oxidized state with an intramolecular disulfide bond between Cys119 and Cys162 that is conserved in vertebrates. The structure of the oxidized state does not affect the conformation of the catalytic site, but alters the docking groove by partially unwinding and displacing the short αD helix due to the movement of Cys119 towards Cys162. The transition between oxidized and reduced conformations provides a mechanism for fine-tuning p38α activity as a function of redox conditions, beyond its activation loop phosphorylation. Moreover, the conformational equilibrium between these redox forms reveals an unexplored cleft for p38α inhibitor design that we describe in detail.

Ubiquinol attenuates γ-radiation induced coronary and aortic changes via PDGF/p38 MAPK/ICAM-1 related pathway.

Endothelial vascular injury is one of the most pivotal disorders emerging during radiotherapy. It is crucial to rely on strong antioxidants to defend against vascular damage. The current study was carried out to investigate the ameliorative effect of ubiquinol (Ubq) against gamma (γ)-radiation induced aortic and coronary changes, with highlighting its role in suppression of p38 mitogen activated protein kinase (MAPK). Exposure to γ-radiation was adopted as a potent detrimental model that induces vascular tissue damage. Concisely, male albino rats were irradiated at a dose level of 7 Gy and treated daily with Ubq (10 mg/kg/day, p.o.) for 7 days pre-and post-irradiation. At the end of the experiment, lipid profile, 8-hydroxydeoxyguanosine (8-OHdG), gene expression of intercellular adhesion molecule (ICAM-1), platelet derived growth factor (PDGF), p38 MAPK and matrix metalloproteinase-9 (MMP-9) were estimated. Exposure to radiation significantly deteriorates aortic and coronary tissues. Conversely, administration of Ubq significantly reduced serum t-cholesterol, LDL and triglycerides (p = 0.001). In addition, Ubq prevented oxidative DNA damage (8-OHdG) (p = 0.1) and reduced serum MMP-9 (p = 0.001) which contributed to the endothelial cells damage. The positive impact of Ubq was more apparent in suppression of both PDGF (p = 0.001) and p38 MAPK (p = 0.1) protein concentrations, leading subsequently in reduction of ICAM-1 (p = 0.001) gene expression. As a conclusion, vascular endothelial damage brought on by γ-radiation is one of the leading causes of coronary and aortic deteriorations which could be successfully mitigated by Ubq.

Mitochondrial p38 Mitogen-Activated Protein Kinase: Insights into Its Regulation of and Role in LONP1-Deficient Nematodes.

p38 Mitogen-Activated Protein Kinase (MAPK) cascades are central regulators of numerous physiological cellular processes, including stress response signaling. In C. elegans, mitochondrial dysfunction activates a PMK-3/p38 MAPK signaling pathway (MAPKmt), but its functional role still remains elusive. Here, we demonstrate the induction of MAPKmt in worms deficient in the lonp-1 gene, which encodes the worm ortholog of mammalian mitochondrial LonP1. This induction is subjected to negative regulation by the ATFS-1 transcription factor through the CREB-binding protein (CBP) ortholog CBP-3, indicating an interplay between both activated MAPKmt and mitochondrial Unfolded Protein Response (UPRmt) surveillance pathways. Our results also reveal a genetic interaction in lonp-1 mutants between PMK-3 kinase and the ZIP-2 transcription factor. ZIP-2 has an established role in innate immunity but can also modulate the lifespan by maintaining mitochondrial homeostasis during ageing. We show that in lonp-1 animals, ZIP-2 is activated in a PMK-3-dependent manner but does not confer increased survival to pathogenic bacteria. However, deletion of zip-2 or pmk-3 shortens the lifespan of lonp-1 mutants, suggesting a possible crosstalk under conditions of mitochondrial perturbation that influences the ageing process. Furthermore, loss of pmk-3 specifically diminished the extreme heat tolerance of lonp-1 worms, highlighting the crucial role of PMK-3 in the heat shock response upon mitochondrial LONP-1 inactivation.